RU2785766C2 - MULTIVALENT Fv-ANTIBODIES - Google Patents

Abstract

FIELD: biochemistry; immunology.

SUBSTANCE: present invention relates to biochemistry and immunology, in particular to a molecule of a tri-specific antibody containing a diabody unit built in a polypeptide chain containing at least six variable domains connected one after another. In some cases, two single-stranded Fv-fragments are connected distally to the diabody unit to form two additional antigen-binding sites. An antigen-binding molecule may be monomer of single polypeptide or multimeric polypeptide.

EFFECT: invention can be used for target impact on two different tumor antigens, wherein the third specificity activates a cytotoxic effect of effector cells, or binding of two effector cells and one tumor antigen; invention can be used in different ways for changing targets of a cytotoxic potential of effector cells of the immune system for destruction of tumor cells or infectious agents.

17 cl, 13 dwg, 1 tbl, 4 ex

Description

The present invention relates to a multivalent and multispecific Fv antibody derivative, in particular an Fv antibody molecule containing a diabody unit.

Bispecific antibodies are used to interact with two different therapeutic targets or perform two different functions. Such antibodies can be used, for example, to recruit immune effector cells, such as T or NK cells, to a particular target cell. Various antibody fragment-based molecules are known and are being investigated, for example in the treatment of cancer.

Bispecific antibodies can be constructed using only the variable domains of antibodies. For example, the linker sequence between the V _H - and V _L -domains can be shortened to such an extent that they could not stack on top of each other and associate in pairs, entering into an intramolecular interaction. Such short linkers, e.g., 2-12 residues, prevent said formation of a monomeric single chain variable fragment (scFv) molecule and favor intermolecular V _H /V _L coupling between complementary variable domains of different polypeptide chains to form a dimeric "diabody" (Holliger et al. , 1993, Proc. Natl. Acad. Sci. USA 90, 6444-6448). Such a diabody can be used to construct bispecific antibodies produced by the non-covalent association of two single chain polypeptide fusion products, each consisting of the V _H domain of one antibody connected by a short linker to the V _L domain of another antibody (or vice versa).

WO 03/025018 describes a bispecific antigen binding molecule whose structure is formed by identical single chain polypeptides with four binding domains. The V _H and V _L domains at the end of each polypeptide chain are linked by a short linker and are intermolecularly associated with the corresponding V _H and V _L domains of another polypeptide chain, while the other V _H and V _L domains of each polypeptide chain are intramolecularly linked to each other. with another within the same chain to form an antigen-binding scFv unit. Such constructs are homodimers, i.e. are composed of identical single-chain polypeptides that associate with each other in pairs.

In addition, trispecific antibodies that target two tumor antigens are desirable, which provides increased selectivity for cancer cells, preservation of healthy tissue, and leads to an increase in the range of doses and therapeutic applicability at which said drug substance can effectively kill cancer cells. For example, one trispecific antibody can be used to target two different tumor antigens, with the third specificity activating the cytotoxic effect of T cells or NK cells.

WO 2009/007124 describes a trispecific single chain Fv (scFv) fusion called a "triple body" containing three scFv fragments arranged in tandem having three different specificities (CD123 and CD33 (tumor markers) and one to CD16 on NK- cells). This molecule is further stabilized by disulfide bonds in the central scFv. Such molecules provide dual targeting to double positive tumor cells and univalent binding to CD16 on NK cells.

The present invention relates to a multivalent Fv antibody containing a diabody unit consisting of two pairs of variable domains associated with two antigen-binding sites. The term "antigen-binding site" refers to the antigen-binding site of an Fv fragment consisting of a VH/VL domain pair, ie. a VH/VL antigen-binding site, or a single-domain antigen-binding site. Each pair of variable domains is linked one after the other in a polypeptide. A diabody unit consists of one polypeptide (single chain diabody unit) or two polypeptides (diabody unit). At least one pair of variable domains is connected in the polypeptide to another variable domain located on the N-terminal side of this pair of variable domains, and to another variable domain - from the C-terminus. Accordingly, such a polypeptide contains at least four variable domains connected one behind the other, with two variable domains located next to each other connected one after the other, representing one pair of variable domains of the diabody unit, and one more variable domain is located on the side N -terminus of this pair of variable domains of the diabody unit, and other additional variable domains are located on the C-terminus side of this pair of variable domains of the diabody unit. The variable domain fused to the N-terminus of this pair of variable domains of the diabody unit may be a light chain variable domain (V _L ) or a heavy chain variable domain (V _H ) as well as a domain fused to the C-terminus of this pair of variable domains. domains of the diabody unit, may be a light chain variable domain (V _L ) or a heavy chain variable domain (V _H ). Such a polypeptide is an Fv polypeptide containing at least four consecutive variable domains, i. a variable domain connected to the N-terminus of a pair of variable domains of the diabody unit, attached at the C-terminus to another variable domain. In specific embodiments, six, eight, or ten variable domains are connected one behind the other in such an Fv polypeptide.

A multivalent Fv antibody is at least tetravalent and contains at least four antigen binding sites. Accordingly, an Fv polypeptide of an Fv antibody comprising at least four variable domains, wherein two of the at least four variable domains are a pair of two adjacent diabody unit variable domains associated with another pair of diabody unit variable domains with the formation of two (first and second) antigen-binding sites, contains at least an additional (third) variable domain at the N-terminus of the Fv polypeptide, which associates with the corresponding variable domain to form an additional (third) antigen-binding site, and at least an additional (fourth) a variable domain at the C-terminus of an Fv polypeptide that associates with the corresponding variable domain to form an additional (fourth) antigen-binding site. In some embodiments, at least four antigen-binding sites of a multivalent Fv antibody are formed by two Fv polypeptides, and in other embodiments, the at least four antigen-binding sites are formed by intramolecular folding of a single Fv polypeptide.

Thus, in some embodiments, each of the other variable domains N-terminally and C-terminally linked to a pair of diabody unit variable domains in the same polypeptide is part of an additional antigen-binding site. Accordingly, one pair of diabody unit variable domains (first pair) is associated with another pair of diabody unit variable domains (second pair) to form two antigen-binding sites (first and second antigen-binding sites), and an additional variable domain located at the N-terminus associates with the corresponding variable domain to form a third antigen-binding site, and an additional C-terminal variable domain associates with the corresponding variable domain to form a fourth antigen-binding site. Thus, such a multivalent Fv antibody is at least tetravalent. In some embodiments, the antigen binding site comprises an additional variable domain N-terminally and/or C-terminally linked to a pair of diabody unit variable domains in the scFv unit or single chain diabody unit. In other embodiments, additional variable domains N-terminally and C-terminally connected to a pair of diabody unit variable domains within the same polypeptide (first polypeptide) are associated with the corresponding V _H or V _L domains of another polypeptide ( second polypeptide) containing the second (other) pair of variable domains of the diabody unit, thereby forming two more (third and fourth) V _H /V _L antigen-binding sites between the variable domains of the two (first and second) polypeptides.

In some embodiments, the two pairs of variable domains of the diabody unit are a pair of light chain variable domains (V _L -V _L ) linked one behind the other in the polypeptide and a pair of heavy chain variable domains (V _H -V _H ) linked one after the other. in a polypeptide, wherein the V _L -V _L pair and the V _H -V _H pair associate into two (first and second) V _L /V _H antigen-binding sites.

In some embodiments, the multivalent antibody molecule contains a diabody unit, i. e. a pair of variable domains of a diabody unit integrated into a polypeptide chain containing at least six, for example six, eight or ten variable domains connected one after the other

The diabody unit consists of a pair of two variable domains connected one behind the other so that these domains cannot form the intramolecular structure of a functional Fv unit, i.e. V _H /V _L antigen-binding site, and instead associate with another pair of two variable domains connected one behind the other to form a bivalent dimer, i.e. diabody, providing two antigen-binding sites. Connecting a pair of light chain variable domains (V _L -V _L ) one after the other and a pair of heavy chain variable domains (V _H -V _H ) one after the other prevents intramolecular pairing of domains within each pair at the expense of domains of the same type, i. V _H -V _H or V _L - V _L . The rigid and compact structure of the diabody unit facilitates the preparation, correct folding (folding) of the multivalent antibody, and enhances the stability of the antibody. Such a diabody unit forms two V _H /V _L antigen-binding sites within the antibody molecule through two covalently linked V _H and V _L domains, which is beneficial to the stability of the antibody molecule as it results in a more compact molecule. In some embodiments, a pair of two variable domains is connected by a short linker.

In some cases, two single chain Fv fragments (scFv fragments) are distally linked to the diabody unit, forming two additional V _H /V _L antigen binding sites (FIGS. 1 and 2). Thus, such a multivalent antibody molecule is at least tetravalent because it contains at least four antigen-binding sites, two from the diabody unit and two from the two scFv units. Each of the distally located scFv fragments can be arranged in the polypeptide in the order V _H -V _L or V _L -V _H .

Such tetravalent antibody molecules are advantageous for the formation of tetravalent, pentavalent or hexavalent trispecific antibodies. These new trispecific antibodies can be used, for example, to recruit effector cells of the immune system to kill target cells, such as tumor cells or virus-infected cells. Since such trispecific antibodies according to the present invention are at least tetravalent, they have enhanced functional activity compared to trivalent and trispecific single chain fragments. The trispecific and tetravalent antibodies of the present invention bind bivalently via two of the four binding sites to both the target cell and effector cells of the immune system. For example, bivalent binding to a target cell increases not only avidity, but also the specificity of targeting if two of the three antigenic specificities are directed to two different antigens, for example, two different tumor antigens on the surface of the target cell. On the other hand, it is possible to modulate, in particular, increase the cytotoxic efficiency of the recruited effector cell of the immune system by divalent binding of the antibody to the effector cell of the immune system. In other cases, such a trispecific antibody may have two specificities for two different antigens on effector cells, and a third specificity for an antigen on a tumor cell, neuron, or virus-infected cell, or bind to and ultimately neutralize a soluble protein, such as a growth factor. cytokines or other ligands not associated with cells.

In some cases, the variable domains of the diabody unit, connected by a short linker that prevents intramolecular pairing, are light chain variable domains (V _L -V _L ) or heavy chain variable domains (V _H -V _H ) (Fig. 1 and 2). This particular domain arrangement has been found to facilitate the correct folding of the multispecific and multivalent antibody molecules of the present invention. In particular, for quadrivalent trispecific or tetraspecific Fv antibody molecules, these measures can be taken to properly associate an Fv antibody molecule containing variable domains for three or more different specificities and to prevent misassociation within a single polypeptide (monomer) or homodimerization between two identical Fv polypeptides instead of the correct heterodimerization of the first and second polypeptide (example 2). The present inventors obtained the correct association between variable domains to obtain three specificities and two different polypeptides containing variable domains by combining into a trispecific antibody molecule, for example, a diabody unit, formed by the first pair of two heavy chain variable domains linked by a short linker associated with the second a pair of two corresponding light chain variable domains connected by a short linker. Thus, such an arrangement of variable domains in a diabody unit, i.e. V _L -V _L in the first variable domain polypeptide and V _H -V _H in the second variable domain polypeptide, ensures the correct association and folding of a tetravalent and trispecific or tetraspecific Fv antibody. The authors of the present invention found that such an arrangement of V _L -V _L / V _H -V _H within a diabody unit or a single-chain diabody unit stimulates the correct folding of a long polypeptide containing more than six variable domains connected one after another to form an Fv antibody molecule ( eg Fig. 1), or correct folding and heterodimerization of two different length polypeptides to form a functional dimeric Fv antibody molecule (eg Figs 2, 5, 6a, 6b).

In further embodiments, at least one single chain diabody unit (scDb) is distally connected to the diabody unit to form at least one additional antigen binding site. Thus, at least one diabody unit polypeptide contains one diabody unit variable domain pair associated with at least one scDb in the polypeptide. For example, two scDb units are distally connected to a diabody unit containing an additional two antigen-binding sites per single chain diabody unit (FIGS. 4 and 5). Thus, such a multivalent antibody molecule is at least tetravalent because it contains at least four antigen-binding sites. Such a multivalent antibody is at least hexavalent in embodiments where at least two scDb units are linked to a diabody unit because two distally oriented scDb units contain four antigen binding sites and two antigen binding sites are in the diabody unit. Variable domains in each of the distal scDb units can be arranged in the order V _H -V _L -V _H -V _L , V _L -V _H -V _L -V _H , V _L -V _L -V _H -V _H or V _H -V _H -V _L -V _L in the polypeptide.

In some embodiments, the diabody unit consists of two pairs of variable domains located on two polypeptides associated with each other to form two antigen-binding sites, each of the two polypeptides containing at least one other single chain fragment and/or a single chain diabody fragment located distal to each of the two pairs of variable domains of the diabody unit. (FIGS. 6A, 6b, 7, 8a and 8b). Thus, such a multivalent antibody molecule is at least heptavalent because it contains at least five antigen-binding sites - two binding sites in a diabody unit, at least two binding sites in two scFv units or scDb units from the N-terminus and the C-terminus from the first pair of variable domains of the diabody unit and at least one binding site from the N-terminus and C-terminus of the other pair of variable domains of the diabody unit of another diabody unit polypeptide.

In some cases, the present invention relates to a multivalent antibody molecule containing a polypeptide containing at least six variable domains connected one behind the other, and the diabody unit containing the first two variable domains of the specified at least six variable domains is built into the specified polypeptide, and said first two variable domains of the diabody unit are associated with the other two variable domains of the diabody unit, i. e. the second two variable domains, with the formation of two antigen-binding sites. Said second two variable domains may be located in the same polypeptide as the first two variable domains, or in a separate second polypeptide associated with the first polypeptide. The two antigen-binding sites contained in the diabody unit are formed between the first and second variable domains, with each variable domain of the first variable domain pair forming an antigen-binding site with a different variable domain of the second variable domain pair. Accordingly, the diabody unit is formed by said first and second two variable domains, the two variable domains being joined by a short peptide linker in the first and also the second two variable domains to prevent intramolecular pairing.

Thus, in some embodiments, the present invention relates to a multivalent antibody molecule containing a polypeptide containing, i. comprising at least six variable domains connected one after another, wherein two variable domains of this polypeptide are linked by a peptide linker that prevents intramolecular pairing, and these two variable domains are associated with other two corresponding variable domains linked by a peptide linker that prevents intramolecular pairing, moreover, these the four variable domains form two antigen-binding sites between the four variable domains. For example, such an antibody is an Fv antibody, in particular a tetravalent and trispecific Fv antibody.

This antibody molecule is multivalent, i. contains more than one antigen-binding site. It is tetravalent if it contains four antigen-binding sites; pentavalent if it contains five antigen-binding sites, and hexavalent if it contains six antigen-binding sites. The term "quadrivalent" refers to an antibody molecule containing, in particular, consisting of four antigen-binding Fv sites, where each of the antigen-binding Fv sites contains a V _H /V _L pair containing a heavy chain variable domain (V _H ) and a light variable domain chains (V _L ) of the same antigenic specificity associated with each other. Thus, such a tetravalent antibody molecule contains at least eight antibody variable domains, namely four heavy chain variable domains (V _H ) and four light chain variable domains (V _L ). Because a tetravalent antigen binding molecule contains at least eight antibody variable domains, its molecular weight is greater than 100 Da, resulting in an increased half-life of such a molecule compared to trivalent and trispecific single chain Fv molecules.

In some cases, the antibody molecule is multispecific, ie. has specificity for different epitopes of antigens. In some cases, the antibody molecule is trispecific.

A trispecific and tetravalent antibody molecule contains an antigen-binding site specific for the first antigen epitope, an antigen-binding site specific for the second antigen epitope, and two antigen-binding sites specific for the third antigen epitope. Thus, such a trispecific and tetravalent antibody molecule has, i. includes different specificities for three different antigen epitopes. For example, such an antigen-binding molecule comprises a first antigen-binding site specific for a first antigen epitope, a second antigen-binding site specific for a second antigen epitope, third and fourth antigen-binding sites specific for a third antigen epitope. A pentavalent and trispecific antibody molecule contains two antigen-binding sites specific for the first antigen epitope, two antigen-binding sites specific for the second antigen epitope, and one antigen-binding site specific for the third antigen epitope. Alternatively, a pentavalent and trispecific antibody molecule may contain three antigen-binding sites against the first antigen epitope and one antigen-binding site each for the second and third antigen epitopes. A hexavalent and trispecific antibody molecule, in some embodiments, contains two antigen-binding sites for each of the three antigen epitopes, or alternatively, one to three antigen-binding sites for each of the three antigen epitopes.

In some instances, the antibody molecule is an Fv antibody molecule. "Fv antibody" refers to an immunoglobulin Fv derivative containing only the variable (V) domains of an antibody, but not containing antibody constant regions or fragments thereof. Each light chain variable domain (V _L ) is associated with a corresponding heavy chain variable domain (V _H ) to form an antigen binding Fv site (VH/V _L antigen binding site). The variable domains of an antibody are connected one after the other in a fusion polypeptide via a peptide linker or peptide bond. Fv antibody, i.e. the antigen-binding molecule of the present invention may be a single polypeptide monomer or a multimeric polypeptide. Multimeric antigen-binding molecule, ie. An Fv antibody, in particular a multivalent Fv antibody, may be, for example, a dimer containing two polypeptides, a trimer containing three polypeptides, or a tetramer containing four polypeptides. A dimer is heterodimeric if it consists of two polypeptides of different amino acid composition, or homodimeric if it consists of two identical polypeptides.

The term "polypeptide" refers to a polymer of amino acid residues linked by amide bonds. Preferably the polypeptide is a single chain fusion straight protein. The variable (Fv) domains of an antibody in a polypeptide are linked one behind the other. The term "Fv-polypeptide" means a hybrid polypeptide in which the variable (Fv) domains of an antibody are linked one behind the other. The polypeptide may contain adjacent amino acid residues in addition to the N-terminus and/or C-terminus. For example, the polypeptide may contain a marker sequence, preferably at the C-terminus, which can be used in purification as well as detection of the polypeptide. An example of a marker sequence is a His marker, e.g., a His marker consisting of six His residues, a FLAG marker, e.g. octapeptide DYKDDDDK (SEQ ID NO:38), or a STREP® II marker, e.g. octapeptide WSHPQFEK (SEQ ID NO:39) , or a C-marker, for example, EPEA tetrapeptide (SEQ ID NO:40). In multimeric antigen-binding molecules, different marker sequences can be used for different polypeptides, for example, a His marker for the first polypeptide and a FLAG marker for the second polypeptide of the dimeric molecule. In some embodiments, the polypeptide may contain variable domains that provide antigen-binding sites, as well as antibody constant domains, such as C _L , C _H and/or Fc domains. For example, such embodiments may include an Fv polypeptide or Fv antibody combined with at least one antibody constant domain, eg, an Fc domain. In further embodiments, the variable domain-containing polypeptide may be coupled to another agent, such as a toxin, immunomodulator, or signal generating agent.

The term "linker" refers to a peptide connecting two adjacent variable domains in a polypeptide between the C-terminus of one domain and the N-terminus of another adjacent domain, or vice versa. From the point of view of the amino acid composition, a peptide is chosen that does not interfere with the formation of Fv-, i.e. VH/VL antigen-binding sites, as well as not interfering with multimerization, for example, dimerization of multispecific, for example, trispecific molecules. For example, a linker containing glycine and serine residues typically provides protease resistance. In some embodiments, peptide linkers (G ₂ S)x are used, in which, for example, x=1-20, for example, peptide linkers are used (G ₂ S), (G ₂ S) ₂ , (G ₂ S) ₃ , (G ₂ S) ₄ , (G ₂ S) ₅ , (G ₂ S) ₆ , (G ₂ S) ₇ or (G ₂ S) ₈ or (G ₃ S)x, in which, for example, x=1 -15, or use peptide linkers (G ₄ S)x, in which, for example, x=1-10, preferably 1-6. The amino acid sequence of the linker can be optimized to improve antigen binding and polypeptide production yield, for example, using phage display techniques.

The length of the linkers affects the flexibility of the antigen-binding polypeptide dimer. The desired flexibility of the antigen-binding polypeptide dimer depends on the density of the target antigen and the availability of the target antigen, i. epitopes of the target antigen. Longer linkers allow for more flexible antigen-binding polypeptides with more flexible antigen-binding sites. The effect of linker length on the formation of dimeric antigen-binding polypeptides is described, for example, in Todorovska et al., 2001 Journal of Immunological Methods 248:47-66; Perisic et al., 1994 Structure 2:1217-1226; Le Gall et al., 2004, Protein Engineering 17:357-366 and WO 94/13804.

The diabody unit is inserted into the polypeptide of the antibody molecule. The term "diabody unit" refers to a divalent Fv molecule consisting of two pairs of variable domains, a first pair and a second pair, which associate to form two VL/VH antigen binding sites. Each pair of variable domains are connected one after the other in a polypeptide. In some embodiments, a divalent Fv module consists of a first and second pair of two variable domains adjacent to each other, with each pair of two variable domains connected by a short peptide linker preventing intramolecular association between the variable domains connected by the short linker. The first pair of variable domains is forced to associate with the second pair of variable domains, forming two antigen-binding Fv sites with two pairs of variable domains. Accordingly, each of the two antigen-binding Fv sites is formed by one variable domain of the first pair of variable domains and one variable domain of the second pair of variable domains. Thus, such a diabody unit contains at least one antigen-binding site of two variable domains indirectly connected by a short peptide linker 3, 3a (FIGS. 1 and 2). Two pairs of variable domains located next to each other are located on two separate polypeptides with the formation of a dimeric unit of the diabody (Fig. (2, 3, 5, 8), or two pairs of variable domains located next to each other are located on one and the same polypeptide to form a single chain diabody unit (FIGS. 1, 4).In each pair of variable domains, a short linker 3,3a connects the C-terminus of one variable domain to the N-terminus of another variable domain, or vice versa.In each pair, the variable domains can be oriented from the N- to the C-terminus, for example, V _L -V _H , V _H -V _L , V _H -V _H or V _L -V _L , and the two variable domains of the pair have specificities against different epitopes of antigens or specificities against one and the same antigen epitope.In some cases, two variable domains are directly linked by a peptide bond between the N-terminus of one variable domain and the C-terminus of the other variable domain of the pair. containing two variable domains in each of the first and second pair of variable domains of the diabody unit, is one that prevents intramolecular association between the variable domains connected by the linker. Such a linker is "short", ie. consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or approximately 12 amino acid residues. In the case of 0 amino acid residues, the linker is a peptide bond. Such a short linker favors proper dimerization between the two pairs of variable domains and the formation of two antigen-binding Fv sites. Shortening the linker to about 12 amino acid residues or less will generally prevent adjacent regions of the same polypeptide chain from interacting with each other. In one embodiment of the present invention, these linkers consist of from about 3 to about 12, for example, from 5 to 10, in particular, from 7 to 9 contiguous amino acid residues. The linker length can be adjusted to obtain a particular orientation of the domains in the diabody unit. For example, a (G ₂ S) ₂ linker can be used for a V _H -V _L pair or a V _L -V _H pair, and a (G ₂ S) ₃ linker can be used for a V _H -V _H pair or a V _L - V _L or a (G ₂ S) ₂ linker can be used for a V _H -V _H pair and a (G ₂ S) ₃ linker can be used for a V _L -V _L pair of a diabody unit (or vice versa). Incidentally, it is in principle possible that two polypeptides containing a linker of more than 12 amino acid residues between the variable domains of an antibody pair will dimerize correctly with each other (see, for example, Le Gall et al., 2004, Protein Engineering 17:357- 366).

In some embodiments, the diabody unit is a single chain diabody unit (FIG. 1). A "single chain diabody unit" consists of a first pair of variable domains connected to a second pair of variable domains by a long linker allowing intramolecular association of the first and second pairs of variable domains, in accordance with the "long linker" used in scFv units and described in the next paragraph. For example, such a long linker may consist of more than 12, in particular from about 15 to about 50, preferably from about 15 to about 35, in particular from about 15 to about 25 contiguous amino acid residues.

In some embodiments, the single-stranded diabody domains in a polypeptide linked one behind the other can be arranged in the order V _L -V _H -V _L -V _H , V _L -V _L -V _H -V _H , V _H -V _H -V _L -V _L or V _H -V _L -V _H -V _L from the N-terminus to the C-terminus of a single-stranded diabody unit.

In some embodiments, an antibody molecule of the present invention consists of a single polypeptide containing a single chain diabody unit (FIGS. 1, 4). In specific embodiments, the antibody molecule comprises at least three single chain diabody units linked one after the other in a polypeptide (FIG. 4). In other embodiments, the antibody molecule of the present invention comprises at least one single chain diabody unit distally linked to the diabody unit (FIG. 5).

"Single chain Fv unit (scFv)" means an antigen-binding Fv site formed by a single chain polypeptide fragment consisting of a light chain variable domain (V _L ) and a heavy chain variable domain (V _H ). Variable domains can be oriented as V _L -V _H or V _H -V _L from the N-terminus to the C-terminus of the scFv unit. The variable domains are connected by a peptide linker between the C-terminus of one variable domain and the N-terminus of another variable domain, or vice versa. The peptide linker is long and flexible (generally about 12 or more amino acid residues) for intramolecular folding and formation of an antigen-binding Fv site. Additional amino acid residues provide additional flexibility. For example, such a long linker may consist of more than 12, in particular from about 15 to about 50, preferably from about 15 to about 35, in particular from about 15 to about 25, contiguous amino acid residues. The linker length can be adjusted for the specific orientation of the domains from the N-terminus to the C-terminus of the scFv unit. For example, a (G ₂ S) ₆ linker can be used for the scFv V _H -V _L unit, and a (G ₂ S) ₇ linker can be used for the scFv V _L -V _H unit.

The scFv unit is linked to the diabody unit by a peptide linker between the variable domain of the scFv unit and the variable domain of the diabody unit. Choose the length of the peptide linker to avoid steric interference between adjacent variable domains and maintain the stability of the molecule; this length may be, for example, from 5 to 50, in particular from 5 to 35, preferably at least 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous amino acid residues.

In some cases, the diabody unit is located in the center of the antibody molecule to facilitate folding and improve the stability of the antibody molecule. In such cases, the diabody unit is N-terminally and C-terminally connected to an additional distal variable domain. In some embodiments, the diabody unit is coupled to two (FIGS. 1, 2, 8a, 8b), three (FIGS. 6a, 6b), or four (FIG. 7) scFv units. In some cases, an antibody molecule, in particular a trispecific Fv antibody molecule, comprises a polypeptide containing at least six variable domains, said polypeptide having an scFv unit at the N-terminus, an scFv unit at the C-terminus, and a first pair of two variable domains of the unit diabody between two scFv units (FIGS. 1 and 2). This first pair of two variable domains of the diabody unit does not associate with the formation of an antigen-binding Fv site. For example, if the diabody unit is a dimeric diabody unit, each of the two variable domains of the first pair of adjacent variable domains is connected to an additional variable domain. In particular, each of the two variable domains of the first pair of adjacent variable domains is connected to an additional variable domain of the scFv unit (FIG. 2). In other cases, one or both of the variable domains of the second pair of adjacent variable domains can be further connected to an additional variable domain (FIGS. 6a, 6b, 7). If the diabody unit is a single chain diabody unit, the first pair of adjacent variable domains is N-terminally connected to an additional variable domain, and the second pair of adjacent variable domains is C-terminally connected to an additional variable domain. domain. In particular, the first pair of adjacent variable domains is N-terminally connected to the additional variable domain of the scFv unit, and the second pair of adjacent variable domains is connected at the C-terminus to the additional variable domain of another scFv unit (Fig. 1 ). In other embodiments, the first pair of variable domains of the first single chain diabody unit is N-terminally connected to an additional variable domain of the second single chain diabody unit, and the second pair of variable domains of the first single chain diabody is connected at the C-terminus to the additional variable domain of the third single chain diabody unit in the polypeptide, containing three single-chain diabody units connected one behind the other (Fig. 4) In other cases, at least one additional variable domain may be located between the first and second pairs of variable domains located next to each other.

In some cases, if the diabody unit is a single chain diabody unit, the multivalent antibody consists of a single polypeptide containing two variable domains adjacent to each other, ie. a first pair of variable domains adjacent to each other associated with two other variable domains of the same polypeptide adjacent to each other, i. e. the second pair of variable domains (Fig. 1). Such an antibody structure favors the production of multispecific, in particular bi-, tri- or tetraspecific antibodies. In some embodiments, such a multivalent, in particular, trispecific Fv antibody contains at least eight variable domains connected one after the other from the N-terminus to the C-terminus, with the first and second variable domains forming an scFv unit at the N-terminus, and said scFv unit at the N-terminus is C-terminally connected to the third variable domain of the first pair of variable domains of the diabody unit containing the third variable domain and the fourth variable domain, said fourth variable domain being C-terminally connected to the fifth variable domain of the second pair of variable domains a diabody unit containing fifth and sixth variable domains, wherein said sixth variable domain is C-terminally connected to the seventh variable domain of the scFv unit at the C-terminus, and said scFv unit is formed by the seventh and eighth variable domains (FIG. 1). Variable domains can be located, for example, in one of the following orientations from the N-terminus to the C-terminus of the polypeptide: V _H -V _L -V _L -V _H -V _L -V _H -V _L -V _H , V _H - V _L -V _H -V _H -V _L -V _L -V _L -V _H , V _L -V _H -V _L -V _H -V _L -V _H -V _H -V _L or V _H -V _L -V _L -V _L -V _H -V _H -V _L -V _H . In a specific embodiment, the orientation of one pair of variable domains of the diabody unit is V _H -V _H and the orientation of the other pair of variable domains of the diabody unit is V _L -V _L (FIG. 1).

Quadrivalent embodiments of an antibody molecule consisting of a single polypeptide containing the eight variable domains described above favor the production of trispecific antibodies. For example, such trispecific antibodies may have first and second specificities for an antigen on a target cell, such as a tumor cell, and a third specificity for an effector cell of the immune system, such as a T or NK cell. In other cases, such a trispecific antibody may have two specificities for different antigens on the effector cells, and a third specificity for the antigen on the tumor cell. In some embodiments, the two distal scFv units may have first and second specificities for a target cell, and the diabody unit located between the two scFv units may have specificity for an effector cell of the immune system; ie, from the N-terminus to the C-terminus, the first and second variable domains have a first specificity for the target cell; the third and fifth variable domains have a third immune system effector cell specificity, the fourth and sixth variable domains have a third immune system effector cell specificity, and the seventh and eighth variable domains have a second target cell specificity (FIG. 1). In other embodiments, the two distal scFv units may have a specificity for an effector cell of the immune system, and the two antigen-binding sites formed by the diabody unit may have two specificities for a target cell; ie, from the N-terminus to the C-terminus, the first and second, as well as the seventh and eighth variable domains have a third specificity for the effector cell of the immune system; the third and fifth variable domains have a first target cell specificity, and the fourth and sixth variable domains have a second target cell specificity. In further alternative embodiments, a single polypeptide may contain more than eight, eg 10, 12 or more variable domains, and contain more than two scFv units and/or more than one diabody unit.

In additional embodiments, a multivalent antibody molecule consisting of a single polypeptide contains three single chain diabody units connected one behind the other (FIG. 4). Such an antibody molecule contains at least 12 variable domains connected one after another from the N-terminus to the C-terminus. In a particular embodiment, the orientation of one pair of variable domains of the second single chain diabody unit is V _H -V _H and the orientation of the other pair of variable domains of the second single chain diabody unit is V _L -V _L (Fig. 4). Such an antibody molecule is hexavalent and may contain antigen binding sites for one to six different antigenic specificities, in particular two or three different antigenic specificities.

In some cases, an antibody molecule, in particular an Fv antibody, contains a diabody unit in the format of a dimeric diabody unit. In such cases, the antibody molecule is a dimer of two polypeptides, with the first pair of two variable domains of the diabody unit, located next to each other, built into the first polypeptide containing at least six variable domains connected one behind the other, and the specified first pair of variable domains , located next to each other, is associated with another second pair of two variable domains located next to each other in the second polypeptide. The first and second polypeptides are preferably non-covalently linked (FIGS. 2, 3, 5, 6a, 6b, 7, 8a and 8b8b). However, in some cases, the first and second polypeptides may be covalently linked, for example, by a disulfide bond or a chemical linker.

In some embodiments, the first polypeptide contains at least six variable domains and the second polypeptide contains at least two variable domains (FIG. 2). In such embodiments, the second polypeptide is contained within a diabody unit and, preferably non-covalently, is associated with another pair of two adjacent variable domains inserted into the first polypeptide. In embodiments where the first polypeptide chain consists of six variable domains and the second polypeptide consists of two variable domains, these variable domains can be arranged from the N-terminus to the C-terminus of the polypeptide, for example, in the following orientation: V _H -V _L - V _H -V _H -V _L -V _H (first polypeptide) and V _L -V _L (second polypeptide); V _L -V _H -V _H -V _H -V _H -V _L (first polypeptide) and V _L -V _L (second polypeptide); V _H -V _L -V _L -V _L -V _H -V _L (first polypeptide) and V _H -V _H (second polypeptide); V _L -V _H -V _L -V _L -V _H -V _L (first polypeptide) and V _H -V _H (second polypeptide) or V _H -V _L -V _L -V _L -V _L -V _H ( first polypeptide) and V _H -V _H (second polypeptide). Diabody units containing one pair of two variable domains in the V _H -V _H orientation and another pair of two variable domains in the V _L -V _L orientation favor the correct folding of, in particular, multispecific, eg, trispecific, antibody molecules.

Quadrivalent embodiments of an antibody molecule comprising a first polypeptide containing at least six variable domains and a second polypeptide containing at least two variable domains, as described above, favor the production of trispecific antibodies. Because of the different size of the first and second polypeptides, these polypeptides can be easily isolated from the supernatant separately. For example, such trispecific antibodies may have first and second specificities for a target cell, such as a tumor cell, and a third specificity for an effector cell of the immune system, such as a T or NK cell. In other embodiments, the trispecific antibody molecules have first and second specificities for a first and second viral antigen or viral antigen epitope, and a third specificity for an effector cell, such as a T or NK cell. In further embodiments, the trispecific antibody molecule has a first specificity for a viral antigen, a second specificity for an antigen on a target cell, and a third specificity for an effector cell, such as a T or NK cell. In other instances, such a trispecific antibody may comprise a first and a second specificity for effector cells, such as NK cells or T cells, and a third specificity for a target cell, such as a tumor antigen on a tumor cell or a viral antigen. The first and second effector cell specificities may correspond to different antigens or epitopes of the same antigen on effector cells of the same type.

In some embodiments, the two distal scFv units formed in the first polypeptide may have first and second specificities for the target cell, and the diabody unit located between these two scFv units and formed by the first and second polypeptides may have specificity for the effector cell immune system; ie, from the N-terminus to the C-terminus in the first polypeptide, the first and second variable domains have the first specificity for the target cell; the third and fourth variable domains have a third immune system effector cell specificity, the fifth and sixth variable domains have a second target cell specificity, and in the second polypeptide, the first and second variable domains have a third immune system effector cell specificity (Fig. 2). In other embodiments, the two distal scFv units in the first polypeptide containing six variable domains may have specificity for an effector cell of the immune system, and the two antigen-binding sites formed by the diabody unit of the first polypeptide, as well as the two variable domains of the second polypeptide, may have different specificities in against the target cell; those. from the N-terminus to the C-terminus of the first polypeptide, the first and second, as well as the fifth and sixth variable domains have a third specificity in relation to the effector cell of the immune system; the third variable domain has a first target cell specificity, the fourth variable domain has a second target cell specificity, and in the second polypeptide the first variable domain has a second target cell specificity and the second variable domain has a first cell specificity -targets.

In additional embodiments, the first polypeptide contains at least six variable domains, and the second polypeptide contains at least four (Fig. 6a, 6b) or six (Fig. 7) variable domains. In such embodiments, the first polypeptide comprises a first pair of diabody unit variable domains and the second polypeptide comprises a second pair of diabody unit variable domains that are preferably non-covalently associated with each other to form two antigen-binding sites between the first and second polypeptides. In embodiments where the first polypeptide chain consists of six variable domains and the second polypeptide consists of four variable domains, a pair of variable domains located adjacent to each other in the diabody unit in the second polypeptide is connected to the scFv unit, forming an additional antigen-binding site on the side N-terminus or C-terminus of the second polypeptide (FIGS. 6a, 6b). Such embodiments are pentavalent and may contain antigen binding sites for one to five different antigenic specificities, in particular two or three different antigenic specificities. Diabody units containing one pair of two variable domains in the V _H -V _H orientation in the first polypeptide and another pair of two variable domains in the V _L -V _L orientation in the second polypeptide favor the correct folding, in particular of multispecific, e.g., trispecific, antibody molecules. .

In other embodiments, where the first polypeptide chain consists of six variable domains and the second polypeptide consists of four variable domains, a pair of diabody unit variable domains located adjacent to each other in the second polypeptide are connected at the N-terminus and C-terminus with units scFv to form two additional antigen binding sites (FIG. 7). Such embodiments are hexavalent and may contain antigen binding sites for one to six different antigenic specificities, in particular two or three different antigenic specificities. In specific embodiments, the diabody unit contains one pair of variable domains in the V _H -V _H orientation in the first polypeptide and another pair of variable domains in the V _L -V _L orientation in the second polypeptide. In further alternative embodiments, a multivalent, eg, trispecific Fv antibody molecule may comprise more than one dimeric diabody unit. In such alternative embodiments, the first polypeptide may contain at least six variable domains, and the second polypeptide may contain at least four (Fig. 3) or six (Fig. 8a, 8b) variable domains. In embodiments where the first polypeptide contains at least six variable domains and the second polypeptide contains four variable domains, the four variable domains of the second polypeptide, located next to each other, are associated with the corresponding four variable domains, located next to each other and built into the first polypeptide, thereby forming a tandem of two diabody units located next to each other. The remaining at least two additional variable domains of the first polypeptide are distal to the tandem diabody units and form the scFv unit (FIGS. 8a, 9b). In other embodiments, where the first polypeptide contains six variable domains and the second polypeptide contains six variable domains, the six variable domains of the first polypeptide are associated with the corresponding six variable domains of the second polypeptide, thereby forming a triplet of diabody units located next to each other (Fig. 3). The latter embodiment, consisting of a first and a second polypeptide, where each polypeptide contains six variable domains, is advantageous in that it may allow the production of a trispecific antibody in a homodimer format, i.e. two identical polypeptides associated with each other and providing bivalent binding for each of the three antigenic specificities.

In some cases, the tetravalent trispecific Fv antibody is presented as a tandem diabody. Such a trispecific Fv antibody molecule consists of a first and a second polypeptide, each polypeptide containing four variable domains linked to each other. In such an Fv antibody molecule, the length of the linker prevents intramolecular pairing of the variable domains so that the molecule cannot fold on itself, but is forced to mate, i.e. associates with complementary domains of another polypeptide. The domains are arranged in such a way that the respective V _H and V _L domains associate with each other during this dimerization. Despite the absence of intermolecular covalent bonds, this dimer is highly stable after formation, remains intact and does not revert back to the monomeric form. In some embodiments, the trispecific Fv antibody molecule comprises a dimeric diabody unit, wherein the orientation of one pair of adjacent variable domains is V _H -V _H , and the orientation of the other pair of adjacent variable domains is V _L -V _L . This orientation of the variable domains in the diabody unit facilitates the correct association of the two trispecific polypeptides. In particular, this orientation makes it possible to obtain molecules of trispecific Fv antibodies in the format of a tandem diabody, since it is a heterodimer of two different polypeptides. Accordingly, this orientation favors the correct heterodimerization of the trispecific tandem diabody. Thus, in some embodiments, the trispecific Fv antibody molecule is a tandem diabody (FIG. 3). In such trispecific tandem diabodies, the variable domains of the first and second polypeptide can be arranged, for example, in the following orientations from the N-terminus to the C-terminus of the polypeptide: V _L -V _H -V _H -V _L (first polypeptide) and V _H -V _L -V _L -V _H (second polypeptide) or vice versa (Fig. 3). Such trispecific antibodies in tandem diabody format may have first and second specificities for a target cell, eg a tumor cell, and a third specificity for an effector cell of the immune system, eg a T or NK cell. In other cases, such a trispecific antibody may have two specificities for different antigens on the effector cells, and a third specificity for the antigen on the tumor cell. In some embodiments, variable domains located on the outer regions of the polypeptides may have first and second specificities for the target cell, and two variable domains located in the central part of the polypeptide between the variable domains located on the outer regions of the polypeptides have a third specificity for effector cells of the immune system; ie, from the N-terminus to the C-terminus in the first polypeptide, the first variable domain has the first specificity for the target cell; the second and third variable domains have a third specificity for an effector cell of the immune system, the fourth variable domain has a second specificity for a target cell, and in the second polypeptide, the first variable domain has a second specificity for a target cell, the second and third variable domains have a third specificity for the effector cell of the immune system, and the fourth variable domain has the first specificity for the target cell (Fig. 3). In other embodiments, variable domains located on the outer regions of the polypeptides may contain a third specificity for the effector cell of the immune system, and two variable domains located in the central part of the polypeptide between the variable domains located on the outer regions of the polypeptides, have the first and second specificities for target cells; those. from the N-terminus to the C-terminus of the first polypeptide, the first variable domain has a third epitope specificity for an effector cell of the immune system; the second variable domain has a first target cell specificity, the third variable domain has a second target cell specificity, the fourth variable domain has a third immune system effector cell specificity, and in the second polypeptide, the first variable domain has a third effector specificity. cells of the immune system, the second variable domain has a second specificity for a target cell, the third variable domain has a first specificity for a target cell, and the fourth variable domain has a third specificity for an effector cell of the immune system. In other embodiments, variable domains containing a third specificity for an effector cell of the immune system are located laterally in the antibody molecule; ie, from the N-terminus to the C-terminus of the first polypeptide, the first and second variable domains have a third specificity for an effector cell of the immune system; the third variable domain has a first target cell specificity, the fourth variable domain has a second target cell specificity, and in the second polypeptide the first variable domain has a second target cell specificity, the second variable domain has a first cell specificity targets, and the third and fourth variable domains have a third specificity for the effector cell of the immune system.

In additional embodiments, the tandem diabody is conjugated to at least one additional antigen-binding domain, in particular at least one scFv unit at the N-terminus or C-terminus of the polypeptide. In preferred embodiments, the tandem diabody is conjugated to at least one scFv unit via a peptide linker. In particular, such an antibody molecule consists of a first and a second polypeptide, both polypeptides consisting of six variable domains, and each of the first polypeptide and the second polypeptide contains an scFv unit from the N-terminus of each polypeptide (Fig. 8b) or the C-terminus of each polypeptide (Fig. 8a). Such antibody molecules are hexavalent and may contain antigen binding sites for one to six different antigenic specificities, in particular two or three different antigenic specificities. In specific embodiments, the diabody unit located in the central part of the antibody molecule contains one pair of variable domains in the V _H -V _H orientation in the first polypeptide and another pair of variable domains in the V _L -V _L orientation in the second polypeptide. In some cases, the multivalent antibody molecule, in particular the multivalent Fv antibody molecule described herein, is at least trispecific and at least tetravalent antibody molecule. Such an antibody molecule contains at least two antigen-binding sites that are specific for the same antigen epitope. Due to this, avidity is increased, i.e. the strength of the interaction between an antigen epitope and an antigen-binding molecule. The avidity of trispecific antibody molecules can be further enhanced by pentavalent and hexavalent embodiments of the present invention. Pentavalent molecules may contain at least two binding sites for two or three epitope specificities of a trispecific antibody, and hexavalent embodiments may contain two antigen binding sites for each of the three epitope specificities. Alternatively, polyspecificity, ie. the number of specificities can be increased by tetravalent, pentavalent and hexavalent implementations. For example, an antibody molecule may be tetraspecific. The benefits of increased avidity are increased stability of interaction and retention on the target. For example, if the target is a cytotoxic effector cell of the immune system, such as a T cell or NK cell, increased avidity may result in an increased cytotoxic potential of the antibody molecule. In yet another example, if the target is a tumor cell, increased avidity improves retention time on the target and reduces the rate of dissociation from the target. In a specific embodiment of the present invention, a trispecific and tetravalent Fv antibody molecule comprises first and second antigen-binding sites specific for two different tumor cell antigen epitopes of the same type, and third and fourth antigen-binding sites specific for an effector antigen epitope. cells of the immune system, such as T cells or NK cells. Such an antibody molecule provides increased specificity as well as avidity for a particular tumor cell type, as well as increased avidity to activate or inhibit a receptor on an effector cell of the immune system, resulting in a beneficially increased specific cytotoxic potential of the antigen-binding molecule Binding to two different tumor antigen epitopes leads to an increase in the specificity of the targeted effect and the expansion of the therapeutic window by reducing non-specific toxicity. Thus, the present invention provides multispecific antibody molecules that advantageously increase avidity and/or biological activity due to the presence of at least two antigen-binding sites for at least one epitope specificity, for example, two binding sites for one, two or three epitope specificities, in other words, targets.

It is important to note that, despite the structural complexity, such multispecific, eg trispecific and multivalent, eg tetravalent, antibody molecules of the present invention are stable.

In other instances, such a multispecific, eg trispecific, antibody may have two specificities for different antigens on the effector cell, eg, NK cell or T cell, and a third specificity for the antigen on the tumor cell.

Thus, the antibody molecule of the present invention can be used in a variety of ways to alter the targets of the cytotoxic potential of effector cells of the immune system to kill tumor cells or infectious agents, such as cells infected with viruses. In some embodiments, a multispecific, eg, trispecific, antibody molecule can bind to two different antigen epitopes on the target. For example, these two different epitopes may be on the same antigen to prevent escape mutants or increase efficiency, or the two epitopes may be on two different target antigens. In some embodiments, a trispecific antibody molecule can bind to two different antigen epitopes on effector cells of the immune system. For example, the first antigen-binding site has specificity for an activating receptor, such as CD16, CD16A, or CD3, and the second antigen-binding site has specificity for a co-stimulatory antigen, such as CD137, OX-40, or CD28. In another example, the first antigen binding site is specific for CD16 or CD16A and the second antigen binding site is specific for another activating receptor on NK cells, eg NKG2D, DNAM, NCR.

In yet another embodiment, a trispecific, in particular Fv antibody molecule comprises a first antigen-binding site specific for an antigen epitope on a tumor cell, a second antigen-binding site specific for an antigen epitope on an effector cell of the immune system, and a third antigen-binding site specific for to an antigen epitope on a soluble protein selected from the group of growth factors, cytokines, chemokines, mitogens and albumins. Examples of such a soluble protein are IL-6, BAFF, APRIL, TGF-beta, IL-10, VEGF-A, TGF-alpha, EGF, HB-EGF, heregulins, angiopoietin-2, and human serum albumin (HSA).

In an alternative embodiment, the antibody molecule comprises one antigen-binding site specific for an antigen epitope present on one cell type and three antigen-binding sites specific for antigen epitopes on one or more cell types.

"Effector cells" are cells of the immune system capable of stimulating or activating cytotoxicity, phagocytosis, antigen presentation, cytokine release. Such effector cells are, for example, T cells, natural killer (NK) cells, gamma delta (gd) T but not limited to α-cells, natural killer T cells (NKT), granulocytes, monocytes, macrophages, dendritic cells, innate lymphoid cells (ILCs), and antigen presenting cells.Examples of specificities for effector cells include CD2, CD3, and CD3 subunits eg CD3s, CD5, CD28 and other components of the T cell receptor (TCR) or CD134 (OX40) for T cells; CD16A, CD25, CD38, CD44, CD56, CD69, CD94, CD335 (NKp46), CD336 (NKp44 ), CD337 (NKp30), NKp80, NKG2A, NKG2C and NKG2D, DNAM, NCR for NK cells; CD18, CD64 and CD89 for granulocytes; CD18, CD32, CD47, CD64, CD89 and mannose receptor for monocytes and macrophages; CD64 and mannose receptor for dendritic cells to; as well as CD35, but are not limited to them. In some embodiments of the present invention, these specificities, ie. cell surface molecules of effector cells are suitable for mediating cell killing upon binding of a multispecific, eg, trispecific antibody molecule to such a cell surface molecule, and thereby inducing cytolysis or apoptosis.

The CD3 antigen is a component of the T cell receptor complex on T cells. In the case where the specificity for the effector cell is CD3, binding of the antigen-binding molecule of the present invention to CD3 causes cytotoxic activity of T cells. Binding of an antibody molecule to CD3 and to a target cell, such as a tumor cell, can induce lysis of the target cell.

The CD16A antigen (FcγRIMA) is a receptor expressed on the surface of NK cells. NK cells have intrinsic cytolytic activity, and binding of an antibody molecule of the present invention to CD16 or CD16A can trigger the cytotoxic activity of the NK cell towards the target.

A "target" is a site at which an antigen epitope is located to which an antibody molecule is to bind. Examples of targets are soluble agents, antigens or cells, infectious agents such as viral or bacterial antigens such as those derived from dengue virus, herpes simplex virus, influenza virus, HIV, HCV, CMV, or antigens on cells that facilitate the entry of viruses and bacteria. , or cells, eg, neurons, displaying antigens, or cells carrying autoimmune targets, eg, IL-2/IL2R, an autoimmune marker or an autoimmune antigen, or tumor cells. In embodiments where at least one of the antigen-binding sites is specific for an effector cell, the target may be a tumor cell against which the activity of the effector cell can be tuned to induce or activate an appropriate biological, eg, immune response.

Suitable specificities in the case of tumor cells can be represented by tumor antigens and cell surface antigens on the corresponding tumor cell, For example, certain tumor markers. The term "tumor antigen" as used herein includes tumor-associated antigen (TAA) and tumor-specific antigen (TSA). The term "tumor-associated antigen" (TAA), as used herein, refers to a protein present on tumor cells as well as on normal cells during the embryonic period (embryonic oncoantigens) and postpartum in certain organs, but in much more lower concentration than on tumor cells. TAAs may also be present in the stroma in close proximity to the tumor cell, but they are expressed in low amounts in the stroma of other parts of the body. In contrast, the term "tumor-specific antigen" (TSA) refers to a protein expressed by tumor cells. The term "cell surface antigen" refers to any antigen molecule or fragment thereof that can be recognized by an antibody on the surface of a cell.

Examples of specificities for tumor cells include CD19, CD20, CD26, CD29, CD30, CD33, CD52, CD200, CD267, EGFR, EGFR2, EGFR3, EGFRvIII, HER2, HER3, IGFR, IGF-1R, Ep-CAM, PLAP, Thomson antigen -Friedenreich (TF), TNFRSF17, gpA33, MUC-1 (mucin), IGFR, CD5, IL-4-R alpha, IL13-R, FcεRI, MHC class I complexes/peptide, and IgE, but are not limited to.

The antibody molecules of the present invention, in which the tumor specificity is directed to the CD19 antigen, can be used for immunotherapy of B-cell malignancies, since the CD19 antigen is expressed on almost all B-line malignancies from lymphoblastic leukemia (ALL, ALL) to non-Hodgkin's lymphoma (NHL ).

Antibody molecules of the present invention, in which the tumor specificity is directed against CD30, may be particularly useful in the treatment of Hodgkin's disease and T-cell lymphomas.

The antibody molecules of the present invention, in which the tumor specificity is directed against the epidermal growth factor receptor (EGFR) or a mutant form of EGFRvIII, can be used in the treatment of gliomas, breast, ovarian, prostate, lung, head and neck tumors; liver diseases, for example, hepatocellular cancer, cirrhosis of the liver or chronic hepatitis.

To increase the half-life of the antibody molecules of the present invention in the body, the antibody molecule can optionally be combined with albumin, such as human serum albumin (HSA), or pegylated, sialylated, pasylated, or glycosylated (see, for example, Stork et al., 2008, J Biol Chem 283:7804-7812). In some embodiments, the antibody molecule is at least trispecific and contains at least one, eg, one or two antigen-binding sites that are specific for albumin, eg, HSA. Such at least trispecific antibody molecule may be, for example, a tetravalent, pentavalent or hexavalent antibody molecule.

Variable domains having specificity for epitopes on target cells or effector cells can be obtained by selecting variable fragments (Fv) specific for the antigens of interest. These procedures can be performed, for example, by screening single chain Fv phage display libraries or using hybridoma technology. For example, IgM-based human scFv phage display libraries can be subjected to several rounds of in vitro selection to be enriched in binding agents specific for the desired antigen. The affinity of the selected scFvs can then be increased by affinity maturation.

In some embodiments of the present invention, at least one, preferably all of the variable domains of an antibody are fully human, humanized, or chimeric domains. Humanized antibodies can be obtained using conventional methods, such as CDR transplantation (see, for example, Antibody engineering: methods and protocols / edited by Benny K.C. Lo; Benny K.C. II Series: Methods in molecular biology (Totowa, NJ.). Thus , a person skilled in the art can easily obtain a humanized or fully human version of the antigen binding molecule and variable domains from non-human sources, such as mice or non-primates, using standard molecular biology techniques known in the art to reduce immunogenicity and improving the efficacy of said antigen binding molecule in the human immune system.In a preferred embodiment of the present invention, all variable domains of an antibody are humanized or fully human, most preferably, the antibody molecule of the present invention is humanized or fully human. Czech. The term "fully human" as used herein means that the amino acid sequences of the variable domains and the peptides that bind the variable domains in the polypeptide originate or can be found in the human body. In some embodiments of the present invention, the variable domains, but not the peptides that bind the variable domains, may be human domains or humanized antibody domains.

One of ordinary skill in the art can easily design and prepare the antibody molecule described herein using established techniques and standard methods known in the art, see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.; The Protein Protocols Handbook, edited by John M. Walker, Humana Press Inc. (2002); or Antibody engineering: methods and protocols / edited by Benny K.C. lo; Benny K.C. II Series: Methods in molecular biology (Totowa, N.J.); Antibody Engineering / edited by Roland E. Kontermann and Stefan Dubel, Springer Verlag Berlin Heidelberg (2010)).

An antibody molecule according to any of the embodiments described herein can be obtained by expression of polynucleotides encoding individual polypeptide chains that form an antibody molecule. Thus, further embodiments of the present invention are polynucleotides, eg DNA or RNA, encoding polypeptides of the antibody molecule described herein above.

Said polynucleotides can be constructed using methods known to those skilled in the art, for example, by combining genes encoding antibody variable domains separated by peptide linkers or directly linked by peptide linkages of polypeptides in a genetic construct operably linked to a suitable promoter and optionally a suitable transcription terminator. , and their expression in bacteria or other appropriate expression system, for example CHO cells. Depending on the vector system and host chosen, any set of suitable transcription and translation elements can be used, including constitutive and inducible promoters. The promoter is chosen to direct the expression of the polynucleotides in the appropriate host cell.

The polynucleotides can be introduced into vectors, preferably expression vectors, which are an additional embodiment of the present invention. These recombinant vectors can be constructed in accordance with methods well known to those skilled in the art.

A variety of expression vector/host systems can be used to insert and express polynucleotides encoding the polypeptide chains of the present invention. Examples of expression vectors for expression in E. coli are pSKK (LeGalI et al., J Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

Thus, the antibody molecule described herein can be obtained by introducing a vector encoding the polypeptides described above into a host cell and culturing said host cell under conditions under which said polypeptides are expressed, they can be isolated and optionally additionally clear.

In a further embodiment, the present invention provides compositions, eg, pharmaceutical compositions, comprising an antibody molecule as described above and at least one additional component.

In further embodiments, the antibody molecules are intended for use as a medicament or diagnostic agent. In particular, antibody molecules are intended for use in immunotherapy. For example, antibody molecules are intended for use in the treatment of a neoplastic, viral, or neurodegenerative disease. Thus, the present invention further includes a method of treating an individual by immunotherapy, in particular an individual suffering from diseases selected from neoplastic, viral or neurodegenerative diseases, comprising the step of administering an antibody molecule in accordance with the present invention.

The present invention further provides a method in which an antibody molecule, in particular a composition comprising the antibody molecule described herein above and at least one additional component, is administered at an effective dose to a subject, e.g., a patient, for the treatment of cancer (e.g. , non-Hodgkin's lymphoma, chronic lymphocytic leukemia). The antigen binding molecule of the present invention may be intended for use as a medicament.

Brief description of drawings:

The figure 1 shows a tetravalent trispecific antigen-binding Fv molecule, consisting of a single polypeptide containing eight variable domains connected one after another in the orientation V _H -V _L -V _H -V _H -V _L -V _L -V _L -V _H from the N-terminus of 7 to the C-terminus of 8 of the polypeptide. This domain orientation is provided as an example only, other arrangements as described above are possible. Two single chain Fv (scFv) units 2 are distally connected to the single chain diabody unit 1 by peptide linkers 5, wherein the light chain variable domain (V _L ) of one scFv 2 unit is connected at the C-terminus by peptide linker 5 to the N-terminus of the heavy chain variable domain (V _H ) of the first pair of variable domains of the diabody unit 1. The V _L of the scFv unit 2 is connected at the N-terminus to the V _H unit of the scFv by a long peptide linker 4. The V _H of the first pair of variable domains is connected by a short peptide linker 3 to another V _H domain of the first variable pair domains. The first pair of variable domains is connected at the C-terminus by a long peptide linker 4 to the N-terminus of the second pair of diabody variable domains, both of which are V _L domains. Two V _L domains of the second pair of variable domains are connected by a short peptide linker 3. The second pair of diabody variable domains is C-terminally attached to the N-terminus of the V _L domain of another scFv 2 unit located at the C-terminus of the polypeptide. The scFv 2 unit at the C-terminus of the polypeptide consists of a V _L domain connected by a long linker 4 to the V _H domain. The two scFv 2 units contain antigen-binding sites with different specificities, and the diabody 1 unit contains two antigen-binding sites with the same specificity.

The figure 2 shows a tetravalent trispecific antigen-binding Fv molecule, consisting of the first polypeptide containing six variable domains connected one after the other in the orientation V _H -V _L -V _H -V _H -V _L -V _H from the N-terminus 7 to C-terminus 8 of the first polypeptide, and a second polypeptide containing two variable domains connected one after another in the V _L -V _L orientation from the N-terminus 7a to the C-terminus 8a of the second polypeptide. This orientation of the domains is given only as an example, and other arrangements described above are possible. The antigen-binding Fv molecule contains a diabody 1 dimeric unit formed by a first pair of two variable domains embedded in a first polypeptide associated with a second pair of two variable domains in a second polypeptide. Each of the two variable domains of the first pair of two variable domains inserted into the first polypeptide is connected to the scFv 2 unit. In each of the scFv 2 units, the variable domains are connected by a long peptide linker 4. The N-terminal scFv 2 unit is connected at the C-terminus by the peptide linker 5 with the N-terminus of the first pair of variable domains of the diabody unit 1. The first pair of variable domains consists of two V _H -domains connected by a short peptide linker 3. The first pair of variable domains of the diabody unit 1 is connected at the C-terminus by a peptide linker 5 with the N-terminus of the unit scFv 2 located at the C-terminus of the first polypeptide. The two scFv 2 units contain antigen-binding sites with different specificities, and the diabody 1 unit contains two antigen-binding sites with the same specificity.

Figure 3 shows a tetravalent trispecific antigen-binding Fv molecule in the format of a trispecific tandem diabody consisting of a first and a second polypeptide forming a non-covalently associated heterodimer. The tandem diabody contains a dimeric diabody unit 1 formed by a pair of V _H -V _H domains located in the central part of the first polypeptide, with a pair of V _L -V _L domains located in the central part of the second polypeptide. The variable domains in the first and second polypeptides are connected one after another by short peptide linkers 3, 3a from the N-terminus 7 to the C-terminus 8 in the orientation V _L -V _H -V _H -V _L in the first polypeptide and from the N-terminus 7a to C-terminus 8a in the V _H -V _L -V _L -V _H orientation in the second polypeptide. The variable domains located at the N-terminus and C-terminus contain antigen-binding sites with different specificities at the N-terminus and C-terminus, and the diabody 1 unit located in the central part contains two antigen-binding sites with the same specificity.

Figure 4 shows a hexavalent antigen-binding Fv molecule consisting of a single polypeptide containing twelve variable domains connected one after the other from the N-terminus 7 to the C-terminus 8 of the polypeptide. Domain orientation is for exemplary purposes only, other arrangements are possible as indicated for each pair of variable domains adjacent to each other. Two units of single-chain diabody 1a, 1b are distally connected to a unit of single-chain diabody 1c by peptide linkers 5, and the variable domain of one unit of single-chain diabody 1a is connected at the C-terminus by peptide linker 5 to the N-terminus of the variable domain of the first pair of variable domains of the diabody unit 1c, and the variable domain the domain of the single chain diabody unit 1b is connected at the N-terminus by linker 5 to the C-terminus of the second pair of variable domains of the single chain diabody unit 1c. The first and second pairs of variable domains of each of the three units of the single-chain diabody 1a, 1b, 1c are connected to each other by a long peptide linker 4. In each of the units of the single-chain diabody 1a, 1b, 1c, one of the variable domains of the first pair of variable domains is connected by a short peptide linker 3 with another variable domain of the first pair of variable domains, and two variable domains of the second pair of variable domains are connected by a short peptide linker 3.

The figure 5 shows a hexavalent antigen-binding Fv molecule, consisting of a first polypeptide containing 10 variable domains connected one after another from the N-terminus 7 to the C-terminus 8 of the first polypeptide, and the second polypeptide containing two variable domains connected one after another from the N-terminus of 7a to the C-terminus of 8a of the second polypeptide. The specific orientation of the domain in this figure is for example only, and other arrangements are indicated for each pair of variable domains located next to each other. The antigen-binding Fv molecule contains a dimeric diabody unit 10 formed by a first pair of two variable domains of the first polypeptide associated with a second pair of two variable domains in the second polypeptide. Each of the two variable domains of the first pair of two variable domains of the first polypeptide is linked to a single chain diabody unit 1a, 1b.

In each of the units of the single-chain diabody 1a, 1b, two pairs of variable domains located next to each other are connected by a long peptide linker 4. diabody unit 10. The first pair of variable domains consists of two V _H domains connected by a short peptide linker 3. The first pair of variable domains of the diabody unit 10 is connected at the C-terminus by a peptide linker 5 to the N-terminus of the single-stranded diabody unit 1b located at the C-terminus first polypeptide.

The figure 6 shows a pentavalent antigen-binding Fv molecule, consisting of a first polypeptide containing six variable domains connected one after another from the N-terminus 7 to the C-terminus 8 of the first polypeptide, and the second polypeptide containing four variable domains connected one after another from the N-terminus of 7a to the C-terminus of 8a of the second polypeptide. Domain orientation is for example only, for each pair of variable domains adjacent to each other other arrangements are possible as shown in the figure. The antigen-binding Fv molecule contains a dimeric diabody unit 10 formed by a first pair of two variable domains of the first polypeptide associated with a second pair of two variable domains in the second polypeptide. Each of the two variable domains of the first pair of two variable domains of the first polypeptide is connected to the scFv 2 unit. In each of the scFv 2 units, the variable domains are connected by a long peptide linker 4. The N-terminal scFv 2 unit is attached at the C-terminus by an N-terminal peptide linker 5 of the first pair of variable domains of the diabody unit 10. The first pair of variable domains consists of two variable domains connected by a short peptide linker 3. The first pair of variable domains of the diabody unit 10 is connected at the C-terminus by a peptide linker 5 to the N-terminus of the scFv unit 2 located on C at the end of the first polypeptide. A) the second polypeptide containing four variable domains consists of a second pair of variable domains of the diabody unit 10 connected to the scFv 2 unit at the C-terminus, and B) the second polypeptide containing four variable domains consists of the second pair of variable domains of the diabody unit 10 connected to the scFv 2 unit at the N-terminus.

The figure 7 shows a hexavalent antigen-binding Fv molecule consisting of a first polypeptide containing six variable domains connected one after another from the N-terminus 7 to the C-terminus 8 of the first polypeptide, and a second polypeptide containing six variable domains connected one after another from the N-terminus of 7a to the C-terminus of 8a of the second polypeptide. Domain orientation is for example only, for each pair of variable domains adjacent to each other other arrangements are possible as shown in the figure. The antigen-binding Fv molecule contains a dimeric diabody unit 10 formed by a first pair of two variable domains of the first polypeptide associated with a second pair of two variable domains in the second polypeptide. Each of the variable domains of the diabody unit 10 is linked to an scFv 2 unit in the first and second polypeptide. In each of the scFv 2 units, the variable domains are connected by a long peptide linker 4. Each of the four scFv 2 units is connected by a peptide linker 5 to the variable domain of the diabody unit 10. This first pair of variable domains consists of two variable domains connected by a short peptide linker 3, and the second the variable domain pair of the diabody unit consists of two variable domains connected by a short 3a peptide linker.

Figure 8 shows a hexavalent antigen-binding Fv molecule containing a tandem diabody unit consisting of a first and a second polypeptide forming a non-covalently associated dimer. The tandem diabody contains a dimeric diabody unit 10a formed by a pair of domains located in the central part of the first polypeptide with a pair of variable domains located in the central part of the second polypeptide. The variable domains in the first and second polypeptide are connected one after another by short peptide linkers 3, 3a from the N-terminus 7 to the C-terminus 8 in each polypeptide. Domain orientation is for example only, for each pair of variable domains adjacent to each other other arrangements are possible as shown in the figure. Each of the first and second polypeptides contains an scFv 2 unit connected by a linker 5 to a tandem diabody unit. A) scFv 2 units are attached at the C-terminus of the first and second polypeptide. C) scFv 2 units are attached at the N-terminus of the first and second polypeptide.

Figure 9 shows the analysis of aTriFlex products by SDS-PAGE. Long and short polypeptides are well expressed (arrows), a) aTriFlex_140 and aTriFlex_142, b) aTriFlex_138 and c) aTriFlex_139.

Figure 10 shows IMAC cleaning of aTriFlex products: a) aTriFlex_140, b) aTriFlex_138 and c) and aTriFlex_142 and d) aTriFlex_139.

Figure 11 shows the analysis of aTriFlex products by SDS-PAGE. Long and short polypeptides are well expressed. A) aTriFlex_101, B) aTriFlex_102, C) aTriFlex_103 and D) aTriFlex_104.

Figure 12 shows the binding of trispecific antibody molecules to single or double positive cell lines. Single-positive CD19+/CD30-Raji and CD19-/CD30+ KARPAS-299 cells, as well as double-positive CD19+/CD30+ MEC-1 cells, were stained with serial dilutions of trispecific antibody molecules; antibodies bound to the cell surface detected the anti-His mAb followed by treatment with FITC-conjugated goat anti-mouse IgG. Average fluorescence intensity values determined by flow cytometry were used to calculate K _D values by non-linear regression. K _D values for trispecific antibody molecules were determined in two independent experiments.

Figure 13 shows the cytotoxic activity of trispecific antibody molecules against single or double positive cell lines. A 4-hour calcein-releasing cytotoxicity assay was performed on single positive CD19+/CD30- Raji and CD19-/CD30+ KARPAS-299 cells, and double positive CD19+/CD30+ MEC-1 target cells using enriched NK cells as effector cells. cells in the presence of serial dilutions of said trispecific antibody molecules. Fluorescent calcein released from lysed target cells was used to calculate antibody-mediated target cell lysis and model sigmoid dose-response curves to determine EC ₅₀ values by non-linear regression. The EC ₅₀ values for the aTriFlex constructs were determined in two independent experiments and plotted.

The examples below further illustrate the present invention without limiting the scope of the present invention.

EXAMPLE 1

Cloning of expression DNA constructs encoding trispecific antigen-binding Fv molecules

For the expression of trispecific constructs in CHO cells, the coding sequences of all molecules were cloned as single or dual gene constructs in an appropriately modified mammalian cell expression system derived from the pcDNA5/FRT vector. A vector with two promoters was used to clone and express two gene constructs (heterodimeric trispecific constructs). For trispecific single gene constructs, the conventional pcDNA5/FRT expression vector was used. Briefly, the sequences of the genes encoding the V _H and V _L domains separated by different linkers (long and short linkers) were synthesized using Life Technologies GeneArt (Regensburg, Germany) and subcloned. The resulting constructs were digested with various restriction enzymes (BamHI, HindIII, XhoI, EcoRV, PacI), or various V _H /V _L domains binding to effectors and targets were amplified by PCR with appropriate primers. Thereafter, the various overlapping DNA fragments and the linearized scaffold 2-promoter vector or the pcDNA5/FRT vector were joined together using a Gibson Assembly in a single isothermal reaction. The design of all expression constructs contained the coding sequences for an N-terminal signal peptide and a C-terminal hexahistidine (6xHis) marker for the first gene cassette or a C-terminal FLAG marker for the second gene cassette to facilitate antibody secretion and purification, respectively. The sequences of all constructs were confirmed by DNA sequencing at GATC (Konstanz, Germany) using a pair of primers 5'-AGAGCTCGTTTAGTGAACCG-3' and 5'-TCATGTCTGGATCCGGCCTTG-3' for 1 gene cassette or 5'-GCCTGGAGACGCCATCC-3' and 5'-GCAGAATTCCACCACACTGG- 3' for gene cassette 2.

Host cell culture

Flp-In CHO cells (Life Technologies), derived from Chinese hamster ovary CHO-KI cells (ATCC, CCL-61) (Kao and Puck, 1968), were cultured in Ham's F-12 medium supplemented with L-glutamine, 10% FBS and 100 μg/ml Zeocin. Adherent cells were separated using 0.25% trypsin-EDTA and subcultured according to standard cell culture protocols provided by Life Technologies.

To adapt to growth in suspension, cells were separated from tissue culture flasks and placed in serum-free HyClone CDM4 CHO medium for subsequent incubation in shake flasks at 37°C, 5% CO ₂ and 120 rpm. The standard culture medium for suspension-adapted Flp-In CHO host cells was HyClone CDM4 CHO supplemented with L-glutamine, HT supplement, penicillin/streptomycin, and 100 μg/ml Zeocin. Suspension-adapted cells were frozen-preserved in 10% DMSO medium and tested for negative Mycoplasma assay using the MycoAlert Mycoplasma Detection Kit (Lonza).

Obtaining pools of stably transfected cells

Flp-In CHO recombinant cell lines stably expressing secreted trispecific antibodies from one or two genes were obtained by transfection of host cells adapted to the suspension. For this, cells were placed in standard medium without zeocin one day prior to co-transfection with expression plasmids (2.5 μg) encoding the desired protein (pcDNA5/FRT) and Flp recombinase (pOG44, Life Technologies) using polyethyleneimine (PEI). Briefly, vector DNA and transfection reagent were mixed at a DNA:PEI ratio of 1:3 (µg/µg) in a total volume of 100 µl OptiMEM I medium and incubated for 10 minutes, and then 2×10 ⁶ Flp-In CHO cells were added, suspended in 1 ml of CHO-S-SFMII medium (Life Technologies). After 24 h of incubation, the selection of stably transfected cells was started by adding 500 μg/ml of hygromycin B after diluting the cultures to a density of 0.1×10 ⁶ viable cells/ml in CHO-S-SFMII medium and seeding into T75 culture flasks. Flp recombinase mediates the insertion of expression constructs into the genome of Flp-In CHO cells at the inserted FRT site by site-specific DNA recombination (O'Gorman et al., 1991). During selection, viable cell density was measured twice a week, cells were centrifuged and resuspended in fresh selective medium at a maximum density of 0.1×10 ⁶ viable cells/ml. Pools of cells stably expressing recombinant protein products were isolated after 2-3 weeks of selection; at this point the cells were transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins was confirmed by protein gel electrophoresis in cell culture supernatant using Criterion Stain-Free technology (Bio-Rad). Stable cell pools were cryopreserved in medium containing 50% ProFreeze (Lonza) and 7.5% DMSO.

Production of recombinant protein during periodic cultivation of CHO cells in suspension with the addition of a substrate

Recombinant proteins were produced during 10-day batch culture of stably transfected CHO cell lines with the addition of substrate by secretion into the cell culture supernatant. For this purpose, pools of cells stably expressing trispecific antibodies were seeded at an initial density of 6⋅10 ⁵ cells/ml in a standard culture medium in polycarbonate Erlenmeyer flasks with gas-permeable lids (Corning) and incubated at 37°C and 5% CO ₂ with stirring at 140 rpm In batch culture with substrate addition, 40 ml/l ActiCHO Feed A and 4 ml/l ActiCHO Feed B (GE Healthcare) were added to the medium on day 0 (the day the culture started) and twice the amount of these additions on days 3, 5 and 7. Cell culture supernatants were harvested after 10 days of culture at >75% culture viability. Sampling of production cultures was performed the day before feeding and cell density and viability were assessed. On the day of collection, the cell culture supernatant was cleared by centrifugation and vacuum filtration (0.22 μm) through Millipore Express PLUS membrane filters (Millipore) before further use.

Purification of trispecific Fv antibodies

Trispecific antibodies were isolated from the clarified supernatant of CHO cell cultures in a two step procedure including IMAC and preparative size exclusion chromatography. If necessary, a third chromatographic step (FLAG affinity chromatography) was used for further fine purification. For IMAC, the clarified non-seed liquid was loaded onto a HisTrap Sepharose column. After washing with Tris/NaCl buffer (pH 7.5), the protein was eluted in a three step gradient using 15 mM, 125 mM and 500 mM imidazole, respectively. Fraction purity was analyzed by size exclusion HPLC and SDS-PAGE. Fractions showing acceptable purity were pooled and subjected to preparative gel filtration using a Superdex 200 preparative column. In some cases, FLAG affinity chromatography was used for further fine purification. Thus, the protein was loaded onto a FLAG affinity column, washed with PBS and eluted using glycine/HCl buffer at pH 3.5. Eluate fractions containing purified trispecific antibodies were pooled, dialyzed against 10 mM sodium acetate (pH 5.0) and concentrated by ultrafiltration to a typical concentration of approximately 1 mg/ml. Purity and homogeneity (approx. 90%) of final samples were assessed by SDS-PAGE under reducing and non-reducing conditions, followed by immunoblot analysis using antibodies specific for the His marker and FLAG marker, as well as analytical size exclusion HPLC. Purified proteins were stored in aliquots at -80°C until use.

Examples of biophysical data for trispecific Fv antibody molecules are described in Example 3; aTriFlex_101 (Example 3a)), aTriFlex_102 (Example 3b)), aTriFlex_103 (Example 3c)) and aTriFlex_104 (Example 3d) are shown in Table 1. Results of SDS-PAGE fraction electrophoresis are shown in Figure 11: All trispecific tetravalent Fv antibody molecules expressed at acceptable titers regardless of domain order, e.g. V _H -V _L -V _H -V _H -V _L -V _H (aTriFlex_101) or V _L -V _H -V _H -V _H -V _H -V _L (aTriFlex_102). The thermal and molecular stability of the trispecific tetravalent Fv antibody was very good and comparable.

EXAMPLE 2

Production of trispecific antibody molecules with different order of domains in a diabody unit

Four different Fv antibody molecules in the format corresponding to Figure 2 were constructed as described in Example 1; they are characterized by a different order of domains in the two pairs of variable domains in the diabody unit (from the N-terminus to the C-terminus). The variable domains of the antibody unit are shown in bold in the tables below:

aTriFlex_138: Antibody unit V _H -V _L /V _H -V _L :

aTriFlex_139: Diabody unit V _L -V _H /V _L -V _H :

aTriFlex_140: Diabody unit V _L -V _L /V _H -V _H :

aTriFlex_142: Diabody unit V _H -V _H /V _L -V _L :

Fv antibody molecules were expressed in CHO cells; analysis of the supernatant of the production cell cultures by SDS-PAGE showed that long polypeptides 1 and short polypeptides 2 were well expressed and allowed separate bands to be obtained on electrophoresis under denaturing conditions (FIGS. 9a, 9b, 9c).

After the one-step (IMAC) purification described in Example 1, 76% purity was achieved for aTriFlex_140 (FIG. 10a) and 78% purity for aTriFlex_142 (FIG. 10c). Accordingly, multispecific Fv antibody molecules with a V _L -V _L /V _H -V _H diabody unit (aTriFlex_140) or a V _H -V _H /V _L -V _L diabody unit (aTriFlex_142) showed a significantly higher proportion of Fv antibody molecules, subjected to the correct folding. At the same time, several overlapping varieties of molecules were obtained, and from aTriFlex_138 and aTriFlex_139 containing domains in diabody units in the alternating order V _H -V _L /V _H -V _L and V _L -V _H /V _L -V _H , respectively, do not heterodimeric antibody molecules were isolated (Fig. 10b, 10d). Accordingly, the orientation of the variable domains in the V _L -V _L /V _H -V _H diabody unit stimulates the association of two different polypeptides 1 and 2 with each other and their heterodimerization into a tetravalent trispecific antibody molecule.

EXAMPLE 3

Received the following molecules of multivalent antibodies containing the variable domains of antibodies against CD16A, CD19 and CD30:

Linkers:

The following peptide linkers were used in the examples:

Linker (4) in scFv unit: (G ₂ S) ₆ for V _H -V _L and (G ₂ S) ₇ for V _L -V _H

Linker (3, 3a) in a diabody unit: (G ₂ S) ₃

Linker (5) connecting the scFv unit to the diabody unit: (G ₂ S) ₃

a) A tetravalent trispecific Fv antibody molecule (aTriFlex_101 shown in Fig. 2) having specificities for CD30, CD19 and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal His marker; and a second polypeptide containing two variable domains and a C-terminal FLAG marker. The dimeric unit of the diabody, located in the central part of the molecule, is formed by a pair of V _H -V _H domains in the first polypeptide and a pair of V _L -V _L domains in the second polypeptide, which ensures the correct heterodimerization of polypeptide 1 and polypeptide 2.

Amino acid sequences:

Polypeptide 1:

Polypeptide 2:

b) A quadrivalent trispecific Fv antibody (aTriFlex_102) having specificities for CD30, CD19 and CD16A and consisting of a first polypeptide containing six variable domains and a C-terminal His marker; and a second polypeptide containing two variable domains and a C-terminal FLAG marker. The dimeric unit of the diabody, located in the central part of the molecule, is formed by a pair of domains V _H -V _H in the first polypeptide and a pair of domains V _L -V _L in the second polypeptide.

Amino acid sequences:

Polypeptide 1:

Polypeptide 2:

c) A quadrivalent trispecific Fv antibody (aTriFlex_103) with specificities for CD30, CD19 and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal FLAG marker; and a second polypeptide containing two variable domains and a C-terminal His marker. The dimeric unit of the diabody, located in the central part of the molecule, is formed by a pair of domains V _H -V _H in the first polypeptide and a pair of domains V _L -V _L in the second polypeptide.

Amino acid sequences:

Polypeptide 1:

Polypeptide 2:

d) A quadrivalent trispecific Fv antibody (aTriFlex_104) with specificities for CD30, CD19 and CD16A and consisting of a first polypeptide containing six variable domains and a C-terminal FLAG marker; and a second polypeptide containing two variable domains and a C-terminal His marker. The dimeric unit of the diabody, located in the central part of the molecule, is formed by a pair of domains V _H -V _H in the first polypeptide and a pair of domains V _L -V _L in the second polypeptide.

Amino acid sequences:

Polypeptide 1:

Polypeptide 2:

Despite the different size of polypeptide 1 (six variable domains) and polypeptide 2 (two variable domains), antibody molecules a), b), c) and d) could be satisfactorily expressed. The thermal stability and stability of the molecules of purified antibodies a)-d) were very good. Constructs a)-d) all bound to the primary NK cell lines Raji, Karpas-299 and MEC-1 by flow cytometry.

e) The quadrivalent trispecific Fv antibody (TeTrisAb_I) shown in FIG. 3 having specificities for CD30, CD19 and CD16A and consisting of a first polypeptide containing four variable domains and a C-terminal His marker; and a second polypeptide containing four variable domains and a C-terminal FLAG marker. The dimeric unit of the diabody, located in the central part of the molecule, is formed by a pair of domains V _H -V _H in the first polypeptide and a pair of domains V _L -V _L in the second polypeptide; this orientation ensures correct heterodimerization of the first and second polypeptide.

Amino acid sequences

Polypeptide 1:

Polypeptide 2:

f) The quadrivalent trispecific Fv antibody (scTriFlex_2) shown in FIG. 1, which has specificities for CD30, CD19 and CD16A and consists of a single polypeptide containing eight variable domains and a His marker at the C-terminus. The dimeric unit of the diabody, located in the central part of the molecule, is formed by the first pair of domains V _H -V _H associated with the second pair of domains V _L -V _L located next to them in the same polypeptide. Pair V _H -V _H connected to a pair of V _L -V _L linker (G ₂ S) ₆ .

Amino acid sequence:

g) A tetravalent trispecific Fv antibody molecule (shown in FIG. 2) having specificities for κCD30, CD19 and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal His marker; and a second polypeptide containing two variable domains and a C-terminal FLAG marker. The dimeric unit of the diabody, located in the central part of the molecule, is formed by a pair of V _L -V _L domains in the first polypeptide and a pair of V _H -V _H domains in the second polypeptide, which ensures the correct heterodimerization of polypeptide 1 and polypeptide 2.

Polypeptide 1:

Polypeptide 2:

h) A pentavalent tetraspecific Fv antibody molecule (shown in Figure 6a) having specificities for κCD30, CD19, EGFRvIII and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal His marker; and a second polypeptide containing four variable domains and a C-terminal FLAG marker. The dimeric unit of the diabody, containing two antigen-binding sites for CD16A, is formed by a pair of V _L -V _L domains in the first polypeptide and a pair of V _H -V _H domains in the second polypeptide, which ensures the correct heterodimerization of polypeptide 1 and polypeptide 2.

Polypeptide 1:

Polypeptide 2:

i) A hexavalent trispecific Fv antibody molecule (shown in FIG. 5) having specificities for κCD30, CD19 and CD16A, consisting of a first polypeptide containing ten variable domains and a C-terminal His marker; and a second polypeptide containing two variable domains and a C-terminal FLAG marker. The dimeric unit of the diabody, located in the central part of the molecule and containing two antigen-binding sites for CD16A, is formed by a pair of V _L -V _L domains in the first polypeptide and a pair of V _H -V _H domains in the second polypeptide, which ensures the correct heterodimerization of polypeptide 1 and polypeptide 2.

Polypeptide 1:

Polypeptide 2:

j) A hexavalent tetraspecific Fv antibody molecule (shown in FIG. 7) having specificities for κCD30, CD19, EGFRvIII and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal His marker; and a second polypeptide containing six variable domains and a C-terminal FLAG marker.

Polypeptide 1:

Polypeptide 2:

k) A pentavalent trispecific Fv antibody molecule (shown in Figure 6b) having specificities for κCD30, CD19 and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal His marker; and a second polypeptide containing four variable domains and a C-terminal FLAG marker. The dimeric unit of the diabody, containing two antigen-binding sites for CD16A, is formed by a pair of V _L -V _L domains in the first polypeptide and a pair of V _H -V _H domains in the second polypeptide, which ensures the correct heterodimerization of polypeptide 1 and polypeptide 2.

Polypeptide 1:

Polypeptide 2:

I) The quadrivalent trispecific Fv antibody shown in FIG. 3 having specificities for κCD30, CD19 and CD16A and consisting of a first polypeptide containing four variable domains and a C-terminal His marker; and a second polypeptide containing four variable domains and a C-terminal FLAG marker. The dimeric unit of the diabody, located in the central part of the molecule, is formed by a pair of domains V _L -V _L in the first polypeptide and a pair of domains V _H -V _H in the second polypeptide; this orientation ensures correct heterodimerization of the first and second polypeptide.

Polypeptide 1:

Polypeptide 2:

m) The hexavalent tetraspecific Fv antibody shown in FIG. 8a, having specificities for κCD30, CD19, EGFRvIII, and CD16A and consisting of a first polypeptide containing six variable domains and a C-terminal His marker; and a second polypeptide containing six variable domains and a C-terminal FLAG marker. Each of the first and second polypeptides contains an scFv unit at the C-terminus.

Polypeptide 1:

Polypeptide 2:

n) The hexavalent tetraspecific Fv antibody shown in FIG. 8b, having specificities for κCD30, CD19, EGFRvIII, and CD16A and consisting of a first polypeptide containing six variable domains and a C-terminal His marker; and a second polypeptide containing six variable domains and a C-terminal FLAG marker. Each of the first and second polypeptides contains an scFv unit at the N-terminus.

Polypeptide 1:

Polypeptide 2:

o) The quadrivalent trispecific Fv antibody shown in FIG. 1, which has specificities for κCD30, CD19 and CD16A and consists of a single polypeptide containing eight variable domains and a His marker at the C-terminus. The single chain diabody unit located in the central part of the molecule is formed by the first pair of V _H -V _H domains associated with the second pair of V _L -V _L domains located next to them in the same polypeptide. Pair V _H -V _H connected to a pair of V _L -V _L linker (G ₂ S) ₆ .

Polypeptide 1

p) The hexavalent trispecific Fv antibody shown in FIG. 4, which has specificities for κCD30, CD19 and CD16A and consists of a single polypeptide containing 12 variable domains and a His marker at the C-terminus. The polypeptide contains three units of a single-stranded diabody connected one after the other.

Polypeptide 1

EXAMPLE 4

Assessment of cell binding and cytotoxic activity mediated by trispecific antibody molecules

Research Procedures:

Cells and cell culture

CD19 ⁺ /CD30 ⁺ MEC-1 cells (DSMZ, cat. no.: ACC 497), CD19 ⁺ /CD30 ^- Raji (DSMZ, cat. no.: ACC 319) and CD19 ^- /CD30 ⁺ KARPAS-299 (DSMZ, cat. no. (cat. no.: ACC 31) were cultured under standard conditions in RPMI 1640 (cat. no.: 21875-034) or IMDM (cat. no.: 12440-053) medium supplemented with 10% thermally inactivated fetal calf serum (FBS) (cat. no.: 10270-106), 100 U/ml penicillin G, 100 µg/ml streptomycin (p/n: 1540-122), and 2 mM L-glutamine (p/n: 25030-024; all reagents purchased from Invitrogen, Karlsruhe, Germany) ), herein referred to as complete RPMI medium, as recommended by the cell line supplier.

MICs were isolated from the leukocyte layer of healthy volunteers (German Red Cross, Mannheim, Germany) by density gradient centrifugation. Buffy coat samples were diluted with two to three volumes of PBS (Invitrogen, cat. no.: 14190-169), layered on Histopaque-1077 (Stemcell Technologies, cat. no.: 07861) and centrifuged at 800×g for 25 min at room temperature without use of braking. PBMC located at the layer boundary were collected and washed 3 times with PBS before being used to enrich NK cells. NK cells were enriched for the PBMC population using the Stemcell Technologies EasySep™ Human NK Cell Enrichment Kit, cat#: 19055) for immunomagnetic isolation of intact human NK cells and the Big Easy EasySep™ Magnet according to the manufacturer's instructions .

Cell Binding and Flow Cytometric Analysis

Aliquots of the indicated cell lines were incubated with 100 µl of serial dilutions of His-tagged antibodies in FACS buffer (PBS, Invitrogen, cat. no.: 14190-169) containing 2% thermally activated ETS (Invitrogen, cat. no.: 10270-106), 0.1% sodium azide (Roth, Karlsruhe, Germany, Cat #: A1430.0100) for 45 minutes on ice. Staining of NK cells was performed in FACS buffer supplemented with 1 mg/ml human polyclonal antibodies (Gammanorm, Octapharma, Langenfeld, Germany, cat#: PZN-2451445) to block antibody binding to Fc receptors.

After washing again with FACS buffer, cell-bound antibodies were detected using 10 μg/mL anti-His mAb 13/45/31-2 (Dianova, Hamburg, Germany, cat. no.: DIA910-1MG) followed by 15 μg/ml ml FITC-conjugated goat anti-mouse antibody (Dianova, catalog # 115-095-062). Cells were then washed again and resuspended in 0.2 ml FACS buffer containing 2 μg/ml propidium iodide (PI) (Sigma, catalog #: P4170) to exclude dead cells. Fluorescence of 2-5×10 ³ live cells was measured using a Beckman-Coulter FC500 MPL flow cytometer and MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer (Merck Millipore, Schwalbach, Germany). The mean fluorescence intensity of cell samples was calculated using CXP software (Beckman-Coulter) or Incyte (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values of cells stained with only secondary and tertiary reagents, the values were used for analysis using GraphPad Prism (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla, CA, USA). The equation for one binding site (hyperbolic) was used to calculate K _D .

Cytotoxicity assay

For the calcein-releasing cytotoxicity assay, target cells were labeled with 10 μM calcein AM (Invitrogen, catalog #: C3100MP) for 30 min in RPMI 1640 medium at 37°C, washed, and plated 1×10 ⁴ cells in individual wells of a 96-well microplate together with effector cells in a total volume of 200 μl at a ratio of effector cells and target cells (E : T) 5:1 in the presence of increasing concentrations of antibodies. After incubation for the indicated time periods at 37° C. in a humidified 5% CO ₂ atmosphere, the fluorescence (F) of calcein released into the supernatant was measured using a plate reader at 520 nm (Victor 3, Perkin Elmer, Turku, Finland, no. catalog: 1420-012). Specific cell lysis was calculated using the formula: [F(sample)-F(spontaneous)]/[F(maximum)-F(spontaneous)]×100%. F(spontaneous) is the fluorescence released from target cells in the absence of effector cells and antibodies, and F(maximum) is complete cell lysis induced by the addition of 1% Triton X 100 (Roth, Karlsruhe, Germany, catalog no: 3051.2 ). Regression curves were fitted to calculate EC ₅₀ (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla, CA, USA).

Activity of trispecific antibody molecules

Binding of the trispecific antibody molecules aTriFlex_101, aTriFlex_102, aTriFlex_103 and aTriFlex_104, described in example 3, representing the trispecific antibody molecule shown in figure 2, containing the variable domains of the same antibody located in a different orientation (see example 3a), 3b), 3c) and 3d)), demonstrated on single positive CD19+/CD30- Raji and CD19-/CD30+ KARPAS-299 cells, as well as double positive CD19+/CD30+ MEC-1 cells by flow cytometry. Dissociation constants for CD19 ranged from 6 to 64 nM and for CD30 from 16 to 45 nM (Figure 12). On double positive MEC-1 cells, dissociation constants ranging from 9 to 47 nM were observed, which generally confirmed the data obtained for single positive Raji and KARPAS-299 cells, respectively.

In cytotoxicity assays, EC ₅₀ values were observed in the range of 89-580 pM on single positive CD30+/CD19- Raji cells and 155-952 pM on CD19-/CD30+ KARPAS-299 cells. On double positive cells CD19+/CD30+ MEC-1 observed values of EC ₅₀ in the range of 67-235 μm, which meant an 11-fold increase in activity (figure 13).

In general, the trispecific antibody molecules showed similar activity against the cell lines tested in this study; however, with other antigen pairs, antigen density and geometry may have a greater effect on the activity of the constructs, leading to a greater increase in apparent affinity for doubly positive cells compared to singly positive cells.

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SEQUENCE LIST

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Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln

645 650 655

Met Pro Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp Pro Gly Asp

660 665 670

Ser Asp Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser

675 680 685

Ala Asp Glu Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys

690 695 700

Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Thr Thr Thr

705 710 715 720

Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

725 730 735

Val Thr Val Ser Ser Ala Ala Ala Gly Ser Asp Tyr Lys Asp Asp Asp

740 745 750

Asp Lys

<210> 8

<211> 260

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 8

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

145 150 155 160

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

180 185 190

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

195 200 205

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Gly Ser His His

245 250 255

His His His His

260

<210> 9

<211> 494

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 9

Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly

100 105 110

Ser Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

115 120 125

Lys Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

130 135 140

Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

145 150 155 160

Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr

165 170 175

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr

180 185 190

Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

195 200 205

Val Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp

210 215 220

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly

225 230 235 240

Gly Ser Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

245 250 255

Lys Lys Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr

260 265 270

Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln

275 280 285

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser

290 295 300

Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser

305 310 315 320

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

325 330 335

Ala Val Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala

340 345 350

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser

355 360 365

Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val

370 375 380

Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn

385 390 395 400

Ile Gly Ser Lys Thr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

405 410 415

Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro

420 425 430

Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile

435 440 445

Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly

450 455 460

Thr Asp Trp Ser Asp His Leu Gln Val Phe Gly Gly Gly Thr Lys Leu

465 470 475 480

Thr Val Leu Ala Ala Ala Gly Ser His His His His His His His

485 490

<210> 10

<211> 499

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 10

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Asn

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Trp Pro Gly Asp Ser Asp Thr Met Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Glu Ser Ile Asn Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser

130 135 140

Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile

145 150 155 160

Gly Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro

165 170 175

Val Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu

180 185 190

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

195 200 205

Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

210 215 220

Asn Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

225 230 235 240

Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser

245 250 255

Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly

260 265 270

His Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly

275 280 285

Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly

290 295 300

Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu

305 310 315 320

Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln

325 330 335

Val Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr

340 345 350

Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gln Val Gln Leu Gln

355 360 365

Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser

370 375 380

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Thr Ile His Trp Val

385 390 395 400

Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile Gly Tyr Ile Asn Pro

405 410 415

Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe Lys Gly Lys Thr Thr

420 425 430

Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr Met Gln Leu Asn Ser

435 440 445

Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Ala Asp

450 455 460

Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr

465 470 475 480

Thr Val Thr Val Ser Ser Ala Ala Ala Gly Ser Asp Tyr Lys Asp Asp

485 490 495

Asp Asp Lys

<210> 11

<211> 1019

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 11

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val

130 135 140

Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

145 150 155 160

Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala

180 185 190

Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

210 215 220

Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

260 265 270

Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

275 280 285

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

290 295 300

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

305 310 315 320

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

325 330 335

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

340 345 350

Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly

355 360 365

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

370 375 380

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

385 390 395 400

Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

405 410 415

Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

420 425 430

Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln

435 440 445

Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr

450 455 460

Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

465 470 475 480

Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp

485 490 495

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser

500 505 510

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu

515 520 525

Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile

530 535 540

Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln

545 550 555 560

Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys

565 570 575

Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn

580 585 590

Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp

595 600 605

Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly

610 615 620

Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser

625 630 635 640

Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln Thr

645 650 655

Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val His

660 665 670

Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln

675 680 685

Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn

690 695 700

Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp

705 710 715 720

Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu Phe

725 730 735

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly

740 745 750

Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro

755 760 765

Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys

770 775 780

Thr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val

785 790 795 800

Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser

805 810 815

Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu

820 825 830

Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr Asp Trp Ser

835 840 845

Asp His Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

850 855 860

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

865 870 875 880

Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

885 890 895

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

900 905 910

Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

915 920 925

Leu Glu Trp Met Gly Met Ile Trp Pro Gly Asp Ser Asp Thr Met Tyr

930 935 940

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Glu Ser Ile

945 950 955 960

Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

965 970 975

Met Tyr Tyr Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr

980 985 990

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

995 1000 1005

Ala Ala Ala Gly Ser His His His His His His

1010 1015

<210> 12

<211> 749

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 12

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val

130 135 140

Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

145 150 155 160

Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala

180 185 190

Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

210 215 220

Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly

260 265 270

Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn

275 280 285

Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile

290 295 300

Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly

305 310 315 320

Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala

325 330 335

Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val

340 345 350

Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly

355 360 365

Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val

370 375 380

Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly

385 390 395 400

Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val

405 410 415

Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg

420 425 430

Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly

435 440 445

Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn

450 455 460

Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

465 470 475 480

Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala

485 490 495

Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

500 505 510

Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro

515 520 525

Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp Pro Gly Asp Ser Asp

530 535 540

Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp

545 550 555 560

Glu Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser

565 570 575

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Thr Thr Thr Val Gly

580 585 590

Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

595 600 605

Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

610 615 620

Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

625 630 635 640

Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile

645 650 655

Gly Ser Lys Thr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

660 665 670

Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu

675 680 685

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

690 695 700

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr

705 710 715 720

Asp Trp Ser Asp His Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr

725 730 735

Val Leu Ala Ala Ala Gly Ser His His His His His His His

740 745

<210> 13

<211> 262

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

145 150 155 160

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

180 185 190

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

195 200 205

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Gly Ser Asp Tyr

245 250 255

Lys Asp Asp Asp Asp Lys

260

<210> 14

<211> 749

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 14

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val

130 135 140

Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

145 150 155 160

Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala

180 185 190

Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

210 215 220

Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly

260 265 270

Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn

275 280 285

Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile

290 295 300

Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly

305 310 315 320

Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala

325 330 335

Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val

340 345 350

Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly

355 360 365

Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val

370 375 380

Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly

385 390 395 400

Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val

405 410 415

Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg

420 425 430

Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly

435 440 445

Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn

450 455 460

Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

465 470 475 480

Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala

485 490 495

Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

500 505 510

Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro

515 520 525

Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp Pro Gly Asp Ser Asp

530 535 540

Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp

545 550 555 560

Glu Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser

565 570 575

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Thr Thr Thr Val Gly

580 585 590

Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

595 600 605

Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

610 615 620

Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

625 630 635 640

Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile

645 650 655

Gly Ser Lys Thr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

660 665 670

Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu

675 680 685

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

690 695 700

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr

705 710 715 720

Asp Trp Ser Asp His Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr

725 730 735

Val Leu Ala Ala Ala Gly Ser His His His His His His His

740 745

<210> 15

<211> 522

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

145 150 155 160

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

180 185 190

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

195 200 205

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly

225 230 235 240

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

245 250 255

Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro

260 265 270

Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln

275 280 285

Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val

290 295 300

Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser

305 310 315 320

Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln

325 330 335

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly

340 345 350

Thr Pro Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly

355 360 365

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

370 375 380

Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

385 390 395 400

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

405 410 415

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

420 425 430

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

435 440 445

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

450 455 460

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

465 470 475 480

Met Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe

485 490 495

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ala Ala

500 505 510

Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys

515 520

<210> 16

<211> 1264

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 16

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met Ser

130 135 140

Thr Ser Val Gly Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln Asn

145 150 155 160

Val Gly Thr Asn Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro

165 170 175

Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp

180 185 190

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His

210 215 220

Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn Gly

225 230 235 240

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

260 265 270

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

275 280 285

Phe Thr Thr Tyr Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp

290 295 300

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr

305 310 315 320

Asn Gln Asn Phe Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser

325 330 335

Asn Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala

340 345 350

Val Tyr Tyr Cys Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr

355 360 365

Trp Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

370 375 380

Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

385 390 395 400

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Thr Val Thr Cys Lys

405 410 415

Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Phe Gln Gln Lys Pro

420 425 430

Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser

435 440 445

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

450 455 460

Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys

465 470 475 480

Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu

485 490 495

Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu

500 505 510

Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile

515 520 525

Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln

530 535 540

Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys

545 550 555 560

Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn

565 570 575

Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp

580 585 590

Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly

595 600 605

Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser

610 615 620

Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln Thr

625 630 635 640

Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val His

645 650 655

Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln

660 665 670

Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn

675 680 685

Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp

690 695 700

Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu Phe

705 710 715 720

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly

725 730 735

Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

740 745 750

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

755 760 765

Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

770 775 780

Trp Met Gly Met Ile Trp Pro Gly Asp Ser Asp Thr Met Tyr Ser Pro

785 790 795 800

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Glu Ser Ile Asn Thr

805 810 815

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

820 825 830

Tyr Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala

835 840 845

Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

850 855 860

Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser

865 870 875 880

Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn

885 890 895

Asn Ile Gly Ser Lys Thr Val His Trp Tyr Gln Gln Lys Pro Gly Gln

900 905 910

Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile

915 920 925

Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr

930 935 940

Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val

945 950 955 960

Gly Thr Asp Trp Ser Asp His Leu Gln Val Phe Gly Gly Gly Thr Lys

965 970 975

Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

980 985 990

Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser

995 1000 1005

Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

1010 1015 1020

Lys Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val

1025 1030 1035

Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp

1040 1045 1050

Pro Gly Asp Ser Asp Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln

1055 1060 1065

Val Thr Ile Ser Ala Asp Glu Ser Ile Asn Thr Ala Tyr Leu Gln

1070 1075 1080

Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala

1085 1090 1095

Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

1100 1105 1110

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser

1115 1120 1125

Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser

1130 1135 1140

Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly

1145 1150 1155

Asn Asn Ile Gly Ser Lys Thr Val His Trp Tyr Gln Gln Lys Pro

1160 1165 1170

Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro

1175 1180 1185

Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr

1190 1195 1200

Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp

1205 1210 1215

Tyr Tyr Cys Gln Val Gly Thr Asp Trp Ser Asp His Leu Gln Val

1220 1225 1230

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly Ser

1235 1240 1245

His His His His His His His Ala Arg Thr Ile Phe Ile Cys Ile Ala

1250 1255 1260

Leu

<210> 17

<211> 262

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

145 150 155 160

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

180 185 190

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

195 200 205

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Gly Ser Asp Tyr

245 250 255

Lys Asp Asp Asp Asp Lys

260

<210> 18

<211> 749

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 18

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val

130 135 140

Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

145 150 155 160

Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala

180 185 190

Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

210 215 220

Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly

260 265 270

Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn

275 280 285

Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile

290 295 300

Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly

305 310 315 320

Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala

325 330 335

Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val

340 345 350

Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly

355 360 365

Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val

370 375 380

Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly

385 390 395 400

Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val

405 410 415

Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg

420 425 430

Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly

435 440 445

Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn

450 455 460

Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

465 470 475 480

Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala

485 490 495

Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

500 505 510

Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro

515 520 525

Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp Pro Gly Asp Ser Asp

530 535 540

Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp

545 550 555 560

Glu Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser

565 570 575

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Thr Thr Thr Val Gly

580 585 590

Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

595 600 605

Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

610 615 620

Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

625 630 635 640

Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile

645 650 655

Gly Ser Lys Thr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

660 665 670

Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu

675 680 685

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

690 695 700

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr

705 710 715 720

Asp Trp Ser Asp His Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr

725 730 735

Val Leu Ala Ala Ala Gly Ser His His His His His His His

740 745

<210> 19

<211> 778

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 19

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val

130 135 140

Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

145 150 155 160

Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala

180 185 190

Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

210 215 220

Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

260 265 270

Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

275 280 285

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

290 295 300

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

305 310 315 320

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

325 330 335

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

340 345 350

Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly

355 360 365

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

370 375 380

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

385 390 395 400

Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

405 410 415

Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

420 425 430

Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln

435 440 445

Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr

450 455 460

Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

465 470 475 480

Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp

485 490 495

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser

500 505 510

Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser

515 520 525

Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys

530 535 540

Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

545 550 555 560

Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe

565 570 575

Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val

580 585 590

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser

595 600 605

Gly Thr Pro Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu

610 615 620

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

625 630 635 640

Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

645 650 655

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

660 665 670

Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

675 680 685

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

690 695 700

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

705 710 715 720

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

725 730 735

Ala Met Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala

740 745 750

Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ala

755 760 765

Ala Gly Ser Asp Tyr Lys Asp Asp Asp Asp

770 775

<210> 20

<211> 749

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 20

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val

130 135 140

Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

145 150 155 160

Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala

180 185 190

Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

210 215 220

Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly

260 265 270

Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn

275 280 285

Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile

290 295 300

Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly

305 310 315 320

Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala

325 330 335

Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val

340 345 350

Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly

355 360 365

Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val

370 375 380

Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly

385 390 395 400

Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val

405 410 415

Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg

420 425 430

Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly

435 440 445

Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn

450 455 460

Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

465 470 475 480

Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala

485 490 495

Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

500 505 510

Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro

515 520 525

Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp Pro Gly Asp Ser Asp

530 535 540

Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp

545 550 555 560

Glu Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser

565 570 575

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Thr Thr Thr Val Gly

580 585 590

Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

595 600 605

Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

610 615 620

Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

625 630 635 640

Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile

645 650 655

Gly Ser Lys Thr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

660 665 670

Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu

675 680 685

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

690 695 700

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr

705 710 715 720

Asp Trp Ser Asp His Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr

725 730 735

Val Leu Ala Ala Ala Gly Ser His His His His His His His

740 745

<210> 21

<211> 519

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 21

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val

130 135 140

Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

145 150 155 160

Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala

180 185 190

Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

210 215 220

Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

260 265 270

Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

275 280 285

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

290 295 300

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

305 310 315 320

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

325 330 335

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

340 345 350

Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly

355 360 365

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

370 375 380

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

385 390 395 400

Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

405 410 415

Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

420 425 430

Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln

435 440 445

Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr

450 455 460

Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

465 470 475 480

Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp

485 490 495

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Gly Ser Asp

500 505 510

Tyr Lys Asp Asp Asp Asp Lys

515

<210> 22

<211> 497

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 22

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val

130 135 140

Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly

145 150 155 160

Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val

165 170 175

Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg

180 185 190

Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly

195 200 205

Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn

210 215 220

Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

225 230 235 240

Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser

245 250 255

Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His

260 265 270

Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln

275 280 285

Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile

290 295 300

Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr

305 310 315 320

Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val

325 330 335

Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val

340 345 350

Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln

355 360 365

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

370 375 380

Lys Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg

385 390 395 400

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp Pro Gly

405 410 415

Asp Ser Asp Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile

420 425 430

Ser Ala Asp Glu Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu

435 440 445

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Thr Thr

450 455 460

Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

465 470 475 480

Leu Val Thr Val Ser Ser Ala Ala Ala Gly Ser His His His His His His

485 490 495

His

<210> 23

<211> 493

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 23

Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Thr Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr Asp Trp Ser Asp His

85 90 95

Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser

100 105 110

Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

115 120 125

Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

130 135 140

Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

145 150 155 160

Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala

165 170 175

Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

180 185 190

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

195 200 205

Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr

210 215 220

Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly

225 230 235 240

Ser Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

245 250 255

Lys Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

260 265 270

Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

275 280 285

Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr

290 295 300

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr

305 310 315 320

Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

325 330 335

Val Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp

340 345 350

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly

355 360 365

Gly Ser Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met

370 375 380

Ser Thr Ser Val Gly Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln

385 390 395 400

Asn Val Gly Thr Asn Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser

405 410 415

Pro Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro

420 425 430

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

435 440 445

Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr

450 455 460

His Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn

465 470 475 480

Ala Ala Ala Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys

485 490

<210> 24

<211> 757

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 24

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val

130 135 140

Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly

145 150 155 160

Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val

165 170 175

Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg

180 185 190

Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly

195 200 205

Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn

210 215 220

Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

225 230 235 240

Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser

245 250 255

Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His

260 265 270

Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln

275 280 285

Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile

290 295 300

Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr

305 310 315 320

Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val

325 330 335

Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val

340 345 350

Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln

355 360 365

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

370 375 380

Lys Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg

385 390 395 400

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp Pro Gly

405 410 415

Asp Ser Asp Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile

420 425 430

Ser Ala Asp Glu Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu

435 440 445

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Thr Thr

450 455 460

Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

465 470 475 480

Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser

485 490 495

Tyr Glu Leu Thr Gln Pro Ser Val Ser Val Ser Pro Gly Gln Thr

500 505 510

Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala Tyr

515 520 525

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Lys

530 535 540

Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Ser

545 550 555 560

Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu Asp

565 570 575

Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly Thr Pro Leu

580 585 590

Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly

595 600 605

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

610 615 620

Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

625 630 635 640

Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser

645 650 655

Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

660 665 670

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser

675 680 685

Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala

690 695 700

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr

705 710 715 720

Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe Asp Ile Trp

725 730 735

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ala Ala Gly Ser His

740 745 750

His His His His His His

755

<210> 25

<211> 753

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 25

Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Thr Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr Asp Trp Ser Asp His

85 90 95

Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser

100 105 110

Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

115 120 125

Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

130 135 140

Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

145 150 155 160

Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala

165 170 175

Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

180 185 190

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

195 200 205

Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr

210 215 220

Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly

225 230 235 240

Ser Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

245 250 255

Lys Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

260 265 270

Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

275 280 285

Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr

290 295 300

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr

305 310 315 320

Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

325 330 335

Val Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp

340 345 350

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly

355 360 365

Gly Ser Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met

370 375 380

Ser Thr Ser Val Gly Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln

385 390 395 400

Asn Val Gly Thr Asn Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser

405 410 415

Pro Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro

420 425 430

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

435 440 445

Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr

450 455 460

His Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn

465 470 475 480

Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro

485 490 495

Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser

500 505 510

Gly Asp Ala Leu Pro Lys Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro

515 520 525

Gly Gln Ala Pro Val Leu Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser

530 535 540

Gly Ile Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr Val Thr

545 550 555 560

Leu Thr Ile Ser Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys

565 570 575

Gln Ser Ala Asp Ser Ser Gly Thr Pro Leu Ile Val Phe Gly Thr Gly

580 585 590

Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

595 600 605

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val

610 615 620

Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser

625 630 635 640

Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val

645 650 655

Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro

660 665 670

Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr

675 680 685

Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser

690 695 700

Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Leu Gly Ser

705 710 715 720

Ser Trp Thr Asn Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val

725 730 735

Thr Val Ser Ser Ala Ala Ala Gly Ser Asp Tyr Lys Asp Asp Asp Asp

740 745 750

Lys

<210> 26

<211> 754

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 26

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe Asp Ile Trp Gly

100 105 110

Gly Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Glu Leu Thr

130 135 140

Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr

145 150 155 160

Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala Tyr Trp Tyr Gln Gln

165 170 175

Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Lys Asp Ser Glu Arg

180 185 190

Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr

195 200 205

Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr

210 215 220

Tyr Cys Gln Ser Ala Asp Ser Ser Gly Thr Pro Leu Ile Val Phe Gly

225 230 235 240

Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly

260 265 270

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr

275 280 285

Tyr Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp

290 295 300

Ile Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn

305 310 315 320

Phe Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala

325 330 335

Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

340 345 350

Cys Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala

355 360 365

Tyr Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly

370 375 380

Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser

385 390 395 400

Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile

405 410 415

Gly Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro

420 425 430

Val Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu

435 440 445

Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser

450 455 460

Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

465 470 475 480

Asn Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

485 490 495

Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser

500 505 510

Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly

515 520 525

His Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly

530 535 540

Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly

545 550 555 560

Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu

565 570 575

Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln

580 585 590

Val Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr

595 600 605

Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val

610 615 620

Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser

625 630 635 640

Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val

645 650 655

Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp Pro

660 665 670

Gly Asp Ser Asp Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr

675 680 685

Ile Ser Ala Asp Glu Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser

690 695 700

Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Thr

705 710 715 720

Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

725 730 735

Thr Leu Val Thr Val Ser Ser Ala Ala Ala Gly Ser His His His His

740 745 750

His His

<210> 27

<211> 750

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 27

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe Asp Ile Trp Gly

100 105 110

Gly Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Glu Leu Thr

130 135 140

Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr

145 150 155 160

Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala Tyr Trp Tyr Gln Gln

165 170 175

Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Lys Asp Ser Glu Arg

180 185 190

Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr

195 200 205

Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr

210 215 220

Tyr Cys Gln Ser Ala Asp Ser Ser Gly Thr Pro Leu Ile Val Phe Gly

225 230 235 240

Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly

260 265 270

Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Thr

275 280 285

Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val

290 295 300

Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly

305 310 315 320

Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala

325 330 335

Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr Asp Trp Ser Asp

340 345 350

His Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

355 360 365

Ser Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

370 375 380

Lys Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

385 390 395 400

Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

405 410 415

Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr

420 425 430

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr

435 440 445

Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

450 455 460

Val Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp

465 470 475 480

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly

485 490 495

Gly Ser Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

500 505 510

Lys Lys Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr

515 520 525

Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln

530 535 540

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser

545 550 555 560

Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser

565 570 575

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

580 585 590

Ala Val Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala

595 600 605

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser

610 615 620

Gly Gly Ser Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Lys Phe

625 630 635 640

Met Ser Thr Ser Val Gly Asp Arg Val Thr Val Thr Cys Lys Ala Ser

645 650 655

Gln Asn Val Gly Thr Asn Val Ala Trp Phe Gln Gln Lys Pro Gly Gln

660 665 670

Ser Pro Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val

675 680 685

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

690 695 700

Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln

705 710 715 720

Tyr His Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

725 730 735

Asn Ala Ala Ala Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys

740 745 750

<210> 28

<211> 1016

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 28

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val

130 135 140

Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

145 150 155 160

Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala

180 185 190

Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

210 215 220

Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

260 265 270

Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

275 280 285

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

290 295 300

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

305 310 315 320

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

325 330 335

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

340 345 350

Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly

355 360 365

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

370 375 380

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

385 390 395 400

Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

405 410 415

Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

420 425 430

Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln

435 440 445

Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr

450 455 460

Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

465 470 475 480

Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp

485 490 495

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser

500 505 510

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu

515 520 525

Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile

530 535 540

Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln

545 550 555 560

Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys

565 570 575

Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn

580 585 590

Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp

595 600 605

Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly

610 615 620

Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser

625 630 635 640

Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln Thr

645 650 655

Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val His

660 665 670

Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln

675 680 685

Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn

690 695 700

Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp

705 710 715 720

Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu Phe

725 730 735

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly

740 745 750

Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

755 760 765

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

770 775 780

Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

785 790 795 800

Trp Met Gly Met Ile Trp Pro Gly Asp Ser Asp Thr Met Tyr Ser Pro

805 810 815

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Glu Ser Ile Asn Thr

820 825 830

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

835 840 845

Tyr Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala

850 855 860

Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

865 870 875 880

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

885 890 895

Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Gln

900 905 910

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Thr Val

915 920 925

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

930 935 940

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

945 950 955 960

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

965 970 975

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr Asp Trp Ser Asp His

980 985 990

Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala

995 1000 1005

Gly Ser His His His His His His

1010 1015

<210> 29

<211> 1521

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 29

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met Ser

130 135 140

Thr Ser Val Gly Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln Asn

145 150 155 160

Val Gly Thr Asn Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro

165 170 175

Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp

180 185 190

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His

210 215 220

Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn Gly

225 230 235 240

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

245 250 255

Ser Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

260 265 270

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

275 280 285

Phe Thr Thr Tyr Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp

290 295 300

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr

305 310 315 320

Asn Gln Asn Phe Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser

325 330 335

Asn Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala

340 345 350

Val Tyr Tyr Cys Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr

355 360 365

Trp Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

370 375 380

Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

385 390 395 400

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Thr Val Thr Cys Lys

405 410 415

Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Phe Gln Gln Lys Pro

420 425 430

Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser

435 440 445

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

450 455 460

Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys

465 470 475 480

Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu

485 490 495

Glu Ile Asn Gly Gly Ser Gly Gly Ser Gly Gly Ser Gln Val Gln Leu

500 505 510

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Val

515 520 525

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp

530 535 540

Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn

545 550 555 560

Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val

565 570 575

Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser

580 585 590

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser

595 600 605

Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln Gly Thr Leu Val

610 615 620

Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gln Val Gln

625 630 635 640

Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys

645 650 655

Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His

660 665 670

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile

675 680 685

Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg

690 695 700

Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu

705 710 715 720

Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly

725 730 735

Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln Gly Thr Leu

740 745 750

Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

755 760 765

Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser

770 775 780

Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His

785 790 795 800

Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln

805 810 815

Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile

820 825 830

Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr

835 840 845

Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val

850 855 860

Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val

865 870 875 880

Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln

885 890 895

Pro Ser Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys

900 905 910

Gly Gly His Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln Gln Arg

915 920 925

Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro

930 935 940

Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala

945 950 955 960

Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr

965 970 975

Cys Gln Val Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys

980 985 990

Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Val Gln

995 1000 1005

Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu

1010 1015 1020

Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp

1025 1030 1035

Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Glu Trp Met

1040 1045 1050

Gly Met Ile Trp Pro Gly Asp Ser Asp Thr Met Tyr Ser Pro Ser

1055 1060 1065

Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Glu Ser Ile Asn Thr

1070 1075 1080

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met

1085 1090 1095

Tyr Tyr Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr

1100 1105 1110

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

1115 1120 1125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr

1130 1135 1140

Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile

1145 1150 1155

Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Thr Val His Trp Tyr

1160 1165 1170

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp

1175 1180 1185

Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn

1190 1195 1200

Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

1205 1210 1215

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Gly Thr Asp Trp Ser Asp

1220 1225 1230

His Leu Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

1235 1240 1245

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

1250 1255 1260

Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu

1265 1270 1275

Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

1280 1285 1290

Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met

1295 1300 1305

Pro Gly Lys Gly Leu Glu Trp Met Gly Met Ile Trp Pro Gly Asp

1310 1315 1320

Ser Asp Thr Met Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile

1325 1330 1335

Ser Ala Asp Glu Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser

1340 1345 1350

Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu

1355 1360 1365

Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly

1370 1375 1380

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser

1385 1390 1395

Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val

1400 1405 1410

Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile

1415 1420 1425

Gly Ser Lys Thr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

1430 1435 1440

Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile

1445 1450 1455

Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu

1460 1465 1470

Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys

1475 1480 1485

Gln Val Gly Thr Asp Trp Ser Asp His Leu Gln Val Phe Gly Gly

1490 1495 1500

Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly Ser His His His

1505 1510 1515

His His His

1520

<210> 30

<211> 767

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 30

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

130 135 140

Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

145 150 155 160

Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

165 170 175

Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile

180 185 190

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

210 215 220

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly

245 250 255

Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val

260 265 270

Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly

275 280 285

Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val

290 295 300

Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg

305 310 315 320

Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly

325 330 335

Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn

340 345 350

Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

355 360 365

Ser Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala

370 375 380

Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

385 390 395 400

Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro

405 410 415

Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp

420 425 430

Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp

435 440 445

Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser

450 455 460

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn

465 470 475 480

Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

485 490 495

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

500 505 510

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

515 520 525

Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Thr Ile His Trp Val Arg Gln

530 535 540

Arg Pro Gly His Asp Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Ser

545 550 555 560

Gly Tyr Ser Asp Tyr Asn Gln Asn Phe Lys Gly Lys Thr Thr Leu Thr

565 570 575

Ala Asp Lys Ser Ser Asn Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr

580 585 590

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Ala Asp Tyr Gly

595 600 605

Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val

610 615 620

Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

625 630 635 640

Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val Met Thr Gln Ser

645 650 655

Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Thr Val Thr Cys

660 665 670

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Phe Gln Gln Lys

675 680 685

Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr

690 695 700

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

705 710 715 720

Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe

725 730 735

Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys

740 745 750

Leu Glu Ile Asn Ala Ala Ala Gly Ser His His His His His His His

755 760 765

<210> 31

<211> 251

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 31

Ser Tyr Glu Leu Thr Gln Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly Thr Pro

85 90 95

Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Ser

100 105 110

Gly Gly Ser Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu

115 120 125

Val Lys Lys Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly

130 135 140

Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly

145 150 155 160

Gln Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr

165 170 175

Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr

180 185 190

Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

195 200 205

Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe

210 215 220

Ala Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala

225 230 235 240

Ala Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys

245 250

<210> 32

<211> 769

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 32

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

130 135 140

Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

145 150 155 160

Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

165 170 175

Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile

180 185 190

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

210 215 220

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly

245 250 255

Ser Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

260 265 270

Lys Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

275 280 285

Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

290 295 300

Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr

305 310 315 320

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr

325 330 335

Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

340 345 350

Val Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp

355 360 365

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly

370 375 380

Gly Ser Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser

385 390 395 400

Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu

405 410 415

Pro Lys Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

420 425 430

Val Leu Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu

435 440 445

Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser

450 455 460

Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp

465 470 475 480

Ser Ser Gly Thr Pro Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr

485 490 495

Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gln Val Gln Leu Gln

500 505 510

Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser

515 520 525

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Thr Ile His Trp Val

530 535 540

Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile Gly Tyr Ile Asn Pro

545 550 555 560

Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe Lys Gly Lys Thr Thr

565 570 575

Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr Met Gln Leu Asn Ser

580 585 590

Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Ala Asp

595 600 605

Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr

610 615 620

Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

625 630 635 640

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val Met Thr

645 650 655

Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Thr Val

660 665 670

Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Phe Gln

675 680 685

Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala Ser Tyr

690 695 700

Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr

705 710 715 720

Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu

725 730 735

Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly Gly Gly

740 745 750

Thr Lys Leu Glu Ile Asn Ala Ala Ala Gly Ser His His His His His

755 760 765

His

<210> 33

<211> 249

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 33

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe Asp Ile Trp Gly

100 105 110

Gly Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro

130 135 140

Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys

145 150 155 160

Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val

165 170 175

Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser

180 185 190

Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln

195 200 205

Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser

210 215 220

Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly

225 230 235 240

Ser Asp Tyr Lys Asp Asp Asp Asp Lys

245

<210> 34

<211> 781

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 34

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

130 135 140

Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

145 150 155 160

Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

165 170 175

Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile

180 185 190

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

210 215 220

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly

245 250 255

Ser Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

260 265 270

Lys Pro Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

275 280 285

Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

290 295 300

Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr

305 310 315 320

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr

325 330 335

Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

340 345 350

Val Tyr Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp

355 360 365

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly

370 375 380

Gly Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

385 390 395 400

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

405 410 415

Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

420 425 430

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

435 440 445

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

450 455 460

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

465 470 475 480

Ala Met Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala

485 490 495

Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly

500 505 510

Ser Gly Gly Ser Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

515 520 525

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser

530 535 540

Gly Tyr Thr Phe Thr Thr Tyr Thr Ile His Trp Val Arg Gln Arg Pro

545 550 555 560

Gly His Asp Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Ser Gly Tyr

565 570 575

Ser Asp Tyr Asn Gln Asn Phe Lys Gly Lys Thr Thr Leu Thr Ala Asp

580 585 590

Lys Ser Ser Asn Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu

595 600 605

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Ala Asp Tyr Gly Asn Tyr

610 615 620

Glu Tyr Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

625 630 635 640

Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

645 650 655

Ser Gly Gly Ser Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Lys

660 665 670

Phe Met Ser Thr Ser Val Gly Asp Arg Val Thr Val Thr Cys Lys Ala

675 680 685

Ser Gln Asn Val Gly Thr Asn Val Ala Trp Phe Gln Gln Lys Pro Gly

690 695 700

Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly

705 710 715 720

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

725 730 735

Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln

740 745 750

Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu

755 760 765

Ile Asn Ala Ala Ala Gly Ser His His His His His His His

770 775 780

<210> 35

<211> 237

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 35

Ser Tyr Glu Leu Thr Gln Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly Thr Pro

85 90 95

Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Ser

100 105 110

Gly Gly Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val

115 120 125

Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn

130 135 140

Ile Gly Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

145 150 155 160

Pro Val Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro

165 170 175

Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile

180 185 190

Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp

195 200 205

Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

210 215 220

Ala Ala Ala Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys

225 230 235

<210> 36

<211> 755

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 36

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly

115 120 125

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

130 135 140

Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

145 150 155 160

Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

165 170 175

Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile

180 185 190

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

210 215 220

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn Gly Gly Ser Gly Gly

245 250 255

Ser Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val

260 265 270

Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly

275 280 285

Ser Lys Asn Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val

290 295 300

Leu Val Ile Tyr Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg

305 310 315 320

Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly

325 330 335

Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn

340 345 350

Tyr Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

355 360 365

Ser Gly Gly Ser Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser

370 375 380

Val Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp

385 390 395 400

Ala Leu Pro Lys Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln

405 410 415

Ala Pro Val Leu Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile

420 425 430

Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr

435 440 445

Ile Ser Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser

450 455 460

Ala Asp Ser Ser Gly Thr Pro Leu Ile Val Phe Gly Thr Gly Thr Lys

465 470 475 480

Leu Thr Val Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gln Val Gln

485 490 495

Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys

500 505 510

Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Thr Ile His

515 520 525

Trp Val Arg Gln Arg Pro Gly His Asp Leu Glu Trp Ile Gly Tyr Ile

530 535 540

Asn Pro Ser Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe Lys Gly Lys

545 550 555 560

Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr Met Gln Leu

565 570 575

Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg

580 585 590

Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr Trp Gly Gln

595 600 605

Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly

610 615 620

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Val

625 630 635 640

Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val

645 650 655

Thr Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp

660 665 670

Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser Ala

675 680 685

Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

690 695 700

Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu

705 710 715 720

Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly

725 730 735

Gly Gly Thr Lys Leu Glu Ile Asn Ala Ala Ala Gly Ser His His His

740 745 750

His His His

755

<210> 37

<211> 263

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 37

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe Asp Ile Trp Gly

100 105 110

Gly Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

145 150 155 160

Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln

180 185 190

Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr

195 200 205

Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

210 215 220

Tyr Cys Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Gly Ser Asp

245 250 255

Tyr Lys Asp Asp Asp Asp Lys

260

<210> 38

<211> 8

<212> PROTEIN

<213> artificial sequence

<220>

<223> FLAG marker

<400> 38

Asp Tyr Lys Asp Asp Asp Asp Lys

fifteen

<210> 39

<211> 8

<212> PROTEIN

<213> artificial sequence

<220>

<223> STREP II marker

<400> 39

Trp Ser His Pro Gln Phe Glu Lys

fifteen

<210> 40

<211> 4

<212> PROTEIN

<213> artificial sequence

<220>

<223> C marker

<400> 40

Glu Pro Glu Ala

one

<---

Claims (30)

1. A multivalent Fv antibody to provide at least four antigen-binding sites for antigen binding, containing a diabody unit, consisting of two pairs of variable domains, where one pair is a pair of light chain variable domains (VL-VL) connected one after another into a polypeptide by a peptide linker consisting of 12 or fewer amino acid residues, and the other pair is a pair of heavy chain variable domains (VH-VH) connected one after another into a polypeptide by a peptide linker consisting of 12 or fewer amino acid residues, wherein the VL-VL pair and the VH-VH pair associate into two antigen-binding sites, and at least one pair of variable domains is inserted into the polypeptide , containing at least six variable domains connected one after another, and I) (a) said one pair of variable domains is inserted into a first polypeptide containing six variable domains, wherein said first polypeptide contains at the N-terminus an scFv unit connected to said one pair of variable domains and at the C-terminus a scFv unit connected to said one pair of variable domains, and said one pair of variable domains associates with said other pair of variable domains in a second polypeptide that consists of two variable domains; (b) said one variable domain pair is inserted into a first polypeptide containing ten variable domains, wherein said first polypeptide comprises at the N-terminus a single chain diabody unit connected to said one pair of variable domains and a single chain diabody unit at the C-terminus connected to said one pair of variable domains, and said one pair of variable domains associates with another pair of variable domains in a second polypeptide that consists of said two variable domains; (c) in a first polypeptide containing six variable domains, the scFv unit is N-terminally fused and the scFv unit is C-terminally fused to said light chain variable domain pair (VL-VL), and in the second polypeptide, the scFv unit is fused at the N-terminus and the scFv unit is connected at the C-terminus to the specified pair of variable heavy chains (VH-VH); or (d) said Fv antibody consists of a single polypeptide containing eight variable domains, and said one pair of two variable domains is integrated into the polypeptide, said polypeptide having at its N-terminus an scFv unit linked to said one pair of variable domains, and at the C-terminus, an scFv unit connected to said other pair of variable domains, wherein said one pair of variable domains is connected at the C-terminus to said other pair of variable domains by a linker of more than 12 amino acid residues, and said one pair of variable domains associates with the other a pair of two variable domains of the specified polypeptide, and at the same time ii) said single chain diabody consists of a first pair of variable domains linked by a linker of more than 12, preferably 15 to 50 amino acid residues, and each pair of variable domains linked by a linker of 12 or fewer amino acid residues; The scFv unit consists of a light chain variable domain and a heavy chain variable domain connected by a linker of 12 or more, preferably 15 to 50 amino acid residues. 2. A multivalent Fv antibody according to claim 1, characterized in that the Fv antibody is tetravalent. 3. Multivalent Fv antibody according to any one of paragraphs. 1 or 2, characterized in that said antibody is trispecific. 4. Multivalent Fv antibody according to any one of paragraphs. 1-3, characterized in that these peptide linkers contain glycine and serine residues. 5. A multivalent Fv antibody according to claim 4, characterized in that said peptide linker is a (G2S)X linker, where x=1-10. 6. Multivalent Fv antibody according to any one of paragraphs. 1-5, characterized in that in the specified unit of a single-stranded diabody, the variable domains are arranged in the order VL-VH-VL-VH, VL-VL-VH-VH, VH-VH-VL-VL or VH-VL-VH-VL. 7. Multivalent Fv antibody according to any one of paragraphs. 1-5, characterized in that the variable domains are located in the scFv unit as VL-VH or VH-VL. 8. A multivalent Fv antibody according to claim 1, characterized in that said polypeptide contains six variable domains connected one after another from the N-terminus to the C-terminus, with the first and second variable domains forming a scFv unit at the N-terminus, where the specified scFv-unit at the N-terminus is connected at the C-terminus to the third variable domain, the specified third variable domain is connected by a peptide linker consisting of 12 or less amino acid residues to the fourth variable domain, the fourth variable domain is connected at the C-terminus to the scFv- unit at the C-terminus, and the specified scFv-unit is formed by the fifth and sixth variable domains, and the variable domains are located from the N-terminus to the C-terminus in an orientation selected from: (a) VH-VL-VH-VH-VL-VH (first polypeptide) and VL-VL (second polypeptide); (b) VL-VH-VH-VH-VH-VL (first polypeptide) and VL-VL (second polypeptide); (c) VH-VL-VL-VL-VH-VL (first polypeptide) and VH-VH (second polypeptide); (d) VL-VH-VL-VL-VH-VL (first polypeptide) and VH-VH (second polypeptide); or (e) VH-VL-VL-VL-VL-VH (first polypeptide) and VH-VH (second polypeptide). 9. Multivalent Fv antibody according to any one of paragraphs. 1-8, characterized in that said variable domains in said diabody unit have the same epitope specificity or different epitope specificities. 10. Multivalent Fv antibody according to any one of paragraphs. 1-9, characterized in that said Fv antibody has specificity for an antigen present on an effector cell of the immune system. 11. A multivalent Fv antibody according to claim 10, characterized in that said Fv antibody contains two antigen-binding sites specific for the same immune system effector cell type. 12. A multivalent Fv antibody according to claim 11, characterized in that said Fv antibody is trispecific and binds divalently to an effector cell of the immune system. 13. Multivalent Fv antibody according to any one of paragraphs. 9-12, characterized in that the antigen is selected from CD3, CD16 and CD16A. 14. Multivalent Fv antibody according to any one of paragraphs. 1-13, characterized in that said Fv antibody has at least one specificity for a tumor antigen. 15. A multivalent Fv antibody according to claim 14, characterized in that said Fv antibody has specificities for two tumor antigens. 16. Multivalent Fv antibody according to any one of paragraphs. 10-15, characterized in that said Fv antibody has at least one specificity for a viral antigen. 17. Multivalent Fv antibody according to any one of paragraphs. 1-16 for use as a medicament.

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