WO2008132174A1 - An immunoglobulin composition - Google Patents

Abstract

An scFv composition comprising a plurality of scFv constructs. At least 60% of the scFv constructs in the composition are in the form of multimers. The multimers have a molecular weight in the range from 316 kDa to 1962 kDa, as measured by Size Exclusion Chromatography.

Description

50887

An Immunoglobulin Composition

Field of the Disclosure

The present disclosure relates to a composition of single chain variable fragment (scFv) constructs and, in particular, scFv constructs which form multimers.

Background Art

A single chain variable fragment (scFv) is a fusion protein comprising the V_H and V_L domains of immunoglobulin, connected by a linker peptide which usually comprises a repeated sequence of serine and glycine residues. Thus scFv constructs have an antigen binding site which is specific for a particular epitope, in the same way as an antibody, but they do not contain the Fc fragment of an antibody. Furthermore, unlike an Fab fragment of an antibody, the light and heavy chains are connected via a peptide bond rather than disulphide bonds.

scFv constructs can be used as the active component of a medicament for treating a pathological condition. Typically, the scFv constructs in a medicament are specific for an epitope of, for example, a bacterial or tumour antigenic protein. Therefore, by administering a medicament, the construct binds the target antigen, thereby preventing the antigenic protein from facilitating the pathology in question.

It is known to attach a polyethylene glycol (PEG) moiety to an scFv construct in order to increase its serum half-life and thus extend the length of time during which the construct is active in a patient. Where the scFv construct is the active component of a medicament, this has the advantage of lengthening the period over which the medicament is therapeutically effective. Many scFv constructs exist as multimers (or "aggregates", the terms will be used interchangeably herein) under native conditions. In particular, all scFv constructs known to have biological activity in homo sapiens form multimers.

It has now been observed that providing scFv constructs in multimeric form also extends their serum half-life. Thus producing medicaments with scFv constructs in multimeric form is an alternative to attaching PEG moieties to constructs in order to increase bioavailability. The multimers become disaggregated after binding their target. Furthermore, it has now been observed that scFv construct multimers of a particular molecular weight range have the highest level of biological activity.

Summary of the Disclosure

According to one aspect of the present disclosure there is provided an scFv composition comprising a plurality of scFv constructs, wherein at least 60% of the scFv constructs in the composition are in the form of multimers having a molecular weight within a designated range, the designated range being from 316 kDa to 1962 kDa, as measured by Size Exclusion Chromatography (SEC).

The percentage of scFv constructs is determined by measuring the area under the curve following SEC analysis.

It is to be appreciated that the range of 316 kDa to 1962 kDa is the average (+/- 2 s.d.) molecular weight of the fractions of scFv constructs found to have the highest level of activity as determined in Example 4 infra.

Details of a suitable protocol for carrying out SEC analysis are provided in the Experimental section infra. An exemplary mobile phase formulation buffer for use in the SEC analysis may be prepared by dissolving 30.00 g urea and 34.8 g L-arginine into approximately 600 mL of molecular water; adjusting the pH to 9.5, making up the volume to 1000 mL using a volumetric flask; filtering the formulation buffer with a 0.2 μm membrane filter and then storing at 4⁰C. Conveniently, the designated range is from 727 kDa to 1550 kDa, as measured by Size Exclusion Chromatography. The range of 727 KDa to 1550 kDa is the average (+/- 1 s.d.) molecular weight of the fractions of scFv constructs found to have the highest level of activity as determined in Example 4 infra.

According to another aspect of the present disclosure, there is provided an scFv composition comprising a plurality of scFv constructs, wherein at least 60% of the scFv constructs in the composition are in the form of multimers consisting of a designated range of number of scFv sub-units, the designated range being between 11 and 73 scFv constructs. That is to say, when considering the scFv multimers in the composition, at least 80% of the scFv constructs are in multimers of between 11 and 73 sub-units. Preferably, the designated range is between 26 and 57.

The composition of the disclosure may comprise components other than scFv constructs and such other components may form a significant part of the composition (e.g. greater than 40%). However, of the components that are scFv constructs in the composition of the disclosure, at least 60% are in the form of multimers within the designated range.

Conveniently, at least 80%, preferably 81%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 99%, more preferably 100% of the scFv constructs in the composition are within the designated range.

It is to be understood that the composition of the disclosure may comprise scFv constructs of different sequences and specific for different epitopes. However, advantageously, the scFv constructs in the composition are all of the same amino acid sequence.

Conveniently, the composition further comprises a pharmaceutically acceptable excipient. Details of suitable excipients are provided in Remington's Pharmaceutical Sciences and US Pharmacopoeia, 1984, Mack Publishing Company, Easton, PA, USA. Exemplary excipients include pharmaceutical grade (Ph Eur) Urea and L-Arginine (Ph Eur). For example, a typical formulation of an scFv composition of the disclosure is lOmg of pure scFv peptide, 150mg of pharmaceutical grade (Ph Eur) Urea and 174mg L-Arginine (Ph Eur) reconstituted in 5 ml water. An scFv composition of the disclosure may be administered in a dosage in the range of 0.1 to 10 mg/kg body weight of the patient. A dosage in the range 0.5 to 5 mg/kg body weight is preferred, with a dosage of around 1 mg/kg being particularly preferred. The scFv composition may be administered orally.

Preferably, the scFv constructs are specific for the epitope comprising the sequence of SEQ. ID NO. 1, which is derived from the hsp90 protein from Candida sp. Such scFv constructs are referred to as Mycograb or mutant Mycograb constructs and thus compositions comprising such constructs are referred to as Mycograb or mutant Mycograb compositions. For example, such constructs may comprise a sequence at least 80% identical to SEQ. ID NO. 2, and preferably at least 90%, 95%, 99% or 100% identical to SEQ. ID NO.2. The sequence of the construct may comprise an additional 6 histidine residues at the C-terminus.

Some Mycograb or mutant Mycograb constructs comprise a V_H domain linked to a V_L domain by an amino acid spacer wherein the V_H domain comprises a sequence with at least 80% sequence identity to SEQ ID NO. 69 and V_L domain comprises a sequence with at least 80% sequence identity to the SEQ ID NO. 71. It is preferred that the amino acid spacer comprises the sequence (GGGGS)_n, typically wherein n is between 3 and 6. The VH and V_L domains may be arranged either the VH domain N-terminal to the V_L domain or vice versa.

Other exemplary Mycograb or mutant Mycograb constructs comprise the sequence of SEQ ID NOS. 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 48, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67.

Alternatively, the scFv constructs are specific for the epitope comprising the sequence of SEQ. ID NO. 3, which is derived from GrfA, an ABC transporter protein from S. aureus. Such scFv constructs are referred to as Aurograb constructs and thus compositions comprising such constructs are referred to as Aurograb compositions.

Conveniently, such scFv constructs comprise a sequence at least 80% identical to SEQ. ID NO. 3, preferably at least 90%, 95%, 99% or 100% identical to SEQ. ID NO. 4. Such an scFv construct is specific for an epitope having the sequence of SEQ. ID NO. 3. The sequence of the construct may comprise an additional 6 histidine residues at the C- terminus.

A specific scFv construct may be readily modified to alter its amino acid sequence whilst presenting the same paratope and retaining its antigen binding specificity. Generally speaking, its structure is arranged (amino to carboxy terminal sequence) as a human immunoglobulin variable heavy domain (V_H) and variable light chain (VL) covalently joined together by a linker and having a His carboxy-terminal sequence.

Each of the variable regions is comprised of the complement determining regions CDR₁, CDR₂ and CDR₃, and they are fundamental in defining the antigen binding specificity of the antibody, i.e. the paratope. Of these regions, the CDR₃ part of the V_H region is the most important in defining antigen specificity. Further teachings about antibodies are widely available in the art, e.g. Harlow, E. and Lane, D., "Using Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1998. With the common general knowledge about the paratope defining portions of an antibody combining site and the above teachings, a person skilled in the art is readily able to modify an scFv construct to produce alternative antibodies with the same paratope and which enable one to achieve the same or similar therapeutic effect.

For example an antigen binding fragment of the antibody of SEQ ID NO: 2 may be readily prepared by simply removing one or more of the carboxy-terminal His residues. Other examples of modifications include the grafting of the hypervariable (complement determining regions) of the antibody of SEQ ID NO: 2 into variable framework regions different to those of SEQ ID NO: 2 such that the resultant antibody still has the same paratope (see e.g. EP 239400 and suchlike).

Preferably, the composition of the disclosure is for use in the therapy or diagnosis of the human or animal body. Details of the therapeutic uses of the Mycograb® and scFvs are disclosed in WO01/76627 and WO05/102386 and for Aurograb® scFvs in WO03/046007, each of which is incorporated herein by reference. However, the composition of the present disclosure will have a higher level of biological activity than that formed by the methods of the prior art. The Mycograb and mutant Mycograb scFv compositions are useful in the treatment of fungal infections e.g. as disclosed in WO01/76627 or WO05/102386. For example, such compositions are useful in the treatment of systemic fungal infections such as invasive candidiasis or invasive aspergillosis or invasive meningitis e.g. virulent Candida species C. albicans, C. tropicalis and C. krusei and the less virulent species C. parapsilosis and Torulopsis glabrata. Such compositions are also useful in the treatment of infections by Candida, Cryptococcus, Histoplasma, Aspergillus, Torulopsis, Mucormycosis, Blastomycosis, Coccidioidomycosis, Paracoccidioidomycosis organism or malaria. Accordingly, the present disclosure provides a method of treating a patient with a fungal infection comprising administering to the patient an effective amount of such a composition.

The Mycograb and mutant Mycograb scFv compositions are particularly useful for combination therapies. Accordingly, in another aspect, the present disclosure provides such a composition and, in addition, an antifungal agent (the "combination partner") such as e.g. a polyene antifungal or an echinocandin antifungal or an azole antifungal. Examples of antifungals useful in such compositions include e.g. amphotericin B, derivatives of amphotericin B such as AmBisome, amphotericin-B lipid complex (Abelcet), amphotericin-B colloidal dispersion (Amphocil) and amphotericin-B intralipid emulsion; nystatin; 5-fluorocytosine; caspofungin, anidulafungin, micafungin, LY303366; azoles such as isavuconazole, voriconazole, itraconazole, fluconazole, miconazole, ketoconazole, posaconazole, anidulafungin, micafungin, griseofulvin, terbinafine. Though such a combination may be a fixed dose combination or composition, generally, the scFv peptide or "construct" and its combination partner are not packaged as fixed dose combinations. The combined preparations of the present disclosure may be for simultaneous, separate or sequential use in the treatment of fungal infections. The compositions of the present disclosure may also comprise more than one antifungal agent, e.g. with amphotericin B and 5-fluorocytosine, a fungin and Amphotericin B or an echinocandin plus azole.

In another embodiment, the present disclosure provides a method of treating a patient with a fungal infection comprising administering to the patient an effective amount of a Mycograb or mutant Mycograb composition of the present disclosure and at least one of the antifungal agents described above. Preferred combination partners are amphotericin B or derivatives of amphotericin B, caspofungin, anidulafungin, micafungin, voriconazole, itraconazole. The combination partners may be administered simultaneously, separately or sequentially.

In one embodiment of the present disclosure, the fungus causing the infection is resistant or partially resistant against an antifungal combination partner of the peptides of the disclosure.

The Mycograb and mutant Mycograb compositions of the present disclosure are also useful in the treatment of cancer, or a condition involving raised levels of TNFα and/or IL-6 such as autoimmune diseases or sepsis e.g. as disclosed in WO06/003384 or WO07/077454 each of which is hereby incorporated by reference. For instance, such compositions are useful in the treatment of leukemia such as e.g. Lymphoid (Lymphocytic) leukaemia (CLL), acute myeloid (myeloblastic) leukeemia (AML), acute Lymphoid (Lymphoblastic) leukemia (ALL), chronic myeloid leukaemia (CML), carcinoma of the breast, carcinoma of the colon, prostate, multiple myeloma; or for the treatment of sepsis targeting human hsp90 (WO07/077454). Accordingly, the present disclosure provides a method of treating a patient with a cancer disease or a condition involving raised levels of TNFα and/or IL-6 (e.g. autoimmune disease, SIRS or sepsis) comprising administering to the patient an effective amount of a Mycograb or mutant Mycograb composition.

In some embodiments, the autoimmune disease is Crohn's disease, rheumatoid arthritis, ulcerative colitis or systemic lupus erythematosus.

The Mycograb and mutant Mycograb compositions of the present disclosure are useful for combination therapies with anticancer agents. Examples of suitable anticancer agents include doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel, cisplatin, imatinib (Gleevec®), paclitaxel, cytarabine or hydroxyurea. Accordingly, the present disclosure provides a composition or a combined preparation comprising a Mycograb or mutant Mycograb composition of the present disclosure, and a anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel, cisplatin, imatinib, paclitaxel and hydroxyurea. Also provided are methods of treating a patient with a cancer disease comprising administering to the patient in need an effective amount of a Mycograb or mutant Mycograb composition of the present disclosure, and at least one of the anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel, cisplatin, imatinib, paclitaxel and hydroxyurea.

The Aurograb compositions of the present disclosure are useful in the treatment of bacterial infections, particularly infections of gram positive bacteria. For example, the Aurograb compositions may be used in the treatment of infections by Staphylococcus such as S. haemolyticus, S. epidermidis, S. saprophytics or S. aureus. Alternatively, the

Aurograb compositions may be used in the treatment of infections of Enterococcus sp. such as Enterococcus faecalis, or Enterococcus faecium; or of Corynebacterium sp. such as Corynebacterium jeikeium or Corynebacterium xerosis. Accordingly, the present disclosure provides a method of treating a patient with a bacterial infection comprising administering to the patient an effective amount of such a composition.

The Aurograb scFv compositions are particularly useful for combination therapies. Accordingly, in another aspect, the present disclosure provides such an Aurograb and, in addition, a glycopeptide antiobiotic such as vancomycin or teicoplanin; or the antiobiotic daptomycin. The Aurograb composition and the antibiotic may be combined in a single composition or may be separate.

In another aspect of the present disclosure, there is provided a method of treating a patient with a bacterial infection comprising administering to the patient an effective amount of an Aurograb composition of the disclosure and at least one antibiotic selected from a glycopeptide antibiotic or daptomycin. The Aurograb composition and the antibiotic may be administered simultaneously, separately or sequentially.

In this specification, sequence identities are determined using the NCBI BLASTN (nucleotide sequence comparisons) or BLASTP (polypeptide comparisons) programs, Version 2.1.2, with default parameters. The NCBI BLAST program is to be found at www.ncbi.nlm.nih.gov/blast/. The term "sequence identity" used herein refers to amino acid residues in optimally aligned sequences which match exactly at corresponding relative positions.

In this specification, the term "construct" when used in relation to an amino acid sequence means a "peptide" comprising the amino acid sequence and the terms are used interchangeably.

Brief Description of the Sequence Listings

The following sequences are referred to in this specification.

SEQ ID NO: 1 is the amino acid sequence of an hsp90 epitope of Candida sp. SEQ ID NO: 2 is the amino acid sequence of an scFv (referred to herein as efungumab, the active component of Mycograb®) specific for the epitope of SEQ ID NO: 1. A C- terminal 6 residue poly-histidine tail may be added to this sequence.

SEQ ID NO: 3 is the amino acid sequence of an epitope of GrfA from S. aureus.

SEQ ID NO: 4 is the amino acid sequence of an scFv (the active component of Aurograb®) specific for the epitope of SEQ ID NO: 3. A C-terminal 6 residue poly- histidine tail may be added to this sequence.

SEQ ID NO: 5 is the amino acid sequence of an extended hsp90 epitope.

SEQ ID NO. 6 is Mycl23

SEQ ID NO. 7 is the peptide sequence encoded by SEQ ID NO. 6 SEQ ID NO. 8 is Mycl 02, Mycograb-6H-TAA

SEQ ID NO. 9 is the peptide sequence encoded by SEQ ID NO. 8

SEQ ID NO. 10 is MyclOl, Mycograb-TAA

SEQ ID NO. 11 is the peptide sequence encoded by SEQ ID NO. 10

SEQ ID NO. 12 is MycC29X-TAA, e.g.: MyclO5, MycC29Y-TAA SEQ ID NO. 13 is the peptide sequence encoded by SEQ ID NO. 12

SEQ ID NO. 14 is MycC29X-6H-TAA, e.g.: MyclOό, MycC29Y-6H-TAA, Mycl 13,

MycoC29S-6H-TAA

SEQ ID NO. 15 is the peptide sequence encoded by SEQ ID NO. 14

SEQ ID NO. 16 is Mycl 07, Myco-4-TAA SEQ ID NO. 17 is the peptide sequence encoded by SEQ ID NO. 16 SEQ ID NO. 18 is MycoC29X-4-TAA, e.g.: MyclO8, MycoC29Y-4-TAA; Mycl l4,

MycoC29S-4-TAA

SEQ ID NO. 19 is the peptide sequence encoded by SEQ ID NO. 18

SEQ ID NO. 20 is MyclO9, N-Myco-4-TAA SEQ ID NO. 21 is the peptide sequence encoded by SEQ ID NO. 20

SEQ ID NO. 22 is N-MycoC29X-4-TAA, e.g.: Mycl 10, N-MycoC29Y-4-TAA

SEQ ID NO. 23 is the peptide sequence encoded by SEQ ID NO. 22

SEQ ID NO. 24 is Mycl 11, N-Myco-6H-TAA

SEQ ID NO. 25 is the peptide sequence encoded by SEQ ID NO. 24 SEQ ID NO. 26 is N-MycoC29X-6H-TAA, e.g.: Mycl 12, N-MycoC29Y-6H-TAA

SEQ ID NO. 27 is the peptide sequence encoded by SEQ ID NO. 26

SEQ ID NO. 28 is Mycl 15, MycYSSS

SEQ ID NO. 29 is the peptide sequence encoded by SEQ ID NO. 28

SEQ ID NO. 30 is Mycl 16, Myc YSIQSS SEQ ID NO. 31 is the peptide sequence encoded by SEQ ID NO. 30

SEQ ID NO. 32 is Mycl 17, MycSIQKS

SEQ ID NO. 33 is the peptide sequence encoded by SEQ ID NO. 32

SEQ ID NO. 34 is Mycl 18, VH-2Bam-2VL

SEQ ID NO. 35 is the peptide sequence encoded by SEQ ID NO. 34 SEQ ID NO. 36 is Mycl 19, VL-2Bam-2VH

SEQ ID NO. 37 is the peptide sequence encoded by SEQ ID NO. 36

SEQ ID NO. 38 is Mycl45, MycC98X-6H-TAA

SEQ ID NO. 39 is the peptide sequence encoded by SEQ ID NO. 38

SEQ ID NO. 40 is Mycl29 (Myc YSRIQSS) SEQ ID NO. 41 is the peptide sequence encoded by SEQ ID NO. 40

SEQ ID NO. 42 is Mycl 30 (Myc YSRSIQS SKS)

SEQ ID NO. 43 is the peptide sequence encoded by SEQ ID NO. 42

SEQ ID NO. 44 is Myc 133

SEQ ID NO. 45 is the peptide sequence encoded by SEQ ID NO. 44 SEQ ID NO. 46 is Myc 134

SEQ ID NO. 47 is the peptide sequence encoded by SEQ ID NO. 46

SEQ ID NO. 48 is Mycl35

SEQ ID NO. 49 is the peptide sequence encoded by SEQ ID NO. 48 SEQ ID NO. 50 is Mycl 36

SEQ ID NO. 51 is the peptide sequence encoded by SEQ ID NO. 50

SEQ ID NO. 52 is Mycl 37

SEQ ID NO. 53 is the peptide sequence encoded by SEQ ID NO. 53 SEQ ID NO. 54 is Myc 138

SEQ ID NO. 55 is the peptide sequence encoded by SEQ ID NO. 54

SEQ ID NO. 56 is Mycl 39

SEQ ID NO. 57 is the peptide sequence encoded by SEQ ID NO. 56

SEQ ID NO. 58 is Myc 140 SEQ ID NO. 59 is the peptide sequence encoded by SEQ ID NO. 58

SEQ ID NO. 60 is Mycl41

SEQ ID NO. 61 is the peptide sequence encoded by SEQ ID NO. 60

SEQ ID NO. 62 is Myc 142

SEQ ID NO. 63 is the peptide sequence encoded by SEQ ID NO. 62 SEQ ID NO. 64 is Myc 143

SEQ ID NO. 65 is the peptide sequence encoded by SEQ ID NO. 64

SEQ ID NO. 66 is Mycl44

SEQ ID NO. 67 is the peptide sequence encoded by SEQ ID NO. 66

SEQ ID NO. 68 is the nucleotide sequence encoding the heavy chain of the wild type Mycl 23 scFv peptide or construct.

SEQ ID NO. 69 is the peptide or construct encoded by SEQ ID NO. 68

SEQ ID NO. 70 is the nucleotide sequence encoding the light chain of the wild type

Myc 123 scFv peptide or construct.

SEQ ID NO. 71 is the peptide or construct encoded by SEQ ID NO. 70

Brief Description of the Figures

The present disclosure will be further apparent from the following description with reference to the accompanying figures, which shows by way of example only the preparation and analysis of certain scFv constructs.

Figure 1 is a graph showing the log of MW of Mycograb scFv aggregates versus retention time following SEC analysis. Figure 2 is a graph showing relative quantity versus time of 4 volumes (20, 40, 80 and 100 μl, respectively) of scFv aggregates from Mycograb batch 300606 following SEC analysis.

Figure 3 is a graph showing the same results as Figure 2, after constant height adjustment.

Figure 4 is a graph showing the relative quantity of batch MG270603 following SEC analysis and divided into 4 fractions.

Figure 5 is a graph showing the relative amounts of the 4 fractions of MG270603 following SEC analysis.

Figure 6 is a graph showing the same results as Figure 5, after constant height adjustment.

Figure 7 is a graph showing the relative quantity of batch MG 071005 following SEC analysis and division into fractions 0-4 (fraction 4 discarded).

Figure 8 is a graph showing the relative amounts of the 4 remaining fractions of MG 071005 following SEC analysis.

Figure 9 is a graph showing the same results as Figure 8, after constant height adjustment.

Figure 10 is a graph showing the relative quantity of batch MG 300606 following SEC analysis and division into fractions 0-11 (fractions 4-11 discarded).

Figure 11 is a graph showing the relative amounts of the 4 remaining fractions of MG 300606 following SEC analysis

Figure 12 is a graph showing the same results as Figure 11, after constant height adjustment.

Figure 13 is a graph of relative amount of aggregates against time, following SEC analysis together with the delineation into fractions. Figure 14 shows size exclusion chromatograms of freshly prepared and freeze/thawed Aurograb batches: A) 2001 batches freshly prepared; B) after freeze/thaw cycle; C) 2004 batches freshly prepared and D) after freeze/thaw cycle.

Figure 15 is a diagram showing schematically the sequence of the wild type Mycograb scFv peptide and Mycograb mutants. Stop codons of the nucleic acid molecules encoding the respective peptides are also shown at the C-terminal end.

Figure 16 is a graph in which the black bars show yield after solubilization with NLS of all investigated mutants. The error bars show the Standard deviation for samples analyzed twice. The white bars are a graphic representation of mass balance after NLS refolds of all investigated mutants. Mutants are ranked according to increasing refolding recovery values.

Figure 17 shows graphs indicating the recovery after refolding for 5 selected mutants when urea and DTT was used as solubilizing agent (A) and when GuHCl and DTT were used as solubilizing agent (B). White bars: Recovery when mass of protein found in the IB. SOL solution was used for calculation (equ. 1) Black bars: Refolding recovery when mass of protein found in the IB.RES solution is used for calculation.

Figure 18 is a graph showing the time required for a visible beginning clarification of a solubilization solution after addition of 4% NLS (white bars) and the time required until no further clarification was observed (black bars) for all tested mutants. Mutants are ranked according to the start time in ascending order.

Figure 19 is a variability chart for the response start of solubilization and indicates number of cysteines, number of linker elements and if the heavy (vh) or light chain (vl) fragment was at the N-terminus. 1: Mutant Myc 106 had the fastest solubilization start but contained 5 cysteines.

Figure 20 shows chromatograms as an overlay of REF end samples of Mutants MYC 135, Myc 130, Myc 133, Myc 119, Myc 123 wild type and Myc 116. Figure 21 shows chromatograms as an overlay of REF end samples of Mutants MYC 134, Myc 137, Myc 138, Myc 106 Myc 136, Myc 123 and Myc 139.

Figure 22 shows scaled estimates and a prediction profiler of the following parameters: linker length, number of cysteines and Vh/Vl arrangement for the response retention time of mutants. The scaled estimates predict to what extent the retention time would shift when the parameter is increased from centerpoint (the red number in the prediction profiler plot on the x-axis) to a higher level.

Figure 23 is a plot of linker length versus retention time measured in RP-HPLC (RPC2) for tested mutants. The early retention time of MYC 130 compared with the other mutants is highlighted.

Figure 24 shows a normalized overlay of all REF.END samples from Figures 21 and 21 for estimation of peak area from peak 2.

Figure 25 shows a RP-HPLC 2 chromatogram overlay of a REF.End sample of MYC 119 solubilized with 8M urea +DTT, 8M urea, 6M GuHCl +DTT and 6M GuHCl dilution was 1:50 with a buffer containing 2OmM Tris, 2mM cysteine, 1% NLS, pH 9.0.

Figure 26 is an RPC 2 chromatogram of a REF.End sample of MYC 119 after solubilization with 6M urea and 5mM DTT and subsequent refolding by a 1 :10 dilution.

Figure 27 is an image of a gel following SDS Page analysis of REF.IM and REF.END sample of MYC 119 after solubilization with 6M urea and refolding by a 1 : 10 and 1 :50 dilution, respectively. Lanes 1-8: non reducing SDS Page, lanes 9-14: reducing SDS Page. R= reducing; n-r = non reducing

Figure 28 is an RP-HPLC chromatogram overlay (RPC 2) of an inclusion body sample from mutant MYC 119 after solubilization with 6 M urea and 4 % NLS. Figure 29 shows images of: left gel: Reducing (r ) SDS-Page for Mutants MYC 118, 119, 130, 133, 134, 135 and 137; and right gel: Non-reducing (n-r) SDS Page of the same samples

Figure 30 shows images of:: left gel: Reducing SDS-Page for Mutants MYC 106, 123 wt, 136, 138, 139 and 140; and right gel: Non-reducing SDS Page of the same samples

Figure 31 is an overlay of SEC HPLC 0.5% NLS chromatograms of REF.End samples for the mutants Myc 118, Myc 119, Myc 130, Myc 133 and Myc 135. IBs from these mutants were isolated at bench scale.

Figure 32 is an overlay of SEC HPLC 0.5% NLS chromatograms of REF.End samples for the mutants Myc 134, Myc 136, Myc 137, Myc 138, Myc 139, Myc 140, Myc 106 and Myc 123 wild type. IBs from these mutants were isolated in the pilot plant.

Figure 33 shows a scatter plot and linear regression (continuous line) of measured MW versus theoretical MW of REF.End samples for all mutants. The 95% confidence interval for the fit is also shown (dashed line). The dot at top left shows MYC 130. The dots within the dashed lines are within the 95% CI and therefore not significantly different from the wildtype. The dots below both dashed lines represent mutants with lower average MW than predicted and the dots above both dashed lines represent mutants where a higher average MW was measured than predicted.

Figure 34 shows SEC-HPLC (formulation) chromatograms for REF.END samples of Myc 119, Myc 106-origami, Myc 123 wt and Myc 137 after UFDF against 5OmM Tris, pH 9.0 buffer. Samples were taken after each volume reconstitution. Sample prior to UFDF treatment (5), after 1^st 'buffer exchange step (2), 2^nd buffer exchange step (3), 3^rd (4 ) and last (5) step.

Figure 35 shows RP-HPLC 2 chromatograms of REF.IM, REF.3T and REF. END samples for all tested mutants: EXPERIMENTAL

The experimental strategy was as follows. Fresh-prepared Mycograb samples were applied to a TSKGEL G6000PWXL column for SEC (size exclusion chromatography). For each batch analysed the peak was collected in 4 fractions. These fractions were separately re-applied to the SEC column. Re-application of the four fractions generated peaks, which were correlated to the original molecular weight of the corresponding fractions.

Materials and Methods

Calibration

Molecular weight reference proteins:

Alcohol dehydrogenase, yeast, 150 kDa MW (Sigma, CAT: A8656)

β-Amylase, sweet potato: 200 kDa (Sigma, CAT: A8781)

Apoferritin, horse spleen: 443 kDa (Sigma, CAT: A3660)

Thyroglobulin, bovine: 669 kDa (Sigma, CAT: T9145)

IgM, human serum: 900 kDa (Sigma, CAT: 18260)

IgM dimer: 1,80O kDa

Mycograb® drug product: batch 270603, 071005, and 300606

SEC Conditions

Instrument: Shimadzu HPLC

Pump: LC-10ADvp

Pump sub-controller: FCV-IOAL

Degasser: DGU- 14A Auto sampler: SIL-HTA

Column oven: CTO-lOACvp

UV-VIS detector: SPD-lOAvp

Fraction collector: FRC-IOA

Column: TSK Gel G6000 PWXL 30cm x 7.8mm ID

Eluent: Ice cold formulation buffer

Flow Rate: 0.5 ml/min

Run Time: 40 minutes

Column oven temperature : 12°C

Detection: 280 run

Formulation Buffer Preparation

Mobile phase formulation buffer was prepared by dissolving 30.00 g urea and 34.8 g L- arginine into approximately 600 mL of molecular water. The pH was adjusted to 9.5 using 10 M (concentrated) hydrochloric acid and the volume is accurately made up to 1000 mL using a volumetric flask. Formulation buffer was then filtered with a 0.2 μm membrane filter and stored at 4⁰C. During SEC analysis, the formulation buffer was pre-chilled in an ice bucket.

Sample Preparations

Reference Protein

Alcohol dehydrogenase, β-Amylase, apoferritin, and thyroglobulin were solubilised in 4⁰C cold formulation buffer (pH 9.5) at 1 mg/mL.

Human IgM was supplied in buffered aqueous solution at 1 mg/mL. IgM dimer was prepared by heating 300 μL of human IgM in a 1.5 mL eppendorf at 8O⁰C for 5 minutes, using a heating block. The sample was then centrifuged at 16,000 xg for 3 minutes and the supernatant was dispensed into an auto-sampler vial for immediate application to the SEC column.

Mycograb® preparation

Mycograb® comprises a humanised scFv recombinant antibody (SEQ ID NO: 2). Details of the formulation of Mycograb® are provided in WO-A-01/76627 (see especially page 11 et seq.) which is hereby incorporated by reference. Each Mycograb® vial was solubilised in 5 ml of molecular grade water (4⁰C) to make up a 2 mg/mL solution. For the analysis of freshly prepared samples, 300 μl aliquots of reconstituted Mycograb® were pipetted into auto-sampler vials before column application.

Data Analysis

Data analysis software: LCsolution (version 1.21 SPl) software base and supplementary GPC postrun analysis software supplied by Shimadzu.

SEC analysis of Mycograb® performed two sets of measurements:

1. Molecular weight determination. Weight average molecular weight MW, and peak molecular weight MW(p) were determined and expressed in Daltons. These values define the molecular weight of the test article.

2. Retention times. RT(s) are absolute values expressed in minutes and define the start of elution envelope of the test article. The RT(p) peak retention time was also determined.

The value MW, the weight average molecular weight is a composite value not derived from a single point on the chromatogram. Instead the chromatogram is sorted into up to 5,000 slices using the Shimadzu postrun analysis software. The molecular weight average is the mean of the weight fraction for each slice (the weight fraction is the weight of the slice, divided by the total weight of all slices, where the weight of the slice is defined by the height x molecular weight at the slice point of the chromatogram). The resultant value gives a weight average for Mycograb® that is representative of the entire chromatogram.

The correlation between molecular weight and retention time has been illustrated in Figure 1. The following data are derived from Figure 1.

Linear: ax+b

a = -0.2713082 b = 11.1025

R^Λ2 = 0.9821346 R = 0.9910270Dispersion = 0.0495613

Example 1 - Application of Mycograb to SEC column

Freshly-prepared Mycograb batch 300606 was applied to the column at 20, 40, 80 and 100 μl respectively. The results of an injection volume of MG 300606 @ 20, 40, 80 and 100 μl are shown in Figure 2 (relative quantity versus time). Figure 3 shows the results of SEC following 4 injection volumes after constant height adjustment. There was no molecular weight difference showing among the 4 injection volumes. In order to obtain enough protein for each fraction, 100 μl injection volumes were used for the subsequent investigations.

Example 2 - Fraction collection and fraction analysis

Mycograb batches 270603, 071005, and 300606 were applied to the column for SEC. 4 fractions (#0-3) were collected from each run for further analysis. Each fraction was reapplied to the column separately and aliquots from each fraction were pooled ("frac- combined"; equal volumes of each fraction) and the mixed pool was also subjected to SEC.

Figure 4 shows the results of SEC analysis of freshly prepared MG 270603. Table 1 shows the GPC calibration of results of the 4 fractions from MG 270603. Table 1

Sample name Mw (Da) MWp (Da) RTs (min) RTp (min)

FP MG 270603 frac-0 3888078 3858189 14.375 16.646

FP MG 270603 frac-1 1506592 1316076 15.367 18.585

FP MG 270603 frac-2 698567 589769 16.365 19.652

FP MG 270603 frac-3 285869 322880 17.955 20.812

FP MG 270603

frac-combined 757664 570959 16.215 19.704

FP MG 270603 848813 589333 13.845 19.653

Figure 5 shows the results of comparisons of the SEC results of the fractions from MG 270603. Figure 6 shows the results of SEC analysis of Fractions 0-3 of MG270603 after constant height adjustment.

Figure 7 shows the results of SEC analysis of freshly prepared MG 071005. Table 2 shows the GPC calibration of results of the 4 fractions from MG 071005.

Table 2

Sample name Mw (Da) MWp (Da) RTs (min) RTp (min)

FP MG 071005 frac-0 7068241 6738976 13.725 15.753

FP MG 071005 frac-1 4247369 3905923 13.875 12.626

FP MG 071005 frac-2 1512577 1210164 14.94 18.502

FP MG 071005 frac-3 530941 447111 17.265 20.096

FP MG 071005 3024593 3325503 14.055 16.884

frac-combined

2941536 3454782 13.395 16.823

FP MG 071005

Figure 8 shows the results of comparisons of the SEC results of the fractions from MG 071005. Figure 9 shows the results of SEC analysis of Fractions 0-3 of MG071005 after constant height adjustment.

Figure 10 shows the results of SEC analysis of freshly prepared MG 300606. Table 3 shows the GPC calibration of results of the 4 fractions from MG 300606. Table 3

Sample name Mw (Da) MWp (Da) RTs (min) RTp (min)

FP MG 300606 frac-0 5179501 5366579 14.375 16.117

FP MG 300606 frac-1 2957084 2601339 14.91 17.277

FP MG 300606 frac-2 1205682 993897 16.055 18.817

FP MG 300606 frac-3 494383 416502 17.97 20.209

FP MG 300606

frac-combined 1528155 1042289 14.565 18.741

FP MG 300606 1738708 1135588 14.01 18.494

Figure 11 shows the results of comparisons of the SEC results of the fractions from MG 300606. Figure 12 shows the results of SEC analysis of Fractions 0-3 of MG300606 after constant height adjustment.

Example 3 - Activity analysis of different size multimeric species of Mycograb separated by SEC-HPLC

Fractions were selected from areas 0, 1, 2 and 3 from batches 270603, 300606 and 071005 as shown in Figure 13 which shows the delineation of fractions from within each batch. The protein concentration as determined from chromatographic profiles was as stated in Table 4.

Table 4

Example 4 - ELISA Assay

Each fraction was then analysed for activity by ELISA by a standard antigen capture ELISA assay.

1. Principle and Source of Procedure

Biotinylated antigen peptide was bound to High bind streptavidin coated microtiter plates. Mycograb® was then overlaid across a dilution series followed by incubation with an Anti-His HRP conjugated mouse monoclonal antibody, which recognised the His tag on the Mycograb® protein. The amount of Mycograb® bound to the plate was then detected by incubation with ABTS® solution, a substrate of the HRP conjugate. The amount of Mycograb® bound to the plate was proportional to the colourimetric absorption at A405nm.

2. Equipment

High bind Streptawell plates supplied by Roche (Cat No 11989685001), an Elisa plate reader (Biorad Model No 680), a Thermo WellWash AC plate washer, 1.5ml Eppendorf tubes (VWR Cat. No. 211-2139).

3. Chemicals and Reagents

Chemicals

AU chemicals were of analytical grade unless otherwise stated.

Tris Sigma Cat No T6791

BSA Sigma Cat No A7030

PBS tablets Sigma Cat No P4417

Concentrated Hydrochloric acid Sigma Cat No 32033-1

Tween 20 Sigma Cat No P7494 Specific biotinylated antigen peptide (biotin-NKILKVIRKNIVKK)... Pepceuticals.

Water, Milli-Q water or better quality.

Anti-His tag HRP mouse monoclonal antibody conjugate... Sigma Cat. No. A7058.

ABTS™ substrate solution Sigma Cat No A3219.

Reagents

Blocking buffer Stock 1 (5% w/v BSA in Milli-0 water)

BSA 2.5g

2.5g of BSA was weighed out and added to 50ml of Milli-Q water. Stored at 4°C for 1 week.

IM Tris buffer pH 7.8

Tris 121.24g

121.24g Tris was weighed out and dissolved in 950ml Milli-Q water with stirring. pH was checked and adjusted with drop-wise addition of Concentrated Hydrochloric Acid until pH was 7.8. This was made up to 1 litre with Milli-Q water. It was then filtered through a 0.22 μm filter (Sartorius) and stored at room temperature for up to 1 month. Sample Diluting Buffer (2OmM Tris pH 7.8 0.1% w/v BSA)

ImL of IM Tris stock solution was added to 48ml of Milli-Q water and ImI of Blocking buffer Stock 1 solution. This was made fresh for each experiment.

Wash Buffer (PBS + Tween 20 0.1% v/v)

5 PBS tablets were dissolved in 90OmL of Milli-Q water, ImI of Tween 20 was added, and stirred until tablets had dissolved. It was made up to 1000ml with Milli-Q water. Stored at 4°C for 1 week.

Blocking buffer Stock 2 (Wash Buffer + 5% w/v BSA)

BSA 2.5g

2.5g of BSA was weighed out and dissolved in 50ml of Wash Buffer. Stored at 4°C for 1 week.

Peptide Diluting Buffer (PBS + 0.1% v/v Tween 20 + 0.1% w/v BSA)

ImI of Blocking buffer Stock 2 was added to 49ml of Wash buffer. Made fresh for each experiment.

Antigenic peptide solution

Biotin-NKILKVIRKNIVKK peptide (SEQ ID NO: 5).

A 2mg/ml solution of the custom synthesised antigenic peptide solution was made up by weighing out lOmg of peptide and dissolving it in 5ml of Milli-Q water. 50-100μl aliquots were dispensed into 1.5ml Eppendorf tubes and stored frozen at -80°C for up to one year. Antigenic peptide Working Solution (4ιιg/ml peptide in Peptide Diluting Buffer)

24μL of Antigenic peptide solution (2mg/ml) was added to 11.976ml of Peptide Diluting Buffer to give a 4μg/ml solution. Made fresh for each experiment.

4. Sample preparation

Test Article

1 x lOmg Mycograb was resuspended for each test batch in 5ml of Milli-Q water, mixed gently to ensure all the powder in the vial was incorporated and dissolved. It was centrifuged at 13,000rpm for 5 minutes to remove any particulate matter. The protein concentration of this solution was then determined according to SOP NTP STO lii. The fractions derived from the SEC analysis were tested at a protein concentration of 0.625 μg/ml and the results expressed as a percentage of the value generated by the unfractionated Mycograb.

5. Control Article

1 x lOmg Mycograb Reference material was resuspended in 5ml of Milli-Q water, and processed in an identical fashion to the Test Article.

6. Procedure

1. An aliquot of the 2mg/ml stock solution of antigenic peptide was removed from the freezer and diluted 1:500 (25μl peptide in 12.5ml buffer) with PBS buffer containing 0.1%(w/v) BSA and 0.1%(v/v) Tween 20 to generate a working solution of 4μg/ml peptide. A 96 well high bind StreptaWell plate (Roche) was coated from rows B-H overnight at 4⁰C with lOOμl of 4μg/ml biotin-NKILKVIRKNIVKK peptide in 0.1% (w/v) BSA PBS-0.1% (v/v) Tween 20. lOOμl per well of 0.1% (w/v) BSA PBS-0.1% (v/v) Tween 20 were added to all wells in Row A.

2. The plate wells were washed 3x 30sec with 200μl of PBS 0.1% (v/v) Tween 20 buffer on a Thermo Well Wash AC.

3. The Mycograb® sample or fraction were prepared to a concentration of 0.625 μg/ml prior to loading by diluting in 2OmM Tris buffer pH 7.8, 0.1% (w/v) BSA. A control set of blank wells contained lOOμl 2OmM Tris pH 7.8, 0.1% (w/v) BSA.

4. The plate was left at room temperature for 1 hour and the wells then washed 3 times with PBS-0.1% (v/v) Tween 20 as described previously.

5. 100 μl of mouse monoclonal Anti-His HRP conjugate (Sigma A7058) was loaded into each well at a concentration of 1:2000 in 0.1% (w/v) BSA PBS-0.1% (v/v) Tween 20 and left for 1 hour at room temperature.

6. Wells were then washed as described above and the bound Mycograb® detected by the addition of lOOμl of ABTS® reagent. The colorimetric development was read at 405nm with readings taken when the absorbance of the highest concentration samples in the second calibration curve reaches 1.3 AU. The concentration of Mycograb® or fraction was proportional to the absorption at A405nm.

7. The ELISA activity was expressed as a fraction of the value obtained by the unfractionated Mycograb control at 0.625 μg/ml as a percentage.

Results

The results have been summarised in Table 5. The fraction 0 contained so little protein that it was impossible to measure in batches 270603 and 300606. The mean values were highest in fraction 2. The mean and standard deviations of fraction 2 from each batch have been listed in Table 6. Thus the average multimer molecular weight of fraction 2 was 1,138,942 Da (standard deviation of 411088.5 Da). Accordingly, the molecular weight range within two standard deviations of the average is 316,765 Da to 1,961,119 Da. Since the molecular weight of a single sub-unit of the Mycograb scFv is approximiately 27.2 kDa, this corresponds to multimers of between 11 and 73 sub-units. The molecular weight range within one standard deviation of the average is 727,853.5 Da to 1,550,030.5 Da. This corresponds to multimers of between 26 and 57 sub-units.

Table 5 Activity of the sub fractions of Mvcograb

na: protein concentration too low for analysis.

Table 6 Description of the fractions of Mycograb

Example 5

Table 7 shows the molecular weights of freshly prepared and freeze/thawed Aurograb® batches. Aurograb® comprises a humanised scFv recombinant antibody the sequence of which is shown in SEQ ID NO: 4. It binds to the immunodominant cell surface antigen, GrfA, a staphylococcal ABC transporter protein (WO 99/50418; Burnie JP et al., Infect Immun. 2000 Jun;68(6):3200-9; PMID: 10816464). The production of Aurograb is reported in further detail in WO-A-03/046007 (see especially section 7) which is hereby incorporated by reference. Figure 14 shows size exclusion chromatograms of freshly prepared and freeze/thawed Aurograb batches: A) 2001 batches freshly prepared and B) after freeze/thaw cycle. C) 2004 batches freshly prepared and D) after freeze/thaw cycle. This figure confirms that aggregate behaviour for Aurograb is the same as for Mycograb.

Table 7

Aurograb batch# MolecularWeight (Da)

Fresh Prep Freeze/Thaw

1^st peak 2^nd peak

3398748 248167 62654

100801 3328361 157100 61987

4467086 195716 64965

070901 4513000 169034 61961

3818755 190639 56929

240801 3871387 148224 51557

629328 148624 59133

070504 606824 138789 60861

836778 169497 57424

210504 808216 193500 70822

672903 58379

280504 668196 130716 58889 Example 6

Summary

A range of Mycograb mutants was prepred. The aim of the Mycograb mutants was to obtain a mutant scFv peptide with improved structural properties compared with the wild type Mycograb. It was believed that through point mutations, especially the replacement of free cysteine by tyrosine, aggregation and formation of incorrect disulfide bonds during down stream processing should be varied. It was also believed that exchanging the orientation of the heavy chain fragment with the light chain fragment and removing the HIS-Tag is be beneficial for formation of a native 3D structure of the Mycograb molecule.

After cloning, the constructs were sequenced prior to fermentation. The fermentation was scaled up to deliver enough material for inclusion body (IB) isolation and for further downstream processing. The expression constructs of the mutants were purified according to the adapted Biomeva process until the refold end step.

The physical parameters of a range of mutant Mycograb peptides was tested as set out in Examples 7 to 14.

An overview of the mutants tested and their mutations is given in Table 8. The wild type was included in the studies for comparison reasons.

Table 8: Name and structural properties of the 12 investigated Mycograb mutants and the wild type (myc 123)

The methodology used in the test assays will now be described.

Inclusion body (IB) isolation

4 L of fermentation broth obtained in LVA (Laborversuchsanstalt) from shake flask culture of Mutants Myc 118,119,130,133 and Myc 135 were disintegrated with a high pressure homogenizer (LAB 40-15 RBFI) in RPP4 at 700 Bar for 2 cycles of 15 min each. The IBs were separated from the cell debris at lab scale with a bottle centrifuge at 10 000 rpms for 20 min at 4°C. IBs were washed twice with water for laboratory use (WFL) and afterwards, a 20% (w/v) suspension in WFL was prepared. The suspension was stored in aliquots at -20°C.

IBs from mutants Myc 134,137,138,106,136,139,140 and Myc 123 (wt) were isolated at pilot scale in RPP4 because fermentation of these mutants was done at 3OL scale in bioreactors. IBs were separated from cell debris with a disc stack centrifuge. A 20% suspension (w/v) in WFI was prepared. This suspension was stored in aliquots at -20°C. Solubilization

Solubilization with NLS (according to adapted Biomeva process). Solubilization of the 20% IB suspension was done by dilution with WFL to a protein concentration of 8 mg/ml followed by a 1:2 dilution with 10OmM Tris/Base, 4% NLS, pH 9.0 buffer. The solution was stirred at room temperature in a beaker until clarification but at least for 30 min. The time until start and end of clarification was recorded.

Alternative Solubilization with Urea, GuHCl, DTT. The alternative solubilization strategy was performed by a 1 : 10 dilution of a respective volume of 20% IB suspension with 2OmM Tris 8M Urea +/- 5mM DTT or 2OmM Tris, 6M GuHCl +/- 5mM DTT both at pH 9.0. The resulting concentration of urea and GuHCl was 7.2M and 5.2M respectively, due to the volume of the IB suspension solution.

Refolding of solubilized IB's

Refolding with NLS. The refold was done by 1:4 dilution of the solubilization solution with a 5OmM Tris/Base buffer. The final concentration of NLS was 0.5%. Refolding was initiated by addition of 50μM CuCl₂. Samples were taken and immediately submitted for RPC2 analysis prior and after CuCl₂ addition, then approximately 24, 48, 72 and 96 hours after CuCl₂ addition.

Refolding after solubilization with Urea, GuHCl. Refolding of a Mycograb solution after solubilization with 8M urea or 6M GuHCl +/-DTT was performed by 1:50 dilution with a buffer containing 2OmM Tris/Base, 1 % NLS and 2mM Cysteine at pH 9.0.

For some mutants, dilution of a Mycograb solution after solubilization with urea by 1 :10 with a buffer containing 2OmM Tris/Base, 0.5M L-arginine and 2mM Cystin at pH 9.0 was also performed.

The refold solution was stirred for 96 hrs at 4°C or 24hrs at RT, respectively. Refolding kinetics

A refold kinetic was recorded for mutant Myc 119 in order to determine the required refolding time. A sample of a 20% IB suspension of Mycl l9 was solubilized as described above. Refolding was performed as described above, but samples were taken at respective time intervals and analyzed by RPC 2.

NLS removal by UF/DF from refolding solution. Mutants Myc 119, Myc 137, Myc 106 and Myc 123 (wt) were solubilized and refolded as described above. After refolding, a buffer exchange of REF.END solution was performed with an Amicon stir cell with 1OkDa molecular weight cut-off. NLS concentration after each turn over volume was determined with RP-HPLC. 50 ml of REF.END solution were concentrated to 25ml and then filled up again to 50ml with diafiltration buffer. This procedure was carried out 4 times. Aggregation tendency after NLS removal was measured with SEC-HPLC running with formulation buffer.

SDS Page analysis

SDS Page was performed using NuPAGE 4-12 BisTris gels and MOPS as running buffer. The run time was 65 minutes at 200 volt. A mass of 0.2-0.4 μg Mycograb was applied on each lane. After electrophoresis, the gels were stained with silver. For reducing SDS Page, 100 mM DTT was added to the sample.

Results

The expression construct of the mutants was analyzed at different stages in the down stream procedure with the analytical methods listed in Table 9: Table 9. Code and description of process intermediates and the respective analytical assay

Table 10 gives a description of the analytical methods listed in Table 9 that were used for evaluation of the mutants. A comment is included describing the specificity of the particular assay.

Table 10: Analytical method, the respective response and Unit of Measurement (UoM) for the assays used to evaluate the mutant samples.

Further details of the respective assays for the investigated mutants are summarized and discussed in the following Examples 7 to 14.

Example 7 - Mass balance after solubilization and refolding - results RPCl titer 0 determination

Protein concentration prior and after solubilization and refolding of IB's was measured with the titer assay. As this assay measures all soluble protein present in the sample, mass balance should yield 100%. Mass balance for solubilization was calculated using 5 equ.1. % Sol = ^mg™^m-^SOL equ. 1

Wg^c₁IB-RES where mg _RPCI IB. SOL is the mass of protein in the solubilzation solution of the IB's calculated from concentration measurement by RPC 1 method and volume of IB SOL solution, mg _RPCI IB. RES is the mass in the 20% IB suspension calculated from concentration measurement by RPC 1 method and volume of the solution after resuspension of the isolated IB's in DI.

Mass balance for refolding was calculated using equ.2 and is expressed as % recovery. IB's solubilized with either 4% NLS, 8 M urea+/- 5mM DTT or 6M GuHCl +/-5mM DTT were diluted and refolded as described in Example 6.

% Ref = ^mgwc> ^{Re f} -^END equ. 2 Wg^₁IB-SOL where mg _RPCI Ref.END is the mass of protein according to concentration measurement with RP-HPLC found in the refolding solution times volume of refolding solution, mg _RPCI IB. SOL is the mass of protein found in the IB solubilisate, % REF is the recovery after refolding.

Mass balance after solubilization with 4% NLS and subsequent refolding of all mutants as calculated by equations 1 and 2 are illustrated in Figure 16. Raw data can be found in Tables 11 and 12.

Table 11 shows the recovery after solubilization with NLS of the IB_RES suspension and recovery after refolding calculated from analytical method RPC I for all tested mutants. Also shown is the protein concentration in the IB-RES solution and protein concentration in the refolding solution determined by analytical method RPCI. Table 12 shows protein concentration determined by RPC I of IB RES, IB_SOL and REF.END samples after solubilization with urea (SOL:urea) and solubilization with GuHCl (SOL:GuHCL).Refolding time was 96hrs at 4° C. The dilution factor was 500 and 50 for IB RES and IB SOL, respectively to yield the REF.END solution. Table 11

Table 12

Mass balance of solubilization was exceeding 100% for 10 of the 12 investigated mutants. This could be due to the fact that the IB suspension was a crude sample type and eventually, the IB's were not completely dissolved when the sample was taken, leading to an inhomogeneous solution and thus underestimating total protein concentration. Data variability is high, with a relative standard deviation for 6 mutants analyzed twice ranging from 2.6% (Myc 138) to 42.9% (Myc 133). % Recovery after refolding was between 72% (Myc 138) and 99% (Myc 134). The expected recovery is 100% (similar to recovery after solubilization). Recoveries after refolding were all lower than 100% and scatter not as much as for recovery after solubilization. This indicates that estimation of protein concentration in IB. SOL and REF.end samples is more accurate than in IB.RES samples. However, calculation of recovery primarily serves as a control for solubilization and refolding experiments.

Refolding yields related to the solubilization solution IB. S OL and the IB suspension IB.RES when 8M urea or 6M GuHCl was used as solubilization agent are shown in Figure 17.

The recovery varies from 44% to 230% reflecting the problems with measurement of protein concentration especially in IB. SOL and IB.RES samples, possibly due to insufficient homogenization prior to sampling.

Concentration in the REF.END samples after urea solubilization was comparable to concentration in REF.END after GuHCl solubilization (see Tables 11 and 12) though by an average factor of 1.2 higher. Dilution was thus consistent.

Example 8 - Solubilization time

Solubilization time with NLS was studied for all mutants. The time until the solution started to become clear and the time until no further clarification could be observed was recorded and is illustrated in Figure 18.

In contrast to using 2% NLS, solubilization with Urea +/- DTT and GuHCl +/-DTT was 2-3 times faster.

A correlation between the number of Cysteines and time required for clarification to start was found. With the exception of mutant 134 (5 cys), mutants with 4 cysteine residues solubilized faster than mutants with 5 cysteine residues. Figure 19 shows a variability chart where START [min] of clarification is plotted versus the 3 categories: alignment of heavy or light chain fragment at the N-terminus, number of linker elements and cysteine residues. With exception of Myc 134, indicated with 1 in Figure 19, data points in category with 4 Cysteines scatter around earlier solubilization start times than compared with data points in the category with 5 cysteines.

A regression model (R²= 0.72 when Myc 134 is excluded) predicted that start of solubilization would decrease from 20 min to 11.3 min for a Mycograb construct with 4 cysteines instead of 5 (model not shown).

Example 9 - RPC 2- NLS refolds

Mycograb REF.END samples solubilized and refolded according to Biomeva adapted process (described in Example 6) mutants were analyzed by RPC 2.

An overlay of REF.END samples from all investigated mutants including the wild type (MYC 123) is shown in Figure 20 and Figure 21. Mutant Myc 116 was screened earlier in lab DSP-DEV 1 and was included in the overlay for comparison reasons.

In Figure 20, chromatograms of REF.END samples generated from IB's isolated at bench scale and in Figure 21, chromatograms of REF.END samples generated from IB's isolated at larger scale in the pilot plant are shown.

Elution profiles were compared with respect to:

1. Retention time peak 1, reflecting hydrophobicity 2. Shape of peak 1, reflecting presence of dimer and homogeneity of monomer species when the peak is sharp

3. Ratio area of monomer/dimer peak (peakl) to aggregate/impurity peak (peak 2), reflecting aggregate/impurity content

Retention time peak 1 , reflecting hydrophobicity

The retention time of the monomer/dimer peak for the tested mutants is listed in Table 13. Table 13: Retention time [min] of peak 1 in RPC2 for the tested mutants and the molecule properties

Retention time varies greatly with molecule construct. There is no trend of retention time (reflecting hydrophobicity) increasing with linker length, as would be expected. One linker element consists of four Glycines and one Serine residue. Glycine is hydrophobic in contrast to Serine, which is hydrophilic. However, the 4x higher Glycine content in the linker seems not to significantly increase the hydrophobicity as measured by retention time in RPC 2.

Retention time of Myc 130 is shorter than for the rest of the mutants. 10 amino acids were replaced by amino acids of more hydrophilic (5 serines) nature, thus decreasing the hydrophobicity of the molecule und consequently resulting in earlier retention time. Excluding this data point from statistical analysis results in a model showing significant difference in retention time when the orientation of the VL is N-terminal compared to an orientation when its C-terminal. Retention time increases by 0.41 min when the VL element is located N-terminal compared with when its located C terminal. Figure 22 shows the scaled estimates and a prediction profiler of the model with number of cysteines, linker length and chain fragment orientation as factors and retention time as response. Note that retention time of Myc 130 was excluded from the model. A plot of retention time versus linker length, shown in Figure 23, demonstrates that retention time for this construct is lower compared to the rest of the mutants because of point mutations resulting in more hydrophilic nature, as mentioned above.

Shape of peak 1

It is assumed that a sharp peak 1 with no or only little shoulders reflects the homogeneity of a monomeric Mycograb. Peak shape was assessed by overlays of the RPC 2 chromatograms of REF.End samples from all mutants and sharpest peaks were determined for the mutants Myc 137, Myc 138 and Myc 139. Peakl was sharper than the wild type Myc 123 and peaks 1 and 2 were almost base-line separated. This may be an indication that these constructs express the monomeric/dimeric protein with greater homogeneity than the wild type.

Ratio area of monomer/dimer peak

Impurity/aggregate content in relation to monomer/dimer was lowest for Myc 116 and MYC 123 wt as shown in Figure 20 (Myc 123 wt). However, analysis of these two samples was performed 3 months earlier by another laboratory and an increase of peak 2 was noticed over time. A chromatogram of REF.End sample from Myc 123 that was prepared and analyzed in the same month as REF.End samples of the tested mutants is shown in Figure 21.

The area ratio of monomer/dimer peak to aggregate peak was determined by normalizing peak 1 to the same peak maximum. The peak area of peak 2 after normalization was ranked according to increasing size using visual area estimation. The normalized overview is shown in Figure 24.

The following ranking could be established:

Myc 116, Myc 139, Myc 136 < Myc 119, Myc 12, Mycl40 < Mycl37, Myc 135, Mycl38 < MyclO6 < Mycl30 < Myc 134 < Mycl 18 < Mycl33 RPC 2 chromatograms of REF.End samples generated from IBs of mutants Myc 106, 134, 136-139 were processed in the pilot plant and showed lower impurity /aggregate peaks (cf Figure 21) than IB's of mutants Myc 118-135 isolated at bench scale.

Example 10 RPC 2 urea/GuHCl refolds

Mutants Myc 118, 119, 130 and Myc 133 were dissolved with 7.6M Urea +/- DTT and 5.6M GuHCl +/-DTT and refolding was initiated by dilution in refolding buffer.

All REF.End samples did not show the monomer/dimer peak (peak 1) in RPC 2. A huge peak 2, assumed to be aggregates and impurities is predominant. A representative RP HPLC chromatogram of a refold end sample from mutant Myc 119 is given in Figure 25. The sample was prepared as described in Example 6.

The monomer was expected to elute at approximately 10.5 min. Peaks eluting earlier are not identified and were not observed in refolds with 0.5% NLS. The huge peak 2 indicates strong aggregation. Similar elution profiles were obtained for all REF.End samples after urea/GuHCl solubilization.

Similar elution profiles were obtained when the refold was done by 1 :10 dilution with a buffer containing 2OmM Tris/Base, 0.5M L-arginine and 2mM Cystin at pH 9.0. A representative chromatogram is shown in Figure 26.

The strong aggregation tendency was confirmed by SDS-Page under non reducing conditions, a huge and intense HMW smear was detected for a REF.End sample, with no monomeric band visible after urea solubilization. This smear then disappeared when the sample was reduced and a monomeric Mycograb band appeared. In Figure 27, SDS Page analysis of a reduced and non- reduced REF.End sample after urea solubilization is shown. Lanes 4-7 show REF.IM and REF.End sample of MYC 119 under non- reducing conditions at 2 different dilutions. Lanes 10-13 show the same sample under reducing conditions. The aggregate smear disappeared when the sample was reduced and the monomeric as well as the dimeric band became visible. Urea and GuHCl were present in the refolding solution at low concentration (0.14M in case of a 1:50 dilution and 0.72M in case of a 1:10 dilution for urea; for GuHCl, it was 0.1 IM in case of a 1 :50 dilution and 0.56M in case of a 1:10 dilution) cannot prevent the protein from aggregation. Using DTT does not seem to have a significant effect on aggregation as RP-HPLC chromatograms (RPC 2) with and without DTT looked comparable, as shown in Figure 25.

In contrast to the REF.End sample, RPC 2 chromatogram of a IB. SOL sample dissolved with urea was comparable in terms of peak shape with a IB. SOL sample dissolved in 2% NLS. Figure 28 shows an overlay of HPLC chromatograms.

SDS-Page analysis of a non-reduced IB. SOL sample showed stronger HMW bands when the sample was dissolved with urea than in case of a IB. SOL sample dissolved in 2% NLS. This is shown in Figure 27: in lane 3, the sample was solubilized with urea and in lane 8, the sample was solubilized with 2% NLS. It can be concluded that peak 2 in RP-HPLC does not give an indication about aggregate content since peak 2 of an chromatogram overlay is even smaller for an IB. SOL sample in urea than in NLS.

Subsequent refolding did not yield a monomeric peak but the protein completely aggregates.

Example 11 - SDS-PAGE reducing and non-reducing

Reducing and non-reducing SDS-Page was performed to determine impurities and aggregates content in REF.End samples. With reducing SDS-Page Mycograb species appeared as monomeric and dimeric band and host cell impurity content in the sample could be distinguished from aggregated species when compared to a non-reducing SDS- Page gel. Non-reducing SDS Page showed Mycograb monomers, dimers and aggregates. Comparing a non-reduced SDS-Page silver stain gel with a reducing SDS- Page analysis, the amount of aggregated species could be evaluated semi-quantitatively. Reducing and non-reducing SDS -Page gels of REF.END samples of all tested mutants are shown in Figure 29 and Figure 30. The gel in Figure 29 on the left side shows a reducing SDS-Page of REF.End samples from mutants MYC 118, 119, 130, 133, 134, 135, 137. The band at 3OkDa is monomeric Mycograb and it is predominant in all samples. According to the migration of the monomeric band, Mycograb expressed in mutant MYC 134 seems to have higher molecular weight than the other mutants analyzed on the gel. The same but to a lesser extend was detected for MYC 135. According to Table 14, MYC 134 has the highest theoretical molecular weight among the mutants shown in Figure 29, followed by MYC 135.

In the non reducing SDS-Page gel, aggregated species as well as the dimer are visible. In lanes 13 (MYC 134 ) and 15 (MYC 137), HMW bands are fainter than in the other lanes. This would indicate a lower content of aggregated species, however, also the bands for the monomer are more faint.

Double bands of different migration time and intensity in comparison with each other were observed for all mutants with exception of Myc 133 shown in Figure 29. Identification of these bands was hardly possible, however it was assumed that it was Mycograb Monomer of native like structure.

Figure 30 shows a reducing and non -reducing SDS Page of REF.End samples from mutants MYC 106, 136,138,139,140 and the wild type, MYC 123. Differences in MW for the different constructs could be determined according to different migration of the monomeric band. The bands in lanes 5 and 7 appeared at a slightly lower MW than the bands in lanes 2,3,6 and 4 which was in agreement with the theoretical MW listed in Table 14.

The mass of protein applied to the gel was not consistent; monomeric bands varied in intensity because protein concentration determination in the REF.End sample was not accurate enough. Thus semi-quanitative analysis of impurity content was not possible. However, the higher impurity content in REF.end sample of Myc 106 was obvious as the thickness of the monomeric band was comparable to one of Myc 140 but the intensity of the other bands was much higher. SDS-Page analysis indicated that a REF.End sample of Myc 106 _orig_ami contained more HCP and product related impurities. However, this was not confirmed by RPC2 analytical method, where the area of peak 2 was in the same range as for the other mutants.

Example 12 - SEC 0.5% NLS : Determination of molecular weight of Mvcograb species in the REF. END sample

All REF.END samples prepared according to the adapted Biomeva process (see Example 6) were analyzed with SEC- HPLC in 0,5% NLS and the average molecular weight was determined. An overlay of the SEC chromatograms is shown in Figure 31 and Figure 32.

The average molecular weight ranged from 48.6kDa to 65.8 kDa. The breadth of the peaks reflects the heterogeneity of species in the sample. Though approximately 80% of the product was monomeric in REF.End samples, dimers and higher MW species were present as well as non-product related impurities, resulting in a broad elution peak.

In Figure 31, the elution profile of Myc 130 sticks out because of increased fronting compared with the other investigated samples. This might have been due to increased heterogeneity in the sample because of the construct' s nature (more hydrophilic) or due to an accidentally different sample treatment.

Samples shown in Figure 31 were prepared simultaneously and stored at 4°C for 5 days prior to analysis. Samples shown in Figure 32 were prepared simultaneously and stored at 4°C over night days prior to analysis. Samples seemed to be stable at 4°C as MW of the constructs were in a similar range.

Fronting of Myc 130 may have beem due to higher amount of aggregated species compared with the other investigated samples. However, peak 2 in the corresponding RPC 2 chromatogram was not outstandingly large but it has been observed that there is only sometimes a correlation between increased MW determined by SEC 0.5% NLS and large peak 2 peak area, determined with RPC2.

Additionally, SDS-page of Myc 130 did not indicate a higher impurity content and increased heterogeneity of the sample. Other factors leading to fronting in SEC such as column overloading and increased temperature during analysis can be excluded as all samples were analyzed on the same day.

There was a very early retention time for Myc 130 in RPC2.

It was assumed that the average MW in a REF.End sample was increasing for an increasing amount of dimers, aggregates and impurities. The calculated MW of a monomeric Mycograb expressed in the different mutants was between 26 and 27kDa because of the different linker length and other mutations. A table listing theoretical MW- calculated from the amino acids-, MW of the REF.End sample determined by SEC, # of amino acids, linker length and number of cysteines is given in Table 14.

Table 14: SEC-HPLC 0,5% NLS results of REF.End samples from all tested mutants. The mutants are ranked according to their theoretical molecular weight.

Theoretical MW was plotted versus MW determined by SEC and a linear relationship with a correlation coefficient of 0.77 was found when the data point for Myc 130 was excluded.

Mutants Myc 106, Myc 138 and Myc 135, represented by dots to the right of the lower dashed line in Figure 33, had a lower average MW than the wild type. Mutants Myc 133, 136 and 139, represented by dots to the left of the higher dashed line had a higher average MW than the wild type. However, assay variability has to be taken into account. Additionally, from Figure 32 it can be seen that the mass of injected protein was not always the same as peak area for some of the samples. Formation of covalent aggregates should be decreased for mutants with 4 cysteines as compared with Mycograb with 5 cysteines because no free cysteine in mutants with 4 cysteines is available after formation of intermolecular SS bridges. Intermolecular covalent aggregates are formed during refolding even with a mutant with only 4 cysteines but it would be likely that the amount is lower than for a mutant with 5 cysteines.

Example 13 - SEC Formulation: NLS removal by UF/DF from refolding solution to measure aggregation tendency

In order to evaluate aggregation tendency of Mycograb mutants, the NLS concentration was lowered by an average factor of 5 from the REF.End solution with ultra/diafiltration using a stir cell. The total buffer volume used during diafiltration divided by the retentate volume is the diafactor and was 2.5.

The rationale of this experiment was to investigate aggregation tendency of the mutants when the dissolving agent NLS is lowered to a concentration at which aggregation cannot be prevented anymore.

It was assumed that formation of aggregates can be assessed with SEC-HPLC formulation. The elution buffer contained 0.5M urea buffer but no NLS to suppress aggregation. Increase in molecular weight was used as a measure of tendency to aggregate and allows the mutants to be compared.

Mutants Myc 137, Myc 106, Myc 119 and the wild type Myc 123 were selected for a first set of experiments. Table 15 shows the molecular weight (MW) in kDa determined by SEC- HPLC running in formulation buffer and concentration of NLS (%) determined by RP-HPLC for REF.End samples from mutants MYC 119,137,106 and MYC 123 before and after UFDF. The NLS reduction factor was calculated from NLS concentration in the sample prior to UFDF (# 2) divided by the concentration of NLS

10 after UFDF (#3). The % increase MW based on SEC HPLC 0,5% NLS was calculated from the MW of the REF.END sample (#1) and the MW after UFDF (#3) for the respective Mutant.

Table 15

15

*The molecular weight was determined with an analytical SEC HPLC method containing 0.% NLS in the running buffer (SEC HPLC 0.5% NLS). ** data from Table 14.

20

The apparent high molecular weight determined by SEC HPLC (running with formulation buffer) of the sample prior to UFDF was due to aggregation of protein during analysis. The sample prior to UFDF still contained 0.5% NLS. As the sample migrated through the column, NLS was more strongly retarded than the protein and consequently aggregation occurred. Therefore this analytical method was not suited to determine molecular weight of samples containing NLS.

In order to determine molecular weight of Mycograb in the REF.End sample, SEC HPLC with 0.5% NLS in the running buffer was used. The increase in MW after removal of NLS in the REF.End sample by UFDF was calculated as described above in relation to Table 15. Values are shown in row 6 of Table 15. MYC 123 showed the smallest increase in MW whereas MYC 119 had the strongest increase, 400%.

MYC 123 has a lower aggregation tendency than MYC 119 based on these data.

In Table 16, the mutants are ranked according to increase in MW after NLS removal together with the mutations. It has to be noted that the two mutants with a 3x linker element have significantly lower % of MW increase compared with mutants with a 4x linker element.

Table 16: Increase of molecular weight after removal of NLS by UFDF for the 4 investigated mutants.

Figure 34 shows an overlay of the SEC HPLC chromatograms obtained from the sample prior to UFDF and after each volume reconstitution. The shape of the elution peaks did not significantly change with reduction of NLS concentration. Consequently, the MW of the sample also remained constant with reduction of NLS. This might be an indication that there is a limit in the concentration of surfactant below which aggregation is initiated but does not proceed further. The NLS concentration was reduced on average (n=5) to a concentration of 0.124% after 5 volume reconstitutions, corresponding to a diavolume of 2.5. Theoretically, for a retention R = O of NLS, the calculated remaining NLS concentration should be 0.020%. Equation 3 was used for calculation of the theoretical remaining NLS concentration:

^Crelenlat ~ ^c feed ^X V^ / ^e1^{U- 3} where c_relentat is the concentration of NLS in the retentate, c_jeed is the concentration of NLS in the feed solution. R is the retention, the fraction of solute that is retained by the membrane. VCF is the volume concentration factor and N is the diavolume which is the total buffer volume introduced to the operation during diafiltration divided by the retentate volume.

The discrepancy between the theoretical and the measured concentration of NLS in the retentate is an indication for retention of NLS by the membrane greater than 0. This might be due to interaction between NLS and the protein, membrane and/or other components. The ability to deform can also cause retention.

Example 14 - Pep-Map analysis

REF.End samples of all mutants were analyzed for disulfide bridging with peptide map. Prior to analysis, the sample was alkylated with iodoacetamide, digested with trypsin and subjected to LC-MS. UV peaks of the single peptides were identified with mass spectrometry. The peptides with free SH groups, correctly and incorrectly formed S-S bonds as well as dimeric peptides were, whenever possible, semi-quantitatively determined.

A correctly folded construct with 5 cysteines forms an S-S bond between Cys 23 and Cys 97 which corresponds to T3 and T9 respectively. The bond T3 -T9 is located in the light chain. The other disulfide bond is on the heavy chain between Cys 159 and Cys 224, corresponding to T12 and T17. The 5^th cysteine is located at Cys28 and corresponds to the T4 peptide. A construct with 4 cysteines always lacks the Cys 28 residue, the correct S-S bridges are similar as for a construct with 5 cysteines.

Mutants 118, 119, 130, 135, 133, 134, 137 and C28Y +HIS (106) as well as C28Y-HIS (108) were analyzed in lab ALL Mutants 106 origami, 136, 138, 139, 140 and the wild type 123 were analyzed in analytical lab AL2 with a different device. The sensitivity of the mass spectrometer in ALl is higher than that in AL2 and therefore, a semiquantitative analysis of mutants analyzed in AL2 could not be obtained. However, it was possible to determine if correctly formed SS bridges are present.

A summary of the obtained data is given in Table 17. The results are compiled in 3 categories: free SH, bridged cysteines and dimeric cysteines. Free cysteines indicate a SH group that did not form a disulfide bond. Bridged cysteines are intramolecular disulfide bonds and dimeric cysteines represent intermolecular disulfides. W indicates that a weak signal for the respective peptide was detected and X represents peptides giving strong signals. Mutants analyzed in AL 2 are labeled with *.

Table 17: Pep Map analysis of all tested mutants

A 'correctly' folded Mycograb with 5 cysteines should give a significant signal for free SH at the T4 peptide and no signal corresponding to other free SH groups. Additionally, a strong signal for the correct disulfides T3-T9 and T 12-Tl 7 is expected and incorrect SS bonds should not be present. Lastly, no intermolecular SS bonds should be present. A 'correctly' folded Mycograb with 4 cysteines should not have any free SH groups. Only the correct S-S bonds T3-T9 and T12-T17 should be detected. Additionally, no intermolecular SS bonds should be present.

Table 17 shows that neither the wild type nor any of the mutants gave strong signals for the correct S-S bonds only. It has to be considered that the REF.End sample consists of a population of differently folded and covalently aggregated species, so that a mixture of all possible combinations of disulfide bonds and free SH groups is present. However, a promising mutant should at least show significant signals for both of the correct S-S bridges which is the case for MYC 137 and the mutants C28Y+HIS and C28Y - HIS.

In 5 cases, only incorrect S-S bonds were found, where no, or only a weak, signal was obtained for the correct disulfide bonds.

Signals for MYC 123, MYC 138, MYC 139 and MYC 140 were extremely weak and reanalysis of the samples did not yield higher signals. Though Mycograb specific peptides were found, the cysteine containing peptides gave no or only a weak signal. This might be due to ineffective digestion of the respective portion of the protein with trypsin because of structurally blocked cleaving sites. It was also noted that mutants with increased linker length (5 and 6X instead of 3X) were more difficult to digest and consequently signals for the late eluting peptides could not or could only hardly be detected.

Interestingly, covalent disulfides were, with one exception, only formed between the two T9 peptides, corresponding to Cys 97 residues.

Mutants Myc 118, 119, 130, 133, 137 and the mutants C28Y+HIS and C28Y - HIS gave stronger signals for correct S-S bonds than the wild type. However, it has to be considered that Myc 123 was analyzed with a different mass spectrometer of lower sensitivity and hence signal intensities cannot be compared. Signals from peptides of Myc 123 can be compared with signals from mutants 106 origami, 136, 138, 139 and 140. For none of these constructs, correct disulfide bridges were obtained. Only signals for incorrect disulfides were found.

Pep Map results for Myc 137, Myc C28Y+HIS and Myc C28Y - HIS were most promising with significant signals for both correct disulfide bridges. Mutants Myc 118, Myc 119 and Myc 133 also showed a certain amount of native like disulfide bridging, however, the signal for T 12-Tl 7 was weak.

Mutant Myc 130 showed strong signals for the T12-T17 SS bond, but the second correct disulfide was not found.

Conclusions

Correlations of analytical results

The best recoveries after refolding were obtained with the MYC 123 (wt) and MYC 134, as determined by the titer assay with Poros column (RPCl).

The solubilization of IBs with chaotropic agents was faster than solubilization with NLS. RPC 2 chromatograms of IBs solubilized with urea or NLS were comparable. However, SDS Page indicated a stronger dissolving power of NLS as the aggregate smear was reduced compared with IBs solubilized with urea, see Figure 27.

Refolding after solubilization with chaotropic agents by dilution and use of different additives such as Cysteine, L-Arginine, 1% NLS and low concentration of urea/ GuHCl did not show a monomeric peak in RPC 2, see Figure 25 and Figure 26. The protein completely aggregated which was confirmed by SDS Page, non reducing and the RPC 2 chromatogram showed a huge peak 2.

The determination of MW with SEC HPLC 0.5% NLS correlated with an R² of 0.77 with the theoretically calculated MW when 1 outlier was excluded. A migration time difference was seen between two mutants of a MW differing by O.όkDa in theory and 14kDa as measured with SEC HPLC (MYC 134 and MYC 118, respectively).

RPC 2 chromatography was able to confirm decreased hydrophobicity of a mutant where 10 hydrophobic amino acids were replaced by more hydrophilic ones (MYC

130). The linker element did not have easy access to binding sites of the stationary phase and did not therefore have influence on retention behavior. It was also observed that Pep Map analysis for constructs with increased linker length gave weaker signals for the peptides of interest compared with constructs with shorter linker. It appeared that the linker element was not easily accessible for the digesting enzyme.

RPC2 chromatograms for MYC 137,138 and 139 showed the sharpest peak which is attributed to a Mycograb Monomer compared with the other tested mutants, indicating increased homogeneity of the sample.

Pep Map analysis showed that almost no free SH group was present in REF.End samples for mutants with 4 Cysteines. This indicates an almost complete formation of disulfide bridges (incorrect and correct ones) as well as formation of covalent aggregates. For mutants with 5 cysteines, weak signals were obtained for various free SH groups but only mutants Myc 118, Myc 119 and Myc 133 had a free SH group at T4, the location of the 5^th cysteine which should remain reduced in the 'native' monomer. This is an indication that the SH group on the T4 peptide preferably forms SS bonds because all mutants with 5 cysteines gave signals for free SH groups but only in 3 of them a free SH group at T4 was detected.

The UFDF experiment showed that NLS is to some extent retained in the sample solution and cannot efficiently be removed.

The results obtained with Pep Map of Mutants MYC C28Y +HIS, C28Y-HIS and MYC 137 indicated reduced aggregation of these mutants. The HIS tag on mutants is required for purification with IMAC, a purification step of high efficiency. Effects of mutations

The number of correct disulfide bonds was increased compared with the removal of the 5^th cysteine. Additionally, solubility of the IBs was enhanced compared with constructs with 5 cysteines.

Exchanging the orientation of the heavy and light chain fragment had a minor effect on retention time in RPC 2 where the retention time decreased when the VL element was C terminal compared with an N-terminal orientation.

The tendency of the peptides to aggregate after NLS removal may be increased with the number of linker elements.

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GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125 GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser Pro 130 135 140

Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys Arg 145 150 155 160

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GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe Thr 195 200 205

Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr Cys 210 215 220

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Met Ala Lys VaI GIn Leu Leu GIn Ser Ala Thr GIu VaI Lys Lys Pro 1 5 10 15

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Lys Phe GIn Asp Arg lie Thr Met Thr Thr Asp Ala Ser Thr Ser Thr 65 70 75 80

Ala Tyr Met GIu Leu Arg Ser Leu Arg Pro Asp Asp Thr Ala VaI Leu 85 90 95

Tyr Cys Ala Arg Asp Arg GIu Asp Ala Ser Leu Leu Arg Asp GIy His 100 105 110

Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser 115 120 125

GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIu 130 135 140

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Leu Ala Trp Tyr GIn GIn Lys Pro GIy GIn Ala Pro Arg Leu Leu lie 180 185 190

Tyr GIy Ala Ser Ser Arg Ala Thr GIy lie Pro Asp Arg Phe Ser GIy 195 200 205

Ser GIy Ser GIy Thr Asp Phe Thr Leu Thr lie Ser Lys Leu GIu Pro 210 215 220

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Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tet ctg cgt ate tet tgc aaa ggt tet ggt tgc ate ate tet 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30 tet tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala , 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gee ace tat tac 672

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GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr He Thr Cys 145 150 155 160 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

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Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg He Ser Cys Lys GIy Ser GIy Cys He He Ser 20 25 30

Ser Tyr Trp He Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro 50 55 60 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp He Lys Arg Ala Ala Ala Leu GIu His His His His His His 245 250 255

<210> 10

<211> 756

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (756)

<400> 10 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gee cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr He Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg gcc gca ctg gaa taa taa 756

VaI Asp He Lys Arg Ala Ala Ala Leu GIu 245 250

<210> 11

<211> 250

<212> PRT

<213> Artificial <220>

<223> Synthetic Construct

<400> 11

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala Ala Ala Leu GIu 245 250

<210> 12

<211> 756

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (756)

<220>

<221> misc_feature

<222> (85) .. (87)

<223> A codon encoding an amino acid other than cysteine

<400> 12 atg get gaa gtt caa ctt gtt gaa tet ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tet ctg cgt ate tet tgc aaa ggt tet ggt nnn ate ate tet 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Xaa lie lie Ser 20 25 30 tet tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gee ace tat tac 672

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys

225 230 235 240 gtg gac ate aaa cgc gcg gcc gca ctg gaa taa taa 756

VaI Asp lie Lys Arg Ala Ala Ala Leu GIu 245 250

<210> 13

<211> 250

<212> PRT

<213> Artificial

<220>

<221> misc_feature

<222> (29).. (29)

<223> The 'Xaa' at location 29 stands for Lys, Asn, Arg, Ser, Thr, lie,

Met, GIu, Asp, GIy, Ala, VaI, GIn, His, Pro, Leu, Tyr, Trp, Cys, or Phe.

<220>

<223> Synthetic Construct

<400> 13

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Xaa lie lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala Ala Ala Leu GIu 245 250

<210> 14

<211> 774

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (774)

<220>

<221> misc_feature

<222> (85) .. (87)

<223> A codon encoding an amino acid other than cysteine

<400> 14 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt nnn ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Xaa lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gcc gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg gcc gca etc gag cac cac cac cac cac cac 768 VaI Asp lie Lys Arg Ala Ala Ala Leu GIu His His His His His His 245 250 255 taa taa 774

<210> 15

<211> 256

<212> PRT

<213> Artificial

<220>

<221> misc_feature

<222> (29) .. (29)

<223> The 'Xaa¹ at location 29 stands for Lys, Asn, Arg, Ser, Thr, lie, Met, GIu, Asp, GIy, Ala, VaI, GIn, His, Pro, Leu, Tyr, Trp, Cys, or Phe.

<220>

<223> Synthetic Construct

<400> 15

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Xaa lie lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 VaI Asp lie Lys Arg Ala Ala Ala Leu GIu His His His His His His 245 250 255

<210> 16

<211> 744

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (744)

<400> 16 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gcc gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gee ace tat tac 672 Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg taa taa 744

VaI Asp lie Lys Arg Ala 245

<210> 17

<211> 246

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 17

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30

Ser Tyr Trp He Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr He Ser Ala Asp Lys Ser He Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 HO

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg He Thr He Thr Cys 145 150 155 160 Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr He Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp He Lys Arg Ala 245

<210> 18

<211> 744

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (744)

<220>

<221> misc_feature

<222> (85) .. (87)

<223> A codon encoding an amino acid other than cysteine

<400> 18 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt nnn ate ate tct 96 GIy GIu Ser Leu Arg He Ser Cys Lys GIy Ser GIy Xaa He He Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp He Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr He Ser Ala Asp Lys Ser He Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 ^■ 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr He Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg taa taa 744

VaI Asp He Lys Arg Ala 245

<210> 19

<211> 246

<212> PRT

<213> Artificial

<220>

<221> misc featu

<222> (29) .. (29)

<223> The 'Xaa' at location 29 stands for Lys, Asn, Arg, Ser, Thr, He, Met, GIu, Asp, GIy, Ala, VaI, GIn, His, Pro, Leu, Tyr, Trp, Cys, or Phe.

<220>

<223> Synthetic Construct

<400> 19

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg He Ser Cys Lys GIy Ser GIy Xaa He He Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala 245

<210> 20

<211> 744

<212> DNA

<213> Artificial

<220>

<223> scFv peptide <220>

<221> CDS

<222> (1) .. (744)

<400> 20 atg gcg gaa gtg cag ctg gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gcc gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg eaa 576 Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg taa taa 744

VaI Asp lie Lys Arg Ala 245

<210> 21

<211> 246

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 21

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala 245

<210> 22

<211> 744

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (744)

<220>

<221> misc_feature

<222> (85) .. (87)

<223> A codon encoding an amino acid other than cysteine

<400> 22 atg gcg gaa gtg cag ctg gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt nnn ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Xaa lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gcc gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr He Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr He Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg taa taa 744

VaI Asp He Lys Arg Ala 245

<210> 23

<211> 246

<212> PRT

<213> Artificial

<220>

<221> misc_feature

<222> (29) .. (29)

<223> The 'Xaa' at location 29 stands for Lys, Asn, Arg, Ser, Thr, He,

Met, GIu, Asp, GIy, Ala, VaI, GIn, His, Pro, Leu, Tyr, Trp, Cys, or Phe.

<220>

<223> Synthetic Construct

<400> 23

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg He Ser Cys Lys GIy Ser GIy Xaa He He Ser 20 25 30

Ser Tyr Trp He Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr He Ser Ala Asp Lys Ser He Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala 245

<210> 24

<211> 774

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (774)

<400> 24 atg gcg gaa gtg cag ctg gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser

20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr

65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336

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

100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys

145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn

180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys

225 230 235 240 gtg gac ate aaa cgc gcg gcc gca etc gag cac cac cac cac cac cac 768

VaI Asp He Lys Arg Ala Ala Ala Leu GIu His His His His His His 245 250 255 taa taa 774

<210> 25

<211> 256

<212> PRT

<213> Artificial <220>

<223> Synthetic Construct

<400> 25

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie He Ser 20 25 30

Ser Tyr Trp He Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr He Ser Ala Asp Lys Ser He Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 HO

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg He Thr He Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr He Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 VaI Asp lie Lys Arg Ala Ala Ala Leu GIu His His His His His His 245 250 255

<210> 26

<211> 774

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (774)

<220>

<221> misc_feature

<222> (85) .. (87)

<223> A codon encoding an amino acid other than cysteine

<400> 26 atg gcg gaa gtg cag ctg gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt nnn ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Xaa lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser

130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gee ace tat tac 672 Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg gcc gca etc gag cac cac cac cac cac cac 768 VaI Asp lie Lys Arg Ala Ala Ala Leu GIu His His His His His His 245 250 255 taa taa 774

<210> 27

<211> 256

<212> PRT

<213> Artificial

<220>

<221> misc_feature

<222> (29) .. (29)

<223> The 'Xaa' at location 29 stands for Lys, Asn, Arg, Ser, Thr, lie,

Met, GIu, Asp, GIy, Ala, VaI, GIn, His, Pro, Leu, Tyr, Trp, Cys, or Phe.

<220>

<223> Synthetic Construct

<400> 27

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Xaa lie lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala Ala Ala Leu GIu His His His His His His 245 250 255

<210> 28

<211> 744

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (744)

<400> 28 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tat age ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gcc gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age tec ctg age gcg age gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Ser Leu Ser Ala Ser VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg taa taa 744

VaI Asp lie Lys Arg Ala 245

<210> 29

<211> 246

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 29

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Ser Leu Ser Ala Ser VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala 245

<210> 30

<211> 744

<212> DNA

<213> Artificial

<220> <223> scFv peptide

<220>

<221> CDS

<222> (1) .. (744)

<400> 30 atg get gaa gtt caa ctt gtt gaa tet ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tet ctg cgt ate tet tgc aaa ggt tet ggt tat age ate tet 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30 tet tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gcc gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat att cag atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp He GIn Met Thr GIn Ser 130 135 140 ccg age tec ctg age gcg age gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Ser Leu Ser Ala Ser VaI GIy Asp Arg He Thr He Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr He Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg taa taa 744

VaI Asp lie Lys Arg Ala 245

<210> 31

<211> 246

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 31

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp lie GIn Met Thr GIn Ser 130 135 140

Pro Ser Ser Leu Ser Ala Ser VaI GIy Asp Arg lie Thr He Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala 245

<210> 32

<211> 744

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (744)

<400> 32 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg age age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gee cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat att cag atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp lie GIn Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa aaa 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Lys 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate age age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr lie Ser Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg taa taa 744

VaI Asp lie Lys Arg Ala 245

<210> 33

<211> 246

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 33

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp He GIn Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg He Thr He Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Lys 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr He Ser Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp He Lys Arg Ala 245

<210> 34

<211> 759

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1)..(759)

<400> 34 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro

1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct 96 GIy GIu Ser Leu Arg He Ser Cys Lys GIy Ser GIy Cys He He Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp He Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc gga tec ggt ggt ggc ggc age ggc ggc ggc ggc age gat gtt 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI 130 135 140 gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt ggt gac cgt 480 VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg 145 150 155 160 ate ace att ace tgc cgc gcc age age ggc ate age cgc tat ctg gcg 528 lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala 165 170 175 tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg ate tat get 576 Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala 180 185 190 gca age ace ctg caa ace ggc gtt ccg age cgt ttt age ggt age ggc 624 Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy 195 200 205 age ggc ace gag ttc ace ctg ace ate aac age ctg caa ccg gag gat 672 Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp 210 215 220 ttt gcc ace tat tac tgc caa cac ctg aat age tat ccg ctg ace ttc 720 Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe 225 230 235 240 ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg taa taa 759

GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala 245 250

<210> 35

<211> 251

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 35

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI 130 135 140

VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg 145 150 155 160

lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala 165 170 175

Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala 180 185 190

Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy 195 200 205

Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp 210 215 220

Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe 225 230 235 240

GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala 245 250

<210> 36

<211> 762

<212> DNA

<213> Artificial

<220>

<223> scFv peptide <220>

<221> CDS

<222> (1)..(762)

<400> 36 atg gat gtt gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt 48

Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15 ggt gac cgt ate ace att ace tgc cgc gee age age ggc ate age cgc 96 GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30 tat ctg gcg tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg 144 Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45 ate tat get gca age ace ctg caa ace ggc gtt ccg age cgt ttt age 192 lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60 ggt age ggc age ggc ace gag ttc ace ctg ace ate aac age ctg caa 240 GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80 ccg gag gat ttt gee ace tat tac tgc caa cac ctg aat age tat ccg 288 Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95 ctg ace ttc ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg age ggt 336 Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy 100 105 110 ggt ggc ggc age ggt ggt ggc gga tec ggt ggt ggc ggc age ggc ggc 384 GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125 ggc ggc age get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa 432 GIy GIy Ser Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys 130 135 140 aaa ccg ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate 480 Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie 145 150 155 160 ate tct tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc 528 lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy 165 170 175 ctg gaa tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac 576 Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr 180 185 190 age ccg age ttt cag ggc cat gtt ace ate age gee gat aaa age att 624 Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie 195 200 205 aac ace get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg 672 Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala 210 215 220 atg tac tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat 720 Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp 225 230 235 240 tac tgg ggt cag ggc ace ctg gtg ace gtg age age taa taa 762 Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser 245 250

<210> 37

<211> 252

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 37

Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15

GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30

Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45

lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60

GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80

Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95

Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy 100 105 110

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125

GIy GIy Ser Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys 130 135 140

Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie 145 150 155 160

lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy 165 170 175

Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr 180 185 190

Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie 195 200 205 Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala 210 215 220

Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp 225 230 235 240

Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser 245 250

<210> 38

<211> 774

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (774)

<220>

<221> misc_feature

<222> (292).. (294)

<223> n is a, c, g, or t

<400> 38 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gcc gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac nnn gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Xaa Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140 ccg age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala

165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg gcc gca etc gag cac cac cac cac cac cac 768 VaI Asp He Lys Arg Ala Ala Ala Leu GIu His His His His His His 245 250 255 taa taa 774

<210> 39

<211> 256

<212> PRT

<213> Artificial

<220>

<221> misc_feature

<222> (98) .. (98)

<223> The 'Xaa' at location 98 stands for Lys, Asn, Arg, Ser, Thr, He,

Met, GIu, Asp, GIy, Ala, VaI, GIn, His, Pro, Leu, Tyr, Trp, Cys, or Phe.

<220>

<223> Synthetic Construct

<400> 39

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg He Ser Cys Lys GIy Ser GIy Cys He He Ser 20 25 30

Ser Tyr Trp He Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr He Ser Ala Asp Lys Ser He Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Xaa Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser 130 135 140

Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala Ala Ala Leu GIu His His His His His His 245 250 255

<210> 40

<211> 744

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (744)

<400> 40 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tat age ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cgt gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy Arg VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gee cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat att cag atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp lie GIn Met Thr GIn Ser 130 135 140 ccg age tec ctg age gcg age gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Ser Leu Ser Ala Ser VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160 cgc gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca 528 Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac 672 Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg taa taa 744

VaI Asp lie Lys Arg Ala 245

<210> 41

<211> 246

<212> PRT

<213> Artificial

<220> <223> Synthetic Construct <400> 41

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy Arg VaI Thr He Ser Ala Asp Lys Ser He Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 HO

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp He GIn Met Thr GIn Ser 130 135 140

Pro Ser Ser Leu Ser Ala Ser VaI GIy Asp Arg He Thr He Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr He Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp He Lys Arg Ala 245 <210> 42

<211> 744

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (744)

<400> 42 atg get gaa gtt caa ctt gtt gaa tet ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tet ctg cgt ate tet tgc aaa ggt tet ggt tat age ate tet 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30 tet tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cgt gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy Arg VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg age age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gee cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc ggc age ggc ggc ggc ggc age gat att cag atg ace cag age 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp He GIn Met Thr GIn Ser 130 135 140 ccg age tec ctg age gcg age gtt ggt gac cgt ate ace att ace tgc 480 Pro Ser Ser Leu Ser Ala Ser VaI GIy Asp Arg He Thr He Thr Cys 145 150 155 160 cgc gee age age ggc ate age cgc tat ctg gcg tgg tat cag caa aaa 528 Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Lys 165 170 175 ccg ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa 576 Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Thr Leu GIn 180 185 190 ace ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc 624 Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205 ace ctg ace ate age age ctg caa ccg gag gat ttt gee ace tat tac 672 Thr Leu Thr lie Ser Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220 tgc caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa 720 Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240 gtg gac ate aaa cgc gcg taa taa 744

VaI Asp lie Lys Arg Ala 245

<210> 43

<211> 246

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 43

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy Arg VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp lie GIn Met Thr GIn Ser 130 135 140

Pro Ser Ser Leu Ser Ala Ser VaI GIy Asp Arg lie Thr lie Thr Cys 145 150 155 160

Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Lys 165 170 175

Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn 180 185 190

Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe 195 200 205

Thr Leu Thr lie Ser Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr 210 215 220

Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys 225 230 235 240

VaI Asp lie Lys Arg Ala 245

<210> 44

<211> 774

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (774)

<400> 44 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr He Ser Ala Asp Lys Ser He Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc gga tec ggt ggt ggc ggc tec ggt ggt ggc ggc age ggc ggc 432

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 130 135 140 ggc ggc age gat gtt gtg atg ace cag age ccg age ttc ctg age gcg 480

GIy GIy Ser Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala

145 150 155 160 ttc gtt ggt gac cgt ate ace att ace tgc cgc gee age age ggc ate 528

Phe VaI GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie

165 170 175 age cgc tat ctg gcg tgg tat cag caa gca ccg ggt aaa gca ccg aaa 576

Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys

180 185 190 ctg ctg ate tat get gca age ace ctg caa ace ggc gtt ccg age cgt 624

Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg 195 200 205 ttt age ggt age ggc age ggc ace gag ttc ace ctg ace ate aac age 672

Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser 210 215 220 ctg caa ccg gag gat ttt gee ace tat tac tgc caa cac ctg aat age 720

Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser

225 230 235 240 tat ccg ctg ace ttc ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg 768

Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala

245 250 255 taa taa 774

<210> 45

<211> 256

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 45

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 130 135 140

GIy GIy Ser Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala 145 150 155 160

Phe VaI GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie 165 170 175

Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys 180 185 190

Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg 195 200 205

Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser 210 215 220

Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser 225 230 235 240

Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala 245 250 255

<210> 46

<211> 789

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (789)

<400> 46 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gee cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc gga tct ggt ggt ggc ggc age ggt ggt ggc gga tec ggt ggt 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 130 135 140 ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age ccg 480 GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser Pro 145 150 155 160 age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc cgc 528 Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys Arg 165 170 175 gee age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca ccg 576 Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala Pro 180 185 190 ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa ace 624 GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn Thr 195 200 205 ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc ace 672 GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe Thr 210 215 220 ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac tgc 720 Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr Cys 225 230 235 240 caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa gtg 768 GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys VaI 245 250 255 gac ate aaa cgc gcg taa taa 789

Asp He Lys Arg Ala 260

<210> 47 <211> 261 <212> PRT <213> Artificial

<220>

<223> Synthetic Construct

<400> 47

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 130 135 140

GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser Pro 145 150 155 160

Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys Arg 165 170 175

Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala Pro 180 185 190

GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn Thr 195 200 205

GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe Thr 210 215 220

Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr Cys 225 230 235 240 GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys VaI 245 250 255

Asp lie Lys Arg Ala 260

<210> 48

<211> 777

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (777)

<400> 48 atg gat gtt gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt 48

Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI

1 5 10 15 ggt gac cgt ate ace att ace tgc cgc gee age age ggc ate age cgc 96 GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30 tat ctg gcg tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg 144 Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45 ate tat get gca age ace ctg caa ace ggc gtt ccg age cgt ttt age 192 lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60 ggt age ggc age ggc ace gag ttc ace ctg ace ate aac age ctg caa 240 GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80 ccg gag gat ttt gee ace tat tac tgc caa cac ctg aat age tat ccg 288 Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95 ctg ace ttc ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg age ggt 336 Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy 100 105 110 ggt ggc ggc age ggt ggt ggc gga tec ggt ggt ggc ggc tec ggt ggt 384 GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125 ggc ggc age ggc ggc ggc ggc age get gaa gtt caa ctt gtt gaa tct 432 GIy GIy Ser GIy GIy GIy GIy Ser Ala GIu VaI GIn Leu VaI GIu Ser 130 135 140 ggt get gaa gtt aaa aaa ccg ggt gaa tct ctg cgt ate tct tgc aaa 480 GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys 145 150 155 160 ggt tct ggt tgc ate ate tct tct tac tgg ate age tgg gtt cgt cag 528 GIy Ser GIy Cys lie lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn 165 170 175 atg ccg ggc aag ggc ctg gaa tgg atg ggt aaa att gat ccg ggc gac 576 Met Pro GIy Lys GIy Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp 180 185 190 age tat att aac tac age ccg age ttt cag ggc cat gtt ace ate age 624 Ser Tyr lie Asn Tyr Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser 195 200 205 gcc gat aaa age att aac ace get tac ctg caa tgg aac age ctg aaa 672 Ala Asp Lys Ser lie Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys 210 215 220 gcg age gac ace gcg atg tac tac tgt gcc cgt ggc ggt cgt gac ttc 720 Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe 225 230 235 240 ggt gat age ttc gat tac tgg ggt cag ggc ace ctg gtg ace gtg age 768 GIy Asp Ser Phe Asp Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser 245 250 255 age taa taa 777

Ser

<210> 49

<211> 257

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 49

Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15

GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30

Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45

lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60

GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80

Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95

Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy 100 105 110

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125 GIy GIy Ser GIy GIy GIy GIy Ser Ala GIu VaI GIn Leu VaI GIu Ser 130 135 140

GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys 145 150 155 160

GIy Ser GIy Cys lie lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn 165 170 175

Met Pro GIy Lys GIy Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp 180 185 190

Ser Tyr lie Asn Tyr Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser 195 200 205

Ala Asp Lys Ser lie Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys 210 215 220

Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe 225 230 235 240

GIy Asp Ser Phe Asp Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser 245 250 255

Ser

<210> 50

<211> 792

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (792)

<400> 50 atg gat gtt gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt 48 Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15 ggt gac cgt ate ace att ace tgc cgc gcc age age ggc ate age cgc 96 GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30 tat ctg gcg tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg 144 Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45 ate tat get gca age ace ctg caa ace ggc gtt ccg age cgt ttt age 192 lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60 ggt age ggc age ggc ace gag ttc ace ctg ace ate aac age ctg caa 240 GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80 ccg gag gat ttt gee ace tat tac tgc caa cac ctg aat age tat ccg 288 Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95 ctg ace ttc ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg age ggt 336 Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy 100 105 110 ggt ggc ggc age ggt ggt ggc gga tct ggt ggt ggc ggc age ggt ggt 384 GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125 ggc gga tec ggt ggt ggc ggc age ggc ggc ggc ggc age get gaa gtt 432 GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Ala GIu VaI 130 135 140 caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg ggt gaa tct ctg 480 GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu 145 150 155 160 cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct tct tac tgg ate 528 Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser Ser Tyr Trp lie 165 170 175 age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa tgg atg ggt aaa 576 Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu Trp Met GIy Lys 180 185 190 att gat ccg ggc gac age tat att aac tac age ccg age ttt cag ggc 624 lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro Ser Phe GIn GIy 195 200 205 cat gtt ace ate age gee gat aaa age att aac ace get tac ctg caa 672 His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr Ala Tyr Leu GIn 210 215 220 tgg aac age ctg aaa gcg age gac ace gcg atg tac tac tgt gee cgt 720 Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg 225 230 235 240 ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg ggt cag ggc ace 768 GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp GIy GIn GIy Thr 245 250 255 ctg gtg ace gtg age age taa taa 792

Leu VaI Thr VaI Ser Ser 260

<210> 51

<211> 262

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 51

Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15

GIy Asp Arg lie Thr He Thr Cys Arg Ala Ser Ser GIy He Ser Arg 20 25 30

Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45

He Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60

GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr He Asn Ser Leu GIn 65 70 75 80

Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95

Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp He Lys Arg Ala Ser GIy 100 105 HO

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125

GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Ala GIu VaI 130 135 140

GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu 145 150 155 160

Arg He Ser Cys Lys GIy Ser GIy Cys He He Ser Ser Tyr Trp He 165 170 175

Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu Trp Met GIy Lys 180 185 190

He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro Ser Phe GIn GIy 195 200 205

His VaI Thr He Ser Ala Asp Lys Ser He Asn Thr Ala Tyr Leu GIn 210 215 220

Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg 225 230 235 240

GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp GIy GIn GIy Thr 245 250 255

Leu VaI Thr VaI Ser Ser 260 <210> 52

<211> 759

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (759)

<400> 52 atg get gaa gtt caa ctt gtt gaa tet ggt get gaa gtt aaa aaa ccg 48

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tet ctg cgt ate tet tgc aaa ggt tet ggt tat age ate tet 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser He Ser 20 25 30 tet tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp He Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr He Ser Ala Asp Lys Ser He Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 HO ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc gga tec ggt ggt ggc ggc age ggc ggc ggc ggc age gat gtt 432 GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI 130 135 140 gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt ggt gac cgt 480 VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg 145 150 155 160 ate ace att ace tgc cgc gcc age age ggc ate age cgc tat ctg gcg 528 He Thr He Thr Cys Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala 165 170 175 tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg ate tat get 576 Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala 180 185 190 gca age ace ctg caa ace ggc gtt ccg age cgt ttt age ggt age ggc 624 Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy 195 200 205 age ggc ace gag ttc ace ctg ace ate aac age ctg caa ccg gag gat 672 Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp 210 215 220 ttt gee ace tat tac tgc caa cac ctg aat age tat ccg ctg ace ttc 720 Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe 225 230 235 240 ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg taa taa 759

GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala 245 250

<210> 53

<211> 251

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 53

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI 130 135 140

VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg 145 150 155 160

lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala 165 170 175 Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala 180 185 190

Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy 195 200 205

Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp 210 215 220

Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe 225 230 235 240

GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala 245 250

<210> 54

<211> 774

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1)..(774)

<400> 54 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48 Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tat age ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144 Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192 Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60 age ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace 240 Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336 Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384 GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125 ggt ggc gga tec ggt ggt ggc ggc tec ggt ggt ggc ggc age ggc ggc 432

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy

130 135 140 ggc ggc age gat gtt gtg atg ace cag age ccg age ttc ctg age gcg 480

GIy GIy Ser Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala

145 150 155 160 ttc gtt ggt gac cgt ate ace att ace tgc cgc gee age age ggc ate 528

Phe VaI GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie

165 170 175 age cgc tat ctg gcg tgg tat cag caa gca ccg ggt aaa gca ccg aaa 576

Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys

180 185 190 ctg ctg ate tat get gca age ace ctg caa ace ggc gtt ccg age cgt 624

Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg

195 200 205 ttt age ggt age ggc age ggc ace gag ttc ace ctg ace ate aac age 672

Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser

210 215 220 ctg caa ccg gag gat ttt gee ace tat tac tgc caa cac ctg aat age 720

Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser

225 230 235 240 tat ccg ctg ace ttc ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg 768

Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala

245 250 255 taa taa 774

<210> 55

<211> 256

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 55

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80 Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 130 135 140

GIy GIy Ser Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala 145 150 155 160

Phe VaI GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie 165 170 175

Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys 180 185 190

Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg 195 200 205

Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser 210 215 220

Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser 225 230 235 240

Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala 245 250 255

<210> 56

<211> 789

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (789)

<400> 56 atg get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg 48

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro

1 5 10 15 ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tat age ate tct 96 GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser He Ser 20 25 30 tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa 144

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu

35 40 45 tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg 192

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro

50 55 60 age ttt cag ggc cat gtt ace ate age gcc gat aaa age att aac ace 240

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr

65 70 75 80 get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac 288

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

85 90 95 tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg 336

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

100 105 110 ggt cag ggc ace ctg gtg ace gtg age age ggt ggt ggc ggc age ggt 384

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy

115 120 125 ggt ggc gga tct ggt ggt ggc ggc age ggt ggt ggc gga tec ggt ggt 432

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy

130 135 140 ggc ggc age ggc ggc ggc ggc age gat gtt gtg atg ace cag age ccg 480

GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser Pro

145 150 155 160 age ttc ctg age gcg ttc gtt ggt gac cgt ate ace att ace tgc cgc 528

Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys Arg

165 170 175 gcc age age ggc ate age cgc tat ctg gcg tgg tat cag caa gca ccg 576

Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala Pro

180 185 190 ggt aaa gca ccg aaa ctg ctg ate tat get gca age ace ctg caa ace 624

GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn Thr

195 200 205 ggc gtt ccg age cgt ttt age ggt age ggc age ggc ace gag ttc ace 672

GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe Thr

210 215 220 ctg ace ate aac age ctg caa ccg gag gat ttt gcc ace tat tac tgc 720

Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr Cys

225 230 235 240 caa cac ctg aat age tat ccg ctg ace ttc ggt ggc ggc ace aaa gtg 768

GIn His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys VaI

245 250 255 gac ate aaa cgc gcg taa taa 789

Asp He Lys Arg Ala 260

<210> 57

<211> 261

<212> PRT

<213> Artificial <220>

<223> Synthetic Construct

<400> 57

Met Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro 1 5 10 15

GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser 20 25 30

Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu 35 40 45

Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro 50 55 60

Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr 65 70 75 80

Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95

Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp 100 105 110

GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy GIy GIy GIy Ser GIy 115 120 125

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 130 135 140

GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI VaI Met Thr GIn Ser Pro 145 150 155 160

Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg lie Thr lie Thr Cys Arg 165 170 175

Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala Trp Tyr GIn GIn Ala Pro 180 185 190

GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala Ala Ser Thr Leu GIn Thr 195 200 205

GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy Ser GIy Thr GIu Phe Thr 210 215 220

Leu Thr lie Asn Ser Leu GIn Pro GIu Asp Phe Ala Thr Tyr Tyr Cys 225 230 235 240 Gin His Leu Asn Ser Tyr Pro Leu Thr Phe GIy GIy GIy Thr Lys VaI 245 250 255

Asp lie Lys Arg Ala 260

<210> 58

<211> 762

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (762)

<400> 58 atg gat gtt gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt 48 Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15 ggt gac cgt ate ace att ace tgc cgc gee age age ggc ate age cgc 96 GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30 tat ctg gcg tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg 144 Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45 ate tat get gca age ace ctg caa ace ggc gtt ccg age cgt ttt age 192 lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60 ggt age ggc age ggc ace gag ttc ace ctg ace ate aac age ctg caa 240 GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80 ccg gag gat ttt gee ace tat tac tgc caa cac ctg aat age tat ccg 288 Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95 ctg ace ttc ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg age ggt 336 Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy 100 105 110 ggt ggc ggc age ggt ggt ggc gga tec ggt ggt ggc ggc age ggc ggc 384 GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125 ggc ggc age get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa 432 GIy GIy Ser Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys 130 135 140 aaa ccg ggt gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tat age 480 Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser 145 150 155 160 ate tct tct tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc 528 lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy 165 170 175 ctg gaa tgg atg ggt aaa att gat ccg ggc gac age tat att aac tac 576

Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr 180 185 190 age ccg age ttt cag ggc cat gtt ace ate age gee gat aaa age att 624

Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie 195 200 205 aac ace get tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg 672

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

210 215 220 atg tac tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat 720

Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp

225 230 235 240 tac tgg ggt cag ggc ace ctg gtg ace gtg age age taa taa 762

Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser 245 250

<210> 59

<211> 252

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 59

Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15

GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30

Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45

lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60

GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80

Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95

Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy 100 105 110

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125

GIy GIy Ser Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys 130 135 140 Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser 145 150 155 160

lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy 165 170 175

Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr 180 185 190

Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser lie 195 200 205

Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala 210 215 220

Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp 225 230 235 240

Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser 245 250

<210> 60

<211> 777

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1) .. (777)

<400> 60 atg gat gtt gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt 48 Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15 ggt gac cgt ate ace att ace tgc cgc gee age age ggc ate age cgc 96 GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30 tat ctg gcg tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg 144 Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45 ate tat get gca age ace ctg caa ace ggc gtt ccg age cgt ttt age 192 lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 ^' 60 ggt age ggc age ggc ace gag ttc ace ctg ace ate aac age ctg caa 240 GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80 ccg gag gat ttt gee ace tat tac tgc caa cac ctg aat age tat ccg 288 Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95 ctg ace ttc ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg age ggt 336

Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy

100 105 110 ggt ggc ggc age ggt ggt ggc gga tec ggt ggt ggc ggc tec ggt ggt 384

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy

115 120 125 ggc ggc age ggc ggc ggc ggc age get gaa gtt caa ctt gtt gaa tct 432

GIy GIy Ser GIy GIy GIy GIy Ser Ala GIu VaI GIn Leu VaI GIu Ser 130 135 140 ggt get gaa gtt aaa aaa ccg ggt gaa tct ctg cgt ate tct tgc aaa 480

GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys 145 150 155 160 ggt tct ggt tat age ate tct tct tac tgg ate age tgg gtt cgt cag 528

GIy Ser GIy Tyr Ser lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn

165 170 175 atg ccg ggc aag ggc ctg gaa tgg atg ggt aaa att gat ccg ggc gac 576

Met Pro GIy Lys GIy Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp

180 185 190 age tat att aac tac age ccg age ttt cag ggc cat gtt ace ate age 624

Ser Tyr lie Asn Tyr Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser

195 200 205 gee gat aaa age att aac ace get tac ctg caa tgg aac age ctg aaa 672

Ala Asp Lys Ser lie Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys 210 215 220 gcg age gac ace gcg atg tac tac tgt gee cgt ggc ggt cgt gac ttc 720

Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe 225 230 235 240 ggt gat age ttc gat tac tgg ggt cag ggc ace ctg gtg ace gtg age 768

GIy Asp Ser Phe Asp Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser

245 250 255 age taa taa 777

Ser

<210> 61

<211> 257

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 61

Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15

GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30

Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45 He Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60

GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr He Asn Ser Leu GIn 65 70 75 80

Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95

Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp He Lys Arg Ala Ser GIy 100 105 HO

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125

GIy GIy Ser GIy GIy GIy GIy Ser Ala GIu VaI GIn Leu VaI GIu Ser 130 135 140

GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu Arg He Ser Cys Lys 145 150 155 160

GIy Ser GIy Tyr Ser He Ser Ser Tyr Trp He Ser Trp VaI Arg GIn 165 170 175

Met Pro GIy Lys GIy Leu GIu Trp Met GIy Lys He Asp Pro GIy Asp 180 185 190

Ser Tyr He Asn Tyr Ser Pro Ser Phe GIn GIy His VaI Thr He Ser 195 200 205

Ala Asp Lys Ser He Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys 210 215 220

Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe 225 230 235 240

GIy Asp Ser Phe Asp Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser 245 250 255

Ser

<210> 62

<211> 792

<212> DNA

<213> Artificial

<220>

<223> scFv peptide <220>

<221> CDS

<222> (1) .. (792)

<400> 62 atg gat gtt gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt 48 Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15 ggt gac cgt ate ace att ace tgc cgc gcc age age ggc ate age cgc 96 GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30 tat ctg gcg tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg 144 Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45 ate tat get gca age ace ctg caa ace ggc gtt ccg age cgt ttt age 192 lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60 ggt age ggc age ggc ace gag ttc ace ctg ace ate aac age ctg caa 240 GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80 ccg gag gat ttt gcc ace tat tac tgc caa cac ctg aat age tat ccg 288 Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95 ctg ace ttc ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg age ggt 336 Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy 100 105 110 ggt ggc ggc age ggt ggt ggc gga tct ggt ggt ggc ggc age ggt ggt 384 GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125 ggc gga tec ggt ggt ggc ggc age ggc ggc ggc ggc age get gaa gtt 432 GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Ala GIu VaI 130 135 140 caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg ggt gaa tct ctg 480 GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu 145 150 155 160 cgt ate tct tgc aaa ggt tct ggt tat age ate tct tct tac tgg ate 528 Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser Ser Tyr Trp lie 165 170 175 age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa tgg atg ggt aaa 576 Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu Trp Met GIy Lys 180 185 190 att gat ccg ggc gac age tat att aac tac age ccg age ttt cag ggc 624 lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro Ser Phe GIn GIy 195 200 205 cat gtt ace ate age gcc gat aaa age att aac ace get tac ctg caa 672 His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr Ala Tyr Leu GIn 210 215 220 tgg aac age ctg aaa gcg age gac ace gcg atg tac tac tgt gcc cgt 720 Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg 225 230 235 240 ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg ggt cag ggc ace 768 GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp GIy GIn GIy Thr 245 250 255 ctg gtg ace gtg age age taa taa 792

Leu VaI Thr VaI Ser Ser 260

<210> 63

<211> 262

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 63

Met Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI 1 5 10 15

GIy Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg 20 25 30

Tyr Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu 35 40 45

lie Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser 50 55 60

GIy Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn 65 70 75 80

Pro GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro 85 90 95

Leu Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ser GIy 100 105 110

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy 115 120 125

GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Ala GIu VaI 130 135 140

GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu 145 150 155 160

Arg lie Ser Cys Lys GIy Ser GIy Tyr Ser lie Ser Ser Tyr Trp lie 165 170 175

Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu Trp Met GIy Lys 180 185 190 lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro Ser Phe GIn GIy 195 200 205

His VaI Thr lie Ser Ala Asp Lys Ser lie Asn Thr Ala Tyr Leu GIn 210 215 220

Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg 225 230 235 240

GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp GIy GIn GIy Thr 245 250 255

Leu VaI Thr VaI Ser Ser 260

<210> 64

<211> 837

<212> DNA

<213> Artificial

<220>

<223> scFv peptide

<220>

<221> CDS

<222> (1)..(837)

<400> 64 atg aaa tac ctg ctg ccg ace get get get ggt ctg ctg etc etc get 48

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala GIy Leu Leu Leu Leu Ala 1 5 10 15 gee cag ccg gcg atg gcc get gaa gtt caa ctt gtt gaa tct ggt get 96 Ala GIn Pro Ala Met Ala Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala 20 25 30 gaa gtt aaa aaa ccg ggt gaa tct ctg cgt ate tct tgc aaa ggt tct 144 GIu VaI Lys Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser 35 40 45 ggt tgc ate ate tct tct tac tgg ate age tgg gtt cgt cag atg ccg 192 GIy Cys lie lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro 50 55 60 ggc aag ggc ctg gaa tgg atg ggt aaa att gat ccg ggc gac age tat 240 GIy Lys GIy Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr 65 70 75 80 att aac tac age ccg age ttt cag ggc cat gtt ace ate age gcc gat 288 lie Asn Tyr Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp 85 90 95 aaa age att aac ace get tac ctg caa tgg aac age ctg aaa gcg age 336 Lys Ser lie Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser 100 105 110 gac ace gcg atg tac tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat 384 Asp Thr Ala Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp 115 120 125 age ttc gat tac tgg ggt cag ggc ace ctg gtg ace gtg age age ggt 432

Ser Phe Asp Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy 130 135 140 ggt ggc ggc age ggt ggt ggc ggc age ggc ggc ggc ggc age gat gtt 480

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI 145 150 155 160 gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt ggt gac cgt 528

VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg

165 170 175 ate ace att ace tgc cgc gee age age ggc ate age cgc tat ctg gcg 576 lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala

180 185 190 tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg ate tat get 624

Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala 195 200 205 gca age ace ctg caa ace ggc gtt ccg age cgt ttt age ggt age ggc 672

Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy 210 215 220 age ggc ace gag ttc ace ctg ace ate aac age ctg caa ccg gag gat 720

Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp 225 230 235 240 ttt gee ace tat tac tgc caa cac ctg aat age tat ccg ctg ace ttc 768

Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe

245 250 255 ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg gee gca etc gag cac 816

GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ala Ala Leu GIu His

260 265 270 cac cac cac cac cac taa taa 837

His His His His His 275

<210> 65

<211> 277

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 65

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala GIy Leu Leu Leu Leu Ala 1 5 10 15

Ala GIn Pro Ala Met Ala Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala 20 25 30

GIu VaI Lys Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser 35 40 45

GIy Cys lie lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro 50 55 60 GIy Lys GIy Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr 65 70 75 80

lie Asn Tyr Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp 85 90 95

Lys Ser lie Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser 100 105 110

Asp Thr Ala Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp 115 120 125

Ser Phe Asp Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy 130 135 140

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI 145 150 155 160

VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg 165 170 175

lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala 180 185 190

Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala 195 200 205

Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy 210 215 220

Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp 225 230 235 240

Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe 245 250 255

GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ala Ala Leu GIu His 260 265 270

His His His His His 275

<210> 66

<211> 837

<212> DNA

<213> Artificial

<220>

<223> scFv peptide <220>

<221> CDS

<222> (1) .. (837)

<400> 66 atg aaa tac ctg ctg ccg ace get get get ggt ctg ctg etc etc get 48 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala GIy Leu Leu Leu Leu Ala 1 5 10 15 gcc cag ccg gcg atg gee get gaa gtt caa ctt gtt gaa tct ggt get 96 Ala GIn Pro Ala Met Ala Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala 20 25 30 gaa gtt aaa aaa ccg ggt gaa tct ctg cgt ate tct tgc aaa ggt tct 144 GIu VaI Lys Lys Pro GIy GIu Ser Leu Arg lie Ser Cys Lys GIy Ser 35 40 45 ggt tat ate ate tct tct tac tgg ate age tgg gtt cgt cag atg ccg 192 GIy Tyr lie lie Ser Ser Tyr Trp lie Ser Trp VaI Arg GIn Met Pro 50 55 60 ggc aag ggc ctg gaa tgg atg ggt aaa att gat ccg ggc gac age tat 240 GIy Lys GIy Leu GIu Trp Met GIy Lys lie Asp Pro GIy Asp Ser Tyr 65 70 75 80 att aac tac age ccg age ttt cag ggc cat gtt ace ate age gcc gat 288 lie Asn Tyr Ser Pro Ser Phe GIn GIy His VaI Thr lie Ser Ala Asp 85 90 95 aaa age att aac ace get tac ctg caa tgg aac age ctg aaa gcg age 336 Lys Ser lie Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser 100 105 110 gac ace gcg atg tac tac tgt gcc cgt ggc ggt cgt gac ttc ggt gat 384 Asp Thr Ala Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp 115 120 125 age ttc gat tac tgg ggt cag ggc ace ctg gtg ace gtg age age ggt 432 Ser Phe Asp Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy 130 135 140 ggt ggc ggc age ggt ggt ggc ggc age ggc ggc ggc ggc age gat gtt 480 GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI 145 150 155 160 gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt ggt gac cgt 528 VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg 165 170 175 ate ace att ace tgc cgc gcc age age ggc ate age cgc tat ctg gcg 576 lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg Tyr Leu Ala 180 185 190 tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg ate tat get 624 Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu lie Tyr Ala 195 200 205 gca age ace ctg caa ace ggc gtt ccg age cgt ttt age ggt age ggc 672 Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy 210 215 220 age ggc ace gag ttc ace ctg ace ate aac age ctg caa ccg gag gat 720 Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn Pro GIu Asp 225 230 235 240 ttt gcc ace tat tac tgc caa cac ctg aat age tat ccg ctg ace ttc 768 Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe 245 250 255 ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg gcc gca etc gag cac 816 GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala Ala Ala Leu GIu His 260 265 270 cac cac cac cac cac taa taa 837

His His His His His 275

<210> 67

<211> 277

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 67

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala GIy Leu Leu Leu Leu Ala 1 5 10 15

Ala GIn Pro Ala Met Ala Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala 20 25 30

GIu VaI Lys Lys Pro GIy GIu Ser Leu Arg He Ser Cys Lys GIy Ser 35 40 45

GIy Tyr He He Ser Ser Tyr Trp He Ser Trp VaI Arg GIn Met Pro 50 55 60

GIy Lys GIy Leu GIu Trp Met GIy Lys He Asp Pro GIy Asp Ser Tyr 65 70 75 80

He Asn Tyr Ser Pro Ser Phe GIn GIy His VaI Thr He Ser Ala Asp 85 90 95

Lys Ser He Asn Thr Ala Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser 100 105 HO

Asp Thr Ala Met Tyr Tyr Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp 115 120 125

Ser Phe Asp Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser GIy 130 135 140

GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Asp VaI 145 150 155 160

VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy Asp Arg 165 170 175 He Thr He Thr Cys Arg Ala Ser Ser GIy He Ser Arg Tyr Leu Ala 180 185 190

Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu He Tyr Ala 195 200 205

Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy Ser GIy 210 215 220

Ser GIy Thr GIu Phe Thr Leu Thr He Asn Ser Leu GIn Pro GIu Asp 225 230 235 240

Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu Thr Phe 245 250 255

GIy GIy GIy Thr Lys VaI Asp He Lys Arg Ala Ala Ala Leu GIu His 260 265 270

His His His His His 275

<210> 68

<211> 363

<212> DNA

<213> Artificial

<220>

<223> Heavy chain of scFv peptide

<220>

<221> CDS

<222> (1) .. (363)

<400> 68 get gaa gtt caa ctt gtt gaa tct ggt get gaa gtt aaa aaa ccg ggt 48

Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro GIy 1 5 10 15 gaa tct ctg cgt ate tct tgc aaa ggt tct ggt tgc ate ate tct tct 96 GIu Ser Leu Arg He Ser Cys Lys GIy Ser GIy Cys He He Ser Ser 20 25 30 tac tgg ate age tgg gtt cgt cag atg ccg ggc aag ggc ctg gaa tgg 144 Tyr Trp He Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu Trp 35 40 45 atg ggt aaa att gat ccg ggc gac age tat att aac tac age ccg age 192 Met GIy Lys He Asp Pro GIy Asp Ser Tyr He Asn Tyr Ser Pro Ser 50 55 60 ttt cag ggc cat gtt ace ate age gee gat aaa age att aac ace get 240 Phe GIn GIy His VaI Thr He Ser Ala Asp Lys Ser He Asn Thr Ala 65 70 75 80 tac ctg caa tgg aac age ctg aaa gcg age gac ace gcg atg tac tac 288 Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr 85 90 95 tgt gcc cgt ggc ggt cgt gac ttc ggt gat age ttc gat tac tgg ggt 336 Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp GIy 100 105 110 cag ggc ace ctg gtg ace gtg age age 363

GIn GIy Thr Leu VaI Thr VaI Ser Ser 115 120

<210> 69

<211> 121

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 69

Ala GIu VaI GIn Leu VaI GIu Ser GIy Ala GIu VaI Lys Lys Pro GIy 1 5 10 15

GIu Ser Leu Arg lie Ser Cys Lys GIy Ser GIy Cys lie lie Ser Ser 20 25 30

Tyr Trp lie Ser Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu Trp 35 40 45

Met GIy Lys lie Asp Pro GIy Asp Ser Tyr lie Asn Tyr Ser Pro Ser 50 55 60

Phe GIn GIy His VaI Thr lie Ser Ala Asp Lys Ser He Asn Thr Ala 65 70 75 80

Tyr Leu GIn Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr 85 90 95

Cys Ala Arg GIy GIy Arg Asp Phe GIy Asp Ser Phe Asp Tyr Trp GIy 100 105 110

GIn GIy Thr Leu VaI Thr VaI Ser Ser 115 120

<210> 70

<211> 327

<212> DNA

<213> Artificial

<220>

<223> Liαht chai

<220>

<221> CDS

<222> (1) .. (327)

<400> 70 gat gtt gtg atg ace cag age ccg age ttc ctg age gcg ttc gtt ggt 48 Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy 1 5 10 15 gac cgt ate ace att ace tgc cgc gee age age ggc ate age cgc tat 96 Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg Tyr 20 25 30 ctg gcg tgg tat cag caa gca ccg ggt aaa gca ccg aaa ctg ctg ate 144 Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu lie 35 40 45 tat get gca age ace ctg caa ace ggc gtt ccg age cgt ttt age ggt 192 Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy 50 55 60 age ggc age ggc ace gag ttc ace ctg ace ate aac age ctg caa ccg 240 Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn Pro 65 70 75 80 gag gat ttt gee ace tat tac tgc caa cac ctg aat age tat ccg ctg 288 GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu 85 90 95 ace ttc ggt ggc ggc ace aaa gtg gac ate aaa cgc gcg 327

Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala 100 105

<210> 71

<211> 109

<212> PRT

<213> Artificial

<220>

<223> Synthetic Construct

<400> 71

Asp VaI VaI Met Thr GIn Ser Pro Ser Phe Leu Ser Ala Phe VaI GIy 1 5 10 15

Asp Arg lie Thr lie Thr Cys Arg Ala Ser Ser GIy lie Ser Arg Tyr 20 25 30

Leu Ala Trp Tyr GIn GIn Ala Pro GIy Lys Ala Pro Lys Leu Leu lie 35 40 45

Tyr Ala Ala Ser Thr Leu GIn Thr GIy VaI Pro Ser Arg Phe Ser GIy 50 55 60

Ser GIy Ser GIy Thr GIu Phe Thr Leu Thr lie Asn Ser Leu GIn Pro 65 70 75 80

GIu Asp Phe Ala Thr Tyr Tyr Cys GIn His Leu Asn Ser Tyr Pro Leu 85 90 95

Thr Phe GIy GIy GIy Thr Lys VaI Asp lie Lys Arg Ala 100 105 

Claims

1. An scFv composition comprising a plurality of scFv constructs, wherein at least 60% of the scFv constructs in the composition are in the form of multimers having a molecular weight within a designated range, the designated range being from 316 kDa to 1962 kDa, as measured by Size Exclusion Chromatography.

2. An scFv composition according to claim 1 wherein the designated range is from 727 kDa to 1550 kDa, as measured by Size Exclusion Chromatography.

3. An scFv composition comprising a plurality of scFv constructs, wherein at least 60% of the scFv constructs in the composition are in the form of multimers consisting of a designated range of number of scFv sub-units, the designated range being between 11 and 73 scFv constructs.

4. An scFv composition according to claim 3 wherein the designated range is between 26 and 57.

5. An scFv composition according to any one of the preceding claims wherein at least 60%, preferably 81%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 99%, more preferably 100% of the scFv constructs in the composition are within the designated range.

6. An scFv composition according to any one of the preceding claims wherein the scFv constructs in the composition are all of the same amino acid sequence.

7. An scFv composition according to any one of the preceding claims further comprising a pharmaceutically acceptable excipient.

8. An scFv composition according to any one of the preceding claims wherein the scFv constructs are specific for the epitope comprising the sequence of SEQ. ID NO. 1.

9. An scFv composition according to claim 8 wherein the scFv construct comprises a sequence at least 80% identical to SEQ. ID NO. 2, preferably at least 90%, 95%, 99% or 100% identical to SEQ. ID NO.2.

10. An scFv composition according to any one of claims 1 to 7 wherein the scFv constructs are specific for the epitope comprising the sequence of SEQ. ID NO. 3.

11. An scFv composition according to claim 10 wherein the scFv constructs comprise a sequence at least 80% identical to SEQ. ID NO. 3, preferably at least 90%, 95%, 99% or 100% identical to SEQ. ID NO. 4.

12. An scFv composition according to any one of the preceding claims for use in the therapy or diagnosis of the human or animal body.