HK1204571B - Protease-regulated antibodies - Google Patents

Description

Protease-regulated antibodies

The present application claims the benefit of serial No. 61/617,837 filed 3/30/2012, which is incorporated herein by reference in its entirety.

All documents cited in this disclosure are incorporated herein by reference in their entirety.

The 64kb text file created on day 3, 26, 2012 and designated "0297301274 sequencing.

Technical Field

The technical field is the treatment of hemophilia and other coagulopathies.

Drawings

FIG. 1 is a schematic illustration of an embodiment of a protease-regulated antibody. VH, heavy chain variable region; VL, light chain variable region; CH, heavy chain constant region; CL, light chain constant region.

FIG. 2 Western blot of two protease-regulated anti-TFPI Fab fragments ("Fab-1" and "Fab-2") with and without thrombin digestion.

FIG. 3 is a graph showing Tissue Factor Pathway Inhibitor (TFPI) binding of Fab-1 and Fab-2 with and without thrombin digestion as determined by ELISA.

FIG. 4 is a graph showing BIACORE binding to TFPI of Fab-1 (Fab 1) and Fab-2 (Fab 2) with and without thrombin digestion^TMAnd (6) measuring.

FIG. 5 SDS-PAGE of purified IgG antibodies expressed in HEK 2936E cells.

FIG. 6 Western blot of parent IgG ("parent IgG"; top) and IgG 2-linker 1 (bottom). Lane 1, digestion with thrombin (1 unit); lane 2, no protease (control); lane 3, digested with bovine plasmin; lane 4, digestion with enterokinase (0.02 μ g); lane 5, digestion with bovine factor Xa (1 μ g); lane 6, digestion with proteolytic enzyme (MTSP) (0.5 μ g); lane 7, digestion with urokinase (uPA) (0.25 μ g); lane 8, digested with human rhinovirus 3C protease (HRV 3C) (2 units).

Figure 7 is a graph showing TFPI binding by ELISA with and without thrombin digested IgG.

FIG. 8 is a graph showing BIACORE binding to TFPI of parent IgG and IgG-linker 1 with and without thrombin digestion^TMAnd (6) measuring.

FIG. 9 Western blot showing protease digestion of IgG-linker 1 and WT antibodies with human protease.

Detailed Description

The present disclosure provides protease-regulated antibodies that specifically bind to Tissue Factor Pathway Inhibitor (TFPI). The antibodies are useful for treating bleeding disorders, such as hemophilia. In some embodiments, the protease-regulated anti-TFPI antibody may be cleaved by thrombin and/or plasmin. By initially inhibiting TFPI, these protease-regulated anti-TFPI antibodies promote the production of thrombin and/or plasmin, which in turn cleave the antibodies and remove or significantly reduce their binding activity to TFPI. This negative feedback allows the antibody to promote clotting within a safe therapeutic window.

1. Protease-regulated anti-TFPI antibodies

The protease-regulated antibodies disclosed herein specifically bind to TFPI, i.e., they bind TFPI with a higher (e.g., at least two-fold higher) affinity than their binding affinity for an unrelated antigen (e.g., BSA, casein). The term "tissue factor pathway inhibitor" or "TFPI" as used herein refers to any variant, isoform and species homolog of human TFPI that is naturally expressed by a cell.

In some embodiments, the protease-regulated antibody binds TFPI, has at least about 10⁵M^-1To about 10¹²M^-1Affinity (e.g. 10)⁵M^-1、10^5.5M^-1、10⁶M^-1、10⁶.⁵M^-1、10⁷M^-1、10⁷.⁵M^-1、10⁸M^-1、10⁸.⁵M^-1、10⁹M^-1、10^9.5M^-1、10¹⁰M^-1、10^10.5M^-1、10¹¹M^-1、10^11.5M^-1、10¹²M^-1). Affinity of antibody binding to antigen (K)_d) Can be determined using any method known in the art, e.g., immunoassays such as enzyme-linked immunospecific assays (ELISAs), Biomolecular Interaction Assays (BIAs) (examples)E.g., Sjolander&Urbaniczky; anal. chem.63:2338-2345, 1991; Szabo, et al, curr. Opin. struct. biol. 5:699-705, 1995), and Fluorescence Activated Cell Sorting (FACS) for quantification of antibody binding on antigen expressing cells. BIA is a technique for real-time analysis of biospecific interactions without labeling any interactors (e.g., BIACORE)^TM). Optical phenomena changes in Surface Plasmon Resonance (SPR) can be used as an indication of real-time reactions between biomolecules.

Protease-regulated anti-TFPI antibodies may be used with substantially full-length immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA), antigen-binding fragments thereof, e.g., Fab or F (ab')₂Or constructs comprising an antigen binding site capable of specifically binding TFPI, such as scFv, Fv, or diabody. The term "antibody" also includes other protein scaffolds capable of accommodating antibody Complementarity Determining Region (CDR) insertions into the same active binding conformation as found in native antibodies, such that the binding to TFPI observed for these chimeric proteins is retained relative to the TFPI binding activity of the native antibody from which the CDRs are derived.

As used herein, an "isolated antibody" is an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that binds TFPI is substantially free of antibodies that bind antigens other than TFPI). However, an isolated antibody that binds an epitope, isoform or variant of human TFPI may have cross-reactivity to other related antigens, such as antigens from other species (e.g., TFPI species homologs). The isolated antibody may be substantially free of other cellular material and/or chemicals.

The protease-regulated antibodies disclosed herein are engineered to comprise a protease cleavage site that is recognized by one or more proteases. As used herein, "protease cleavage site" refers to an amino acid sequence that is recognized and cleaved by a protease. In some embodiments, the protease cleavage site is located at a position between its variable and constant regions. In some embodiments, the protease-regulated anti-TFPI antibody includes one or more protease cleavage sites that can be cleaved by thrombin, plasmin, and/or factor Xa. In some embodiments, the amino acid sequence between the variable and constant regions of the protease-modulated anti-TFPI antibody comprises a polypeptide linker in addition to the protease cleavage site (as illustrated, for example, in fig. 1). The linker may be a single amino acid or a polypeptide sequence (e.g., up to 100 amino acids). For example, the linker may be GGGGS (SEQ ID NO: 149). Other useful linkers include SEQ ID NOs: 151, 176. In other embodiments, no linker is present, and the cleavage site itself is interposed between the variable and constant regions.

At least two optimal thrombin cleavage sites have been identified: (1) x₁-X₂-P-R-X₃-X₄(SEQ ID NO:147), wherein X₁And X₂Is a hydrophobic amino acid, and X₃And X₄Is a non-acidic amino acid, and (2) GRG. Thrombin specifically cleaves after an arginine residue. Plasmin can also cleave both cleavage sites, but with lower specificity compared to thrombin. Other thrombin cleavage sites that may be used are provided as SEQ ID NO: 1-60. Other plasmin cleavage sites available are provided as SEQ ID NO: 12. 47, 48, 53 and 61-130. In some embodiments, the cleavage site is LVPRGS (SEQ ID NO: 137).

In some embodiments, a factor Xa cleavage site is used, e.g., I- (E or D) -G-R (SEQ ID NO: 148). Other useful factor Xa cleavage sites are provided as SEQ ID NO: 29. 59, and 61-69. Other thrombin and FXa cleavage sites or sequences can be found in previous publications, the authors being Bianchini [ Bianchini EP et al 2002 JBC ]. A protease-regulated antibody may include more than one protease cleavage site.

In addition to the cleavage site, a second protease binding site, referred to as exosite (exosite), may be introduced into the protease-regulated TFPI antibody to make cleavage more efficient. The exosites of thrombin may be derived from the natural exosites of protease substrates or inhibitors such as PAR1, fibrinogen and hirudin. The exosite may also be a derivative of the native exosite.

Protease-regulated TFPI antibodies may be synthesized or recombinantly produced. Antibodies can be produced using a number of techniques. For example, antibodies can be generated using phage-antibody technology (Knappik)Etc., J. mol. biol. 296:57-86, 2000). Another method for obtaining antibodies is to screen DNA libraries from B cells, for example as described in WO 91/17271 and WO 92/01047. In these methods, a phage library is generated, in which the members display different antibodies on their outer surface. Antibodies are typically displayed as Fv or Fab fragments. Phage-displayed antibodies that bind the selected protein are selected by affinity enrichment. Antibodies can also be produced using a trioma technique (e.g., Oestberg)Etc., Hybridoma 2361, 367, 1983, 4,634,664, 4,634,666).

The antibody may also be purified from any cell expressing the antibody, including host cells that have been transfected with an expression construct encoding the antibody. The host cell may be cultured under conditions from which the antibody is expressed. Using methods well known in the art, the purified antibody can be separated from other cellular components that can be associated with the antibody in the cell, such as certain proteins, carbohydrates, or lipids. These methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. The purity of the preparation can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. A preparation of purified antibodies may comprise more than one type of antibody.

Alternatively, protease-regulated anti-TFPI antibodies may be generated using chemical methods to synthesize their amino acid sequences, such as by direct peptide synthesis using solid phase techniques (e.g., Merrifield, J). Am. Chem. Soc. 85:2149-2154, 1963; RobergeEtc. Science 269:202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Optionally, antibody fragments may be usedChemical methods were synthesized separately and combined to produce full-length molecules.

Protease-regulated anti-TFPI antibodies may also be constructed as "single chain fv (scFv) versions," in which a protease cleavage site is inserted in or near the peptide linker between the light chain variable region and the heavy chain variable region of the scFv. Because a peptide linker is necessary to hold the two variable regions of the scFv together for antigen binding, cleavage of the peptide linker or flanking region inactivates the protease of interest or down regulates the binding of the scFv to its antigen. Amino acid sequence SEQ ID NO:179 (which may be encoded by SEQ ID NO: 180) is an example of a protease-regulated anti-TFPI antibody in the form of an scFv.

In some embodiments, the protease-regulated TFPI antibody is constructed as an "IgG form" having two binding sites, and may comprise one, two, three, or four protease cleavage sites on the heavy chain, the light chain, or both. In each case, the protease cleavage site may be flanked by linkers at either or both ends. Furthermore, the cleavage sites may be the same or different in each case. "IgG-linker 1" (example 5) and "IgG-linker 2" are examples of protease-regulated anti-TFPI antibodies in the form of IgG:

。

2. negative feedback

Protease-regulated antibodies may comprise a protease cleavage site for a protease that is up-regulated or produced as a result of the function of the antibody. These proteases can then cleave the protease-regulated antibody, thereby providing a negative feedback loop that can be used to prevent over-activity of the protease-regulated antibody.

For example, anti-TFPI antibodies that can be modulated by thrombin and/or plasmin-cleaved proteases may exhibit such negative feedback effects. Such protease-regulated anti-TFPI antibodies promote the production of thrombin and/or plasmin. Thrombin and plasmin in turn cleave protease-regulated anti-TFPI antibodies and remove or significantly reduce their binding activity to TFPI. This negative feedback allows the antibody to promote clotting within a safe therapeutic window.

3. Polynucleotide

The disclosure also provides polynucleotides encoding the protease-regulated antibodies. These polynucleotides can be used, for example, to generate antibody amounts for therapeutic use.

cDNA molecules encoding the antibodies can be prepared using standard molecular biology techniques using mRNA as a template. Thereafter, cDNA molecules can be replicated using Molecular biology techniques known in the art and disclosed in handbooks (e.g., Sambrook, et al, Molecular Cloning: Arabidopsis Manual, (second edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989), volumes 1-3). Additional copies of the polynucleotide may be obtained using amplification techniques, such as PCR. Alternatively, polynucleotides encoding protease-regulated anti-TFPI antibodies may be synthesized using synthetic chemistry techniques.

For expression of the polynucleotide encoding the antibody, the polynucleotide may be inserted into an expression vector that contains the necessary elements for transcription and translation of the inserted coding sequence. Expression vectors comprising antibody-encoding sequences and appropriate transcriptional and translational control elements can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. These techniques are described, for example, in Sambrook et al (1989) and Ausubel et al, (Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1995).

A variety of expression vector/host systems may be utilized to contain and express the antibody-encoding sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with a yeast expression vector; insect cell systems transfected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV); or bacterial expression vectors (e.g., Ti or pBR22 plasmids), or animal cell systems.

Control elements or regulatory sequences are those vectors of the untranslated regions-enhancer, promoter, 5 'and 3' untranslated region-it and host cell protein interactions to transcription and response. The strength and specificity of these elements can vary. Depending on the vector system and host, any number of suitable transcription and translation elements may be used, including constitutive and inducible promoters. For example, when cloning in a bacterial system, inducible promoters may be used. Baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genome of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or promoters or enhancers derived from plant viruses (e.g., viral promoters or leaders) may be cloned into vectors. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses may be used. If it is necessary to generate a cell line comprising multiple copies of the nucleotide sequence encoding the antibody, an SV40 or EBV-based vector with an appropriate selectable marker may be used.

General documents describing further useful Molecular Biology Techniques, including the preparation of Antibodies, are Berger and Kimmel (Guide to Molecular Cloning technologies, Methods in Enzymology, Vol.152, Academic Press, Inc.); Sambrook, et al, (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol.1-3); Current Protocols in Molecular Biology, (F. M. Austabel et al, [ Eds. ], Current Protocols, a J.upright Green purifying plants, Inc. wild et al, (wild et al; wild animal, U.S.; wild animal et al; wild animal research, U.S.; Harbor plant et al; wild animal research, U.S.; Harbor plant et al; road plant & S.; road plant et al; road plant, plant et al; road plant, cold Spring Harbor Laboratory Press (1998)).

4. Pharmaceutical composition

The protease-modulated anti-TFPI antibodies may be provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is preferably non-pyrogenic. The pharmaceutical compositions comprising the protease-regulated anti-TFPI antibodies may be administered alone or in combination with at least one other agent (e.g., a stabilizing composition), which may be used in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, glucose, and water. Various aqueous carriers can be employed, such as 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter. These solutions can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The composition may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting agents and buffers, and the like. Depending on the particular mode of administration selected, the concentration of protease-regulated anti-TFPI antibody in the pharmaceutical composition may vary widely, i.e., from less than about 0.5%, typically at or at least about 1% up to 15% or 20% by weight, and will be selected based primarily on fluid volume, viscosity, etc. See U.S. patent No. 5,851,525. If desired, more than one different protease-regulated anti-TFPI antibody may be included in the pharmaceutical composition.

In addition to the active ingredient, the pharmaceutical composition may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the composition into preparations which can be used as medicaments. The pharmaceutical compositions may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.

After the pharmaceutical compositions have been prepared, they may be placed in an appropriate container and marked for treatment indicative of the condition. Such indicia will include the amount, frequency and method of administration.

5. Method of producing a composite material

Pharmaceutical compositions comprising one or more protease-regulated anti-TFPI antibodies may be administered to a patient alone, or in combination with other agents, drugs, or clotting factors to treat hemophilia or other clotting disorders. A "therapeutically effective dose" of a protease-modulated anti-TFPI antibody refers to an amount of protease-modulated anti-TFPI antibody that will promote blood clotting or reduce bleeding time. Determination of a therapeutically effective dose is well within the capability of those skilled in the art.

The therapeutically effective dose can be initially evaluated in cell culture assays or in animal models (typically rat, mouse, rabbit, dog or pig). Animal models can be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine useful doses and routes for administration in humans.

The therapeutic efficacy and toxicity of protease-regulated anti-TFPI antibodies can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED₅₀(therapeutically effective dose in 50% of the population) and LD₅₀(dose lethal to 50% of the population). The dose ratio of toxicity to therapeutic effect is the therapeutic index, and it can be expressed as the ratio LD₅₀/ED₅₀。

Pharmaceutical compositions exhibiting a large therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used in dosage ranges formulated for human use. The dosages contained in these compositions are preferably within the circulating concentration range, which includes the ED with little or no toxicity₅₀. The dosage will vary within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the physician, depending on factors related to the patient in need of treatment. The dosage and administration is adjusted to provide sufficient levels of protease-modulated TFPI antibody or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the general health of the subject, the age, weight and sex of the subject, diet, time and frequency of administration, combination of one or more drugs, response sensitivity and tolerance/response to treatment. Long acting pharmaceutical compositions may be administered once every 3 to 4 days, weekly, or biweekly, depending on the half-life and clearance of the particular formulation.

In some embodiments, the in vivo dose of a therapeutically effective protease-modulated anti-TFPI antibody ranges from about 5 μ g to about 100mg/kg, from about 1mg to about 50mg/kg, from about 10mg to about 50mg/kg of patient body weight.

The mode of administration of the pharmaceutical composition comprising the protease-regulated anti-TFPI antibody may be any suitable route that delivers the antibody to the host (e.g., subcutaneous, intramuscular, intravenous, or intranasal administration).

In some embodiments, the protease-modulated anti-TFPI antibody is administered without an additional therapeutic agent. In some embodiments, the protease-regulated anti-TFPI antibody is administered in combination with other agents (e.g., drugs or clotting factors) to enhance the initial production of thrombin while ensuring that thrombin levels remain below the range that may cause thrombosis in some people with clotting dysfunction. Administration of the protease-regulated anti-TFPI antibody may be before, after, or substantially simultaneous with the administration of the other agent.

Nothing in this specification should be taken as limiting the scope of the disclosure. All examples presented are representative and non-limiting. Modifications or variations of the above-described embodiments may be made by persons skilled in the art in light of the teachings. It is therefore to be understood that within the scope of the appended claims and their equivalents, the embodiments disclosed herein may be practiced otherwise than as specifically described.

Example 1

Construction of protease-regulated anti-TFPI Fab fragments

Two protease-regulated anti-TFPI Fab, "Fab-1" and "Fab-2" are based on the anti-TFPI antibody sequences shown in SEQ ID NO:177 (heavy chain) and SEQ ID NO:178 (light chain). Both Fab's have a thrombin/plasmin protease cleavage site LVPRGS (SEQ ID NO:137) inserted C-terminal to both variable domains. The cleavage site in Fab-1 is flanked by (Gly)₄A Ser linker. Fab-2 contains only six amino acid cleavage sites, and no linker is present. Thrombin and plasmin will cleave the C-terminus relative to the Arg (R) residue of LVPRGS (SEQ ID NO: 137). DNA encoding Fab was synthesized by GenScript with optimized codons for bacterial expression. The amino acid and DNA sequences for the Fab are identified in the table below.

Using restriction enzymesBsaI andHindIII (New England Biolabs) digestion of the Fab coding region. The DNA fragment was purified using an agarose gel and subcloned into pBADmycHisA (Invitrogen). Cloned DNA is ligated and transformed using standard techniques. Positive clones were confirmed by DNA sequencing and used for BL21 e.coli expression.

Example 2

Western blot of thrombin-cleaved Fab

Approximately 2.5 μ g of the crude purified Fab-1 and Fab-2 were digested with 0, 2 or 10 units of thrombin (Novagen) at 37 ℃ for 1 hour. The digestate is in the range of 4-15% criseon^TMTGX^TMRun on gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes and probed with anti-human Fab antibodies (Southern Biotech).

Western blots of Fab-1 and Fab-2 cleavage are shown in FIG. 2. All samples were reduced. When treated with thrombin, a band of about 12kDA was observed for both fabs. The absence of these bands in the sample without thrombin digestion indicates that the product of thrombin cleavage is a small size protein.

Example 3

TFPI ELISA of thrombin-digested Fab-1 and Fab-2.

About 2.5 μ g of partially purified Fab-1 and Fab-2 were digested with 2 units of biotinylated thrombin (Novagen) overnight at 23 ℃. Streptavidin agarose beads (100 μ L) were added to the digests to deplete thrombin. The digested Fab sample was applied to a column that captured the agarose bead/thrombin complex. The eluate contained digested Fab.

MAXISORP^®96-well plates (Nunc) were coated overnight with 1 μ g/mL TFPI in PBS at 4 ℃. Plates were incubated at room temperature with 5% defatted dry milk PBS/0.5% TWEEN-20^®(PBS-T) for 1 hour. Serial two-fold dilutions of undigested and digested Fab-1 and Fab-2 were added to wells (100 μ L/well) and incubated for 1 hour at RT. Plates were washed 5 times with PBS-T. Secondary HRP-conjugated anti-Fab antibody (100 μ L, 1:10,000 dilution) was added to the reaction mixture with AMPLEX^®Red (Invitrogen) solution assay. As shown in fig. 3, thrombin digestion significantly reduced the signal for TFPI binding.

Example 4

BIACORE OF THROMBIN-DIGESTING Fab-1 AND Fab-2 ^TM Measuring

Approximately 2.5 μ g of the crude purified Fab-1 and Fab-2 were digested with 2 units of biotinylated thrombin (Novagen) overnight at 23 ℃. Streptavidin agarose beads (100 μ L) were added to the digests to deplete thrombin. The digested Fab sample was applied to a column that captured the agarose bead/thrombin complex. The eluate contained digested Fab.

For BIACORE^TMAssay human TFPI (American Diagnostica) was immobilized at a target level of 100 Relative Units (RU) using amine coupling and in mobile phaseAntibodies were injected. HBS-p was used as running buffer. A reference channel was prepared using a blank immobilization of proteins without any immobilization in which the surface was activated and then inactivated. Manual runs were performed to measure elevated RU for Fab and buffer control.

As shown in figure 4, the parent Fab without the cleavable linker produced 24.6RU compared to undigested Fab-1 (25.6 RU) and Fab2 (27.9 RU). After thrombin cleavage, the binding signals of Fab-1 and Fab-2 decreased by more than 50%, indicating that the cleaved Fab-1 and Fab-2 lost not only the constant domain but also the binding activity to TFPI.

Example 5

IgG expression and purification

A protease-regulated anti-TFPI immunoglobulin molecule, "IgG-linker 1", was constructed based on the parental anti-TFPI antibody sequences shown in SEQ ID NO:177 (heavy chain) and SEQ ID NO:178 (light chain). To facilitate molecular cloning, theBlpThe I site is introduced into the heavy chain coding sequence. The introduction of the BlpI site shifted the linker position of IgG-linker 1 by two amino acids towards the constant region compared to the position of the protease cleavable linker in Fab-1.

HEK 2936E cells were transfected with the constructed IgG expression vector and culture supernatants containing IgG antibodies were harvested. Using MABSELECT SURE^TMThe affinity column is then passed through SUPERDEX^TMThe antibody was purified by 200 chromatography. IgG-linker 1 purified on SDS-PAGE is shown in FIG. 5.

Example 6

Western blot of parent IgG and IgG-linker 1

Purified parent IgG and IgG-linker 1 (0.5 μ g) were digested with thrombin, bovine plasmin, bovine factor Xa, proteolytic enzyme (MTSP), urokinase (uPA), or human rhinovirus 3C protease (HRV 3C). Antibodies in combination with proteases 3Incubate at 7 ℃ for 1 hour. In the range of 4-20% CRISTEREON^TMTGX^TMDigests were run on gels (Bio-Rad). Proteins were transferred to nitrocellulose membranes and probed with anti-human IgG heavy and light chain antibodies (Pierce). Western blots of IgG2 and IgG 2-linker 1 are shown in FIG. 6. All samples were reduced.

Intact IgG produced two bands under reducing conditions. The two bands correspond to the heavy chain (50 kD) and light chain (25 kD). The digested antibody IgG-linker 1 shows a shift in molecular weight relative to the undigested antibody. The following proteases were used to digest the antibodies: thrombin, plasmin, bovine factor Xa, MTSP and uPA. Digested IgG-linker 1 antibody shows a molecular weight shift of the heavy chain from 50kD to 37 kD. This size shift is associated with the loss of the VH domain from the heavy chain. There is also a molecular weight shift of the 25kD light chain to a faint band of about 16kDa, indicating that the VL domain is cleaved from the light chain. The protease did not cleave the parent IgG, indicating that the molecular weight loss was due to protease digestion engineered into the cleavage site of the antibody.

Example 7

Thrombin digested parental IgG and IgG-linker 1 TFPI-binding ELISA

One microgram of full length antibody, parent IgG and IgG-linker 1 was digested with 1 unit of biotinylated thrombin (Novagen) at 37 ℃ for 1 hour. Then 50 μ L of streptavidin agarose beads were added to the digests to deplete thrombin. The digested IgG sample was applied to a column that captured the agarose bead/thrombin complex. The eluate contained digested IgG.

MAXISORP^®96-well plates (Nunc) were coated overnight with 1 μ g/mL TFPI in PBS at 4 ℃. Plates were incubated at Room Temperature (RT) in 5% defatted dry milk PBS/0.5% TWEEN-20^®(PBS-T) for 1 hour. Serial two-fold dilutions of undigested and digested parental IgG and IgG 1-linker 1 were added to wells (100 μ L/well) and incubated for 1 hour at RT. Plates were washed 5 times in PBS-T. Addition of Secondary HRP-conjugated anti-Fab antibody(100 μ L, 1:10,000 dilution) for use with AMPLEX^®Red (Invitrogen) solution assay.

The results of TFPI binding ELISA are shown in figure 7. Showing loss of TFPI binding of thrombin digested IgG linker-1. TFPI binding of undigested IgG-linker 1 is similar to that of undigested and thrombin-digested parent IgG.

Example 8

BIACORE OF parental IgG AND IgG-linker 1 ^TM Analysis of

CM4 sensor chips were immobilized with low density human TFPI using an amine coupling kit (GE HealthCare). Kinetic measurements of parent IgG and IgG-linker 1 were performed using different concentrations of antibody, followed by regeneration with glycine buffer pH 1.5. Parental IgG and IgG-linker 1 antibody at a concentration of 1 μ g were digested with 1 unit of biotinylated thrombin (Novagen) at 37 ℃ for 1 hour. Injecting digested or undigested antibody into BIACORE^TMThe system was used for TFPI binding analysis. The lower panel shows the signal generated from injection of 45 μ l of 6.25 μ g/ml antibody or control sample.

In the kinetic assay, parent IgG and IgG 2-linker 1 each had 1.536x10⁶/Ms and 1.902 x10⁶The binding rate of/Ms (ka). The two antibodies did not have measurable dissociation within 30 minutes. This indicates that the insertion of linker 1 did not significantly alter the binding activity of the antibody.

The effect of thrombin cleavage on the antibody is shown in figure 8. Thrombin digestion slightly reduced the binding of the parent IgG to TFPI, whereas thrombin digestion reduced the binding of IgG 2-linker 1 to TFPI from 20RU to 5RU, a 75% reduction.

Example 9

Western blot of protease/coagulation factor treated IgG-linker 1 and parent IgG

80nM of IgG-linker 1 and WT antibodies were digested with the following human protease/clotting factor: thrombin (0.1. mu.M), plasmin (0.1. mu.M), factor VIIa (0.01. mu.M), factor IXa (0.089. mu.M), factor Xa (13. mu.M), factor XIa (0.031. mu.M), factor XIIIa (0.03. mu.M). At 4-15% CRISTEREON^TMThe treated material was run on TGX gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes and probed with anti-human IgG antibodies (Pierce).

Human thrombin, plasmin and factor Xa digested IgG-linker 1, thrombin digestion was most efficient (fig. 9), as shown by the appearance of the 37kDa band. The protease did not cleave the parent IgG, indicating that the molecular weight loss was due to protease digestion engineered into the cleavage site of the antibody.

Sequence listing

<110>TBD

<120> protease-regulated antibodies

<130>012973.01274

<160>180

<170> FastSEQ for Windows Version 4.0

<210>1

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

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Ala His Pro Arg Ile Ile Ser Ala

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<210>2

<211>8

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<213> Artificial sequence

<220>

<223> protease cleavage site

<400>2

Ala Arg Thr Arg Ala Arg Arg Pro

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<213> Artificial sequence

<220>

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Ala Ser Ala Arg Thr Thr Gly Ser

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<400>4

Ala Thr Pro Arg Gly Ala Ala Pro

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Ala Val Val Arg Thr Pro Pro Lys

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<211>8

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<213> Artificial sequence

<220>

<223> protease cleavage site

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Glu Arg Thr Arg Ser Phe Gln Leu

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Phe Gly Leu Arg Phe Tyr Ala Tyr

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<213> Artificial sequence

<220>

<223> protease cleavage site

<400>8

Phe Asn Pro Arg Thr Phe Gly Ser

1 5

<210>9

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>9

Phe Arg Pro Lys His Thr Arg Ile

1 5

<210>10

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>10

Phe Ser Ala Arg Gly His Arg Pro

1 5

<210>11

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>11

Gly Asp Ile Arg Gly Pro Arg Ile

1 5

<210>12

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>12

Gly Gly Val Arg Gly Pro Arg Val

1 5

<210>13

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>13

Gly Ser Phe Arg Ala Gly Leu Phe

1 5

<210>14

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>14

His Lys Gly Arg Ser Ala Leu Val

1 5

<210>15

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>15

Ile Ala Gly Arg Ser Leu Asn Pro

1 5

<210>16

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>16

Ile Asp Gly Arg Ile Val Glu Gly

1 5

<210>17

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>17

Ile Glu Pro Arg Ser Phe Ser Gln

1 5

<210>18

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>18

Ile Lys Pro Arg Ile Val Gly Gly

1 5

<210>19

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>19

Ile Gln Ile Arg Ser Val Ala Lys

1 5

<210>20

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>20

Ile Gln Ile Arg Ser Val Ala Lys

1 5

<210>21

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>21

Lys Asn Val Lys Ser Lys Ile Gly

15

<210>22

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>22

Lys Pro Lys Asp Ser Ser Val Asp

1 5

<210>23

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>23

Leu Asp Pro Arg Ser Phe Leu Leu

1 5

<210>24

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>24

Leu Gly Ile Arg Ser Phe Arg Asn

1 5

<210>25

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>25

Leu Pro Ile Lys Thr Phe Arg Gly

1 5

<210>26

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>26

Leu Arg Pro Arg Phe Lys Ile Ile

1 5

<210>27

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>27

Leu Arg Pro Arg Ile Ile Gly Gly

1 5

<210>28

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>28

Leu Ser Pro Arg Gly Val His Ile

1 5

<210>29

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>29

Leu Ser Pro Arg Thr Phe His Pro

1 5

<210>30

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>30

Met Thr Pro Arg Ser Glu Gly Ser

1 5

<210>31

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>31

Met Thr Pro Arg Ser Gly Gly Ser

1 5

<210>32

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>32

Met Thr Pro Arg Ser Arg Gly Ser

1 5

<210>33

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>33

Met Val Pro Arg Ala Val Tyr Leu

1 5

<210>34

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>34

Asn Val Pro Arg Ile Leu Ser Pro

1 5

<210>35

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>35

Pro Ala Pro Arg Gly Tyr Pro Gly

1 5

<210>36

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>36

Pro Asp Leu Arg Ser Cys Val Asn

1 5

<210>37

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>37

Pro Gly Pro Arg Gly Pro Pro Pro

1 5

<210>38

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>38

Pro Gly Ser Arg Ser Arg Thr Pro

1 5

<210>39

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>39

Pro Gln Gly Arg Ile Val Gly Gly

1 5

<210>40

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>40

Pro Gln Gly Arg Thr Thr Ala His

1 5

<210>41

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>41

Pro Arg Ser Phe Leu Leu Arg Asn

1 5

<210>42

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>42

Gln Ser Pro Arg Ser Phe Gln Lys

1 5

<210>43

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>43

Gln Ser Pro Arg Ser Phe Gln Lys

1 5

<210>44

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>44

Gln Tyr Leu Arg Val Pro Leu Val

1 5

<210>45

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>45

Ser Glu Phe Arg Cys Leu Thr Pro

1 5

<210>46

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>46

Ser Ile Gly Arg Ala Ser Leu His

1 5

<210>47

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>47

Ser Lys Gly Arg Ser Leu Ile Gly

1 5

<210>48

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>48

Ser Arg Leu Arg Ala Tyr Leu Leu

1 5

<210>49

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>49

Thr Cys Leu Arg Ser Thr Lys Phe

1 5

<210>50

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>50

Val Cys Leu Arg Ser Phe Gln Thr

1 5

<210>51

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>51

Val Asp Pro Arg Leu Ile Asp Gly

1 5

<210>52

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>52

Val Glu Val Lys Ser Glu Lys Leu

1 5

<210>53

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>53

Val Ile Pro Lys Arg Ile Ser Pro

1 5

<210>54

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>54

Val Ile Pro Arg Ser Gly Gly Ser

1 5

<210>55

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>55

Val Asn Pro Arg Gly Ile Val Thr

1 5

<210>56

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>56

Val Gln Pro Arg Ala Gln Lys Ile

1 5

<210>57

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>57

Val Ser Pro Arg Ala Ser Ala Ser

1 5

<210>58

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>58

Val Val Pro Arg Gly Val Asn Leu

1 5

<210>59

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>59

Trp Tyr Leu Arg Ser Asn Asn Gly

1 5

<210>60

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>60

Trp Tyr Leu Arg Ser Asn Thr Gly

1 5

<210>61

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>61

Ala Phe Trp Lys Thr Asp Ala Ser

1 5

<210>62

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>62

Ile Glu Gly Arg Thr Ala Thr Ser

1 5

<210>63

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>63

Thr Ala Ala Arg Gln Ser Thr Asn

1 5

<210>64

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>64

Ile Asp Gly Arg Ile Val Glu Gly

1 5

<210>65

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>65

Phe Asn Pro Arg Thr Phe Gly Ser

1 5

<210>66

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>66

Arg Val Pro Lys Ser Phe Pro Phe

1 5

<210>67

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>67

Pro Gln Leu Arg Met Lys Asn Asn

1 5

<210>68

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>68

Ser Ser Trp Arg Leu Thr Ser Ser

1 5

<210>69

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>69

Leu Gly Ile Arg Ser Phe Arg Asn

1 5

<210>70

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>70

Ala Met Ser Arg Met Ser Leu Ser

1 5

<210>71

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>71

Ala Asn Asn Arg Asp Asn Thr Tyr

1 5

<210>72

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>72

Ala Asn Val Arg Arg Lys Arg Tyr

1 5

<210>73

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>73

Ala Arg Gly Arg Ala Phe Pro Gln

1 5

<210>74

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>74

Ala Thr Leu Lys Ser Arg Lys Met

1 5

<210>75

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>75

Ala Thr Gln Lys Lys Val Glu Arg

1 5

<210>76

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>76

Ala Thr Trp Lys Thr Arg Trp Tyr

1 5

<210>77

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>77

Asp Val Gly Glu Tyr Asn Val Phe

1 5

<210>78

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>78

Glu Ala Arg Gly Ser Val Ile Leu

1 5

<210>79

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>79

Glu Ala Tyr Arg Arg Phe Tyr Gly

1 5

<210>80

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>80

Glu Asp Asn Arg Asp Ser Ser Met

1 5

<210>81

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>81

Glu Thr Leu Lys Val Ile Asp Glu

1 5

<210>82

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>82

Phe Arg Ala Arg Ala Tyr Gly Phe

1 5

<210>83

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>83

Gly Glu Ala Arg Gly Ser Val Ile

1 5

<210>84

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>84

Gly Gly Tyr Arg Ala Arg Pro Ala

1 5

<210>85

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>85

Gly Ile Leu Lys Glu Asn Ala Ala

1 5

<210>86

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>86

Gly Pro Lys Arg Gly Thr Glu Pro

1 5

<210>87

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>87

Ile Ile Arg Arg Ser Ile Gln Ile

1 5

<210>88

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>88

Ile Thr Phe Arg Met Asn Val Ala

1 5

<210>89

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>89

Lys His Ser Lys Arg His Ile His

15

<210>90

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>90

Lys Lys Asp Arg Ala Arg Gln Glu

1 5

<210>91

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>91

Lys Lys Pro Arg Cys Gly Val Pro

1 5

<210>92

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>92

Lys Gln Val Lys Asp Asn Glu Asn

1 5

<210>93

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>93

Leu Asp Pro Arg Ser Phe Leu Leu

1 5

<210>94

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>94

Leu Pro Pro Lys Ser Gln Pro Pro

1 5

<210>95

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>95

Leu Ser Phe Arg Ala Arg Ala Tyr

1 5

<210>96

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>96

Met Ser Met Arg Val Arg Arg His

1 5

<210>97

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>97

Asn Ser Gly Arg Ala Val Thr Tyr

1 5

<210>98

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>98

Pro Ala Pro Arg Gly Tyr Pro Gly

1 5

<210>99

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>99

Pro Glu Ala Lys Ala Ser Cys Tyr

1 5

<210>100

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>100

Pro Glu Ser Lys Ala Thr Asn Ala

1 5

<210>101

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>101

Pro Gly Pro Lys Arg Gly Thr Glu

1 5

<210>102

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>102

Pro Lys Ala Lys Ser His Ala Pro

1 5

<210>103

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>103

Pro Leu Asp Lys Lys Arg Glu Glu

1 5

<210>104

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>104

Pro Leu Gln Lys Gln Leu Pro Ala

1 5

<210>105

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>105

Pro Gln Phe Arg Ile Lys Gly Gly

1 5

<210>106

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>106

Pro Gln Leu Arg Leu Pro His Thr

1 5

<210>107

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>107

Pro Gln Leu Arg Arg Gly Trp Arg

1 5

<210>108

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>108

Pro Gln Ser Arg Ser Val Pro Pro

1 5

<210>109

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>109

Pro Arg Phe Lys Ile Ile Gly Gly

1 5

<210>110

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>110

Pro Tyr Leu Lys Val Phe Asn Pro

1 5

<210>111

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>111

Gln Lys Ser Arg Asn Gly Leu Arg

1 5

<210>112

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>112

Gln Leu Ile Lys Ala Ile Gln Leu

1 5

<210>113

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>113

Gln Arg Tyr Lys Val Asp Tyr Glu

1 5

<210>114

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>114

Arg Ala Gln Arg Ser Ala Gly Ala

1 5

<210>115

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>115

Arg Gly Pro Arg Val Val Glu Arg

1 5

<210>116

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>116

Arg Pro Ala Lys Ala Ala Ala Thr

1 5

<210>117

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>117

Arg Arg Lys Arg Tyr Ala Ile Gln

1 5

<210>118

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>118

Arg Ser Ser Lys Gly Arg Ser Leu

1 5

<210>119

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>119

Arg Ser Thr Arg Phe Ala Ala Thr

1 5

<210>120

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>120

Ser Cys Asp Lys Thr His Thr Cys

1 5

<210>121

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>121

Ser Ile Asn Lys Ser Ser Pro Leu

1 5

<210>122

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>122

Ser Gln Pro Pro Glu Lys Thr Glu

1 5

<210>123

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>123

Ser Gln Arg Lys His Ser Lys Arg

1 5

<210>124

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>124

Ser Ser Met Lys Leu Ser Phe Arg

1 5

<210>125

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>125

Thr Glu Pro Lys Val Lys Leu Pro

1 5

<210>126

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>126

Thr Glu Tyr Arg Leu Val Ser Ile

1 5

<210>127

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>127

Thr His Glu Lys Gly Arg Gln Ser

1 5

<210>128

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>128

Thr Tyr Ser Lys Ala Ser Thr Pro

1 5

<210>129

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>129

Thr Tyr Ser Arg Ser Arg Tyr Leu

1 5

<210>130

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>130

Val Ser Asn Lys Val Ser Met Ser

1 5

<210>131

<211>228

<212>PRT

<213> Artificial sequence

<220>

<223> Fab-1 light chain

<400>131

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Pro Arg Gly Ser Gly Gly Gly Gly Ser Gly Gln ProLys Ala Ala

115 120 125

Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn

130 135 140

Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val

145 150 155 160

Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu

165 170 175

Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser

180 185 190

Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser

195 200 205

Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro

210 215 220

Thr Glu Cys Ser

225

<210>132

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site and linker

<400>132

Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>133

<211>245

<212>PRT

<213> Artificial sequence

<220>

<223> Fab-1 heavy chain

<400>133

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 7075 80

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

85 90 95

Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser

115 120 125

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

130 135 140

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

145 150 155 160

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

165 170 175

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

180 185 190

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

195 200 205

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

210 215 220

Thr Lys Val Asp Lys Thr Val Asp Tyr Lys Asp Asp Asp Asp Lys His

225 230 235 240

His His His His His

245

<210>134

<211>1590

<212>DNA

<213> Artificial sequence

<220>

<223> sequence encoding Fab-1

<400>134

ggtctcacat gaaaaaaacc gctatcgcta tcgccgtcgc actggctggc ttcgcaaccg 60

tggcacaggc atcctatgaa ctgacccaac cgccgagtgt ctccgtgtca ccgggtcaga 120

cggcacgtat tacctgcagc ggtgataacc tgccgaaata ttacgcgcat tggtatcagc 180

aaaaaccggg ccaagccccg gtggttgtca tcttttatga cgttaatcgt ccgtccggta 240

tcccggaacg cttctcgggc agcaactctg gtaatacggc aaccctgacg atcagcggca 300

cccaggcaat ggatgaagct gactattact gtcaagcatg gtggagctct acgccggtgt 360

ttggcggtgg cacgaaactg accgtgctgg gtggcggtgg ctctctggtt ccgcgtggct 420

ccggtggcgg tggctcaggc cagccgaaag cagcaccgag tgttaccctg tttccgccga 480

gttccgaaga actgcaagca aacaaagcta ccctggtgtg cctgattagc gatttctatc 540

cgggcgcagt tacggtcgcg tggaaagccg actcatcgcc ggtgaaagct ggtgttgaaa 600

ccacgacccc gtcaaaacag tcgaacaata aatatgcagc tagctcttac ctgtctctga 660

ccccggaaca gtggaaaagt catcgcagtt actcctgtca agttacgcac gaaggctcca 720

cggtcgaaaa aaccgtggca ccgacggaat gctcatgata agcatgcgta ggagaaaata 780

aaatgaaaca gtctaccatc gcactggcac tgctgccgct gctgtttacg ccggtgacca 840

aagcagaagt tcagctggtc gaaagtggtg gcggtctggt tcaaccgggc ggttcactgc 900

gtctgtcgtg cgcagcaagc ggttttacct tcagttccta tggtatggat tgggtccgtc 960

aggcaccggg taaaggtctg gaatgggtgt catcgattcg tggcagccgc ggttctacct 1020

attacgccga ttcagttaaa ggccgtttta ccatctctcg cgacaacagt aaaaatacgc 1080

tgtatctgca gatgaacagc ctgcgcgcgg aagataccgc cgtgtattac tgtgcccgtc 1140

tgtatcgcta ctggttcgac tactggggcc agggtacgct ggtgaccgtt agctctggcg 1200

gtggcggttc gctggtcccg cgtggcagcg gcggtggcgg ttcagcgagc accaaaggtc 1260

cgagcgtgtt tccgctggca ccgtgcagcc gctctaccag tgaatccacg gcagctctgg 1320

gttgtctggt gaaagattat tttccggaac cggtcaccgt gagttggaac tccggcgcac 1380

tgacctcggg tgttcatacg ttcccggctg tcctgcagag ttccggcctg tatagcctgt 1440

catcggtggt taccgttccg agctctaatt tcggcaccca aacgtacacc tgcaacgtcg 1500

atcacaaacc gtctaatacc aaagttgaca aaacggttga ttacaaagac gacgacgaca 1560

aacaccacca ccaccaccat tgataagctt 1590

<210>135

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> Fab-2 light chain

<400>135

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe

35 40 45

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

50 55 60

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

6570 75 80

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

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Leu Val Pro Arg Gly Ser

100 105 110

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

115 120 125

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

130 135 140

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

145 150 155 160

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

165 170 175

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

180 185 190

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

195 200 205

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210>136

<211>235

<212>PRT

<213> Artificial sequence

<220>

<223> Fab-2 heavy chain

<400>136

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Leu Val Pro Arg Gly Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

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

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

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

180 185 190

Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Asp Tyr Lys

210 215 220

Asp Asp Asp Asp Lys His His His His His His

225 230 235

<210>137

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavage site

<400>137

Leu Val Pro Arg Gly Ser

1 5

<210>138

<211>1530

<212>DNA

<213> Artificial sequence

<220>

<223> Fab-2 coding sequence

<400>138

ggtctcacat gaaaaaaacg gctatcgcaa tcgctgtggc actggcaggc ttcgcaacgg 60

tcgcgcaggc atcctatgaa ctgacgcaac cgccgagtgt ctccgtgtca ccgggtcaga 120

cggcacgtat tacctgctct ggtgataacc tgccgaaata ttacgcgcat tggtatcagc 180

aaaaaccggg ccaagccccg gtggttgtca tcttttatga cgttaatcgt ccgtcgggta 240

tcccggaacg cttctcgggc agcaactctg gtaatacggc caccctgacg atctcaggca 300

cccaggcaat ggatgaagct gactattact gccaagcatg gtggagctct acgccggtgt 360

ttggcggtgg cacgaaactg accgttctgc tggtcccgcgtggctccggt cagccgaaag 420

cagccccgtc agttaccctg tttccgccga gttccgaaga actgcaagca aacaaagcta 480

ccctggtgtg tctgattagc gatttctatc cgggcgcagt tacggtcgcg tggaaagccg 540

actcatcgcc ggtcaaagct ggtgtggaaa ccaccacccc gtcaaaacag tcgaacaata 600

aatatgcagc tagctcttac ctgtcgctga ccccggaaca gtggaaaagc catcgcagtt 660

actcctgcca agttacgcac gaaggctcta cggttgaaaa aaccgtcgca ccgacggaat 720

gcagttgata agcatgcgta ggagaaaata aaatgaaaca gagcaccatc gcactggcac 780

tgctgccgct gctgtttacg ccggtcacca aagcagaagt gcagctggtt gaatctggtg 840

gcggtctggt gcaaccgggc ggttcactgc gtctgtcgtg tgcggccagc ggctttacct 900

tcagttccta tggtatggat tgggtccgtc aggcaccggg taaaggtctg gaatgggtgt 960

catcgattcg tggcagccgc ggttctacct attacgccga ttccgttaaa ggccgtttca 1020

ccatctctcg cgacaacagt aaaaatacgc tgtatctgca gatgaacagt ctgcgcgcgg 1080

aagataccgc cgtgtattac tgcgcccgtc tgtatcgcta ctggtttgac tactggggcc 1140

agggtacgct ggtgaccgtt agctctctgg ttccgcgtgg ctcagcgagc accaaaggtc 1200

cgagtgtctt cccgctggca ccgtgcagcc gctctaccag tgaatccacg gcagctctgg 1260

gttgtctggt gaaagattat tttccggaac cggtcaccgt gagttggaac tccggcgcac 1320

tgacctccgg tgtgcatacgttcccggctg ttctgcagag ttccggcctg tattcactgt 1380

catcggtggt taccgtgccg agctctaatt ttggcaccca aacgtacacc tgtaacgttg 1440

atcacaaacc gagcaatacc aaagttgaca aaaccgttga ctacaaagac gacgacgaca 1500

aacaccacca ccaccaccac tgataagctt 1530

<210>139

<211>228

<212>PRT

<213> Artificial sequence

<220>

<223> IgG 2-linker 1, light chain amino acid sequence

<400>139

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Pro Arg Gly Ser Gly Gly Gly Gly Ser Gly Gln Pro Lys Ala Ala

115 120 125

Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn

130 135 140

Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val

145 150 155 160

Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu

165 170 175

Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser

180 185 190

Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser

195 200 205

Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro

210 215 220

Thr Glu Cys Ser

225

<210>140

<211>458

<212>PRT

<213> Artificial sequence

<220>

<223> IgG 2-linker 1, heavy chain amino acid sequence

<400>140

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Leu Val Pro Arg

115 120 125

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

130 135 140

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

145 150 155 160

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

165 170 175

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

180 185 190

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

195 200 205

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

210 215220

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

225 230 235 240

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455

<210>141

<211>684

<212>DNA

<213> Artificial sequence

<220>

<223> IgG 2-linker 1, light chain DNA sequence

<400>141

tcctacgagc tgacccagcc cccttccgtg tccgtgtctc ctggccagac cgcccggatc 60

acctgttccg gcgacaacct gcccaagtac tacgcccact ggtatcagca gaagcccggc 120

caggcccccg tggtggtcat cttctacgac gtgaaccggc cctccggcat ccccgagaga 180

ttctccggct ccaactccgg caacaccgcc accctgacca tctccggcac ccaggccatg 240

gacgaggccg actactactg ccaggcttgg tggtcctcca cccccgtgtt tggcggcgga 300

acaaagttaa ccgtgctggg cggtggagga tcactggttc cgcgtggctc tggcggtgga 360

ggatcaggcc agcccaaggc cgctccttcc gtgaccctgt tccccccatc ctccgaggaa 420

ctgcaggcca acaaggccac cctggtctgc ctgatctccg acttctaccc tggcgccgtg 480

accgtggcct ggaaggccga cagctctcct gtgaaggccg gcgtggaaac caccaccccc 540

tccaagcagt ccaacaacaa atacgccgcc tcctcctacc tgtccctgac ccccgagcag 600

tggaagtccc accggtccta cagctgccag gtcacacacg agggctccac cgtggaaaag 660

acagtggccc ccaccgagtg ctct 684

<210>142

<211>1374

<212>DNA

<213> Artificial sequence

<220>

<223> IgG 2-linker 1, heavy chain DNA sequence

<400>142

gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgagactg 60

tcctgcgccg cctccggctt caccttctcc agctacggca tggactgggt ccgacaggcc 120

cctggcaagg gcctggaatg ggtgtcctcc atccggggct ctcggggctc cacctactac 180

gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc cagactgtac 300

cggtattggt tcgactactg gggccagggc accctggtca ccgtcagctc agcttctggc 360

ggaggcggct ctctggtgcc tagaggatct ggcggcggag gctccaccaa gggcccttcc 420

gtgttccctc tggccccttg ctcccggtcc acctccgagt ctaccgccgc tctgggctgc 480

ctggtcaagg attacttccc cgagcccgtg accgtgtcct ggaactctgg cgccctgacc 540

agcggcgtgc acaccttccc tgccgtgctg cagtcctccg gcctgtactc cctgtcctcc 600

gtcgtgacag tgccctcctc caacttcggc acccagacct acacctgtaa cgtggaccac 660

aagccctcca acaccaaggt ggacaagacc gtggaacgga agtgctgcgt ggaatgcccc 720

ccctgtcctg cacctcctgt ggctggacct agcgtgttcc tgttcccccc aaagcccaag 780

gacaccctga tgatctcccg gacccccgaa gtgacctgcg tggtggtgga cgtgtcccac 840

gaggaccccg aggtgcagtt caattggtac gtggacggcg tggaagtgca caacgccaag 900

accaagccca gagaggaaca gttcaactcc accttccggg tggtgtccgt gctgaccgtg 960

gtgcaccagg actggctgaa cggcaaagag tacaagtgca aggtctccaa caagggcctg 1020

cctgccccca tcgaaaagac catcagcaag accaagggcc agccccgcga gccccaggtg 1080

tacacactgc cacctagccg ggaagagatg accaagaacc aggtgtccct gacctgtctg 1140

gtcaagggct tctacccatc cgacattgcc gtggaatggg agtccaacgg ccagcccgag 1200

aacaactaca agaccacccc ccccatgctg gactccgacg gctcattctt cctgtactcc 1260

aagctgacag tggacaagtc ccggtggcag cagggcaacg tgttctcctg ctccgtgatg 1320

cacgaggccc tgcacaacca ctacacccag aagtccctgt ccctgagccc cggc 1374

<210>143

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> IgG 2-linker 2, light chain amino acid sequence

<400>143

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Leu Val Pro Arg Gly Ser

100 105 110

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

115 120 125

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

130 135 140

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

145 150 155 160

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

165 170 175

Lys Tyr Ala Ala Ser Ser Tyr LeuSer Leu Thr Pro Glu Gln Trp Lys

180 185 190

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

195 200 205

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210>144

<211>448

<212>PRT

<213> Artificial sequence

<220>

<223> IgG 2-linker 2, heavy chain amino acid sequence

<400>144

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Leu Val Pro Arg Gly Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

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

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

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

180 185 190

Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys

210 215 220

Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

290 295 300

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210>145

<211>654

<212>DNA

<213> Artificial sequence

<220>

<223> IgG 2-linker 2, light chain DNA sequence

<400>145

tcctacgagc tgacccagcc cccttccgtg tccgtgtctc ctggccagac cgcccggatc 60

acctgttccg gcgacaacct gcccaagtac tacgcccact ggtatcagca gaagcccggc 120

caggcccccg tggtggtcat cttctacgac gtgaaccggc cctccggcat ccccgagaga 180

ttctccggct ccaactccgg caacaccgcc accctgacca tctccggcac ccaggccatg 240

gacgaggccg actactactg ccaggcttgg tggtcctcca cccccgtgtt tggcggcgga 300

acaaagttaa ccgtgctgct ggttccgcgt ggctctggcc agcccaaggc cgctccttcc 360

gtgaccctgt tccccccatc ctccgaggaa ctgcaggcca acaaggccac cctggtctgc 420

ctgatctccg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagctctcct 480

gtgaaggccg gcgtggaaac caccaccccc tccaagcagt ccaacaacaa atacgccgcc 540

tcctcctacc tgtccctgac ccccgagcag tggaagtccc accggtccta cagctgccag 600

gtcacacacg agggctccac cgtggaaaag acagtggccc ccaccgagtg ctct 654

<210>146

<211>1344

<212>DNA

<213> Artificial sequence

<220>

<223> IgG 2-linker 2, heavy chain DNA sequence

<400>146

gaggtgcagc tggtggaatccggcggaggc ctggtccagc ctggcggatc tctgagactg 60

tcctgcgccg cctccggctt caccttctcc agctacggca tggactgggt ccgacaggcc 120

cctggcaagg gcctggaatg ggtgtcctcc atccggggct ctcggggctc cacctactac 180

gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc cagactgtac 300

cggtattggt tcgactactg gggccagggc accctggtca ccgtcagctc agcttctctg 360

gtgcctagag gatctaccaa gggcccttcc gtgttccctc tggccccttg ctcccggtcc 420

acctccgagt ctaccgccgc tctgggctgc ctggtcaagg attacttccc cgagcccgtg 480

accgtgtcct ggaactctgg cgccctgacc agcggcgtgc acaccttccc tgccgtgctg 540

cagtcctccg gcctgtactc cctgtcctcc gtcgtgacag tgccctcctc caacttcggc 600

acccagacct acacctgtaa cgtggaccac aagccctcca acaccaaggt ggacaagacc 660

gtggaacgga agtgctgcgt ggaatgcccc ccctgtcctg cacctcctgt ggctggacct 720

agcgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa 780

gtgacctgcg tggtggtgga cgtgtcccac gaggaccccg aggtgcagtt caattggtac 840

gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gttcaactcc 900

accttccggg tggtgtccgt gctgaccgtg gtgcaccagg actggctgaa cggcaaagag 960

tacaagtgca aggtctccaa caagggcctg cctgccccca tcgaaaagac catcagcaag 1020

accaagggcc agccccgcga gccccaggtg tacacactgc cacctagccg ggaagagatg 1080

accaagaacc aggtgtccct gacctgtctg gtcaagggct tctacccatc cgacattgcc 1140

gtggaatggg agtccaacgg ccagcccgag aacaactaca agaccacccc ccccatgctg 1200

gactccgacg gctcattctt cctgtactcc aagctgacag tggacaagtc ccggtggcag 1260

cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320

aagtccctgt ccctgagccc cggc 1344

<210>147

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> Thrombin cleavage site

<221> variants

<222>(1)...(2)

<223> Xaa = hydrophobic amino acid

<221> variants

<222>(5)...(6)

<223> Xaa = non-acidic amino acid

<400>147

Xaa Xaa Pro Arg Xaa Xaa

1 5

<210>148

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> factor Xa cleavage site

<221> variants

<222>(2)...(2)

<223> Xaa = Glu or Asp

<400>148

Ile Xaa Gly Arg

1

<210>149

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>149

Gly Gly Gly Gly Ser

1 5

<210>150

<211>58

<212>PRT

<213> Artificial sequence

<220>

<223> amino acids 95-152 of SEQ ID NO:133

<400>150

Tyr Cys Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly

1 5 10 15

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

20 25 30

Pro Arg Gly Ser Gly Gly Gly Gly Ser Thr Lys Gly Pro Ser Val Phe

35 40 45

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser

50 55

<210>151

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>151

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

1 5 10 15

<210>152

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>152

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

1 5 10 15

Val

<210>153

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>153

Ala Lys Thr Thr Pro Lys Leu Gly Gly

1 5

<210>154

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>154

Ser Ala Lys Thr Thr Pro Lys Leu Gly Gly

1 5 10

<210>155

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>155

Ser Ala Lys Thr Thr Pro

1 5

<210>156

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>156

Arg Ala Asp Ala Ala Pro

1 5

<210>157

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>157

Arg Ala Asp Ala Ala Pro Thr Val Ser

1 5

<210>158

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>158

Arg Ala Asp Ala Ala Ala Ala Gly Gly Pro Gly Ser

1 5 10

<210>159

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>159

Arg Ala Asp Ala Ala Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210>160

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>160

Ser Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala

1 5 10 15

Arg Val

<210>161

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>161

Ala Asp Ala Ala Pro

1 5

<210>162

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>162

Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro

1 5 10

<210>163

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>163

Thr Val Ala Ala Pro

1 5

<210>164

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>164

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

1 5 10

<210>165

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>165

Gln Pro Lys Ala Ala Pro

1 5

<210>166

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>166

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro

1 5 10

<210>167

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>167

Ala Lys Thr Thr Pro Pro

1 5

<210>168

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>168

Ala Lys Thr Thr Pro Pro Ser Val Thr Pro Leu Ala Pro

1 5 10

<210>169

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>169

Ala Lys Thr Thr Ala Pro

1 5

<210>170

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>170

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro

1 5 10

<210>171

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>171

Ala Ser Thr Lys Gly Pro

1 5

<210>172

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>172

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

1 5 10

<210>173

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>173

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

1 5 10 15

<210>174

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>174

Gly Glu Asn Lys Val Glu Tyr Ala Pro Ala Leu Met Ala Leu Ser

1 5 10 15

<210>175

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>175

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

1 5 10 15

<210>176

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> amino acid linker

<400>176

Gly His Glu Ala Ala Ala Val Met Gln Val Gln Tyr Pro Ala Ser

1 5 10 15

<210>177

<211>442

<212>PRT

<213> Intelligent people

<400>177

Glu ValGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

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

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

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

325 330 335

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

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210>178

<211>212

<212>PRT

<213> Intelligent people

<400>178

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe

35 40 45

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

50 55 60

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

65 70 75 80

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

85 9095

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala

100 105 110

Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn

115 120 125

Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val

130 135 140

Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu

145 150 155 160

Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser

165 170 175

Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser

180 185 190

Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro

195 200 205

Thr Glu Cys Ser

210

<210>179

<211>239

<212>PRT

<213> Artificial sequence

<220>

<223> protease cleavable anti-TFPI scFv antibody

<400>179

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235

<210>180

<211>717

<212>DNA

<213> Artificial sequence

<220>

<223> protease cleavable anti-TFPI scFv antibody coding sequence

<400>180

gaagttcagc tggtcgaaag tggtggcggt ctggttcaac cgggcggttc actgcgtctg 60

tcgtgcgcag caagcggttt taccttcagt tcctatggta tggattgggt ccgtcaggca 120

ccgggtaaag gtctggaatg ggtgtcatcg attcgtggca gccgcggttc tacctattac 180

gccgattcag ttaaaggccg ttttaccatc tctcgcgaca acagtaaaaa tacgctgtat 240

ctgcagatga acagcctgcg cgcggaagat accgccgtgt attactgtgc ccgtctgtat 300

cgctactggt tcgactactg gggccagggt acgctggtga ccgttagctc tggtggcggt 360

ggctctctgg ttccgcgtgg ctccggtggc ggtggctcat cctatgaact gacccaaccg 420

ccgagtgtct ccgtgtcacc gggtcagacg gcacgtatta cctgcagcgg tgataacctg 480

ccgaaatatt acgcgcattg gtatcagcaa aaaccgggcc aagccccggt ggttgtcatc 540

ttttatgacg ttaatcgtcc gtccggtatc ccggaacgct tctcgggcag caactctggt 600

aatacggcaa ccctgacgat cagcggcacc caggcaatgg atgaagctga ctattactgt 660

caagcatggt ggagctctac gccggtgttt ggcggtggca cgaaactgac cgtgctg 717

Claims (18)

1. A protease-regulated antibody that specifically binds Tissue Factor Pathway Inhibitor (TFPI), comprising a variable domain, a constant domain, and an amino acid sequence linking the variable domain to the constant domain, wherein the amino acid sequence comprises a protease cleavage site,

the protease-regulated antibody comprises an amino acid sequence selected from the group consisting of:

(1) 131 of the Fab-1 light chain and 133 of the Fab-1 heavy chain, amino acids 1-239 of the SEQ ID NO;

(2) the Fab-2 light chain shown in SEQ ID NO. 135 and the Fab-2 heavy chain shown in amino acids 1-229 of SEQ ID NO. 136;

(3) an IgG 2-linker 1 light chain amino acid sequence as shown in SEQ ID NO. 139 and an IgG 2-linker 1 heavy chain amino acid sequence as shown in SEQ ID NO. 140; or

(4) The IgG 2-linker 2 light chain amino acid sequence shown in SEQ ID NO. 143 and the IgG 2-linker 2 heavy chain amino acid sequence shown in SEQ ID NO. 144.

2. The protease-regulated antibody of claim 1, wherein the protease cleavage site is a thrombin cleavage site.

3. The protease-regulated antibody of claim 1, wherein the protease cleavage site is a plasmin cleavage site.

4. The protease-regulated antibody of claim 1, wherein the protease cleavage site is a factor Xa cleavage site.

5. The protease-regulated antibody of any one of claims 1-4, wherein the amino acid sequence further comprises an amino acid linker.

6. The protease-regulated antibody of any one of claims 1-4 which is a full-length antibody.

7. The protease-regulated antibody of any one of claims 1-4 which is an antibody fragment.

8. A pharmaceutical composition comprising the protease-regulated antibody of any one of claims 1-7 and a pharmaceutically acceptable vehicle.

9. An isolated nucleic acid molecule encoding the protease-regulated antibody according to any one of claims 1-7.

10. Use of the protease-regulated antibody of any one of claims 1-7 in the manufacture of a medicament for the treatment of coagulation disorders.

11. The use of claim 10, wherein the coagulation disorder is hemophilia.

12. A method of modulating procoagulant activity of an anti-TFPI antibody comprising adding a protease cleavage site to the anti-TFPI antibody,

the anti-TFPI antibody comprises an amino acid sequence selected from the group consisting of:

(1) 131 of the Fab-1 light chain and 133 of the Fab-1 heavy chain, amino acids 1-239 of the SEQ ID NO;

(2) the Fab-2 light chain shown in SEQ ID NO. 135 and the Fab-2 heavy chain shown in amino acids 1-229 of SEQ ID NO. 136;

(3) an IgG 2-linker 1 light chain amino acid sequence as shown in SEQ ID NO. 139 and an IgG 2-linker 1 heavy chain amino acid sequence as shown in SEQ ID NO. 140; or

(4) The IgG 2-linker 2 light chain amino acid sequence shown in SEQ ID NO. 143 and the IgG 2-linker 2 heavy chain amino acid sequence shown in SEQ ID NO. 144.

13. The method of claim 12, wherein the protease cleavage site is a thrombin cleavage site.

14. The method of claim 12, wherein said protease cleavage site is a plasmin cleavage site.

15. The method of claim 12, wherein the protease cleavage site is a factor Xa cleavage site.

16. Use of the protease-regulated antibody of any of claims 1-7 in the manufacture of a medicament for promoting the production of thrombin or plasmin.

17. A protease-regulated antibody that specifically binds Tissue Factor Pathway Inhibitor (TFPI), comprising:

(a) 131, comprising the light chain variable domain and the light chain constant domain of the protease-regulated antibody;

(b) 133, comprising the heavy chain variable domain and the heavy chain constant domain of the protease-regulated antibody; and

(c) a protease cleavage site comprising SEQ ID NO 131 and a portion of SEQ ID NO 133,

wherein the protease-regulated antibody binds to TFPI and promotes the production of thrombin or plasmin.

18. The protease-regulated antibody according to claim 17, wherein the amino acid sequence of the protease cleavage site is set forth in SEQ ID NO. 137.