Patent application title: PROTEASE-REGULATED ANTIBODIES
Inventors:
IPC8 Class: AC07K1638FI
USPC Class:
1 1
Class name:
Publication date: 2018-09-06
Patent application number: 20180251570
Abstract:
This disclosure provides protease-regulated antibodies which specifically
bind to tissue factor pathway inhibitor (TFPI). The antibodies are useful
for treating bleeding disorders such as hemophilia.Claims:
1. A protease-regulated antibody which specifically binds to tissue
factor pathway inhibitor (TFPI), comprising a variable domain, a constant
domain, and an amino acid sequence joining the variable domain to the
constant domain, wherein the amino acid sequence comprises a protease
cleavage site.
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 claim 1 which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 131, amino acids 1-239 of SEQ ID NO: 133, SEQ :ID NO: 135, amino acids 1-229 of SEQ ID NO: 136, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 143, and SEQ ID NO: 144.
6. The protease-regulated antibody of claim 1, wherein the amino acid sequence further comprises an amino acid linker.
7. The protease-regulated antibody of claim 1, wherein the protease-regulated antibody is a full-length antibody.
8. The protease-regulated antibody of claim 1, wherein the protease-regulated antibody is an antibody fragment.
9. A pharmaceutical composition comprising the protease-regulated antibody of claim 1, and a pharmaceutically acceptable vehicle.
10. An isolated nucleic acid molecule encoding the protease-regulated antibody of claim 1.
11. A method of treating a coagulopathy, comprising administering to a patient in need thereof a therapeutically effective amount of the protease-regulated antibody of claim 1.
12. The method of claim 11, wherein the coagulopathy is hemophilia.
13. A method of modulating procoagulant activity of an anti-TFPI antibody, comprising adding a protease cleavage site into the anti-TFPI antibody.
14. The method of claim 13, wherein the protease cleave site is a thrombin cleavage site.
15. The method of claim 13, wherein the protease cleavage site is a plasmin cleavage site.
16. The method of claim 13, wherein the protease cleavage site is a Factor Xa cleavage site.
17. A method of promoting generation of thrombin or plasmin, comprising contacting TFPI with a protease-regulated antibody of claim 1.
Description:
[0001] This application claims the benefit of Ser. No. 61/617,837 filed on
Mar. 30, 2012, which is incorporated herein by reference in its entirety.
[0002] All documents cited in this disclosure are incorporated herein by reference in their entireties.
[0003] This application incorporates by reference a 64 kb text file created on Mar. 26, 2012 and named "0297301274sequencelisting.txt," which is the sequence listing for this application.
TECHNICAL HELD
[0004] The technical field is treatment of hemophilia and other coagulopathies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1. Illustration of an embodiment of a protease-regulated antibody. VI, heavy chain variable region; VT, light chain variable region; CH, heavy chain constant region; CL, light chain constant region,
[0006] FIG. 2. Western blot of two protease-regulated anti-TFPI Fab fragments ("Fab-1" and "Fab-2"), with and without thrombin digestion.
[0007] FIG. 3. Graph showing tissue factor pathway inhibitor (TFPI) binding of Fab-1 and Fab-2, with and without thrombin digestion, assayed by ELISA.
[0008] FIG. 4. Graph showing BIACORE.TM. measurement of TFPI binding of Fab-1 (Fab1) and Fab-2 (Fab2) with and without thrombin digestion.
[0009] FIG. 5. SDS-PAGE of purified IgG antibody expressed in HEK293 6.E cells.
[0010] FIG. 6. Western Blot of parental IgG ("Parent IgG;" top) and IgG2-linker1 (bottom) Lane 1, digestion with thrombin (1 unit); lane 2, no protease (control); lane 3, digestion with bovine plasmin; lane 4, digestion with enterokinase (0.02 .mu.g); lane 5, digestion with bovine Factor Xa (1 .mu.g); lane 6, digestion with matriptase (MTSP) (0.5 .mu.g); lane 7, digestion with urokinase (uPA) (0.25 .mu.g) lane 8, digestion with human rhinovirus 3C protease (HRV 3C) (2 units).
[0011] FIG. 7. Graph showing TFPI binding of IgG with and without thrombin digestion, assayed by ELISA.
[0012] FIG. 8. Graph showing BIACORE.TM. measurement of TFPI binding of parental IgG and IgG-linker1, with and without thrombin digestion.
[0013] FIG. 9. Western blot showing protease digestion of IgG-Linker1 and WT antibody with human proteases.
DETAILED DESCRIPTION
[0014] This disclosure provides protease-regulated antibodies which specifically bind to tissue factor pathway inhibitor (TFPI). The antibodies are useful for treating bleeding disorders such as hemophilia. In some embodiments, protease-regulated anti-TFPI antibodies can be cleaved by thrombin and/or plasmin. By initially inhibiting TFPI, such protease-regulated anti-TFPI antibodies promote the generation of thrombin and/or plasmin, which in turn cleaves the antibodies and removes or significantly reduces their binding activity to TFPI. This negative feedback allows the antibodies to promote coagulation within a safe therapeutic window.
[0015] 1. Protease-Regulated Anti-TFPI Antibodies
[0016] Protease-regulated antibodies disclosed herein specifically bind to TFPI; i.e. they bind to TFPI with an affinity that is higher (e.g., at least two-fold higher) than their binding affinity for an irrelevant 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 cells.
[0017] In some embodiments, protease-regulated antibodies bind to TFPI with an affinity of at least about 10.sup.5 M.sup.-1 to about 10.sup.12 M.sup.-1 (e.g., 10.sup.5 M.sup.-1, 10.sup.5.5 M.sup.-1, 10.sup.6 M.sup.-1, 10.sup.5.6 M.sup.-1, 10.sup.7 M.sup.-1, 10.sup.7.5 M.sup.-1, 10.sup.8 M.sup.-1, 10.sup.8.5 M.sup.-1, 10.sup.9 M.sup.-1, 10.sup.9.5 M.sup.-1, 10.sup.10 M.sup.-1, 10.sup.10.5 M.sup.-1, 10.sup.11 M.sup.-1, 10.sup.11.5 M.sup.-1, 10.sup.12 M.sup.-1). The affinity (KO of antibody binding to an antigen can be assayed using any method known in the art including, for example, immunoassays such as enzyme-linked immununospecific assay (ELISA), Bimolecular Interaction Analysis (BIA) (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 to cells that express an. antigen. BIA is a technology for analyzing biospecific interactions in real time, without labeling any of the interactants (e.g., BIACORE.TM.), Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
[0018] A protease-regulated anti-TFPI antibody can be constructed using a substantially full-length immunoglobulin molecule (e.g., IgG 1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA), an antigen binding fragment thereof, such as a Fab or. F(ab').sub.2, or a construct containing an antigen binding site, such as a scFv, Fv, or diabody, which is capable of specific binding to TFPI. The term "antibody" also includes other protein scaffolds that are able to orient antibody complementarity-determining region (CDR) inserts into the same active binding conformation as that found in natural antibodies such that the binding to TFPI observed with these chimeric proteins is maintained relative to the TFPI binding activity of the natural antibody from which the CDRs were derived.
[0019] An "isolated antibody" as used herein is an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds to TFPI is substantially free of antibodies that bind antigens other than TFPI). An isolated antibody that binds to an epitope, isoform, or variant of human TFPI may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., TFPI species homologs). An isolated antibody can be substantially free of other cellular material and/or chemicals.
[0020] The protease-regulated antibodies disclosed herein are engineered to comprise a protease cleavage site 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 positioned between its variable and constant regions. In some embodiments, protease-regulated anti-TFPI antibodies include 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 a protease-regulated anti-TFPI antibody comprises a polypeptide linker in addition to the protease cleavage site (as illustrated, for example, in FIG. 1). The linker can be a single amino acid or a polypeptide sequence (e.g., up to 100 amino acids). For example, the tinker can be GGGGS (SEQ ID NO:149). Other useful linkers include those shown in SEQ ID NOS:151-176. In other embodiments, no linker is present, and the cleavage site itself is inserted between the variable and constant regions.
[0021] At least two optimal cleavage sites for thrombin have been determined: (1) X.sub.1-X.sub.2-P-R-X.sub.3-X.sub.4 (SEQ ID NO:147), Where X.sub.1 and X.sub.2 are hydrophobic amino acids and X.sub.3 and X.sub.4 are nonacidic amino acids; and (2) GRG. Thrombin specifically cleaves after the arginine residue. Plasmin can also cleave the two aforementioned cleavage sites, however with less specificity as compared to thrombin. Other useful thrombin cleavage sites are provided as SEQ ID NOS:1-60. Other useful plasmin cleavages sites are provided as SEQ ID NOS:12, 47, 18, 53, and 61-130. In some embodiments, the cleavage site is LVPRGS (SEQ ID NO:137).
[0022] In some embodiments, a Factor Xa cleavage site, such as I-(E or D)-G-R (SEQ ID NO:148), is used. Other useful Factor Xa cleavage sites are provided as SEQ ID NOS:29, 59, and 61-69. Other thrombin and FXa cleavage sites or sequences can be found from previous publication authored by Bianchini [Bianchini E P et al 2002 :IBC]. One protease-regulated antibody may comprise more than one protease cleavage sites.
[0023] In addition to cleavage site, a second binding site of protease, so-called exosite, can be introduced into a protease-regulated TFPI antibody to make the cleavage more efficient. The exosite of thrombin can be from the native exosite of protease substrates or inibitor, such as PAR1, fibrinogen and hirudin. The exosite can also be a derivative of native exosite.
[0024] Protease-regulated TFPI antibodies can be produced synthetically or recombinantly. A number of technologies are available to produce antibodies. For example, phage-antibody technology can be used to generate antibodies (Knappik et al., J. Mol. Biol. 296:57-86, 2000), Another approach for obtaining antibodies is to screen a DNA library from B cells as described in WO 91/17271 and WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies are selected by affinity enrichment for binding to a selected protein. Antibodies can also be produced using trioma methodology Oestberg et al., Hybridoma 2:361-367, 1983; U.S. Pat. No. 4,634,664; U.S. Pat. No. 4,634,666).
[0025] Antibodies can also be purified from any cell that expresses the antibodies, including host cells that have been transfected with antibody-encoding expression constructs. The host cells can be cultured under conditions whereby the antibodies are expressed. Purified antibody can be separated from other cellular components that can associate with the antibody in the cell, such as certain proteins, carbohydrates, or lipids, using methods well known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. A preparation of purified antibodies can contain more than one type of antibody.
[0026] Alternatively, protease-regulated anti-TFPI antibodies can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963; Roberge et al., Science 269:202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Optionally, fragments of antibodies can be separately synthesized and combined using chemical methods to produce a full-length molecule.
[0027] A protease-regulated anti-TFPI antibody can also be constructed in a "single chain PV (scFv) format," in which a protease cleavage site is inserted in or around a peptide linker between the variable region of light chain and the variable region of heavy chain of a scFv. As the peptide linker is necessary to hold together the two variable regions of a scFv for antigen binding, cleavage of the peptide linker or flanking region allows a protease of interest to inactivate or to down-regulate the binding of scFv to its antigen. The amino acid sequence SEQ ID NO:179 (which can be encoded by SEQ 11) NO:180) is an example of a protease-regulated anti-TFPI antibodies in scFv format.
[0028] In some embodiments, protease-regulated TFPI antibodies are constructed in "IgG format," having two binding sites, and can comprise one, two, three, or four protease cleavage sites on the heavy chain, light chain, or both. In each case, a protease cleavage site can be flanked on either or both sides by a linker. Further, in each case, the cleavage sites can be the same or different. "IgG-linker1" (Example 5) and "IgG-linker2" are examples of protease-regulated anti-TFPI antibodies in IgG format:
TABLE-US-00001 light chain heavy chain amino acid DNA amino acid DNA IgG-linker1 SEQ ID SEQ ID SEQ ID SEQ ID NO: 139 NO: 141 NO: 140 NO: 142 IgG-linker2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 143 NO: 145 NO: 144 NO: 146
[0029] 2. Negative Feedback
[0030] Protease-regulated antibodies can include a protease cleavage site of a protease upregulated or generated as a result of the function of the antibody. Such protease can then cleave the protease-regulated antibody thereby providing a negative feedback loop which can be useful in preventing excess activity of the protease-regulated. antibody.
[0031] For example, protease-regulated anti-TFPI antibodies which can be cleaved by thrombin and/or plasmin can exhibit such a negative feedback effect. Such protease-regulated anti-TFPI antibodies promote the generation of thrombin and/or plasmin. Thrombin and plasmin will in turn cleave the protease-regulated anti-TFPI antibodies and remove or significantly reduce their binding activity to TFPI. This negative feedback allows the antibodies to promote coagulation within a safe therapeutic window.
[0032] 3. Polynucleotides
[0033] This disclosure also provides polynucleotides encoding protease-regulated antibodies. These polynucleotides can be used, for example, to produce quantities of the antibodies for therapeutic use.
[0034] Antibody-encoding cDNA molecules can be made with 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 manuals such as Sambrook, et al., (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3). An amplification technique, such as PCR, can be used to obtain additional copies of the polynucleotides. Alternatively, synthetic chemistry techniques can be used to synthesize polynucleotides encoding protease-regulated anti-TFPI antibodies.
[0035] To express a polynucleotide encoding an antibody, the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding antibodies and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, et al. (1989) and in Ausubel, et al., (Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1995).
[0036] A variety of expression vector/host systems can be utilized to contain and express sequences encoding antibodies. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with vines expression vectors e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV); or bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
[0037] The control elements or regulatory sequences are those non-translated regions of the vector enhancers, promoters, 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in strength and specificity. Depending on the vector system and host, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses can be used. If it is necessary to generate a cell line that contains Multiple copies of a nucleotide sequence encoding an antibody, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
[0038] General texts describing additional useful molecular biological techniques, including the preparation of antibodies, are Berger and Kimmel (Guide to Molecular Cloning Techniques, 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. Ausabel et al. [Eds.], Current Protocols, a joint venture between Green Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 2000)); Harlow et al., (Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988), Paul [Ed.]); Fundamental Immunology, (Lippincott & Wilkins (1998)); and Harlow, et al. (Using Antibodies: A Laboratory Manual, Cold. Spring Harbor Laboratory Press (1998)).
[0039] 4. Pharmaceutical Compositions
[0040] A protease-regulated anti-TFPI antibody can be provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier preferably is non-pyrogenic. A pharmaceutical composition comprising a protease-regulated anti-TFPI antibody can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. A variety of aqueous carriers can be employed, e.g., 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 compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of protease-regulated anti-TFPI antibody in a pharmaceutical composition can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected. Sec U.S. Pat. No. 5,851,525. If desired, more than one different protease-regulated anti-TFPI antibody can be included in a pharmaceutical composition.
[0041] In addition to the active ingredients, pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the compositions into preparations which can be used pharmaceutically. Pharmaceutical compositions can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
[0042] After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
[0043] 5. Methods
[0044] Pharmaceutical compositions comprising one or more protease-regulated anti-TFPI antibodies can be administered to a patient alone, or in combination with other agents, drugs or coagulation factors, to treat hemophilia or other clotting disorders. A "therapeutically effective dose" of protease-regulated anti-TFPI antibody refers to that amount of protease-regulated anti-TFPI antibody that will promote coagulation or reduce bleeding time. The determination of a therapeutically effective dose is well within the capability of those skilled in the art.
[0045] A therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually rats, mice, rabbits, dogs, or pigs. An animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
[0046] Therapeutic efficacy and toxicity, e.g., ED.sub.50 (the dose therapeutically effective in 50% of the population) and LD.sub.50 (the dose lethal to 50% of the population) of a protease-regulated anti-TFPI antibody can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD.sub.50/ED.sub.50.
[0047] Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
[0048] The exact dosage will be determined by the practitioner, in light of factors related to the patient who requires treatment. Dosage and administration are adjusted to provide sufficient levels of the protease-regulated TFPI antibody or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
[0049] In some embodiments, therapeutically effective in vivo dosages of a protease-regulated anti-TFPI antibody are in the range of about 5 .mu.g to about 100 mg/kg, about 1 mg to about 50 mg/kg, about 10 mg to about 50 mg/kg of patient body weight.
[0050] The mode of administration of a pharmaceutical composition comprising a protease-regulated anti-TFPI antibody can be any suitable route which delivers the antibody to the host (e.g., subcutaneous, intramuscular, intravenous, or intranasal administration).
[0051] In some embodiments, a protease-regulated anti-TFPI antibody is administered without other therapeutic agents. In some embodiments, a protease-regulated anti-TFPI antibody is administered in combination with other agents, such as drugs or coagulation factors, to enhance initial production of thrombin while ensuring that the thrombin level stays below the range that may cause thrombosis in some people with coagulopathy. The administration of the protease-regulated anti-TFPI antibody can be before, after, or at substantially the same time as the administration of other agents.
[0052] Nothing in this specification should be considered as limiting the scope of this disclosure. All examples presented are representative and non-limiting. The above-described embodiments can be modified or varied, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the embodiments disclosed herein can be practiced otherwise than as specifically described.
EXAMPLE 1
Construction of Protease-Regulated Anti-TFPI Fab Fragments
[0053] Two protease-regulated anti-TFPI Fabs, "Fab-1" and "Fab-2," were based on the anti-TFPI antibody sequences shown in SEQ ID NO: 177 (heavy chain) and SEQ NO:178 (light chain). Both Fabs have the 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 a (Gly).sub.4Ser linker. Fab-2 contains the six amino acid cleavage site alone, no linker is present. Thrombin and plasmin will cleave C-terminal to the Arg (R) residue of LVPRGS (SEQ ID NO:137). DNA encoding the Fabs was synthesized by GenScript with optimized codons for bacterial expression. The amino acid and DNA sequences for the Fabs are identified in the table below.
TABLE-US-00002 Fab-1 variable and SEQ ID NO: 131 constant light amino acid sequence Fab-1 variable and SEQ ID NO: 133 constant heavy amino acid sequence Fab-1 DNA Sequence SEQ ID NO: 134 Fab-2 variable and SEQ ID NO: 135 constant light amino acid sequence Fab-2 variable and SEQ ID NO: 136 constant heavy amino acid sequence Fab-2 DNA Sequence SEQ ID NO: 138
[0054] The Fab coding regions were digested with the restriction enzymes BsaI and HindIII (New England Biolabs). The DNA fragments were purified using an agarose gel and subcloned into pBADmycHisA (Invitrogen). The cloned DNA was 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 Fabs
[0055] Approximately 2.5 .mu.g of the crudely purified Fab-1 and Fah-2 were digested with 0, 2 or 10 units of thrombin (Novagen) for 1 hr at 37.degree. C. Digests were run on a 4-15% CRITEREON.TM. TGX.TM. (Bio-Rad). Protein were transferred to a nitrocellulose membrane and probed with an anti-human Fab antibody (Southern Biotech).
[0056] The Western blot of Fab-1 and Fab-2 cleavage is shown in FIG. 2. All samples were reduced. Bands at approximately 12 kDA were observed for both Fabs when treated with thrombin. These bands were not present in the samples without thrombin digestion, indicating that the small size protein was the product of thrombin cleavage.
EXAMPLE 3
TFPI ELISA of Thrombin Digested Fab-1 and Fab-2
[0057] Approximately 2.5 .mu.g of partially purified Fab-1 and Fab-2 were digested with 2 units of biotinylated thrombin (Novagen) at 23.degree. C. overnight. Streptavidin sepharose beads (100 .mu.L) were added to the digestion to deplete the thrombin. The digested Fab samples were applied to a column that captures the sepharose bead/thrombin complex. The eluate contained the digested Fabs.
[0058] A MAXISORP.RTM. 96-well plate (Nunc) was coated with 1 .mu.g/mL of TFPI in PBS overnight at 4.degree. C. The plate was blocked for 1 hr at room temperature (RT) in 5% non-fat dry milk PBS/0.5% TWEEN-20.RTM. (PBS-T). Serial two-fold dilutions of undigested and digested Fab-1 and Fab-2 were added to the wells (100 .mu.L/well) and incubated for 1 hr at RT. The plates were washed five times with PBS-T. A secondary HRP-conjugated anti-Fab antibody was added (100 .mu.L of a 1:10000 dilution) for detection with an AMPLEX.RTM. Red (Invitrogen) solution. As shown in FIG. 3, thrombin digestion significantly reduced the signal of TFPI binding.
EXAMPLE 4
BIACORE.TM. Measurements of Thrombin-Digested FAB-1 and FAB-2
[0059] Approximately 2.5 .mu.g of crudely purified. Fab-1 and Fab-2 were digested with 2 units of biotinylated thrombin (Novagen) at 23.degree. C. overnight. Streptavidin sepharose beads (100 .mu.L) were added to the digestion to deplete the thrombin. The digested Fab samples were applied to a column that captures the sepharose bead/thrombin complex. The eluate contained the digested Fabs.
[0060] For BIACORE.TM. analysis, human TFPI (American Diagnostica) was immobilized using amine coupling at targeted level of 100 relative units (RU), and the antibodies were injected in the mobile phase. HBS-p was used as the running buffer. A reference channel was prepared using blank immobilization where the surface was activated and then inactivated without any immobilized protein. A manual run was performed to measure elevated RU of the Fabs and a buffer control.
[0061] As shown in FIG. 4. The parental Fab without cleavable linker generated 24.6 comparable to undigested Fab-1 (25.6 RU) and Fab2 (27.9 RU). After thrombin cleavage, the binding signal of Fab-1 and Fah-2 was reduced by more than 50%, indicating that cleaved Fab-1 and Fab-2 not only lost the constant domain, but also lost binding activity to TFPI.
EXAMPLE 5
IgG Expression and Purification
[0062] A protease-regulated anti-TFPI immunoglobulin molecules, "IgG-linker1," was constructed based on parental anti-TFPI antibody sequences shown in SEQ ID NO:177 (heavy chain) and SEQ ID NO: 178 (light chain). To facilitate molecular cloning, a BlpI site was introduced in the heavy chain coding sequence. Compared to the position of protease cleavable linker in Fab-1, the introduction of BlpI site shifts the linker position of IgG-linker1 two amino acids to the constant region.
[0063] HEK293 6E cells were transfected with constructed IgG expression vectors, and the culture supernatant containing the IgG antibodies was harvested. The antibodies were purified using an affinity column of MABSELECT SURE.TM. followed by SUPERDEX.TM.200 chromatography. The purified IgG-linker1 on SDS-PAGE is shown FIG. 5.
EXAMPLE 6
Western Blot of Parental IgG and IgG-Linker1
[0064] Purified parental IgG and IgG-linker1 (0.5 .mu.g) were digested with thrombin, bovine plasmin, bovine Factor Xa, matriptase (MTSP), urokinase (uPA), or human rhinovirus 3C protease WWI 3C). The antibodies were incubated with the proteases for 1 hr at 37.degree. C. Digests were run on a 4-20% CRITEREON.TM. TGX.TM. gel (Bio-Rad). Protein was transferred to a nitrocellulose membrane and probed with an anti-human IgG heavy and light chain antibody (Pierce). Western blots of IgG2 and IgG2-linker1 are shown in FIG. 6. All samples were reduced.
[0065] Intact IgG produced two bands under reducing conditions. The two bands correspond to the heavy chain (50 kD) and the light chain (25 kD). Digested antibody IgG-linker1 showed a shift in molecular weight vs. undigested antibody. The following proteases were used to digests the antibodies: thrombin, plasmin, bovine Factor Xa, MTSP, and uPA. The digested IgG-linker1 antibody showed a shift in the molecular weight of the heavy chain from 50 kD to 37 kD. This size shift correlates with the loss of the VH domain from the heavy chain. There was also a molecular weight shift of the 25 kD light chain to a faint band around 16 kDa, which indicates cleavage of the VL domain from the light chain. The proteases did not cleave parental IgG, indicating that molecular weight loss was a result of the protease digestion due to the cleavage site engineered into the antibody.
EXAMPLE 7
TFPI-Binding ELISA of Thrombin Digested Parental IgG and IgG-Linker1
[0066] One microgram of the full length antibodies, parental IgG and IgG-linker1, were digested with 1 unit of biotinylated thrombin (Novagen) for 1 hr at 37.degree. C. Then 50 .mu.L of streptavidin sepharose beads were added to the digestion to deplete the thrombin. The digested IgG samples were applied to a column that captures the sepharose bead/thrombin complex. The dilate contains the digested IgGs.
[0067] A MAXISORP.RTM. 96-well plate (Nuns) was coated with 1 .mu.g/mile of TFPI in PBS over night at 4.degree. C. The plate was blocked for 1 hr at room temperature (RT) in 5% non-fat dry milk PBS/0.5% TWEEN.RTM. 20 (PBS-T). Serial two-fold dilutions of undigested and digested parental IgG and IgG-linker1 were added to the wells (100 .mu.L/well) and incubated for 1 hr at RT. The plate was washed five times in PBS-T. A secondary HRP conjugated anti-Fab-antibody was added (100 .mu.L of a 1:10,000 dilution) for detection with an AMPLEX RED.RTM. (Invitrogen) solution.
[0068] The results of the TFPI binding ELISA are shown in FIG. 7. Loss of TFPI binding by thrombin digested IgG Linker-1 is shown. The TFPI binding of undigested IgG-linker1 was similar to the TFPI binding of the undigested and thrombin-digested parental IgG.
EXAMPLE 8
BIACORE.TM. Analysis of Parental IgG and IgG-Linker1
[0069] A CM4 sensor chip was immobilized with a low density of human TFPI using an amine coupling kit (GE HealthCare). Kinetic assays of parental IgG and IgG-linker1 were conducted using different concentration of the antibodies, followed by regeneration with pH 1.5 glycine buffer. The parental IgG and IgG-linker1 antibodies at a concentration of 1 .mu.g were digested with 1 unit of biotinylated thrombin (Novagen) for 1 hr at 37.degree. C. The antibodies with or without digestion were injected to a BIACORE.TM. system for TFPI-binding analysis. The following figure shows the signal generated from injection 45 .mu.l of 6.25 .mu.g/ml antibodies or control samples.
[0070] In kinetics assay, parental IgG and IgG2-linker1 have association rates (ka) of 1.536.times.10.sup.6/Ms and 1.902.times.10.sup.6/Ms, respectively. The two antibodies did not have measurable dissociation in 30 minutes. This indicates that the insertion of linker1 did not significantly change binding activity of the antibody.
[0071] The effect of thrombin cleavage on the antibodies is shown in FIG. S. Thrombin digestion slightly decreased the parental IgG binding on TFPI, whereas thrombin digestion reduced IgG2-linker1 binding on TFPI from 20 RU to 5 RU, a 75% decrease.
EXAMPLE 9
Western Blot of Protease/Coagulation Factor Treated IgG-Linker1 and Parental IgG
[0072] The following human proteases/coagulation factors were used to digest 80 nM of IgG-Linker1 and the WT antibody: thrombin (0.1 .mu.M), plasmin (0.1 .mu.M), Factor Vila (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). The treated material was run on a 4-15% CRITEREON.TM. TGX gel (Bio-Rad). Proteins were transferred to a nitrocellulose membrane and probed with an anti-human IgG antibody (Pierce).
[0073] Human thrombin, plasmin and FactorXa digested IgG-Linker1, thrombin digesting most efficiently (FIG. 9) as shown by the appearance of a 37 kDa band. The proteases did not cleave the parental IgG, indicating that molecular weight loss was a result of the protease digestion due to the cleavage site engineered into the antibody.
Sequence CWU
1
1
18018PRTArtificial Sequenceprotease cleavage site 1Ala His Pro Arg Ile Ile
Ser Ala1 528PRTArtificial Sequenceprotease cleavage site
2Ala Arg Thr Arg Ala Arg Arg Pro1 538PRTArtificial
Sequenceprotease cleavage site 3Ala Ser Ala Arg Thr Thr Gly Ser1
548PRTArtificial Sequenceprotease cleavage site 4Ala Thr Pro Arg Gly
Ala Ala Pro1 558PRTArtificial Sequenceprotease cleavage
site 5Ala Val Val Arg Thr Pro Pro Lys1 568PRTArtificial
Sequenceprotease cleavage site 6Glu Arg Thr Arg Ser Phe Gln Leu1
578PRTArtificial Sequenceprotease cleavage site 7Phe Gly Leu Arg Phe
Tyr Ala Tyr1 588PRTArtificial Sequenceprotease cleavage
site 8Phe Asn Pro Arg Thr Phe Gly Ser1 598PRTArtificial
Sequenceprotease cleavage site 9Phe Arg Pro Lys His Thr Arg Ile1
5108PRTArtificial Sequenceprotease cleavage site 10Phe Ser Ala Arg
Gly His Arg Pro1 5118PRTArtificial Sequenceprotease
cleavage site 11Gly Asp Ile Arg Gly Pro Arg Ile1
5128PRTArtificial Sequenceprotease cleavage site 12Gly Gly Val Arg Gly
Pro Arg Val1 5138PRTArtificial Sequenceprotease cleavage
site 13Gly Ser Phe Arg Ala Gly Leu Phe1 5148PRTArtificial
Sequenceprotease cleavage site 14His Lys Gly Arg Ser Ala Leu Val1
5158PRTArtificial Sequenceprotease cleavage site 15Ile Ala Gly Arg
Ser Leu Asn Pro1 5168PRTArtificial Sequenceprotease
cleavage site 16Ile Asp Gly Arg Ile Val Glu Gly1
5178PRTArtificial Sequenceprotease cleavage site 17Ile Glu Pro Arg Ser
Phe Ser Gln1 5188PRTArtificial Sequenceprotease cleavage
site 18Ile Lys Pro Arg Ile Val Gly Gly1 5198PRTArtificial
Sequenceprotease cleavage site 19Ile Gln Ile Arg Ser Val Ala Lys1
5208PRTArtificial Sequenceprotease cleavage site 20Ile Gln Ile Arg
Ser Val Ala Lys1 5218PRTArtificial Sequenceprotease
cleavage site 21Lys Asn Val Lys Ser Lys Ile Gly1
5228PRTArtificial Sequenceprotease cleavage site 22Lys Pro Lys Asp Ser
Ser Val Asp1 5238PRTArtificial Sequenceprotease cleavage
site 23Leu Asp Pro Arg Ser Phe Leu Leu1 5248PRTArtificial
Sequenceprotease cleavage site 24Leu Gly Ile Arg Ser Phe Arg Asn1
5258PRTArtificial Sequenceprotease cleavage site 25Leu Pro Ile Lys
Thr Phe Arg Gly1 5268PRTArtificial Sequenceprotease
cleavage site 26Leu Arg Pro Arg Phe Lys Ile Ile1
5278PRTArtificial Sequenceprotease cleavage site 27Leu Arg Pro Arg Ile
Ile Gly Gly1 5288PRTArtificial Sequenceprotease cleavage
site 28Leu Ser Pro Arg Gly Val His Ile1 5298PRTArtificial
Sequenceprotease cleavage site 29Leu Ser Pro Arg Thr Phe His Pro1
5308PRTArtificial Sequenceprotease cleavage site 30Met Thr Pro Arg
Ser Glu Gly Ser1 5318PRTArtificial Sequenceprotease
cleavage site 31Met Thr Pro Arg Ser Gly Gly Ser1
5328PRTArtificial Sequenceprotease cleavage site 32Met Thr Pro Arg Ser
Arg Gly Ser1 5338PRTArtificial Sequenceprotease cleavage
site 33Met Val Pro Arg Ala Val Tyr Leu1 5348PRTArtificial
Sequenceprotease cleavage site 34Asn Val Pro Arg Ile Leu Ser Pro1
5358PRTArtificial Sequenceprotease cleavage site 35Pro Ala Pro Arg
Gly Tyr Pro Gly1 5368PRTArtificial Sequenceprotease
cleavage site 36Pro Asp Leu Arg Ser Cys Val Asn1
5378PRTArtificial Sequenceprotease cleavage site 37Pro Gly Pro Arg Gly
Pro Pro Pro1 5388PRTArtificial Sequenceprotease cleavage
site 38Pro Gly Ser Arg Ser Arg Thr Pro1 5398PRTArtificial
Sequenceprotease cleavage site 39Pro Gln Gly Arg Ile Val Gly Gly1
5408PRTArtificial Sequenceprotease cleavage site 40Pro Gln Gly Arg
Thr Thr Ala His1 5418PRTArtificial Sequenceprotease
cleavage site 41Pro Arg Ser Phe Leu Leu Arg Asn1
5428PRTArtificial Sequenceprotease cleavage site 42Gln Ser Pro Arg Ser
Phe Gln Lys1 5438PRTArtificial Sequenceprotease cleavage
site 43Gln Ser Pro Arg Ser Phe Gln Lys1 5448PRTArtificial
Sequenceprotease cleavage site 44Gln Tyr Leu Arg Val Pro Leu Val1
5458PRTArtificial Sequenceprotease cleavage site 45Ser Glu Phe Arg
Cys Leu Thr Pro1 5468PRTArtificial Sequenceprotease
cleavage site 46Ser Ile Gly Arg Ala Ser Leu His1
5478PRTArtificial Sequenceprotease cleavage site 47Ser Lys Gly Arg Ser
Leu Ile Gly1 5488PRTArtificial Sequenceprotease cleavage
site 48Ser Arg Leu Arg Ala Tyr Leu Leu1 5498PRTArtificial
Sequenceprotease cleavage site 49Thr Cys Leu Arg Ser Thr Lys Phe1
5508PRTArtificial Sequenceprotease cleavage site 50Val Cys Leu Arg
Ser Phe Gln Thr1 5518PRTArtificial Sequenceprotease
cleavage site 51Val Asp Pro Arg Leu Ile Asp Gly1
5528PRTArtificial Sequenceprotease cleavage site 52Val Glu Val Lys Ser
Glu Lys Leu1 5538PRTArtificial Sequenceprotease cleavage
site 53Val Ile Pro Lys Arg Ile Ser Pro1 5548PRTArtificial
Sequenceprotease cleavage site 54Val Ile Pro Arg Ser Gly Gly Ser1
5558PRTArtificial Sequenceprotease cleavage site 55Val Asn Pro Arg
Gly Ile Val Thr1 5568PRTArtificial Sequenceprotease
cleavage site 56Val Gln Pro Arg Ala Gln Lys Ile1
5578PRTArtificial Sequenceprotease cleavage site 57Val Ser Pro Arg Ala
Ser Ala Ser1 5588PRTArtificial Sequenceprotease cleavage
site 58Val Val Pro Arg Gly Val Asn Leu1 5598PRTArtificial
Sequenceprotease cleavage site 59Trp Tyr Leu Arg Ser Asn Asn Gly1
5608PRTArtificial Sequenceprotease cleavage site 60Trp Tyr Leu Arg
Ser Asn Thr Gly1 5618PRTArtificial Sequenceprotease
cleavage site 61Ala Phe Trp Lys Thr Asp Ala Ser1
5628PRTArtificial Sequenceprotease cleavage site 62Ile Glu Gly Arg Thr
Ala Thr Ser1 5638PRTArtificial Sequenceprotease cleavage
site 63Thr Ala Ala Arg Gln Ser Thr Asn1 5648PRTArtificial
Sequenceprotease cleavage site 64Ile Asp Gly Arg Ile Val Glu Gly1
5658PRTArtificial Sequenceprotease cleavage site 65Phe Asn Pro Arg
Thr Phe Gly Ser1 5668PRTArtificial Sequenceprotease
cleavage site 66Arg Val Pro Lys Ser Phe Pro Phe1
5678PRTArtificial Sequenceprotease cleavage site 67Pro Gln Leu Arg Met
Lys Asn Asn1 5688PRTArtificial Sequenceprotease cleavage
site 68Ser Ser Trp Arg Leu Thr Ser Ser1 5698PRTArtificial
Sequenceprotease cleavage site 69Leu Gly Ile Arg Ser Phe Arg Asn1
5708PRTArtificial Sequenceprotease cleavage site 70Ala Met Ser Arg
Met Ser Leu Ser1 5718PRTArtificial Sequenceprotease
cleavage site 71Ala Asn Asn Arg Asp Asn Thr Tyr1
5728PRTArtificial Sequenceprotease cleavage site 72Ala Asn Val Arg Arg
Lys Arg Tyr1 5738PRTArtificial Sequenceprotease cleavage
site 73Ala Arg Gly Arg Ala Phe Pro Gln1 5748PRTArtificial
Sequenceprotease cleavage site 74Ala Thr Leu Lys Ser Arg Lys Met1
5758PRTArtificial Sequenceprotease cleavage site 75Ala Thr Gln Lys
Lys Val Glu Arg1 5768PRTArtificial Sequenceprotease
cleavage site 76Ala Thr Trp Lys Thr Arg Trp Tyr1
5778PRTArtificial Sequenceprotease cleavage site 77Asp Val Gly Glu Tyr
Asn Val Phe1 5788PRTArtificial Sequenceprotease cleavage
site 78Glu Ala Arg Gly Ser Val Ile Leu1 5798PRTArtificial
Sequenceprotease cleavage site 79Glu Ala Tyr Arg Arg Phe Tyr Gly1
5808PRTArtificial Sequenceprotease cleavage site 80Glu Asp Asn Arg
Asp Ser Ser Met1 5818PRTArtificial Sequenceprotease
cleavage site 81Glu Thr Leu Lys Val Ile Asp Glu1
5828PRTArtificial Sequenceprotease cleavage site 82Phe Arg Ala Arg Ala
Tyr Gly Phe1 5838PRTArtificial Sequenceprotease cleavage
site 83Gly Glu Ala Arg Gly Ser Val Ile1 5848PRTArtificial
Sequenceprotease cleavage site 84Gly Gly Tyr Arg Ala Arg Pro Ala1
5858PRTArtificial Sequenceprotease cleavage site 85Gly Ile Leu Lys
Glu Asn Ala Ala1 5868PRTArtificial Sequenceprotease
cleavage site 86Gly Pro Lys Arg Gly Thr Glu Pro1
5878PRTArtificial Sequenceprotease cleavage site 87Ile Ile Arg Arg Ser
Ile Gln Ile1 5888PRTArtificial Sequenceprotease cleavage
site 88Ile Thr Phe Arg Met Asn Val Ala1 5898PRTArtificial
Sequenceprotease cleavage site 89Lys His Ser Lys Arg His Ile His1
5908PRTArtificial Sequenceprotease cleavage site 90Lys Lys Asp Arg
Ala Arg Gln Glu1 5918PRTArtificial Sequenceprotease
cleavage site 91Lys Lys Pro Arg Cys Gly Val Pro1
5928PRTArtificial Sequenceprotease cleavage site 92Lys Gln Val Lys Asp
Asn Glu Asn1 5938PRTArtificial Sequenceprotease cleavage
site 93Leu Asp Pro Arg Ser Phe Leu Leu1 5948PRTArtificial
Sequenceprotease cleavage site 94Leu Pro Pro Lys Ser Gln Pro Pro1
5958PRTArtificial Sequenceprotease cleavage site 95Leu Ser Phe Arg
Ala Arg Ala Tyr1 5968PRTArtificial Sequenceprotease
cleavage site 96Met Ser Met Arg Val Arg Arg His1
5978PRTArtificial Sequenceprotease cleavage site 97Asn Ser Gly Arg Ala
Val Thr Tyr1 5988PRTArtificial Sequenceprotease cleavage
site 98Pro Ala Pro Arg Gly Tyr Pro Gly1 5998PRTArtificial
Sequenceprotease cleavage site 99Pro Glu Ala Lys Ala Ser Cys Tyr1
51008PRTArtificial Sequenceprotease cleavage site 100Pro Glu Ser
Lys Ala Thr Asn Ala1 51018PRTArtificial Sequenceprotease
cleavage site 101Pro Gly Pro Lys Arg Gly Thr Glu1
51028PRTArtificial Sequenceprotease cleavage site 102Pro Lys Ala Lys Ser
His Ala Pro1 51038PRTArtificial Sequenceprotease cleavage
site 103Pro Leu Asp Lys Lys Arg Glu Glu1 51048PRTArtificial
Sequenceprotease cleavage site 104Pro Leu Gln Lys Gln Leu Pro Ala1
51058PRTArtificial Sequenceprotease cleavage site 105Pro Gln Phe
Arg Ile Lys Gly Gly1 51068PRTArtificial Sequenceprotease
cleavage site 106Pro Gln Leu Arg Leu Pro His Thr1
51078PRTArtificial Sequenceprotease cleavage site 107Pro Gln Leu Arg Arg
Gly Trp Arg1 51088PRTArtificial Sequenceprotease cleavage
site 108Pro Gln Ser Arg Ser Val Pro Pro1 51098PRTArtificial
Sequenceprotease cleavage site 109Pro Arg Phe Lys Ile Ile Gly Gly1
51108PRTArtificial Sequenceprotease cleavage site 110Pro Tyr Leu
Lys Val Phe Asn Pro1 51118PRTArtificial Sequenceprotease
cleavage site 111Gln Lys Ser Arg Asn Gly Leu Arg1
51128PRTArtificial Sequenceprotease cleavage site 112Gln Leu Ile Lys Ala
Ile Gln Leu1 51138PRTArtificial Sequenceprotease cleavage
site 113Gln Arg Tyr Lys Val Asp Tyr Glu1 51148PRTArtificial
Sequenceprotease cleavage site 114Arg Ala Gln Arg Ser Ala Gly Ala1
51158PRTArtificial Sequenceprotease cleavage site 115Arg Gly Pro
Arg Val Val Glu Arg1 51168PRTArtificial Sequenceprotease
cleavage site 116Arg Pro Ala Lys Ala Ala Ala Thr1
51178PRTArtificial Sequenceprotease cleavage site 117Arg Arg Lys Arg Tyr
Ala Ile Gln1 51188PRTArtificial Sequenceprotease cleavage
site 118Arg Ser Ser Lys Gly Arg Ser Leu1 51198PRTArtificial
Sequenceprotease cleavage site 119Arg Ser Thr Arg Phe Ala Ala Thr1
51208PRTArtificial Sequenceprotease cleavage site 120Ser Cys Asp
Lys Thr His Thr Cys1 51218PRTArtificial Sequenceprotease
cleavage site 121Ser Ile Asn Lys Ser Ser Pro Leu1
51228PRTArtificial Sequenceprotease cleavage site 122Ser Gln Pro Pro Glu
Lys Thr Glu1 51238PRTArtificial Sequenceprotease cleavage
site 123Ser Gln Arg Lys His Ser Lys Arg1 51248PRTArtificial
Sequenceprotease cleavage site 124Ser Ser Met Lys Leu Ser Phe Arg1
51258PRTArtificial Sequenceprotease cleavage site 125Thr Glu Pro
Lys Val Lys Leu Pro1 51268PRTArtificial Sequenceprotease
cleavage site 126Thr Glu Tyr Arg Leu Val Ser Ile1
51278PRTArtificial Sequenceprotease cleavage site 127Thr His Glu Lys Gly
Arg Gln Ser1 51288PRTArtificial Sequenceprotease cleavage
site 128Thr Tyr Ser Lys Ala Ser Thr Pro1 51298PRTArtificial
Sequenceprotease cleavage site 129Thr Tyr Ser Arg Ser Arg Tyr Leu1
51308PRTArtificial Sequenceprotease cleavage site 130Val Ser Asn
Lys Val Ser Met Ser1 5131228PRTArtificial SequenceFab-1
light chain 131Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro
Gly Gln1 5 10 15Thr Ala
Arg Ile Thr Cys Ser Gly Asp Asn Leu Pro Lys Tyr Tyr Ala 20
25 30His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Val Val Val Ile Phe 35 40
45Tyr Asp Val Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50
55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr
Ile Ser Gly Thr Gln Ala Met65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Trp Ser Ser Thr
Pro Val 85 90 95Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Leu 100
105 110Val Pro Arg Gly Ser Gly Gly Gly Gly
Ser Gly Gln Pro Lys Ala Ala 115 120
125Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
130 135 140Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp Phe Tyr Pro Gly Ala Val145 150
155 160Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
Ala Gly Val Glu 165 170
175Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser
180 185 190Tyr Leu Ser Leu Thr Pro
Glu Gln Trp Lys Ser His Arg Ser Tyr Ser 195 200
205Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
Ala Pro 210 215 220Thr Glu Cys
Ser22513216PRTArtificial Sequenceprotease cleavage site and linker 132Gly
Gly Gly Gly Ser Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser1
5 10 15133245PRTArtificial
SequenceFab-1 heavy chain 133Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met Asp Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Leu Val Pro Arg Gly Ser 115 120
125Gly Gly Gly Gly Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 130 135 140Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys145 150
155 160Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 165 170
175Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
180 185 190Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 195
200 205Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn 210 215 220Thr Lys Val
Asp Lys Thr Val Asp Tyr Lys Asp Asp Asp Asp Lys His225
230 235 240His His His His His
2451341590DNAArtificial Sequencesequence encoding Fab-1 134ggtctcacat
gaaaaaaacc gctatcgcta tcgccgtcgc actggctggc ttcgcaaccg 60tggcacaggc
atcctatgaa ctgacccaac cgccgagtgt ctccgtgtca ccgggtcaga 120cggcacgtat
tacctgcagc ggtgataacc tgccgaaata ttacgcgcat tggtatcagc 180aaaaaccggg
ccaagccccg gtggttgtca tcttttatga cgttaatcgt ccgtccggta 240tcccggaacg
cttctcgggc agcaactctg gtaatacggc aaccctgacg atcagcggca 300cccaggcaat
ggatgaagct gactattact gtcaagcatg gtggagctct acgccggtgt 360ttggcggtgg
cacgaaactg accgtgctgg gtggcggtgg ctctctggtt ccgcgtggct 420ccggtggcgg
tggctcaggc cagccgaaag cagcaccgag tgttaccctg tttccgccga 480gttccgaaga
actgcaagca aacaaagcta ccctggtgtg cctgattagc gatttctatc 540cgggcgcagt
tacggtcgcg tggaaagccg actcatcgcc ggtgaaagct ggtgttgaaa 600ccacgacccc
gtcaaaacag tcgaacaata aatatgcagc tagctcttac ctgtctctga 660ccccggaaca
gtggaaaagt catcgcagtt actcctgtca agttacgcac gaaggctcca 720cggtcgaaaa
aaccgtggca ccgacggaat gctcatgata agcatgcgta ggagaaaata 780aaatgaaaca
gtctaccatc gcactggcac tgctgccgct gctgtttacg ccggtgacca 840aagcagaagt
tcagctggtc gaaagtggtg gcggtctggt tcaaccgggc ggttcactgc 900gtctgtcgtg
cgcagcaagc ggttttacct tcagttccta tggtatggat tgggtccgtc 960aggcaccggg
taaaggtctg gaatgggtgt catcgattcg tggcagccgc ggttctacct 1020attacgccga
ttcagttaaa ggccgtttta ccatctctcg cgacaacagt aaaaatacgc 1080tgtatctgca
gatgaacagc ctgcgcgcgg aagataccgc cgtgtattac tgtgcccgtc 1140tgtatcgcta
ctggttcgac tactggggcc agggtacgct ggtgaccgtt agctctggcg 1200gtggcggttc
gctggtcccg cgtggcagcg gcggtggcgg ttcagcgagc accaaaggtc 1260cgagcgtgtt
tccgctggca ccgtgcagcc gctctaccag tgaatccacg gcagctctgg 1320gttgtctggt
gaaagattat tttccggaac cggtcaccgt gagttggaac tccggcgcac 1380tgacctcggg
tgttcatacg ttcccggctg tcctgcagag ttccggcctg tatagcctgt 1440catcggtggt
taccgttccg agctctaatt tcggcaccca aacgtacacc tgcaacgtcg 1500atcacaaacc
gtctaatacc aaagttgaca aaacggttga ttacaaagac gacgacgaca 1560aacaccacca
ccaccaccat tgataagctt
1590135218PRTArtificial SequenceFab-2 light chain 135Ser Tyr Glu Leu Thr
Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5
10 15Thr Ala Arg Ile Thr Cys Ser Gly Asp Asn Leu
Pro Lys Tyr Tyr Ala 20 25
30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe
35 40 45Tyr Asp Val Asn Arg Pro Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser 50 55
60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65
70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Gln Ala Trp Trp Ser Ser Thr Pro Val 85
90 95Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Leu
Val Pro Arg Gly Ser 100 105
110Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
115 120 125Glu Glu Leu Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135
140Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser
Pro145 150 155 160Val Lys
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
165 170 175Lys Tyr Ala Ala Ser Ser Tyr
Leu Ser Leu Thr Pro Glu Gln Trp Lys 180 185
190Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser
Thr Val 195 200 205Glu Lys Thr Val
Ala Pro Thr Glu Cys Ser 210 215136235PRTArtificial
SequenceFab-2 Heavy Chain 136Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met Asp Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110Val Thr Val Ser Ser Leu
Val Pro Arg Gly Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser 130 135 140Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150
155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe 165 170
175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190Thr Val Pro Ser Ser
Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val 195
200 205Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr
Val Asp Tyr Lys 210 215 220Asp Asp Asp
Asp Lys His His His His His His225 230
2351376PRTArtificial Sequenceprotease cleavage site 137Leu Val Pro Arg
Gly Ser1 51381530DNAArtificial SequenceFab-2 coding
sequence 138ggtctcacat gaaaaaaacg gctatcgcaa tcgctgtggc actggcaggc
ttcgcaacgg 60tcgcgcaggc atcctatgaa ctgacgcaac cgccgagtgt ctccgtgtca
ccgggtcaga 120cggcacgtat tacctgctct ggtgataacc tgccgaaata ttacgcgcat
tggtatcagc 180aaaaaccggg ccaagccccg gtggttgtca tcttttatga cgttaatcgt
ccgtcgggta 240tcccggaacg cttctcgggc agcaactctg gtaatacggc caccctgacg
atctcaggca 300cccaggcaat ggatgaagct gactattact gccaagcatg gtggagctct
acgccggtgt 360ttggcggtgg cacgaaactg accgttctgc tggtcccgcg tggctccggt
cagccgaaag 420cagccccgtc agttaccctg tttccgccga gttccgaaga actgcaagca
aacaaagcta 480ccctggtgtg tctgattagc gatttctatc cgggcgcagt tacggtcgcg
tggaaagccg 540actcatcgcc ggtcaaagct ggtgtggaaa ccaccacccc gtcaaaacag
tcgaacaata 600aatatgcagc tagctcttac ctgtcgctga ccccggaaca gtggaaaagc
catcgcagtt 660actcctgcca agttacgcac gaaggctcta cggttgaaaa aaccgtcgca
ccgacggaat 720gcagttgata agcatgcgta ggagaaaata aaatgaaaca gagcaccatc
gcactggcac 780tgctgccgct gctgtttacg ccggtcacca aagcagaagt gcagctggtt
gaatctggtg 840gcggtctggt gcaaccgggc ggttcactgc gtctgtcgtg tgcggccagc
ggctttacct 900tcagttccta tggtatggat tgggtccgtc aggcaccggg taaaggtctg
gaatgggtgt 960catcgattcg tggcagccgc ggttctacct attacgccga ttccgttaaa
ggccgtttca 1020ccatctctcg cgacaacagt aaaaatacgc tgtatctgca gatgaacagt
ctgcgcgcgg 1080aagataccgc cgtgtattac tgcgcccgtc tgtatcgcta ctggtttgac
tactggggcc 1140agggtacgct ggtgaccgtt agctctctgg ttccgcgtgg ctcagcgagc
accaaaggtc 1200cgagtgtctt cccgctggca ccgtgcagcc gctctaccag tgaatccacg
gcagctctgg 1260gttgtctggt gaaagattat tttccggaac cggtcaccgt gagttggaac
tccggcgcac 1320tgacctccgg tgtgcatacg ttcccggctg ttctgcagag ttccggcctg
tattcactgt 1380catcggtggt taccgtgccg agctctaatt ttggcaccca aacgtacacc
tgtaacgttg 1440atcacaaacc gagcaatacc aaagttgaca aaaccgttga ctacaaagac
gacgacgaca 1500aacaccacca ccaccaccac tgataagctt
1530139228PRTArtificial SequenceIgG2-linker 1, Light chain
amino acid sequence 139Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val
Ser Pro Gly Gln1 5 10
15Thr Ala Arg Ile Thr Cys Ser Gly Asp Asn Leu Pro Lys Tyr Tyr Ala
20 25 30His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Val Val Ile Phe 35 40
45Tyr Asp Val Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60Asn Ser Gly Asn Thr Ala
Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65 70
75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Trp
Ser Ser Thr Pro Val 85 90
95Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Leu
100 105 110Val Pro Arg Gly Ser Gly
Gly Gly Gly Ser Gly Gln Pro Lys Ala Ala 115 120
125Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
Ala Asn 130 135 140Lys Ala Thr Leu Val
Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val145 150
155 160Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
Val Lys Ala Gly Val Glu 165 170
175Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser
180 185 190Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser 195
200 205Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
Thr Val Ala Pro 210 215 220Thr Glu Cys
Ser225140458PRTArtificial SequenceIgG2-linker1, heavy chain amino acid
sequence 140Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30Gly Met Asp Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95Ala Arg
Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110Val Thr Val Ser Ser Ala Ser Gly Gly
Gly Gly Ser Leu Val Pro Arg 115 120
125Gly Ser Gly Gly Gly Gly Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
130 135 140Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys145 150
155 160Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser 165 170
175Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
180 185 190Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn 195 200
205Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn 210 215 220Thr Lys Val Asp Lys
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro225 230
235 240Pro Cys Pro Ala Pro Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro 245 250
255Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn 275
280 285Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg 290 295 300Glu Glu Gln
Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val305
310 315 320Val His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser 325
330 335Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys 340 345 350Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 355
360 365Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe 370 375
380Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu385
390 395 400Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 405
410 415Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly 420 425
430Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 450 455141684DNAArtificial
SequenceIgG2-linker 1, Light chain DNA sequence 141tcctacgagc tgacccagcc
cccttccgtg tccgtgtctc ctggccagac cgcccggatc 60acctgttccg gcgacaacct
gcccaagtac tacgcccact ggtatcagca gaagcccggc 120caggcccccg tggtggtcat
cttctacgac gtgaaccggc cctccggcat ccccgagaga 180ttctccggct ccaactccgg
caacaccgcc accctgacca tctccggcac ccaggccatg 240gacgaggccg actactactg
ccaggcttgg tggtcctcca cccccgtgtt tggcggcgga 300acaaagttaa ccgtgctggg
cggtggagga tcactggttc cgcgtggctc tggcggtgga 360ggatcaggcc agcccaaggc
cgctccttcc gtgaccctgt tccccccatc ctccgaggaa 420ctgcaggcca acaaggccac
cctggtctgc ctgatctccg acttctaccc tggcgccgtg 480accgtggcct ggaaggccga
cagctctcct gtgaaggccg gcgtggaaac caccaccccc 540tccaagcagt ccaacaacaa
atacgccgcc tcctcctacc tgtccctgac ccccgagcag 600tggaagtccc accggtccta
cagctgccag gtcacacacg agggctccac cgtggaaaag 660acagtggccc ccaccgagtg
ctct 6841421374DNAArtificial
SequenceIgG2-linker 1, Heavy chain DNA sequence 142gaggtgcagc tggtggaatc
cggcggaggc ctggtccagc ctggcggatc tctgagactg 60tcctgcgccg cctccggctt
caccttctcc agctacggca tggactgggt ccgacaggcc 120cctggcaagg gcctggaatg
ggtgtcctcc atccggggct ctcggggctc cacctactac 180gccgactccg tgaagggccg
gttcaccatc tcccgggaca actccaagaa caccctgtac 240ctgcagatga actccctgcg
ggccgaggac accgccgtgt actactgcgc cagactgtac 300cggtattggt tcgactactg
gggccagggc accctggtca ccgtcagctc agcttctggc 360ggaggcggct ctctggtgcc
tagaggatct ggcggcggag gctccaccaa gggcccttcc 420gtgttccctc tggccccttg
ctcccggtcc acctccgagt ctaccgccgc tctgggctgc 480ctggtcaagg attacttccc
cgagcccgtg accgtgtcct ggaactctgg cgccctgacc 540agcggcgtgc acaccttccc
tgccgtgctg cagtcctccg gcctgtactc cctgtcctcc 600gtcgtgacag tgccctcctc
caacttcggc acccagacct acacctgtaa cgtggaccac 660aagccctcca acaccaaggt
ggacaagacc gtggaacgga agtgctgcgt ggaatgcccc 720ccctgtcctg cacctcctgt
ggctggacct agcgtgttcc tgttcccccc aaagcccaag 780gacaccctga tgatctcccg
gacccccgaa gtgacctgcg tggtggtgga cgtgtcccac 840gaggaccccg aggtgcagtt
caattggtac gtggacggcg tggaagtgca caacgccaag 900accaagccca gagaggaaca
gttcaactcc accttccggg tggtgtccgt gctgaccgtg 960gtgcaccagg actggctgaa
cggcaaagag tacaagtgca aggtctccaa caagggcctg 1020cctgccccca tcgaaaagac
catcagcaag accaagggcc agccccgcga gccccaggtg 1080tacacactgc cacctagccg
ggaagagatg accaagaacc aggtgtccct gacctgtctg 1140gtcaagggct tctacccatc
cgacattgcc gtggaatggg agtccaacgg ccagcccgag 1200aacaactaca agaccacccc
ccccatgctg gactccgacg gctcattctt cctgtactcc 1260aagctgacag tggacaagtc
ccggtggcag cagggcaacg tgttctcctg ctccgtgatg 1320cacgaggccc tgcacaacca
ctacacccag aagtccctgt ccctgagccc cggc 1374143218PRTArtificial
SequenceIgG2-linker 2, Light chain amino acid sequence 143Ser Tyr Glu Leu
Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5
10 15Thr Ala Arg Ile Thr Cys Ser Gly Asp Asn
Leu Pro Lys Tyr Tyr Ala 20 25
30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe
35 40 45Tyr Asp Val Asn Arg Pro Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser 50 55
60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65
70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Gln Ala Trp Trp Ser Ser Thr Pro Val 85
90 95Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Leu
Val Pro Arg Gly Ser 100 105
110Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
115 120 125Glu Glu Leu Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135
140Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser
Pro145 150 155 160Val Lys
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
165 170 175Lys Tyr Ala Ala Ser Ser Tyr
Leu Ser Leu Thr Pro Glu Gln Trp Lys 180 185
190Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser
Thr Val 195 200 205Glu Lys Thr Val
Ala Pro Thr Glu Cys Ser 210 215144448PRTArtificial
SequenceIgG2-linker 2, heavy chain amino acid sequence 144Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Ile Arg Gly Ser Arg Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser Ala Ser Leu Val Pro Arg Gly Ser Thr Lys Gly
115 120 125Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135
140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val145 150 155 160Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185
190Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys
Asn Val 195 200 205Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys 210
215 220Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
Val Ala Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val 290
295 300Val Ser Val Leu Thr Val Val His Gln Asp Trp
Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340
345 350Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370
375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440
445145654DNAArtificial SequenceIgG2-linker 2, Light chain DNA sequence
145tcctacgagc tgacccagcc cccttccgtg tccgtgtctc ctggccagac cgcccggatc
60acctgttccg gcgacaacct gcccaagtac tacgcccact ggtatcagca gaagcccggc
120caggcccccg tggtggtcat cttctacgac gtgaaccggc cctccggcat ccccgagaga
180ttctccggct ccaactccgg caacaccgcc accctgacca tctccggcac ccaggccatg
240gacgaggccg actactactg ccaggcttgg tggtcctcca cccccgtgtt tggcggcgga
300acaaagttaa ccgtgctgct ggttccgcgt ggctctggcc agcccaaggc cgctccttcc
360gtgaccctgt tccccccatc ctccgaggaa ctgcaggcca acaaggccac cctggtctgc
420ctgatctccg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagctctcct
480gtgaaggccg gcgtggaaac caccaccccc tccaagcagt ccaacaacaa atacgccgcc
540tcctcctacc tgtccctgac ccccgagcag tggaagtccc accggtccta cagctgccag
600gtcacacacg agggctccac cgtggaaaag acagtggccc ccaccgagtg ctct
6541461344DNAArtificial SequenceIgG2-linker 2, Heavy chain DNA sequence
146gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgagactg
60tcctgcgccg cctccggctt caccttctcc agctacggca tggactgggt ccgacaggcc
120cctggcaagg gcctggaatg ggtgtcctcc atccggggct ctcggggctc cacctactac
180gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac
240ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc cagactgtac
300cggtattggt tcgactactg gggccagggc accctggtca ccgtcagctc agcttctctg
360gtgcctagag gatctaccaa gggcccttcc gtgttccctc tggccccttg ctcccggtcc
420acctccgagt ctaccgccgc tctgggctgc ctggtcaagg attacttccc cgagcccgtg
480accgtgtcct ggaactctgg cgccctgacc agcggcgtgc acaccttccc tgccgtgctg
540cagtcctccg gcctgtactc cctgtcctcc gtcgtgacag tgccctcctc caacttcggc
600acccagacct acacctgtaa cgtggaccac aagccctcca acaccaaggt ggacaagacc
660gtggaacgga agtgctgcgt ggaatgcccc ccctgtcctg cacctcctgt ggctggacct
720agcgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa
780gtgacctgcg tggtggtgga cgtgtcccac gaggaccccg aggtgcagtt caattggtac
840gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gttcaactcc
900accttccggg tggtgtccgt gctgaccgtg gtgcaccagg actggctgaa cggcaaagag
960tacaagtgca aggtctccaa caagggcctg cctgccccca tcgaaaagac catcagcaag
1020accaagggcc agccccgcga gccccaggtg tacacactgc cacctagccg ggaagagatg
1080accaagaacc aggtgtccct gacctgtctg gtcaagggct tctacccatc cgacattgcc
1140gtggaatggg agtccaacgg ccagcccgag aacaactaca agaccacccc ccccatgctg
1200gactccgacg gctcattctt cctgtactcc aagctgacag tggacaagtc ccggtggcag
1260cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag
1320aagtccctgt ccctgagccc cggc
13441476PRTArtificial SequenceThrombin cleavage siteVARIANT(1)...(2)Xaa =
hydrophobic amino acidVARIANT(5)...(6)Xaa = nonacidic amino acid 147Xaa
Xaa Pro Arg Xaa Xaa1 51484PRTArtificial SequenceFactor Xa
cleavage siteVARIANT(2)...(2)Xaa = Glu or Asp 148Ile Xaa Gly
Arg11495PRTArtificial Sequenceamino acid linker 149Gly Gly Gly Gly Ser1
515058PRTArtificial Sequenceamino acids 95-152 of SEQ ID
NO133 150Tyr Cys Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly1
5 10 15Thr Leu Val Thr
Val Ser Ser Ala Ser Gly Ser Gly Gly Gly Leu Val 20
25 30Pro Arg Gly Ser Gly Gly Gly Gly Ser Thr Lys
Gly Pro Ser Val Phe 35 40 45Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser 50
5515116PRTArtificial Sequenceamino acid linker 151Ala Lys Thr Thr Pro Lys
Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg1 5
10 1515217PRTArtificial Sequenceamino acid linker
152Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg1
5 10 15Val 1539PRTArtificial
Sequenceamino acid linker 153Ala Lys Thr Thr Pro Lys Leu Gly Gly1
515410PRTArtificial Sequenceamino acid linker 154Ser Ala Lys Thr
Thr Pro Lys Leu Gly Gly1 5
101556PRTArtificial Sequenceamino acid linker 155Ser Ala Lys Thr Thr Pro1
51566PRTArtificial Sequenceamino acid linker 156Arg Ala Asp
Ala Ala Pro1 51579PRTArtificial Sequenceamino acid linker
157Arg Ala Asp Ala Ala Pro Thr Val Ser1 515812PRTArtificial
Sequenceamino acid linker 158Arg Ala Asp Ala Ala Ala Ala Gly Gly Pro Gly
Ser1 5 1015927PRTArtificial Sequenceamino
acid linker 159Arg Ala Asp Ala Ala Ala Ala Gly Gly Gly Gly Ser Gly Gly
Gly Gly1 5 10 15Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 20
2516018PRTArtificial Sequenceamino acid linker 160Ser Ala Lys Thr Thr Pro
Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala1 5
10 15Arg Val 1615PRTArtificial Sequenceamino acid
linker 161Ala Asp Ala Ala Pro1 516212PRTArtificial
Sequenceamino acid linker 162Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro
Pro1 5 101635PRTArtificial Sequenceamino
acid linker 163Thr Val Ala Ala Pro1 516412PRTArtificial
Sequenceamino acid linker 164Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro1 5 101656PRTArtificial Sequenceamino
acid linker 165Gln Pro Lys Ala Ala Pro1 516613PRTArtificial
Sequenceamino acid linker 166Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro1 5 101676PRTArtificial
Sequenceamino acid linker 167Ala Lys Thr Thr Pro Pro1
516813PRTArtificial Sequenceamino acid linker 168Ala Lys Thr Thr Pro Pro
Ser Val Thr Pro Leu Ala Pro1 5
101696PRTArtificial Sequenceamino acid linker 169Ala Lys Thr Thr Ala Pro1
517013PRTArtificial Sequenceamino acid linker 170Ala Lys
Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro1 5
101716PRTArtificial Sequenceamino acid linker 171Ala Ser Thr Lys Gly
Pro1 517213PRTArtificial Sequenceamino acid linker 172Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro1 5
1017315PRTArtificial Sequenceamino acid linker 173Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5
10 1517415PRTArtificial Sequenceamino acid
linker 174Gly Glu Asn Lys Val Glu Tyr Ala Pro Ala Leu Met Ala Leu Ser1
5 10 1517515PRTArtificial
Sequenceamino acid linker 175Gly Pro Ala Lys Glu Leu Thr Pro Leu Lys Glu
Ala Lys Val Ser1 5 10
1517615PRTArtificial Sequenceamino acid linker 176Gly His Glu Ala Ala Ala
Val Met Gln Val Gln Tyr Pro Ala Ser1 5 10
15177442PRTHomo sapiens 177Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Gly Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr
Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145
150 155 160Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165
170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn 180 185
190Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205Thr Lys Val Asp Lys Thr Val
Glu Arg Lys Cys Cys Val Glu Cys Pro 210 215
220Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro225 230 235 240Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
245 250 255Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Asn 260 265
270Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg 275 280 285Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 290
295 300Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser305 310 315
320Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
325 330 335Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340
345 350Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe 355 360 365Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370
375 380Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe385 390 395
400Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
405 410 415Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420
425 430Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440178212PRTHomo sapiens 178Ser Tyr Glu Leu Thr Gln
Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5
10 15Thr Ala Arg Ile Thr Cys Ser Gly Asp Asn Leu Pro
Lys Tyr Tyr Ala 20 25 30His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe 35
40 45Tyr Asp Val Asn Arg Pro Ser Gly Ile
Pro Glu Arg Phe Ser Gly Ser 50 55
60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65
70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Gln Ala Trp Trp Ser Ser Thr Pro Val 85
90 95Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Gln Pro Lys Ala Ala 100 105
110Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
115 120 125Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp Phe Tyr Pro Gly Ala Val 130 135
140Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val
Glu145 150 155 160Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser
165 170 175Tyr Leu Ser Leu Thr Pro Glu
Gln Trp Lys Ser His Arg Ser Tyr Ser 180 185
190Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
Ala Pro 195 200 205Thr Glu Cys Ser
210179239PRTArtificial Sequenceprotease-cleavable anti-TFPI scFv
antibody 179Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30Gly Met Asp Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95Ala Arg
Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Leu Val Pro Arg Gly Ser 115 120
125Gly Gly Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser
130 135 140Val Ser Pro Gly Gln Thr Ala
Arg Ile Thr Cys Ser Gly Asp Asn Leu145 150
155 160Pro Lys Tyr Tyr Ala His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro 165 170
175Val Val Val Ile Phe Tyr Asp Val Asn Arg Pro Ser Gly Ile Pro Glu
180 185 190Arg Phe Ser Gly Ser Asn
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser 195 200
205Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Ala
Trp Trp 210 215 220Ser Ser Thr Pro Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu225 230
235180717DNAArtificial Sequenceprotease-cleavable anti-TFPI scFv
antibody coding sequence 180gaagttcagc tggtcgaaag tggtggcggt
ctggttcaac cgggcggttc actgcgtctg 60tcgtgcgcag caagcggttt taccttcagt
tcctatggta tggattgggt ccgtcaggca 120ccgggtaaag gtctggaatg ggtgtcatcg
attcgtggca gccgcggttc tacctattac 180gccgattcag ttaaaggccg ttttaccatc
tctcgcgaca acagtaaaaa tacgctgtat 240ctgcagatga acagcctgcg cgcggaagat
accgccgtgt attactgtgc ccgtctgtat 300cgctactggt tcgactactg gggccagggt
acgctggtga ccgttagctc tggtggcggt 360ggctctctgg ttccgcgtgg ctccggtggc
ggtggctcat cctatgaact gacccaaccg 420ccgagtgtct ccgtgtcacc gggtcagacg
gcacgtatta cctgcagcgg tgataacctg 480ccgaaatatt acgcgcattg gtatcagcaa
aaaccgggcc aagccccggt ggttgtcatc 540ttttatgacg ttaatcgtcc gtccggtatc
ccggaacgct tctcgggcag caactctggt 600aatacggcaa ccctgacgat cagcggcacc
caggcaatgg atgaagctga ctattactgt 660caagcatggt ggagctctac gccggtgttt
ggcggtggca cgaaactgac cgtgctg 717
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