RU2785766C9 - 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 to 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 specific target cell. Various molecules based on antibody fragments are known and are being studied, 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 cannot stack on top of each other and associate in pairs, entering into intramolecular interactions. Such short linkers, for example of 2-12 residues, prevent said formation of a monomeric single chain variable fragment (scFv) molecule and favor intermolecular _VH / _VL 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 obtained through the non-covalent association of two single-chain polypeptide hybrid products, each of which consists 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 terminal part of each polypeptide chain are connected by a short linker and are intermolecularly associated with the corresponding V _H and V _L domains of another polypeptide chain, and the other V _H and V _L domains of each polypeptide chain are intramolecularly associated with each other with each other within the same chain to form an antigen-binding scFv unit. Such constructs are homodimers, i.e. consist of identical single-chain polypeptides that associate with each other in pairs.

In addition, trispecific antibodies targeting two tumor antigens are desirable, providing increased selectivity for cancer cells, sparing healthy tissue, and resulting in an expanded range of dosages and therapeutic applicability at which the drug can effectively kill cancer cells. For example, a single trispecific antibody can be used to target two different tumor antigens, with the third specificity activating the cytotoxic effects of T cells or NK cells.

WO 2009/007124 describes a trispecific single chain Fv (scFv) hybrid, 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 of double positive tumor cells and monovalent binding to CD16 on NK cells.

The present invention relates to a multivalent Fv antibody comprising 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 the Fv fragment, consisting of a pair of VH/VL domains, i.e. VH/VL antigen-binding site, or 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 at the N-terminus of this pair of variable domains, and with another variable domain at the C-terminus. Accordingly, such a polypeptide contains at least four variable domains connected to each other, where two adjacent variable domains connected to each other constitute one pair of variable domains of the diabody unit, and another variable domain is located on the N side -terminus of this pair of variable domains of the diabody unit, and other additional variable domains are located at the C-terminus of this pair of variable domains of the diabody unit. The variable domain connected to the N-terminus of this pair of variable domains of the diabody unit may be a light chain variable domain ( _VL ) or a heavy chain variable domain ( _VH ), as well as a domain connected to the C-terminus of this pair of variable domains. domains of a diabody unit may be a light chain variable domain ( _VL ) or a heavy chain variable domain ( _VH ). Such a polypeptide is an Fv polypeptide containing at least four variable domains connected one after another, i.e. a variable domain joined at the N-terminus of a pair of variable domains of a diabody unit joined at the C-terminus to another variable domain. In specific embodiments, six, eight, or ten variable domains are linked together within 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 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, associated 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, the 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 variable domains of a diabody unit (the first pair) associates with another pair of variable domains of a diabody unit (the second pair) to form two antigen-binding sites (the first and second antigen-binding sites), and an additional variable domain located at the N-terminus associates with a corresponding variable domain to form a third antigen binding site, and an additional variable domain located at the C-terminus associates with a 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 linked at the N-terminus and/or C-terminus to a pair of variable domains of the diabody unit in the scFv unit or single-chain diabody unit. In other embodiments, additional variable domains connected at the N-terminus and C-terminus to a pair of variable domains of a diabody unit of the same polypeptide (the first polypeptide) are associated with the corresponding V _H - or V _L -domains of another polypeptide ( a second polypeptide) containing a 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 a diabody unit are a pair of light chain variable domains (V _L -V _L ) connected to each other in the polypeptide, and a pair of heavy chain variable domains (V _H -V _H ) connected to each other in a polypeptide, and the V _L -V _L pair and the V _H -V _H pair associate in two (first and second) V _L /V _H antigen-binding sites.

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

The diabody unit consists of a pair of two variable domains connected one after the other in such a way that these domains cannot form the intramolecular structure of the functional Fv unit, i.e. The _VH / _VL antigen-binding site is instead associated with another pair of two variable domains linked one behind the other to form a divalent dimer, i.e. diabodies, providing two antigen-binding sites. Connecting a pair of light chain variable domains (V _L -V _L ) one after another and a pair of heavy chain variable domains (V _H -V _H ) one after another prevents intramolecular pairing of domains within each pair due to domains of the same type, i.e. V _H -V _H or V _L -V _L . The rigid and compact structure of the diabody unit facilitates the preparation and correct folding of the multivalent antibody, and increases the stability of the antibody. Such a diabody unit forms two V _H /V _L antigen-binding sites within the antibody molecule due to two covalently linked V _H and V _L domains, which is beneficial for the stability of the antibody molecule, since it leads to the formation of 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, since it contains at least four antigen binding sites - two due to the diabody unit and two due to 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 cells infected with a virus. Since such trispecific antibodies of the present invention are at least tetravalent, they have enhanced functional activity compared to trivalent and trispecific single chain fragments. The trispecific and quadrivalent antibodies of the present invention bind divalently through two of the four binding sites to both target and effector cells of the immune system. For example, divalent binding to a target cell increases not only avidity but also specificity of targeting if two of the three antigen 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 to increase, the cytotoxic efficacy of a recruited immune effector cell upon divalent binding of the antibody to the immune effector cell. 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 preventing intramolecular pairing, are light chain variable domains (V _L -V _L ) or heavy chain variable domains (V _H -V _H ) (FIGS. 1 and 2). This particular arrangement of domains has been found to facilitate the correct folding of the multispecific and multivalent antibody molecules of the present invention. In particular, for tetravalent trispecific or tetraspecific Fv antibody molecules, these measures can be taken to ensure correct association of an Fv antibody molecule containing variable domains for three or more different specificities and to prevent incorrect association within a single polypeptide (monomer) or homodimerization between two identical Fv polypeptides instead of correct heterodimerization of the first and second polypeptides (example 2). The present inventors obtained the correct association between variable domains to obtain three specificities and two different variable domain-containing polypeptides 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 correct association and folding of the tetravalent and trispecific or tetraspecific Fv antibodies. The present inventors have discovered that this 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 linked one after another to form an Fv antibody molecule ( for example, Fig. 1), or the correct folding and heterodimerization of two polypeptides of different lengths to form a functional dimeric Fv antibody molecule (for example, Figs. 2, 5, 6a, 6b).

In additional embodiments, at least one single chain diabody unit (scDb) is distally coupled to the diabody unit to form at least one additional antigen binding site. Thus, the at least one diabody unit polypeptide contains one pair of diabody unit variable domains linked to at least one scDb in the polypeptide. For example, two scDb units are distally linked to a diabody unit containing an additional two antigen binding sites for each single-chain diabody unit (FIGS. 4 and 5). Thus, such a multivalent antibody molecule is at least tetravalent in that 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 coupled to a diabody unit because two distally oriented scDb units contain four antigen binding sites and two antigen binding sites are located in a diabody unit. The 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 a polypeptide.

In some embodiments, a diabody unit consists of two pairs of variable domains located on two polypeptides that associate with each other to form two antigen binding sites, wherein each of the two polypeptides contains 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 heptivalent because it contains at least five antigen binding sites - two binding sites in the diabody unit, at least two binding sites in two scFv units or scDb units at the N-terminus and a C-terminus from the first pair of variable domains of the diabody unit and at least one binding site from the N-terminus and the C-terminus from the other pair of variable domains of the diabody unit of another polypeptide of the diabody unit.

In some cases, the present invention provides a multivalent antibody molecule comprising a polypeptide comprising at least six variable domains linked one after another, wherein a diabody unit containing the first two variable domains of said at least six variable domains is embedded in said 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, forming 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. Two antigen binding sites contained in a diabody unit are formed between the first and second variable domains, with each variable domain of the first pair of variable domains forming an antigen binding site with another variable domain of the second pair of variable domains. Accordingly, the diabody unit is formed by said first and second two variable domains, the two variable domains being connected by a short peptide linker in the first as well as the second two variable domains to prevent intramolecular pairing.

Thus, in some embodiments, the present invention provides a multivalent antibody molecule comprising a polypeptide containing, i.e. comprising at least six variable domains linked one after another, wherein two variable domains of a given polypeptide are linked by an intramolecular pairing preventing peptide linker, and said two variable domains are associated with another two corresponding variable domains linked by an intramolecular pairing preventing peptide linker, wherein said 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.e. 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 "tetravalent" refers to an antibody molecule comprising, in particular, four antigen-binding Fv sites, wherein 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 chain variable domain chains (V _L ) of the same antigen specificity associated with each other. Thus, such a tetravalent antibody molecule contains at least eight antibody variable domains, namely four heavy chain variable domains ( _VH ) and four light chain variable domains (VL ₎ . 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 the molecule compared to trivalent and trispecific single-chain Fv molecules.

In some cases, the antibody molecule is multispecific, i.e. has specificity for various antigen epitopes. In some cases, the antibody molecule is trispecific.

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

In some cases, the antibody molecule is an Fv antibody molecule. "Fv antibody" refers to an Fv derivative of an immunoglobulin containing only the variable (V) domains of the antibody, but not the antibody constant regions or fragments thereof. Each light chain variable domain (V _L ) is associated with a corresponding heavy chain variable domain (V _H ), forming an Fv antigen binding site (VH/V _L antigen binding site). The variable domains of an antibody are linked together in a hybrid polypeptide via a peptide linker or peptide bond. Fv antibody, i.e. the antigen binding molecule of the present invention may be a monomer of a single polypeptide or a multimeric polypeptide. Multimeric antigen-binding molecule, i.e. 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, unbranched fusion protein. The variable (Fv) domains of an antibody in a polypeptide are connected one after another. The term "Fv polypeptide" refers to a hybrid polypeptide in which the variable (Fv) domains of an antibody are linked one after another. The polypeptide may contain contiguous 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, such as a His marker consisting of six His residues, a FLAG marker, such as the DYKDDDDK octapeptide (SEQ ID NO:38), or a STREP® II marker, such as the WSHPQFEK octapeptide (SEQ ID NO:39) , or a C marker, for example, tetrapeptide EPEA (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 _CL , _CH , and/or Fc domains. For example, such embodiments may include an Fv polypeptide or Fv antibody combined with at least one antibody constant domain, such as an Fc domain. In additional 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 amino acid composition, a peptide is chosen that does not interfere with the formation of Fv-, i.e. VH/VL antigen-binding sites, and also does not interfere 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, (G ₂ S)x peptide linkers are used, in which, for example, x=1-20, for example, (G ₂ S), (G ₂ S) ₂ , (G ₂ S) ₃ peptide linkers are used, (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 methods.

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.e. 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 an 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 is connected one after another in a polypeptide. In some embodiments, the divalent Fv module consists of a first and second pair of two variable domains adjacent to each other, wherein in each pair the two variable domains are connected by a short peptide linker that prevents 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 adjacent to each other are located on two separate polypeptides to form a dimeric diabody unit (Fig. (2, 3, 5, 8), or two pairs of variable domains located adjacent to each other are located on one and the same polypeptide to form a single-chain diabody unit (Fig. 1, 4). In each pair of variable domains, a short linker 3, 3a connects the C-terminus of one variable domain and the N-terminus of another variable domain, or vice versa. In each pair, the variable domains can be oriented from N- to C-terminus, for example, V _L -V _H , V _H -V _L , V _H -V _H or V _L -V _L , with the two variable domains of the pair having specificities against different antigen epitopes or specificities against one and the same epitope of the antigen. 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. The length of the short peptide linker connecting the two variable domains in each of the first and second pair of variable domains diabody unit is one that allows intramolecular association between variable domains connected by a linker to be prevented. Such a linker is “short”, i.e. 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 two pairs of variable domains and the formation of two antigen-binding Fv sites. Shortening the linker to approximately 12 amino acid residues or less generally prevents 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 achieve a specific orientation of the domains within 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 - pair 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 antibody variable domains of a pair 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, consistent 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 domains of a single chain diabody in a polypeptide connected one after another may 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 the single-chain diabody unit.

In some embodiments, the 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 contains at least three single-chain diabody units linked together in a polypeptide (FIG. 4). In other embodiments, the antibody molecule of the present invention contains at least one single-chain diabody unit distally coupled to the diabody unit (FIG. 5).

"Single-chain Fv (scFv) unit" refers to the 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 ). The variable domains can be oriented V _L -V _H or V _H -V _L from the N-terminus to the C-terminus of the scFv unit. 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 consisting of approximately 12 or more amino acid residues) for intramolecular folding and formation of the Fv antigen binding 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, linker (G ₂ S) ₆ can be used for the scFv V _H -V _L unit, and linker (G ₂ S) ₇ 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. The length of the peptide linker is selected to avoid steric interference between adjacent variable domains and to maintain the stability of the molecule; this length can 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 at the center of the antibody molecule to facilitate folding and improve stability of the antibody molecule. In such cases, the diabody unit is connected at the N-terminus and C-terminus to an additional distal variable domain. In some embodiments, a 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, particularly a trispecific Fv antibody molecule, comprises a polypeptide comprising at least six variable domains, said polypeptide comprising an N-terminal scFv unit, a C-terminal scFv unit, and a first pair of two variable domains of the unit diabodies 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 the Fv antigen-binding 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 variable domains of a second pair of adjacent variable domains may be further coupled to an additional variable domain (FIGS. 6a, 6b, 7). If the diabody unit is a single chain diabody unit, the first pair of variable domains adjacent to each other is connected at the N-terminus to the additional variable domain, and the second pair of variable domains located adjacent to each other is connected at the C-terminus to the additional variable domain domain. Specifically, the first pair of contiguous variable domains is N-terminally linked to an additional variable domain of a scFv unit, and the second pair of contiguous variable domains is C-terminally linked to an additional variable domain of yet 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 C-terminally connected to the additional variable domain of a third single-chain diabody unit in the polypeptide, containing three single chain diabody units connected one after another (FIG. 4). In other cases, at least one additional variable domain may be located between the first and second pairs of variable domains adjacent 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, i.e. the first pair of variable domains located next to each other, associated with two other variable domains of this polypeptide located next to each other, i.e. the second pair of variable domains (Fig. 1). This antibody structure favors the production of multispecific, in particular bi-, tri- or tetraspecific antibodies. In some embodiments, such a multivalent, particularly trispecific Fv antibody comprises at least eight variable domains linked one after the other from the N-terminus to the C-terminus, the first and second variable domains forming a scFv unit at the N-terminus, and said scFv unit is N-terminally connected at its C-terminus to a third variable domain of a first pair of variable domains of a diabody unit comprising a third variable domain and a fourth variable domain, wherein said fourth variable domain is C-terminally connected to a fifth variable domain of a second variable domain pair a diabody unit containing a fifth and a sixth variable domain, wherein said sixth variable domain is C-terminally connected to a 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 arranged, 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 particular embodiment, the orientation of one pair of variable domains of a diabody unit is V _H -V _H and the orientation of the other pair of variable domains of a 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 facilitate 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 effector cells, and a third specificity for an antigen on a tumor cell. In some embodiments, two distal scFv units may have first and second target cell specificities, and a diabody unit located between the two scFv units may have immune effector cell specificity; that is, from the N-terminus to the C-terminus, the first and second variable domains have a first target cell specificity; the third and fifth variable domains have a third specificity for an immune system effector cell, the fourth and sixth variable domains have a third specificity for an immune system effector cell, and the seventh and eighth variable domains have a second specificity for a target cell (FIG. 1). In other embodiments, the two distal scFv units may have immune effector cell specificities, and the two antigen binding sites formed by the diabody unit may have two target cell specificities; that is, 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 an 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, for example, 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 linked one after another (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 antigen specificities, in particular two or three different antigen specificities.

In some cases, an antibody molecule, particularly 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, wherein a first pair of two variable domains of a diabody unit adjacent to each other is embedded in a first polypeptide containing at least six variable domains connected one after another, and said first pair of variable domains , located adjacent to each other, is associated with yet another second pair of two variable domains located adjacent 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, such as 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 included in the diabody unit and is preferably non-covalently associated with another pair of two variable domains adjacent to each other embedded in 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, facilitate the production of trispecific antibodies. Due to the different sizes of the first and second polypeptides, these polypeptides are 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, 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 additional 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 cases, such a trispecific antibody may comprise first and second specificities 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, two distal scFv units formed in the first polypeptide may have first and second target cell specificities, and a diabody unit located between the two scFv units and formed by the first and second polypeptides may have effector cell specificities immune system; that is, from the N-terminus to the C-terminus in the first polypeptide, the first and second variable domains have a first target cell specificity; the third and fourth variable domains have a third specificity for an immune effector cell, the fifth and sixth variable domains have a second specificity for a target cell, and in the second polypeptide, the first and second variable domains have a third specificity for an immune effector cell (FIG. 2). In other embodiments, the two distal scFv units in the first polypeptide containing six variable domains may have specificities for an immune system effector cell, 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 target cell relationship; 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 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 and the second variable domain has a first cell specificity -targets.

In further 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 (Fig. 6a, 6b). Such embodiments are pentavalent and may contain antigen binding sites for one to five different antigen specificities, in particular two or three different antigen 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 of, in particular, multispecific, for example, 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 adjacent to each other in the second polypeptide are N-terminally and C-terminally connected to the units scFv with the formation of two additional antigen-binding sites (Fig. 7). Such embodiments are hexavalent and may contain antigen binding sites for one to six different antigen specificities, in particular two or three different antigen specificities. In specific embodiments, the diabody unit comprises 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 contain 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 adjacent to each other are associated with the corresponding four variable domains located adjacent to each other and embedded in 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 located distal to the tandem diabody units and form the scFv unit (Fig. 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 adjacent 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 can 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 antigen 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 pair, i.e. associates with complementary domains of another polypeptide. The domains are arranged in such a way that the corresponding 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 transform back into 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 within the diabody unit facilitates the correct association of the two trispecific polypeptides. In particular, this orientation makes it possible to obtain trispecific Fv antibody molecules in a tandem diabody format, since it is a heterodimer of two different polypeptides. Accordingly, this orientation favors proper 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 N-terminus to 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, such as a tumor cell, and a third specificity for an immune effector cell, such as a T or NK cell. In other cases, such a trispecific antibody may have two specificities for different antigens on effector cells, and a third specificity for an antigen on a tumor cell. In some embodiments, variable domains located in the outer regions of the polypeptides may have first and second specificities for a target cell, and two variable domains located in the central portion of the polypeptide between variable domains located in the outer regions of the polypeptides have a third specificity for effector cell of the immune system; that is, from the N-terminus to the C-terminus in the first polypeptide, the first variable domain has a first specificity for a target cell; the second and third variable domains have a third specificity for an immune system effector cell, 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 an effector cell of the immune system, and the fourth variable domain has a first specificity for a target cell (FIG. 3). In other embodiments, variable domains located in the external regions of the polypeptides may contain a third specificity for an effector cell of the immune system, and two variable domains located in the central part of the polypeptide between the variable domains located in the external regions of the polypeptides can have a first and second specificity 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 specificity for a target cell, the third variable domain has a second specificity for a target cell, the fourth variable domain has a third specificity for an effector cell of the immune system, and in the second polypeptide the first variable domain has a third specificity for an effector cell 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; that is, 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 an effector cell of the immune system.

In further embodiments, the tandem diabody is conjugated to at least one additional antigen binding domain, particularly at least one scFv unit at the N-terminal or C-terminal side 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 containing a scFv unit at 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 antigen specificities, in particular two or three different antigen specificities. In specific embodiments, the diabody unit located in the central portion of the antibody molecule comprises 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, particularly the multivalent Fv antibody molecule described herein, is at least a trispecific and at least a tetravalent antibody molecule. Such an antibody molecule contains at least two antigen-binding sites that have specificity for the same antigen epitope. Due to this, avidity increases, i.e. the strength of interaction between an antigen epitope and an antigen-binding molecule. The avidity of trispecific antibody molecules can be further increased 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 the trispecific antibody, and hexavalent embodiments may contain two antigen binding sites for each of the three epitope specificities. Alternatively, polyspecificity, i.e. the number of specificities can be increased through tetravalent, pentavalent and hexavalent implementation options. For example, the 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 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 certain embodiment of the present invention, the trispecific and tetravalent Fv antibody molecule contains first and second antigen binding sites specific for two different epitopes of tumor cell antigens of the same type, and third and fourth antigen binding sites specific for an epitope of the effector antigen. 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 increased specificity of targeted effects and expansion of the therapeutic window by reducing nonspecific 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 cases, such a multispecific, eg trispecific antibody may have two specificities for different antigens on an effector cell, such as an NK cell or T cell, and a third specificity for an antigen on a tumor cell.

Thus, the antibody molecule of the present invention can be used in various ways to modify 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 a target. For example, the two different epitopes may be on the same antigen to prevent escape mutants or improve efficiency, or the two epitopes may be on two different target antigens. In some embodiments, the 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 costimulatory 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, particularly Fv antibody molecule comprises a first antigen binding site having specificity for an epitope of an antigen on a tumor cell, a second antigen binding site having specificity for an epitope of an antigen on an effector cell of the immune system, and a third antigen binding site having specificity to an antigen epitope on a soluble protein selected from the group of growth factors, cytokines, chemokines, mitogens and albumins. Examples of such 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 contains one antigen binding site that is specific for an epitope of an antigen present on one cell type, and three antigen binding sites that are specific for epitopes of antigens 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 -cells, natural killer T cells (NKT), granulocytes, monocytes, macrophages, dendritic cells, innate lymphoid cells (ILC) 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; as well as CD35, but not limited to them. In some embodiments of the present invention, the specified specificities, i.e. cell surface molecules of effector cells are suitable for mediating cell killing following binding of a multispecific, eg, trispecific antibody molecule to such a cell surface molecule, thereby inducing cytolysis or apoptosis.

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 the 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.

CD16A antigen (FcγRIMA) is a receptor expressed on the surface of NK cells. NK cells have characteristic 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.

The "target" is the site at which the epitope of the antigen is located, to which the antibody molecule must 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 bearing autoimmune targets, eg IL-2/IL2R, an autoimmune marker or 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 toward which the activity of the effector cell can be tuned to induce or activate an appropriate biological response, such as an 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 after birth in certain organs, but significantly more lower concentration than on tumor cells. TAAs may also be present in the stroma in the immediate vicinity of the tumor cell, but are expressed at low levels 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 tumor cell specificities 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/peptide complexes 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-cell malignancies from lymphoblastic leukemia (ALL) to non-Hodgkin's lymphoma (NHL ).

The 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, such as hepatocellular cancer, liver cirrhosis 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 an albumin, such as human serum albumin (HSA), or pegylated, sialylated, pasylated, or glycosylated (see, e.g., 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, such as one or two, antigen binding sites that are specific for albumin, such as 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 desired antigens. 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 sequence phage display libraries can be subjected to multiple rounds of in vitro selection to enrich for binding agents specific for the desired antigen. The affinity of selected scFvs can then be increased by affinity maturation.

In some embodiments of the present invention, at least one, preferably all, of the antibody variable domains are fully human, humanized, or chimeric domains. Humanized antibodies can be obtained using conventional methods, for example, 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 One skilled in the art can readily obtain a humanized or fully human version of the antigen binding molecule and variable domains from non-human sources, such as mice or non-primate animals, using standard molecular biology techniques known in the art to reduce immunogenicity and improving the effectiveness of said antigen binding molecule in the human immune system.In a preferred embodiment of the present invention, all variable domains of the antibody are humanized or fully human, most preferably, the antibody molecule of the present invention is humanized or fully human. The term “fully human” as used herein means that the amino acid sequences of the variable domains and variable domain-binding peptides in the polypeptide are or can be found in humans. In some embodiments of the present invention, the variable domains, but not the variable domain binding peptides, may be human domains or humanized antibody domains.

One skilled in the art can easily design and produce 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)).

The antibody molecule of any of the embodiments described herein can be produced by expressing polynucleotides encoding the individual polypeptide chains that make up the antibody molecule. Thus, additional embodiments of the present invention are polynucleotides, such as DNA or RNA, encoding polypeptides of the antibody molecule described above herein.

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

The polynucleotides can be incorporated into vectors, preferably expression vectors, which are a further 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 incorporate 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 produced 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, isolated, and optionally further clear.

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

In additional embodiments, the antibody molecules are 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 tumor, viral or neurodegenerative disease. Thus, the present invention further includes a method of treating an individual through 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, particularly a composition comprising the antibody molecule described hereinabove and at least one additional component, is administered at an effective dose to a subject, e.g., a patient, for the treatment of cancer (eg , 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:

Figure 1 shows a tetravalent trispecific antigen-binding Fv molecule consisting of a single polypeptide containing eight variable domains linked 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 7 to the C-terminus 8 of the polypeptide. This domain orientation is given as an example only; other arrangements described above are also possible. Two single-chain Fv (scFv) units 2 are connected distally to the single-chain diabody unit 1 by peptide linkers 5, with the light chain variable domain (V _L ) of one scFv 2 unit 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 diabody unit 1. V _L of scFv unit 2 is connected at the N-terminus to the V _H unit of scFv by a long peptide linker 4. 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 pair of variable 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 variable domains of the diabody is attached at the C-terminus 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 a V _H domain. Two scFv 2 units contain antigen binding sites with different specificities, and diabody unit 1 contains two antigen binding sites with the same specificity.

Figure 2 shows a tetravalent trispecific antigen-binding Fv molecule consisting of a first polypeptide containing six variable domains linked one after another in an 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 a 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; other arrangement options described above are also 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 embedded in the first polypeptide is connected to a 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 a peptide linker 5 with the N-terminus of the first pair of variable domains of 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 diabody unit 1 is connected at the C-terminus by peptide linker 5 to the N-terminus of the unit scFv 2, located at the C-terminus of the first polypeptide. Two scFv 2 units contain antigen binding sites with different specificities, and diabody unit 1 contains two antigen binding sites with the same specificity.

Figure 3 shows a tetravalent trispecific antigen-binding Fv molecule in a trispecific tandem diabody format consisting of a first and 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 polypeptide 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 The C-terminus of 8a is 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 unit 1, 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 another from the N-terminus 7 to the C-terminus 8 of the polypeptide. The domain orientation is given as an example only, other arrangements are possible and are indicated for each pair of variable domains located adjacent to each other. Two units of single-chain diabody 1a, 1b are distally connected to 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 unit 1c, and the variable the single-chain diabody unit domain 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.

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 a second polypeptide containing two variable domains connected one after another from the N-terminus 7a to the C-terminus 8a of the second polypeptide. The specific domain orientation in this figure is for illustrative purposes only, and other arrangements are possible, as indicated for each pair of variable domains adjacent to each other. The antigen-binding Fv molecule contains a dimeric diabody unit 10 formed by a first pair of two variable domains of 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 of the first polypeptide is connected to a single chain diabody unit 1a, 1b.

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

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 a second polypeptide containing four variable domains connected one after another from the N-terminus 7a to the C-terminus 8a of the second polypeptide. The domain orientation is given as an example only; for each pair of variable domains located adjacent to each other, other arrangements shown in the figure are possible. The antigen-binding Fv molecule contains a dimeric diabody unit 10 formed by a first pair of two variable domains of 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 of the first polypeptide is connected to a 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 a peptide linker 5 to the N-terminus the first pair of variable domains of 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 diabody unit 10 is connected at the C-terminus by peptide linker 5 to the N-terminus of the scFv 2 unit located at C -end of the first polypeptide. A) a second four variable domain polypeptide consisting of a second pair of diabody unit 10 variable domains linked to the scFv 2 unit at the C-terminus, and B) a second four variable domain polypeptide consisting of a second pair of diabody unit 10 variable domains , connected to the scFv 2 unit at the N-terminus.

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

Figure 8 shows a hexavalent antigen-binding Fv molecule containing a tandem diabody unit consisting of a first and 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. The domain orientation is given as an example only; for each pair of variable domains located adjacent to each other, other arrangements shown in the figure are possible. Each of the first and second polypeptides contains a 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 polypeptides. B) scFv 2 units are attached at the N-terminus of the first and second polypeptides.

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 the IMAC purification of aTriFlex products: a) aTriFlex_140, b) aTriFlex_138 and c) 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 and 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 were detected by anti-His mAb, followed by treatment with FITC-conjugated goat anti-mouse IgG. Average fluorescence intensities determined by flow cytometry were used to calculate K _D values by nonlinear regression. _KD values for trispecific antibody molecules were determined in two independent experiments.

Figure 13 shows the cytotoxic activity of trispecific antibody molecules against single and double positive cell lines. A 4-hour calcein release cytotoxicity assay was performed on CD19+/CD30- Raji and CD19-/CD30+ KARPAS-299 single-positive cells and CD19+/CD30+ MEC-1 double-positive target cells using enriched NK cells as effectors cells in the presence of serial dilutions of the indicated 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 through nonlinear regression. EC ₅₀ values for the aTriFlex constructs were determined in two independent experiments and are plotted.

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

EXAMPLE 1

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

For expression of trispecific constructs in CHO cells, the coding sequences of all molecules were cloned as one- or two-gene constructs in a mammalian cell expression system suitably modified and derived from the pcDNA5/FRT vector. A vector with two promoters was used for cloning and expression of two-gene constructs (heterodimeric trispecific constructs). For trispecific single-gene constructs, the conventional pcDNA5/FRT expression vector was used. Briefly, gene sequences 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 _VH / _VL domains binding to effectors and targets were amplified by PCR with appropriate primers. The various overlapping DNA fragments and the linearized backbone 2-promoter vector or pcDNA5/FRT vector were then joined together using Gibson Assembly in a single isothermal reaction. The design of all expression constructs contained coding sequences for an N-terminal signal peptide and a C-terminal hexahistidine (6xHis) tag for the first gene cassette or a C-terminal FLAG tag 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 the primer pair 5'-AGAGCTCGTTTAGTGAACCG-3' and 5'-TCATGTCTGGATCCGGCCTTG-3' for 1 gene cassette or 5'-GCCTGGAGACGCCATCC-3' and 5'-GCAGAATTCCACCACACTGG- 3' for 2 gene cassette.

Host cell culture

Flp-In CHO cells (Life Technologies), derived from Chinese hamster ovary cells CHO-KI (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 removed from tissue culture flasks and placed in serum-free HyClone CDM4 CHO medium for subsequent incubation in shaking 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 freeze-preserved in 10% DMSO and checked for negative Mycoplasma assays using the MycoAlert Mycoplasma detection kit (Lonza).

Preparation of pools of stably transfected cells

Recombinant Flp-In CHO cell lines stably expressing secreted trispecific antibodies from one or two genes were generated by transfecting suspension-adapted host cells. For this purpose, cells were placed in standard medium without Zeocin one day before co-transfection with expression plasmids (2.5 μg) encoding the protein of interest (pcDNA5/FRT) and Flp recombinase (pOG44, Life Technologies) using polyethylenimine (PEI). Briefly, vector DNA and transfection reagent were mixed at a DNA:PEI ratio of 1:3 (μg/μg) in a total volume of OptiMEM I medium of 100 μl 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 hours of incubation, the selection of stably transfected cells was started by adding 500 μg/ml hygromycin B after diluting the cultures to a density of 0.1⋅10 ⁶ viable cells/ml in CHO-S-SFMII medium and inoculating into T75 culture flasks. Flp recombinase mediates the insertion of expression constructs into the genome of Flp-In CHO cells at the integrated FRT site due to site-specific DNA recombination (O'Gorman et al., 1991). During selection, the viable cell density was measured twice a week, the cells were centrifuged and resuspended in fresh selection 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 shaking flasks. Expression of recombinant secreted proteins was confirmed by gel electrophoresis of proteins in cell culture supernatants 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 substrate

Recombinant proteins were produced by 10-day batch cultivation of stably transfected CHO cell lines with the addition of substrate by secretion into the cell culture supernatant. To do this, 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, the medium was supplemented with 40 ml/L ActiCHO Feed A and 4 ml/L ActiCHO Feed B (GE Healthcare) on day 0 (the day the culture began) and double the amount of these additives on days 3, 5 and 7. The cell culture supernatant was collected after 10 days of cultivation when the culture viability was >75%. Sampling of producing cultures was performed every other day before feeding, and cell density and viability were assessed. On the day of collection, cell culture supernatants were 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 cleared supernatant of CHO cell cultures in a two-step procedure that included IMAC and preparative size exclusion chromatography. If necessary, a third chromatographic step (FLAG affinity chromatography) was used for additional fine purification. For IMAC, the cleared non-seating 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. The purity of the fractions was analyzed by size exclusion HPLC and SDS-PAGE. Fractions exhibiting 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. The purity and homogeneity (ca. 90%) of the final samples were assessed by SDS-PAGE under reducing and non-reducing conditions, followed by immunoblotting using antibodies specific for His marker and FLAG marker, and 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. The results of SDS-PAGE fractions are shown in Figure 11: All molecules of trispecific tetravalent Fv antibodies expressed with 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 stability and stability of the trispecific tetravalent Fv antibody molecules were very good and comparable.

EXAMPLE 2

Production of trispecific antibody molecules with different domain orders per 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 two pairs of variable domains per diabody unit (from N-terminus to C-terminus). The variable domains of the antibody unit are shown in the tables below in bold:

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 culture supernatants of producing cells by SDS-PAGE showed that long polypeptides 1 and short polypeptides 2 were well expressed and produced distinct bands when electrophoresed 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 high proportion of Fv antibody molecules subjected to 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, 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 the two different polypeptides 1 and 2 with each other and their heterodimerization into a tetravalent trispecific antibody molecule.

EXAMPLE 3

We obtained the following molecules of multivalent antibodies containing 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) per 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 correct heterodimerization of polypeptide 1 and polypeptide 2.

Amino acid sequences:

Polypeptide 1:

Polypeptide 2:

b) 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 V _H -V _H domains in the first polypeptide and a pair of V _L -V _L domains in the second polypeptide.

Amino acid sequences:

Polypeptide 1:

Polypeptide 2:

c) Quadrivalent trispecific Fv antibody (aTriFlex_103), having 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 V _H -V _H domains in the first polypeptide and a pair of V _L -V _L domains in the second polypeptide.

Amino acid sequences:

Polypeptide 1:

Polypeptide 2:

d) Quadrivalent trispecific Fv antibody (aTriFlex_104), having 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 V _H -V _H domains in the first polypeptide and a pair of V _L -V _L domains in the second polypeptide.

Amino acid sequences:

Polypeptide 1:

Polypeptide 2:

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

e) 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 V _H -V _H domains in the first polypeptide and a pair of V _L -V _L domains in the second polypeptide; this orientation ensures proper heterodimerization of the first and second polypeptides.

Amino acid sequences

Polypeptide 1:

Polypeptide 2:

f) 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 V _H -V _H domains associated with the second pair of V _L -V _L domains located next to them in the same polypeptide. The V _H -V _H pair is connected to the V _L -V _L pair by a linker (G ₂ S) ₆ .

Amino acid sequence:

g) A tetravalent trispecific Fv antibody molecule (shown in FIG. 2) having the specificities κCD30, CD19 and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal His tag; 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 correct heterodimerization of polypeptide 1 and polypeptide 2.

Polypeptide 1:

Polypeptide 2:

h) A pentavalent tetraspecific Fv antibody molecule (shown in Fig. 6a) having the specificities κCD30, CD19, EGFRvIII and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal His tag; 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 the specificities κCD30, CD19 and CD16A, consisting of a first polypeptide containing ten variable domains and a C-terminal His tag; 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 the specificities κCD30, CD19, EGFRvIII and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal His tag; 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 Fig. 6b) having the specificities κCD30, CD19 and CD16A, consisting of a first polypeptide containing six variable domains and a C-terminal His tag; 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) Quadrivalent trispecific Fv antibody shown in FIG. 3, having the specificities 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 V _L -V _L domains in the first polypeptide and a pair of V _H -V _H domains in the second polypeptide; this orientation ensures proper heterodimerization of the first and second polypeptides.

Polypeptide 1:

Polypeptide 2:

m) Hexavalent tetraspecific Fv antibody shown in FIG. 8a, having the specificities 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 a scFv unit at the C-terminus.

Polypeptide 1:

Polypeptide 2:

n) Hexavalent tetraspecific Fv antibody shown in FIG. 8b, having the specificities 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 a scFv unit at the N-terminus.

Polypeptide 1:

Polypeptide 2:

o) The tetravalent trispecific Fv antibody shown in FIG. 1, having the specificities κCD30, CD19 and CD16A and consisting 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 a first pair of V _H -V _H domains associated with a second pair of V _L -V _L domains located adjacent to them in the same polypeptide. The V _H -V _H pair is connected to the V _L -V _L pair by a linker (G ₂ S) ₆ .

Polypeptide 1

p) Hexavalent trispecific Fv antibody shown in FIG. 4, having the specificities κCD30, CD19 and CD16A and consisting of a single polypeptide containing 12 variable domains and a His marker at the C-terminus. The polypeptide contains three single-chain diabody units connected one after another.

Polypeptide 1

EXAMPLE 4

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

Study procedures:

Cells and cell culture

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

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

Cell binding assay 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, catalog no.: 14190-169) containing 2% thermally activated FBS (Invitrogen, catalog no.: 10270-106). 0.1% sodium azide (Roth, Karlsruhe, Germany, catalog no.: A1430.0100) for 45 min on ice. NK cell staining was performed in FACS buffer supplemented with 1 mg/ml human polyclonal antibody (Gammanorm, Octapharma, Langenfeld, Germany, Cat. No.: PZN-2451445) to block antibody binding to Fc receptors.

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

Cytotoxicity Assay

For the calcein release cytotoxicity assay, target cells were labeled with 10 μM calcein AM (Invitrogen, catalog no.: C3100MP) for 30 min in RPMI 1640 medium at 37°C, washed and seeded with 1 x 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 to target cells (E:T) of 5:1 in the presence of increasing concentrations of the antibody. After incubation for the indicated periods of time at 37°C in a humidified atmosphere of 5% CO ₂ , fluorescence (F) of calcein released into the supernatant was measured using a plate reader at 520 nm (Victor 3, Perkin Elmer, Turku, Finland, no. according to catalog: 1420-012). Specific cell lysis was calculated using the formula: [F(sample)-F(spontaneous)]/[F(maximum)-F(spontaneous)]×100%. F(spontaneous) represents the fluorescence released from target cells in the absence of effector cells and antibodies, and F(maximum) represents complete cell lysis caused 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)) were demonstrated on single-positive CD19+/CD30- Raji and CD19-/CD30+ KARPAS-299 cells, as well as double-positive CD19+/CD30+ MEC-1 cells using flow cytometry. Dissociation constants for CD19 ranged from 6 to 64 nM and for CD30 from 16 to 45 nM (Figure 12). Dissociation constants ranging from 9 to 47 nM were observed in doubly positive MEC-1 cells, which generally confirmed the data obtained for singly positive Raji and KARPAS-299 cells, respectively.

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

Overall, the trispecific antibody molecules showed similar activity against the cell lines tested in this study; however, when other antigen pairs are used, antigen density and geometry may have a greater effect on the activity of the constructs, resulting in a greater increase in apparent affinity for double-positive cells compared to singly positive cells.

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LIST OF SEQUENCES

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

610 615 620

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

625 630 635 640

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 Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly 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

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 Gln 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

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 Gln 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 Gln 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

Gln 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

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 Gln 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

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 Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly 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 Gln 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

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 Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly 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 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 Gln 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 Gln 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 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 Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly 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 Gln 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

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 Gln 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

Gln 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 Gln 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

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 Gln 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

Gln 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 Gln 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

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 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 Gln 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 Gln 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 Gln 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 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

755

<210> 25

<211> 753

<212> PROTEIN

<213> artificial sequence

<220>

<223> Fv polypeptide

<400> 25

Ser Tyr Val Leu Thr Gln Pro 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 Gln 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 Gln 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

Gln 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 Gln 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

Gln 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 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 Gln 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 Gln 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

Gln 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 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

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 Gln 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 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

Gln 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 Gln 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

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 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 Gln 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 Gln 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

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

Gln 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 Gln 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 Gln 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

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 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 Gln 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

Gln 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

15

<210> 39

<211> 8

<212> PROTEIN

<213> artificial sequence

<220>

<223> STREP II marker

<400> 39

Trp Ser His Pro Gln Phe Glu Lys

15

<210> 40

<211> 4

<212> PROTEIN

<213> artificial sequence

<220>

<223> C-marker

<400> 40

Glu Pro Glu Ala

1

<---

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, one pair being a pair of light chain variable domains (V _L -V _L ) linked together to form a polypeptide by a peptide linker of 12 or fewer amino acid residues, and the other pair being a pair of variable domains heavy chain (V _H -V _H ), joined one after another into a polypeptide by a peptide linker consisting of 12 or fewer amino acid residues, wherein the V _L -V _L pair and the V _H -V _H pair associate into two antigen-binding sites, and at least at least one pair of variable domains is embedded in a polypeptide containing at least six variable domains connected one after another, wherein I) (a) said one pair of variable domains is embedded in a first polypeptide containing six variable domains, wherein said first polypeptide contains at the N-terminus a scFv unit linked to said one pair of variable domains and at the C-terminus a scFv unit linked 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 pair of variable domains is embedded in a first polypeptide containing ten variable domains, wherein said first polypeptide contains 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 the first polypeptide containing six variable domains, the scFv unit is N-terminally linked and the scFv unit is C-terminally linked to said pair of light chain variable domains (V _L -V _L ), and in the second polypeptide scFv- unit is N-terminally linked and the scFv unit is C-terminally linked to said variable heavy chain pair ( _{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 embedded in the polypeptide, wherein said polypeptide contains at the N-terminus a 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 C-terminally 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 said polypeptide, and at the same time ii) said single-chain diabody consists of a first pair of variable domains connected by a linker of more than 12, preferably 15 to 50 amino acid residues, and each pair of variable domains is connected by a linker of 12 or less 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. 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 said peptide linkers contain glycine and serine residues. 5. 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 by the fact that in the specified unit of a single-chain diabody the variable domains are 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 . 7. Multivalent Fv antibody according to any one of paragraphs. 1-5, characterized by the fact that in the scFv unit the variable domains are located as V _L -V _H or V _H -V _L . 8. 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, wherein said scFv unit at the N-terminus is C-terminally connected to a third variable domain, said third variable domain is connected by a peptide linker of 12 or fewer amino acid residues to a fourth variable domain, the fourth variable domain is C-terminally connected to the scFv- unit at the C-terminus, and said scFv unit is formed by fifth and sixth variable domains, the variable domains being arranged from N-terminus to C-terminus in an orientation selected from: (a) V _H -V _L -V _H -V _H -V _L -V _H (first polypeptide) and V _L -V _L (second polypeptide); (b) V _L -V _H -V _H -V _H -V _H -V _L (first polypeptide) and V _L -V _L (second polypeptide); (c) V _H -V _L -V _L -V _L -V _H -V _L (first polypeptide) and V _H -V _H (second polypeptide); (d) V _L -V _H -V _L -V _L -V _H -V _L (first polypeptide) and V _H -V _H (second polypeptide); or (e) V _H -V _L -V _L -V _L -V _L -V _H (first polypeptide) and V _H -V _H (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. The multivalent Fv antibody according to claim 10, characterized in that said Fv antibody contains two antigen binding sites that have specificity for the same type of effector cell of the immune system. 12. 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. 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 medicine.