KR101804988B1 - Novel Antibody Binding to TFPI, and Composition Comprising the Same - Google Patents

Abstract

The present invention relates to an antibody specifically binding to TFPI (Tissue factor pathway inhibitor), a nucleic acid encoding the antibody, a vector containing the nucleic acid, a host cell, a method for producing the antibody, The present invention relates to a therapeutic pharmaceutical composition.
The antibody specifically binding to TFPI according to the present invention can activate the exogenous pathway of blood clotting by blocking TFPI, so that it is useful for the treatment of hemophilia patients with antibody and for the prevention of blood coagulation diseases of hemophilia-A or hemophilia-B patients Lt; / RTI >

Description

Novel Antibody Binding to TFPI, and Composition Comprising the Same < RTI ID = 0.0 >

The present invention relates to an antibody specifically binding to TFPI (Tissue factor pathway inhibitor), a nucleic acid encoding the antibody, a vector containing the nucleic acid, a host cell, a method for producing the antibody, The present invention relates to a therapeutic pharmaceutical composition.

About 30% of patients with hemophilia (Hemophilia A and B) are known to have antibodies to FVIII (Factor VIII, Factor VIII) or FIX (Factor IX, Factor IX) As an alternative, active Factor VIIa or aPCC (planta derived activated prothrombin complex concentrate) is administered.

Hemophilia patients are receiving intravenous administration of the recombinant protein more than twice a week, but the inconvenience caused by repeated administration is constantly being raised. Therefore, studies on recombinant proteins that are long-acting for a long time by increasing half-life are actively under way.

Recently, approaches to TFPI (Tissue factor pathway inhibitor) have been attempted in the hemophilia model. TFPI is involved in the extrinsic pathway in blood clotting and blocks Factor X activation by TF / FVIIa to block blood clotting (see FIG. 1). Thus, inhibition of TFPI through antibodies to TFPI can activate blood clotting during bleeding through an exogenous pathway.

TFPI consists of three KPIs (Kunitz-type domain, Kunitz domain) and KPI-2 (Kunitz domain 2) binds directly to FXa to inhibit FXa (see FIG. 2). This means that they constitute a complex of TF / FVIIa / FXa / TFPI and consequently have a mechanism of action which directly inhibits the production of FXa.

The antibody of TFPI can also be used for patients who have i) antibodies against FVIII or FIX protein, and ii) because of the nature of the antibody, it has a very long half-life (~ 2 weeks).

The development of hemophilic cures for TFPI is mostly in the research phase or in the early development stage. For example, humanized mAb mAb2021 from Novo Nordisk is a humanized antibody (IgG4) with anti-TFPI monoclonal antibody, which has entered clinical phase 1. Also, Baxter's ARC19499 is in preclinical phase as a PEGylated Aptamer targeting TFPI. And, Baxter & 3B Pharmaceuticals' JBT2329 is a pegylated anti-human TFPI 20mer peptide in preclinical phase.

The need for a new hemophilia treatment has been constantly being addressed, and the development of therapeutic agents in addition to bypassing agents such as FVIIa is urgently needed, and access to drugs that block the TFPI pathway is desirable. In hemophilia patients receiving blood coagulation factors, a number of patients resistant to the factor are present and new drugs are needed, but medical problems such as Ag-Ab complex clearance should also be considered.

Thus, the present inventors have made intensive efforts to prepare a novel antibody specifically binding to TFPI. As a result, it has been confirmed that when the antibody is used, the anticoagulant mechanism of TFPI can be blocked and the exogenous pathway of blood coagulation can be activated , Thereby completing the present invention.

An object of the present invention is to provide a novel antibody that specifically binds to TFPI, a nucleic acid encoding the antibody, a vector containing the nucleic acid, a host cell, a method for producing the same, and TFPI containing the antibody as an active ingredient, Which is capable of activating the exogenous pathway of hemophilia-A and hemophilia-B patients.

In order to achieve the above object, the present invention provides an antibody that specifically binds to TFPI (Tissue Factor Pathway Inhibitor) shown in SEQ ID NO: 165.

The invention also relates to a nucleic acid encoding an antibody to TFPI; A vector containing the nucleic acid; And a cell into which the vector is introduced.

The present invention also provides a pharmaceutical composition for the treatment of hemophilia comprising an anti-TFPI antibody as an active ingredient.

The antibody specifically binding to TFPI according to the present invention can activate the exogenous pathway of blood clotting by blocking TFPI, so that it is useful for the treatment of hemophilia patients with antibody and for the prevention of blood coagulation diseases of hemophilia-A or hemophilia-B patients Lt; / RTI >

Figs. 1 to 9 show results of evaluating the potency of anti-TFPI antibodies according to affinity maturation through Fxa activity measurement test.
FIGS. 10 to 14 show results of evaluating the efficacy of anti-TFPI antibodies according to affinity maturation through TF / FVIIa / FX complex test.
Fig. 15 shows the results of evaluation of the efficacy of anti-TFPI antibodies according to affinity maturation through thrombin production assay.

It has been reported that TFPI (Tissue Factor Pathway Inhibitor) is involved in the extrinsic pathway in blood coagulation and blocks Factor X activation by TF / FVIIa to block blood coagulation. Respectively. In particular, activating the exogenous pathway of blood clotting through an antibody that blocks KPI-2 in TFPI is at the heart of the present invention. The targeted antigenic site is the KPI-2 domain of TFPI, which is more than 90% identical to the human-rabbit-monkey amino acid sequence, thus facilitating the design of animal experiments and introducing a simple model to measure the rate of blood clotting .

As used herein, the term " Tissue Factor Pathway Inhibitor "or" TFPI (Tissue Factor Pathway Inhibitor) "refers to any variant, isoform and species homologue of human TFPI that is naturally expressed by the cell . In a preferred embodiment of the invention, binding of an antibody of the invention to TFPI reduces blood coagulation time.

In one embodiment of the present invention, the isolated monoclonal antibodies "clone 1001 "," clone 1015 ", "clone 1021 "," clone 1021 ", and the like, which are structurally characterized to specifically bind to TFPI (SEQ ID NO: Clone 1224 "," clone 1224 "," clone 1224 "," clone 1224 "," Clone 1232, Clone 1234, Clone 1238, Clone 1243, Clone 1248, Clone 3007, Clone 3016, Clone 3024, Clone 3115, Clone 3120, Quot; Clone 3131 ", "clone 3203 "," clone 3241 ", "clone 4011 "," clone 4017 ", "clone 4034 "," clone 4041 & Clone 4 "," Clone 5 "," Clone 6 "," Clone 7 "," Clone 2 "," Clone 14, Clone 14, Clone 10, Clone 11, Clone 12, Clone 13, Clone 14, Clone 15, Clone 16, Clone 17, Clone 18 "," Clone 19 "," Clone 20 "," Clone 21, "Clone 22, Clone 23, Clone A24, Clone A25, Clone A52, Clone A63, Clone A67, Clone A71 and Clone A74 . The amino acid sequences for heavy chain CDRs and light chain CDRs of each antibody are as shown in Tables 5 and 8 below. As shown in the following Tables 4 and 7, the anti-TFPI antibody may comprise the amino acid sequence of the heavy chain variable region and the light chain variable region or a sequence having homology thereto.

In another embodiment of the present invention, the quantitative binding force of the purified antibodies, clone 12, clone 1023, clone 1202 and clone 3241 to recombinant human TFPI was measured using a Biacore T-200 (GE Healthcare, USA) (Example 4). As a result, as shown in Table 9, all of the clone antibodies produced showed a slight affinity but showed affinity.

Thus, in one aspect, the present invention relates to an antibody that specifically binds to a tissue factor pathway inhibitor (TFPI) represented by SEQ ID NO: 165.

In the present invention, the antibody comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 110, 118, 124, 133, 142, 143, 144, 149, 163 or 165; A heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 111, 116, 120, 123, 126, 127, 128, 129, 134, 145, 147, 148, 162 or 164; And SEQ ID NO can be characterized in that it contains a heavy chain variable region that comprises a heavy chain CDR3 comprising the amino acid sequence of 112, 117, 131, 135, 136 or 146.

In the present invention, the antibody comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 113, 119, 121, 130, 132, 137, 138 or 139; A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 114; And a light chain variable region comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 115, 122, 125, 140 or 141.

In the present invention, the antibody may be selected from the group consisting of SEQ ID NOS: 56, 58, 59, 60, 61, 63, 65, 66, 68, 70, 71, 73, 74, 75, 76, 78, 79, 80, 84, 85, 86, 87, 88, 89, 90, 92, 93, 94, 95, 96, 97, 98, 99, 102, 103, 104, 105, 106, 107, 109, 156, 158, 159, A heavy chain variable region comprising a sequence having 80% or more homology, preferably 90% or more homology, more preferably 100% homology with the amino acid sequence of SEQ ID NO: 160 or 161, Preferably has 90% or more homology, more preferably 100% or more homology with the amino acid sequence of SEQ ID NO: 57, 62, 64, 67, 69, 72, 77, 83, 91, 100, % ≪ / RTI > of the light chain variable region.

In the present invention, the antibody may be selected from the group consisting of SEQ ID NOS: 56, 58, 59, 60, 61, 63, 65, 66, 68, 70, 71, 73, 74, 75, 76, 78, 79, 80, 84, 85, 86, 87, 88, 89, 90, 92, 93, 94, 95, 96, 97, 98, 99, 102, 103, 104, 105, 106, 107, 109, 156, 158, 159, 160 or 161 and a light chain variable region of SEQ ID NOs: 57, 62, 64, 67, 69, 72, 77, 83, 91, 100, 101, 108 or 157, The antibody may be a human monoclonal antibody, but is not limited thereto.

The antibody may be substituted for the amino acid sequence of the antibody through conservative substitution. "Conservative substitution" refers to a modification of a polypeptide comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not cause loss of the biological or biochemical function of the polypeptide. A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. A class of amino acid residues having similar side chains is defined in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (Such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains, such as, for example, asparagine, glutamine, serine, threonine, tyrosine, cysteine (E.g., threonine, valine, isoleucine) and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is contemplated that the antibodies of the present invention may have conservative amino acid substitutions and still retain activity.

In the case of nucleic acids and polypeptides, the term "substantial homology" means that two nucleotides or two polypeptides, or at least two of the nucleotides or amino acids, if they have the appropriate nucleotide or amino acid insertions or deletions, At least about 85%, preferably about 90%, 91%, 92%, 93%, 94% or 95%, more preferably at least about 96%, 97% 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5%. Alternatively, if the fragment is hybridized with the complement of the strand under selective hybridization conditions, there is substantial homology to the nucleic acid. The invention encompasses nucleic acid sequences and polypeptide sequences that have substantial homology to the particular nucleic acid sequences and amino acid sequences referred to herein.

(V _H ) CDR1, 2 and 3 sequences and light chain (V _L ) CDR1, 2 and 3 sequences described in Table 5 and Table 8 are structurally similar to the heavy chain (V _H ) (V _H ) / light chain (V _L ) pairs of CDRs 1, 2 and 3 can be formed by mixing a light chain (V _L ) sequence and a light chain (V _L ) sequence.

As used herein, the term " antibody "or" antibody composition "refers to a preparation of antibody molecules in a single molecular composition. Wherein the monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity, with variable and constant regions derived from the human intermuscular immunoglobulin sequence. Human antibodies of the invention can be introduced by amino acid residues that are not encoded by the human guinea pig immunoglobulin sequence (e. G. , By random or site directed mutagenesis in vitro or by somatic mutation in vivo ) Lt; / RTI > mutants).

As used herein, the term "antibody" means an immunoglobulin molecule that is immunologically reactive with a specific antigen, and refers to a protein molecule that serves as a receptor that specifically recognizes an antigen, and includes polyclonal antibodies and monoclonal antibodies Monoclonal antibody, and whole antibody and antibody fragments. It may also include chimeric antibodies (e. G., Humanized murine antibodies) and bivalent or bispecific molecules (e. G., Bispecific antibodies), diabodies, triabodies and tetrabodies.

The whole antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4. The antibody fragment means a fragment having an antigen-binding function, and includes Fab, Fab ', F (ab') 2, scFv, Fv and the like.

The Fab has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1 domain) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond.

The Fv (variable fragment) means the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. The double-stranded Fv (dsFv) is linked to a light chain variable region and a light chain variable region by a disulfide bond. The short chain Fv (scFv) is generally linked to a variable region of a heavy chain and a variable region of a light chain through a peptide linker through a covalent bond . Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, a Fab can be obtained by restriction of the whole antibody to papain, and F (ab ') 2 fragment can be obtained by cleavage with pepsin) (For example, DNA encoding the heavy chain of the antibody or its variable region and DNA coding for the light chain or the variable region thereof) is used as a template and amplified by PCR (Polymerase Chain Reaction) using primer pairs , A DNA encoding a peptide linker, and a pair of primers that allow both ends to be linked to the heavy chain or its variable region and the light chain or variable region thereof, respectively).

Immunoglobulins have a heavy chain and a light chain, and each heavy and light chain includes a constant region and a variable region (also known as a domain). The variable region of the light chain and the heavy chain comprises three variable regions and four framework regions called complementarity-determining regions (CDRs). The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, CDR3 starting from the N-terminus and sequentially identified by the chain in which the particular CDR is located.

As used herein, a monoclonal antibody refers to a single molecule composition of an antibody molecule obtained in a substantially identical population of antibodies, and may exhibit a single binding specificity and affinity for a particular epitope.

As used herein, a monoclonal antibody (monoclonal antibody) is a molecule derived from a human immunoglobulin, wherein all of the amino acid sequences constituting the antibody including the complementarity determining region and the structural region are composed of the amino acid sequence of human immunoglobulin . Human antibodies are typically used in the treatment of human disease, i) better interacting with the human immune system, for example, to produce complement-dependent cytotoxicity (CDC) or antibody-dependent cellular (Ii) the human immune system does not recognize the antibody as exogenous, and (iii) less amount, more preferably less than It is an advantage in that the half-life in the human circulatory system is similar to the naturally occurring antibody even when a small amount of drug is administered.

Taking this into consideration, the antibody according to the present invention is a monoclonal antibody that specifically binds to TFPI and can exhibit excellent affinity and specificity for TFPI, and exhibits low immunogenicity because it is derived from human. Therefore, (Hemophilia-A or hemophilia-B).

As used herein, the terms "clone 1001", "clone 1015", "clone 1021", "clone 1023", "clone 1024", "clone 1104", "clone 1123" The term "clone 1202", "clone 1208", "clone 1214", "clone 1216", "clone 1223", "clone 1224", "clone 1232", "clone 1234", "clone 1238" Clone 3016 "," clone 3011 "," clone 3115 "," clone 3120 "," clone 3131 "," clone 3203 "," clone 3241 "," clone 4011 " Clone 4017, Clone 4041, Clone 4141, Clone 4146, Clone 4206, Clone 4208, Clone 4278, Clone 4287, Clone 1, Clone 7, Clone 8, Clone 9, Clone 10, Clone 11, Clone 5, Clone 6, Clone 7, Clone 8, Clone 3, Clone 12, Clone 13, Clone 14, Clone 15, Clone 16, Clone 17, Clone 18, Clone 19, Clone 20, Clone 21, "Clone 22", "Clone 23", "Clone A24", "Clone A25", "Clone A52", "Clone A63", "Clone A67" "The clone A74" refers to antibodies that bind to TFPI results in inhibition of the biological activity of TFPI, and can be used interchangeably with the term -TFPI antibody.

The K _D (equilibrium dissociation constant) of the antibody to the TFPI is, for example: (1) 9.009 x 10 ^-12 M or less, preferably 8.59 x 10 ^-12 M or less, x10 ^-12 M in the case of less than or equal to number, and (2) clones 1023, 3.31x10 ^-11 M or less, preferably 3.16x10 ^-11 M or less, more preferably 3.01x10 ^-11 M can be less than, 3, clone 1202 If, 10.42x10 ^-12 M or less, preferably may be less than or equal to 9.94x10 ^-12 M or less, more preferably 9.47x10 ^-12 M, (4) for clone 3241, 8.14x10 ^-11 M or less, preferably 7.77x10 ^{- 11} M or less, and more preferably 7.4 x ¹⁰ < ^-11 > M or less.

In another embodiment of the present invention, the human heavy chain variable region and light chain variable region genes that bind to human TFPI are identified and then the heavy chain variable region genes are human immunoglobulin type 4 And the light chain variable region gene is ligated to a human light chain constant region and then inserted into a protein expression vector for animal cells to construct a vector Then, these cells were transfected and cultured in Expi293 cell line to produce antibodies, which were then purified with protein A to prepare antibodies (Examples 2 and 3).

Thus, in another aspect, the invention relates to a nucleic acid encoding said antibody. As used herein, the nucleic acid may be present in a cell, a cell lysate, or in a partially purified form or in a substantially pure form. The nucleic acid may be separated from other cellular components or other contaminants, e. G., By a standard technique including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. Quot; isolated "or" substantially pure "when purified from the nucleic acid or protein of a cell. The nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not include an intron sequence.

The present invention In yet another aspect, the invention relates to a vector comprising the nucleic acid. For expression of antibodies or antibody fragments thereof, DNA encoding light chains and heavy chains that are partial or full length can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing the antibody of interest) , And DNA can be "coupled to" into transcription and translation control sequences and inserted into an expression vector.

As used herein, the term " operably linked "may mean that the antibody gene is ligated into the vector such that transcriptional and translational control sequences within the vector serve the intended function of regulating transcription and translation of the antibody gene. The expression vector and expression control sequences are selected so as to be compatible with the host cell used for expression. The light chain gene of the antibody and the heavy chain gene of the antibody are inserted into separate vectors, or both genes are inserted into the same expression vector. Antibodies are inserted into expression vectors by standard methods (e. G., Ligation of complementary restriction enzyme sites on the antibody gene fragment and vector, or blunt end ligation if no restriction enzyme site is present). Optionally, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector such that the signal peptide is attached to the amino terminus of the antibody chain gene in frame. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i. E., A signal peptide derived from an immunoglobulin protein). In addition, the recombinant expression vector has a regulatory sequence that controls the expression of the antibody chain gene in the host cell. "Regulatory sequences" may include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control transcription or translation of the antibody chain gene. Those skilled in the art will appreciate that the design of the expression vector may be varied by selecting different regulatory sequences depending on factors such as the choice of host cell to be transformed, the level of expression of the protein, and the like.

In another aspect, the present invention relates to a nucleic acid or a host cell comprising the vector. The nucleic acid or the vector is transfected or transfected into a host cell. Various techniques commonly used to introduce foreign nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for "transfection" or "transfection" include electrophoresis, calcium phosphate precipitation, DEAE Dextran transfection or lipofection, etc. The antibody according to the present invention can be expressed in a eukaryotic cell, preferably a mammalian host cell, taking into consideration the possibility of application to a mammalian cell. Suitable mammalian host cells for expression of antibodies include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells used, for example, with DHFR selectable markers), NSO myeloma cells, COS cells or SP2 cells And the like.

In another aspect, the present invention relates to a method for producing an antibody comprising culturing a host cell to express the antibody. When the recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody may be administered to the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell, or more preferably to cause the antibody to be secreted into the culture medium in which the host cell is cultured By incubating the host cells for a sufficient period of time.

Optionally, the expressed antibody may be isolated from the host cell and purified to homogeneity. The separation or purification of the antibody can be carried out by separation and purification methods used in conventional proteins, for example, chromatography. The chromatography may include, for example, affinity chromatography involving a protein A column, a protein G column, ion exchange chromatography or hydrophobic chromatography. In addition to the above chromatography, the antibody can be separated and purified by further combining filtration, ultrafiltration, salting out, dialysis, and the like.

In another embodiment of the present invention, a FXa activity measurement test was performed to evaluate the effect of anti-TFPI antibodies (Example 5). As a result, as shown in Figs. 1 to 9, the efficacy of the antibodies according to affinity maturation in anti-TFPI candidate antibodies was confirmed. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced.

In another embodiment of the present invention, the TF / FVIIa / FXa complex test was performed to evaluate the effect of anti-TFPI antibodies (Example 6). That is, the degree to which FXa was produced and inhibited by the TF / FVIIa complex in the presence of TFPI with or without the anti-TFPI antibody was evaluated by the degree of FXa activity. As a result, as shown in Fig. 10 to Fig. 14 and Table 12 to Table 21, the efficacy of the antibodies according to the affinity maturation in the MG1113 candidate antibody as an anti-TFPI antibody was confirmed. As a result, it was confirmed that the absorbance was increased depending on the antibody treatment concentration, so that all of the candidate antibodies showed an effect of inhibiting TFPI in a concentration dependent manner.

In another embodiment of the present invention, a test for measurement of thrombin production was performed on anti-TFPI antibodies selected through FXa activity measurement test TF / FVIIa / FXa complex test (Example 7). As a result, as shown in FIG. 15, among the antibody candidates according to the affinity maturation selected through the Fxa activity measurement test and the TF / FVIIa / FXa complex test, For the 1023 antibody, a comparative test for measurement of thrombin production using a T417 chimeric antibody was performed. Compared to the blank treated with the sample diluent alone, the T417 antibody at about 2.5 nM contained about 335%, 1023 antibody showed about thrombin peak value of about 401%. In the case of ETP showing the total production amount of thrombin, about 293% in the case of the T417 antibody in the 2.5 nM treated sample compared with the negative control (antibody member) And that of 1023 antibody was increased to about 309%. Comparison of the two antibodies revealed that the antibody derived from the affinity maturation. 1023 antibody was more effective than the T417 antibody.

Accordingly, the present invention relates to a pharmaceutical composition for the treatment of hemophilia comprising an antibody as an active ingredient from a different viewpoint.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-TFPI antibody and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" is a substance that can be added to the active ingredient to help formulate or stabilize the formulation and does not cause significant toxic effects on the patient.

The carrier refers to a carrier or diluent that does not irritate the patient and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier that is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added and formulated into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Other carriers are described, for example, in Remington ' s Pharmaceutical Sciences (E. W. Martin). Such compositions will contain a therapeutically effective amount of one or more anti-TFPI antibodies.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutical active materials is well known in the art. The composition is preferably formulated for parenteral use. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In some cases, the composition may include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent, optionally together with one or a combination of the ingredients described above, followed by sterile microfiltration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those described above. In the case of sterile powders for the preparation of sterile injectable solutions, some preparative methods involve vacuum drying and freeze-drying (freeze-drying), which produces powders of the active ingredient and any additional desired ingredients from a pre-sterilized- )to be.

Anti-TFPI antibodies can be used for therapeutic purposes to treat genetic and acquired deficiencies or defects in clotting. For example, the antibody may be used to block the interaction of TFPI with FXa or to prevent TFPI-dependent inhibition of TF / FVIIa activity. In addition, the antibody may also be used to reverse the lack of FVIII- or FIX-dependent amplification of FXa by restoring the production of TF / FVIIa-driven FXa.

Such antibodies have therapeutic uses in the treatment of hemostatic disorders such as thrombocytopenia, platelet disorders and bleeding disorders (e. G., Hemophilia A and haemophilia B). Such disorders can be treated by administering a therapeutically effective amount of an anti-TFPI antibody to a patient in need of such treatment. The antibody also has therapeutic use in the treatment of uncontrolled bleeding in indications such as trauma and hemorrhagic stroke. Accordingly, there is provided a method of reducing bleeding time, comprising administering a therapeutically effective amount of an anti-TFPI antibody to a patient in need of reducing bleeding time.

The antibody can be used as a monotherapy or in combination with other therapies to resolve hemostatic disorders. For example, co-administration of one or more antibodies and blood clotting factors of the invention, such as TF (Tissue factor), FVII (Factor VII) or FX (Factor X), is considered useful in the treatment of hemophilia. The simultaneous administration or combination therapy of the antibody and the blood coagulation factor refers to the administration of two therapeutic drugs separately formulated or formulated together in one composition and can be administered at about the same time or different times It is administered over the treatment period.

The pharmaceutical composition can be administered orally or parenterally at a dose and frequency that can vary depending on the severity of the bleeding episode to a subject suffering from hemophilia A or B, or in the case of prophylaxis, depending on the severity of the patient's blood clotting deficiency have. The composition may be administered to the patient as a bolus or as a continuous infusion if desired. For example, bolus administration of an antibody of the invention, presented as a Fab fragment, can be in the range of 0.0025 to 100 mg / kg body weight, 0.025 to 0.25 mg / kg, 0.010 to 0.10 mg / kg, or 0.10 to 0.50 mg / kg . In the case of continuous infusion, the antibodies of the present invention, presented as Fab fragments, can be administered at a dose of 0.001-100 mg / kg body weight / min, 0.0125-1.25 mg / kg / min, 0.010-0.75 mg / Min or 0.10 to 0.50 mg / kg / min for 1 hour to 24 hours, 1 hour to 12 hours, 2 hours to 12 hours, 6 hours to 12 hours, 2 hours to 8 hours, or 1 hour to 2 hours ≪ / RTI > When administering an antibody of the present invention presented as a full length antibody (having a fully constant region), the dosage may be about 1 to 10 mg / kg body weight, 2 to 8 mg / kg, or 5 to 6 mg / kg. Such full-length antibodies are typically administered via infusion that lasts from 30 minutes to 35 minutes. The frequency of administration depends on the severity of the condition. The frequency can range from three times per week to once a week or once every two weeks.

In addition, the composition can be administered to a patient via subcutaneous injection. For example, a 10 to 100 mg dose of anti-TFPI antibody can be administered to a patient via weekly, biweekly or monthly subcutaneous injection.

As used herein, a "therapeutically effective amount" refers to an amount of an anti-TFPI antibody or antibody and TF (Tissue factor) that is required to effectively increase in vivo blood coagulation time or cause in vivo measurable benefit to a patient in need. , FVII (Factor VII), or FX (Factor X). The exact amount will depend on a number of factors including, but not limited to, the composition and physical characteristics of the therapeutic composition, the intended patient population, individual patient considerations, etc., and can be readily determined by those skilled in the art. When fully considering these factors, it is important to administer a minimal amount sufficient to achieve maximum efficacy without adverse effects, and this dose can be readily determined by an expert in the field.

The dosage of the pharmaceutical composition of the present invention is not particularly limited, but may be varied depending on various factors including the health condition and the weight of the patient, the severity of the disease, the kind of the drug, administration route and administration time. The compositions may be administered orally, rectally, rectally, intravenously, intramuscularly, intraperitoneally, intraperitoneally, intramuscularly, intraperitoneally, intraperitoneally, intramuscularly, intraperitoneally, , Subcutaneously, intrauterinely, or intracerebrovascularly. ≪ RTI ID = 0.0 >

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1: Prediction of binding structure between 308-4 clone, an anti-TFPI antibody, and Kunitz domain-2

TFPI (Tissue Factor Pathway Inhibitor), which inhibits Factor X activity, was designed to produce antibodies for the treatment of hemophilia or preventive antibodies that can block the clotting of blood.

Blood clotting is induced by an intrinsic pathway and an extrinsic pathway. Both pathways activate thrombin through a common pathway that activates Factor X in common and ultimately activates fibrin Fibrin is formed to induce blood coagulation. In addition, TFPI is composed of Kunitz 1 (K1), Kunitz 2 (K2) and Kunitz 3 (K3) domains. The K1 domain binds to FVIIa and the K2 domain binds to FXa.

308-4 clone, an anti-TFPI antibody, was prepared in "New anti-TFPI antibody and composition containing the same" described in Korean Patent Application No. 10-2015-0026555. In the case of the 308-4 clone, K _D it was found that the 2.64x10 ^-11 M or less, preferably 2.52x10 ^-11 M or less, more preferably 2.4x10 ^-11 M or less.

In the present invention, an antibody having higher affinity (affinity) to TFPI was prepared through affinity maturation of the 308-4 clone.

First, to predict the binding structure between the anti-TFPI antibody 308-4 and Kunitz domain-2, Igblast (http://blast.ncbi.nlm.nih.gov/Blast. cgi). As a result, it was confirmed that 3QOS (PDB number) structure was similar. Based on 3QOS, the structure of 308-4 clones was designed using homology modeling of the bioluminate module (Schrodinger, Germany). The designed structure was predicted by using docking simulation with kunitz domain-2 using PIPER, a protein-protein binding prediction program. In order to select the paratope from the ensured binding structure, the amino acid residues of the 308-4 clone producing non-covalent bonds were selected by analyzing the interaction characteristics between the amino acid residues of 308-4 clones and Kunitz domain-2 One). The binding energy of each paratope was calculated using the affinity maturation in the bioluminate module, and the binding energy of the selected paratope was predicted by substitution with other amino acids. Amino acids with stable binding energy values were selected and reflected in the primer design (Table 2).

Table 1 summarizes the amino acid sequences of 308-4 clones that produce noncovalent binding by analyzing the interaction characteristics between the 308-4 clone, an anti-TFPI antibody, and the amino acid residues of Kunitz domain-2.

Table 2 shows selected amino acids of Table 1 having stable binding energy values of the paratope using affinity maturation.

Variable area Selected amino acids (on a kabat basis) Heavy chain S31, T52a, Y56, E64, N98 Light chain S31a, T92, H93

Variable area Selected amino acids VH_S31 H, K, R, T, Y, I, L VH_T52a F, Y, L, H, K, R, I VH_Y56 H, R, K VH_E64 Q, D, H VH_N98 F, H, K, Q, R, Y VL_S31a I, L, N, Q, R, F, K, T, V VL_T92 F, Y, I, N VL_H93 Y, L, I, Q, N, K

Example  2: Yeast display scFv  Affinity of 308-4 clone using library Methods for the preparation of novel antibodies by affinity maturation

2-1: Construction of yeast display scFv library (yeast display scFv library)

In order to introduce mutations into the yeast library, first, polymerase chain reaction (PCR) was performed by dividing into 3 light chain variable regions and 2 light chain variable regions. The heavy chain variable region fragment No. 1 contains a forward primer (SEQ ID NO: 1) and a reverse primer (SEQ ID NO: 2 to 9) in the heavy chain variable region gene sequence of the anti-TFPI 308-4 clone as a template And the heavy chain variable region fragment 2 was added with a forward primer (Table 3: SEQ ID NO: 10) and a reverse primer (SEQ ID NO: 11 to 22) in the heavy chain variable region gene sequence of the anti-TFPI 308-4 clone as a template And heavy chain variable region fragment 3 was obtained by adding a forward primer (Table 3: SEQ ID NO: 23) and a reverse primer (Table 3: SEQ ID NOS: 24 to 30) to the heavy chain variable region gene sequence of anti-TFPI 308-4 clone as a template Each fragment was subjected to PCR using AccuPower Pfu PCR PreMix (CAT.No.K-2015, Bioneer). The PCR conditions were 95 ° C for 30 minutes, 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 60 seconds, 30 cycles, and 72 ° C for 10 minutes. The amplified genes were identified by DNA bands of the expected size in 1% agarose gel, and they were separated using a gel extraction kit (QIAquick Gel Extraction Kit, CAT No. 28706, QIAGEN). The light chain variable region fragment 1 was prepared by adding the forward primer (SEQ ID NO: 33) and the reverse primer (SEQ ID NO: 34 to SEQ ID NO: 43) to the light chain variable region gene sequence of the anti-TFPI 308-4 clone, The variable region fragment 2 was prepared by adding the forward primer (SEQ ID NO: 44) and the reverse primer (SEQ ID NO: 45 to SEQ ID NO: 48) to the light chain variable region gene sequence of the anti-TFPI 308-4 clone as a template, fragment was subjected to PCR using AccuPower Pfu PCR PreMix (Bioneer). The PCR conditions were 95 ° C for 30 minutes, 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 60 seconds, 30 cycles, and 72 ° C for 10 minutes. The amplified gene was confirmed to have a DNA band of a size expected from 1% agarose gel, which was separated using a QIAquick Gel Extraction Kit (QIAGEN).

The reverse primer (Table 3: SEQ ID NO: 31) and the reverse primer (Table 3: SEQ ID NO: 32) were added to the obtained heavy chain variable region fragment gene at a molar ratio of 1: star PCR premix (CAT.NO.R040B, Takara). The PCR conditions were as follows: 95 ° C for 2 minutes, 95 ° C for 10 seconds, 55 ° C for 20 seconds, 72 ° C for 30 seconds, 20 cycles, and 72 ° C for 5 minutes. The amplified gene was confirmed to have a DNA band of the expected size in 1% agarose gel, and the amplified gene was isolated using a QIAquick Gel Extraction Kit (QIAGEN) to obtain a heavy chain variable region gene.

(SEQ ID NO: 52), reverse primer (Table 3: SEQ ID NO: 53) was added to the Takara primer star PCR premix using a 1: 1 molar ratio of the obtained light chain variable region fragment gene as a template CAT.No.R040B, Takara). The PCR conditions were as follows: 95 ° C for 2 minutes, 95 ° C for 10 seconds, 55 ° C for 30 seconds, 72 ° C for 40 seconds, 20 cycles, and 72 ° C for 5 minutes. The amplified gene was confirmed to have a DNA band of an expected size in 1% agarose gel, and the DNA band was separated using a QIAquick Gel Extraction Kit (QIAGEN) to obtain a light chain variable region gene.

The forward primer (SEQ ID NO: 54) and the reverse primer (Table 3: SEQ ID NO: 55) were added to the obtained heavy chain variable region gene and the light chain variable region gene at a 1: star PCR premix (Takara). The PCR conditions were as follows: 95 ° C for 2 minutes, 95 ° C for 10 seconds, 55 ° C for 20 seconds, 72 ° C for 30 seconds, 20 cycles, and 72 ° C for 5 minutes. The amplified gene was identified with a DNA band of the expected size in 1% agarose gel. After separation by using a QIAquick Gel Extraction Kit (QIAGEN), 308-4 affinity maturation scFv library The gene was completed. 200 ng of the completed library gene was mixed with 1 μg of the linear pCTCON gene treated with restriction enzymes NheI (CAT.No.R0131L, NEB) and BamHI (CAT.No.R0136L, NEB), followed by transforming yeast (EBY100 electro- competent cell. The transformed yeast was suspended in 100 mL of YPD medium, shake cultured at 30 DEG C and 200 rpm for 1 hour, inoculated into 1 L of SD medium, cultured at 30 DEG C and 200 rpm for 12 hours or longer, centrifuged to discard the supernatant, Resuspended in storage buffer and stored at -70 ° C. The size of the library was determined by colony counting after culturing at 100 ° C for 1 hour after the transformation, plating on SD plate in a stepwise dilution manner, culturing at 30 ° C for 12 hours or more, and then performing colony counting.

Table 3 shows the primers for constructing the yeast display scFv library.

name Base sequence SEQ ID NO: VH FR1 Fo GAA GTC CAG CTG GTG GAG TCT GGA GGT One VH FR1 Re_S CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA GCT GCT GAA GGT GAA GCC GCT CGC TGC 2 VH FR1 Re_H CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA ATG GCT GAA GGT GAA GCC GCT CGC TGC 3 VH FR1 Re_K CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA TTT GCT GAA GGT GAA GCC GCT CGC TGC 4 VH FR1 Re_R CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA TCT GCT GAA GGT GAA GCC GCT CGC TGC 5 VH FR1 Re_T CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA AGT GCT GAA GGT GAA GCC GCT CGC TGC 6 VH FR1 Re_Y CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA ATA GCT GAA GGT GAA GCC GCT CGC TGC 7 VH FR1 Re_I CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA AAT GCT GAA GGT GAA GCC GCT CGC TGC 8 VH FR1 Re_L CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA AAG GCT GAA GGT GAA GCC GCT CGC TGC 9 VH FR2 Fo TAT GCC ATG AAC TGG GTT CGT CAG GCC 10 VH FR2 Re_T-YH-EQDH GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC GCT ATC GGC GTA GTA GGT GTX1 TGA CCC ACC GGT TGT GAT GGT GCT GAC CCA TTC CAA GCC 11 VH FR2 Re_T-RK-EQDH GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC GGT TGT GAT GGT GCT GAC CCA TTC CAA GCC 12 VH FR2 Re_FYLH-YH-EQDH GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC GCT ATC GGC GTA GTA GGT GTX1 TGA CCC ACC TWR TGT GAT GGT GCT GAC CCA TTC CAA GCC 13 VH FR2 Re_FYLH-RK-EQDH GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC TWR TGT GAT GGT GCT GAC CCA TTC CAA GCC 14 VH FR2 Re_KRI-YH-EQDH GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC GCT ATC GGC GTA GTA GGT GTX1 TGA CCC ACC THT TGT GAT GGT GCT GAC CCA TTC CAA GCC 15 VH FR2 Re_KRI-RK-EQDH GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC THT TGT GAT GGT GCT GAC CCA TTC CAA GCC 16 VH FR2 Re_T-YH-EQDH_ # 2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC GCT ATC GGC GTA GTA GGT ATR TGA CCC ACC GGT TGT GAT GGT GCT GAC CCA TTC CAA GCC 17 VH FR2 Re_T-RK-EQDH_ # 2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC GGT TGT GAT GGT GCT GAC CCA TTC CAA GCC 18 VH FR2 Re_FYLH-YH-EQDH_ # 2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC GCT ATC GGC GTA GTA GGT ATR TGA CCC ACC TWR TGT GAT GGT GCT GAC CCA TTC CAA GCC 19 VH FR2 Re_FYLH-RK-EQDH_ # 2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC TWR TGT GAT GGT GCT GAC CCA TTC CAA GCC 20 VH FR2 Re_KRI-YH-EQDH_ # 2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC GCT ATC GGC GTA GTA GGT ATR TGA CCC ACC THT TGT GAT GGT GCT GAC CCA TTC CAA GCC 21 VH FR2 Re_KRI-RK-EQDH_ # 2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC THT TGT GAT GGT GCT GAC CCA TTC CAA GCC 22 VH FR3 Fo GGG CGC TTC ACC ATT TCC CGC GAT AAC 23 VH FR3 Re_N GCC CTG GCC CCA ATA ATC CAT CAG AAA ATT GCC ATC CTG GCG CGC GCA ATA ATA TAC CGC 24 VH FR3 Re_F GCC CTG GCC CCA ATA ATC CAT CAG AAA AAA GCC ATC CTG GCG CGC GCA ATA ATA TAC CGC 25 VH FR3 Re_H GCC CTG GCC CCA ATA ATC CAT CAG AAA ATG GCC ATC CTG GCG CGC GCA ATA ATA TAC CGC 26 VH FR3 Re_K GCC CTG GCC CCA ATA ATC CAT CAG AAA TTT GCC ATC CTG GCG CGC GCA ATA ATA TAC CGC 27 VH FR3 Re_Q GCC CTG GCC CCA ATA ATC CAT CAG AAA TTG GCC ATC CTG GCG CGC GCA ATA ATA TAC CGC 28 VH FR3 Re_R GCC CTG GCC CCA ATA ATC CAT CAG AAA TCT GCC ATC CTG GCG CGC GCA ATA ATA TAC CGC 29 VH FR3 Re_Y GCC CTG GCC CCA ATA ATC CAT CAG AAA ATA GCC ATC CTG GCG CGC GCA ATA ATA TAC CGC 30 VH Final Fo GGT TCT GGT GGT GGT GGT TCT GCT AGC GAC GTG GTG ATG ACA CAG ACG CCG CTG 31 VH Final Re GGA GCT CAC AGT CAC CAG CGT GCC CTG GCC CCA ATA ATC CAT CAG AAA 32 VL FR1 Fo GAC GTG GTG ATG ACA CAG ACG CCG CTG 33 VL FR1 Re_S GAG CCA ATT CAG ATA CGT CTT GCC GTC GGA GTC CAG CAG CGA CTG GCT TGA TTT GCA 34 VL FR1 Re_I GAG CCA ATT CAG ATA CGT CTT GCC GTC AAT GTC CAG CAG CGA CTG GCT TGA TTT GCA 35 VL FR1 Re_L GAG CCA ATT CAG ATA CGT CTT GCC GTC AAG GTC CAG CAG CGA CTG GCT TGA TTT GCA 36 VL FR1 Re_N GAG CCA ATT CAG ATA CGT CTT GCC GTC AGC GTC CAG CAG CGA CTG GCT TGA TTT GCA 37 VL FR1 Re_Q GAG CCA ATT CAG ATA CGT CTT GCC GTC TTG GTC CAG CAG CGA CTG GCT TGA TTT GCA 38 VL FR1 Re_R GAG CCA ATT CAG ATA CGT CTT GCC GTC TCT GTC CAG CAG CGA CTG GCT TGA TTT GCA 39 VL FR1 Re_F GAG CCA ATT CAG ATA CGT CTT GCC GTC AAA GTC CAG CAG CGA CTG GCT TGA TTT GCA 40 VL FR1 Re_K GAG CCA ATT CAG ATA CGT CTT GCC GTC TTT GTC CAG CAG CGA CTG GCT TGA TTT GCA 41 VL FR1 Re_T GAG CCA ATT CAG ATA CGT CTT GCC GTC AGT GTC CAG CAG CGA CTG GCT TGA TTT GCA 42 VL FR1 Re_V GAG CCA ATT CAG ATA CGT CTT GCC GTC AAC GTC CAG CAG CGA CTG GCT TGA TTT GCA 43 VL FR2 Fo GAC GGC AAG ACG TAT CTG AAT TGG CTC CAG 44 VL FR2 Re_T-YH GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA CGT AAA CGG AAA GTR GGT GCC CTG CCA GCA ATA GTA GAC GCC 45 VL FR2 Re_T-LIHQNK GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA CGT AAA CGG AAA WWK GGT GCC CTG CCA GCA ATA GTA GAC GCC 46 VL FR2 Re_FYIN-YH GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA CGT AAA CGG AAA GTR AWW GCC CTG CCA GCA ATA GTA GAC GCC 47 VL FR2 Re_FYIN-LIHQNK GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA CGT AAA CGG AAA WWK AWW GCC CTG CCA GCA ATA GTA GAC GCC 48 VL Final Re GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA CGT AAA 49 VL Final Fo SfiI Cgtggcccaggcggcc GAC GTG GTG ATG ACA CAG ACG CCG CTG 50 VL Final Fo NruI CTA TCG CGA TTG CAG TGG CAC TGG CTG GTT TCG 51 VL Overlapping Fo GGC ACG CTG GTG ACT GTG AGC TCC GGA ggc ggc gga agt ggc gga gga ggc agc ggc gga ggc ggg agt GAC GTG GTG ATG ACA CAG ACG CCG CTG 52 VL Final Re GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA CGT AAA 53 VH Homologous recombination GCT CTG CAG GCT AGT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT gct agc 54 VL Homologous recombination TTG TTA TCA GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC 55

2-2: Antibody screening

The library yeast cells constructed in Example 2-1 were inoculated into an SD medium, cultured at 30 DEG C and 200 rpm for 12 hours or more, and then the medium was replaced with an SG medium. The medium was cultured at 25 DEG C and 200 rpm for 12 hours or more, Antibodies were expressed. Then, the yeast cells recovered by centrifugation were washed with PBSM (PBS containing 3% BSA) buffer, resuspended in 1 mL PBSM buffer, and reacted with biotin-conjugated recombinant human TFPI protein at room temperature for 1 hour. Yeast cells reacted with the recombinant human TFPI protein were washed with PBSM and reacted with streptavidin microbead (CAT.NO.130-048-101, Miltenyi biotech) on ice for 15 minutes. Then, it was washed once with PBSM buffer, resuspended in PBSM buffer, and passed through a MACS column (CAT.NO.130-042-901, Milternyi biotech) to isolate yeast cells bound to TFPI protein. Separated yeast cells were inoculated into SD medium and incubated for more than 48 hours. The above procedure was repeated twice to select antibodies.

2-3: Securing individual clones by FACS method

A single colony of the final amplified population was collected from the yeast display library and then cultured in SD medium at 30 ° C and 200 rpm for at least 12 hours and then exchanged with SG medium and incubated at 25 ° C and 200 rpm for more than 12 hours to remove the antibody Lt; / RTI > Then, centrifuged and recovered yeast cells were washed with PBSF (PBS containing 1% BSA) buffer, resuspended in 50 μl of PBSF buffer, and then the biotin-conjugated recombinant human TFPI protein and anti-c-myc mice (CAT.No.M4439, Sigma) for 30 minutes at room temperature. The reacted yeast cells were washed with PBSF and resuspended in 50 μl of PBSF buffer. Then, streptavidin conjugated with FITC-conjugated anti-mouse antibody (CAT.No.F0257, Sigma) and PE was blocked with light and blocked with 15 Lt; / RTI > Then, the cells were washed with PBSF buffer, resuspended in 500 μl of PBSF buffer, and clones showing high values in both FITC and PE wavelength ranges were selected using FACS to obtain individual clones.

As a result, as shown in Table 4, clones that specifically bind to human TFPI could be selected, and their amino acid sequences were confirmed. Among the antibodies described in Korean Patent Application No. 10-2015-0026555, the antibody used in the present invention is described as '2015-26555_ (Priority Sequence No.)'.

Table 5 shows the CDR amino acid sequences of the clone antibodies of Table 4 on the basis of Kabat numbering.

Clone Variable area Amino acid sequence SEQ ID NO: 1001 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 56 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 1015 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 58 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 1021 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 59 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 1023 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 60 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 1024 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAM S WVRQAPGKGLEWVSTITTGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 61 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD L DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 62 1104 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGS H TYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 63 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 1123 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 65 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 1202 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGSYTYYADSV D GRFTISRDNAKNSLYLKMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 66 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 1208






Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 65 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTYLPFTFGQGTKVEIKR 67 1214 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 166 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 1216 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS H YAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 68 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHLPFTFGQGTKVEIKR 69 1223 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 70 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 1224 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTITTGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 61 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 1232 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 59 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 1234 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 71 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTL E ISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 72 1238 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 70 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 1243





Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSVHGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 73 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 1248 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 74 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 3007 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTF T SYAMNWVRQAPGKGLEWVSTIT L GGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 75 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD L DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 62 3016 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 76 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTH L PFTFGQGTKVEIKR 77 3024 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAM S WVRQAPGKGLEWVSTITTGGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 78 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTH L PFTFGQGTKVEIKR 77 3115 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGS H TYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 78 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 3120 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG Y FLMDYWGQGTLVTVSS 79 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 3131 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAM S WVRQAPGKGLEWVSTITTGGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG Q FLMDYWGQGTLVTVSS 80 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 3203






Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAM S WVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 81 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD L DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 62 3241 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 166 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 4011 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTF Y SYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 82 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTH L PFTFGQGTKVEIKR 83 4017 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG Y FLMDYWGQGTLVTVSS 84 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 4034 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG Y FLMDYWGQGTLVTVSS 85 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 4041 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAM S WVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG Y FLMDYWGQGTLVTVSS 86 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 4141 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG Y FLMDYWGQGTLVTVSS 87 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 4146 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG Y FLMDYWGQGTLVTVSS 88 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 4206





Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDNAKNSLYLQM D SLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 89 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTH L PFTFGQGTKVEIKR 83 4208 Heavy chain EVQLVESGGGLVK S GGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV G TITTGGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 90 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD T DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 91 4278 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS K YAMNW F RQAPGKGLEWVSTIT L GGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQ YL DGNFLMDYWGQGTLVTVSS 92 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 4287 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS K YAMNW F RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQ HPY GNFLMDYWGQGTLVTVSS 93 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 One Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 60 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 167 2 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 166 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 3 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGS H TYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 78 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 4 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 94 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDVDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 5






Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 56 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 167 6 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 95 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 167 7







Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 60 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 8 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 166 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 9 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 96 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 10 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 71 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 11 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 97 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 12 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 98 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 13 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWV G TITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 99 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 14 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS K YAMNW F RQAPGKGLEWVSTITLGGSYTYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQ YL DGNFLMDYWGQGTLVTVSS 92 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGT Y FPFTFGQGTKVEIKR 100 15 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS K YAMNW F RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQ HPY GNFLMDYWGQGTLVTVSS 93 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQG FY FPFTFGQGTKVEIKR 101 16 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS H YAMTWVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 102 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 17 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGLEWVSTITTGGS H TYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 103 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 18 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS H YAMTWVRQAPGKGLEWVSTITTGGS H TYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 104 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 19 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS Q YAMNWVRQAPGKGLEWVSTIT KK GS F TYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG E FLMDYWGQGTLVTVSS 105 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 167 20 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS Q YAMNWVRQAPGKGLEWVSTI KK GGS F TYYADSVDGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG E FLMDYWGQGTLVTVSS 106 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 167 21







Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTIT K GGSYTYYADSVDGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 107 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 167 22 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 70 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTYFPFTFGQGTKVEIKR 108 23







Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFS H YAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDG H FLMDYWGQGTLVTVSS 109 Light chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTH L PFTFGQGTKVEIKR 69

Clone Variable area CDR1 amino acid sequence SEQ ID NO: CDR2 amino acid sequence SEQ ID NO: CDR3 amino acid sequence SEQ ID NO: 1001 Heavy chain SYAMN 110 TITTGGSYTYYADSV D G 111 QDGNFLMDY 112 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1015 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDGNFLMDY 112 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1021 Heavy chain SYAMN 110 TITTGGSYTYYADSV D G 111 QDG H FLMDY 117 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1023 Heavy chain SYAMN 110 TITTGGSYTYYADSVDG 111 QDGNFLMDY 112 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1024 Heavy chain SYAM S 118 TITTGGSYTYYADSV D G 111 QDGNFLMDY 112 Light chain KSSQSLLD L DGKTYLN 119 LVSKLDS 114 WQGTHFPF 115 1104 Heavy chain SYAMN 110 TITTGGS H TYYADSV Q G 120 QDGNFLMDY 112 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTHFPF 115 1123 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDG H FLMDY 117 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1202 Heavy chain SYAMN 110 TITTGGSYTYYADSV D G 111 QDGNFLMDY 112 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1208 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDG H FLMDY 117 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTYLPF 122 1214 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDGNFLMDY 112 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTHFPF 115 1216 Heavy chain H YAMN 124 TITTGGSYTYYADSVEG 123 QDG H FLMDY 117 Light chain KSSQSLLD I DGKTYL 113 LVSKLDS 114 WQGTHLPF 125 1223 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDG H FLMDY 117 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1224




Heavy chain SYAMS 118 TITTGGSYTYYADSV D G 111 QDGNFLMDY 112 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1232 Heavy chain SYAMN 110 TITTGGSYTYYADSV D G 111 QDG H FLMDY 117 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTHFPF 115 1234 Heavy chain SYAMN 110 TITTGGSYTYYADSV Q G 126 QDGNFLMDY 112 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1238 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDG H FLMDY 117 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTHFPF 115 1243 Heavy chain SYAMN 110 TITTGGS H TYYADSVHG 127 QDG H FLMDY 117 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 1248 Heavy chain SYAMN 110 TITTGGS H TYYADSV D G 128 QDG H FLMDY 117 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 3007 Heavy chain SYAMN 110 TIT L GGSYTYYADSV Q G 129 QDGNFLMDY 112 Light chain KSSQSLLD L DGKTYLN 119 LVSKLDS 114 WQGTHFPF 115 3016 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDGNFLMDY 112 Light chain KSSQSLLDSDGKTYLN 130 LVSKLDS 114 WQGTH L PF 125 3024 Heavy chain SYAM S 118 TITTGGSYTYYADSV Q G 126 QDGNFLMDY 112 Light chain KSSQSLLDSDGKTYLN 130 LVSKLDS 114 WQGTH L PF 125 3115 Heavy chain SYAMN 110 TITTGGS H TYYADSV D G 128 QDGNFLMDY 112 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTHFPF 115 3120 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDG Y FLMDY 131 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 3131 Heavy chain SYAM S 118 TITTGGSYTYYADSV Q G 126 QDG Q FLMDY 132 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 3203




Heavy chain SYAM S 118 TITTGGSYTYYADSVEG 123 QDGNFLMDY 112 Light chain KSSQSLLD L DGKTYLN 119 LVSKLDS 114 WQGTHFPF 115 3241 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDGNFLMDY 112 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 4011 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDGNFLMDY 112 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTH L PF 125 4017 Heavy chain SYAMN 110 TITTGGSYTYYADSV Q G 126 QDG Y FLMDY 131 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 4034 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDG Y FLMDY 131 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 4041 Heavy chain SYAM S 118 TITTGGS H TYYADSVEG 116 QDG Y FLMDY 131 Light chain KSSQSLLD V DGKTYL 121 LVSKLDS 114 WQGTHFPF 115 4141 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDG Y FLMDY 131 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTHFPF 115 4146 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDG Y FLMDY 131 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 4206 Heavy chain SYAMN 110 TITTGGSHTYYADSVEG 116 QDGNFLMDY 112 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTH L PF 125 4208 Heavy chain SYAMS 118 TITTGGSYTYYADSV Q G 126 QDGNFLMDY 112 Light chain KSSQSLLD T DGKTYLN 132 LVSKLDS 114 WQGTHFPF 115 4278 Heavy chain K YAMN 133 TIT L GGSYTYYADSV D G 134 Q YL DGNFLMDY 135 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 4287 Heavy chain K YAMN 133 TITTGGSYTYYADSVEG 123 Q HPY GNFLMDY 136 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 One




Heavy chain SYAMN 110 TITTGGSYTYYADSV D G 111 QDGNFLMDY 112 Light chain KSSPSLLDSDGKTYLN 137 LVSKLDS 114 WQGTHFPF 115 2 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDGNFLMDY 112 Light chain KSSPSLLD I DGKTYLN 138 LVSKLDS 114 WQGTHFPF 115 3 Heavy chain SYAMN 110 TITTGGS H TYYADSV D G 128 QDGNFLMDY 112 Light chain KSSPSLLD V DGKTYLN 139 LVSKLDS 114 WQGTHFPF 115 4 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDGNFLMDY 112 Light chain KSSPSLLDVDGKTYLN 139 LVSKLDS 114 WQGTHFPF 115 5 Heavy chain SYAMN 110 TITTGGSYTYYADSV D G 111 QDGNFLMDY 112 Light chain KSSPSLLDSDGKTYLN 137 LVSKLDS 114 WQGTHFPF 115 6 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDGNFLMDY 112 Light chain KSSPSLLDSDGKTYLN 137 LVSKLDS 114 WQGTHFPF 115 7 Heavy chain SYAMN 110 TITTGGSYTYYADSV D G 111 QDGNFLMDY 112 Light chain KSSPSLLD V DGKTYLN 139 LVSKLDS 114 WQGTHFPF 115 8 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDGNFLMDY 112 Light chain KSSPSLLD V DGKTYLN 139 LVSKLDS 114 WQGTHFPF 115 9 Heavy chain SYAMN 110 TITTGGSYTYYADSV Q G 126 QDGNFLMDY 112 Light chain KSSPSLLD V DGKTYLN 139 LVSKLDS 114 WQGTHFPF 115 10 Heavy chain SYAMN 110 TITTGGSYTYYADSV Q G 126 QDGNFLMDY 112 Light chain KSSPSLLD V DGKTYLN 139 LVSKLDS 114 WQGTHFPF 115 11 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDGNFLMDY 112 Light chain KSSPSLLD I DGKTYLN 138 LVSKLDS 114 WQGTHFPF 115 12 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDG H FLMDY 117 Light chain KSSPSLLD I DGKTYLN 138 LVSKLDS 114 WQGTHFPF 115 13




Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDGNFLMDY 112 Light chain KSSPSLLD I DGKTYLN 138 LVSKLDS 114 WQGTHFPF 115 14 Heavy chain K YAMN 133 TITLGGSYTYYADSV D G 134 Q YL DGNFLMDY 135 Light chain KSSPSLLDSDGKTYLN 137 LVSKLDS 114 WQGT Y FPF 140 15 Heavy chain K YAMN 133 TITTGGSYTYYADSVEG 123 Q HPY GNFLMDY 136 Light chain KSSPSLLDSDGKTYLN 137 LVSKLDS 114 WQG FY FPF 141 16 Heavy chain H YAMT 142 TITTGGS H TYYADSVEG 116 QDGNFLMDY 112 Light chain KSSPSLLD I DGKTYLN 138 LVSKLDS 114 WQGTHFPF 115 17 Heavy chain SYAMT 143 TITTGGS H TYYADSV Q G 120 QDGNFLMDY 112 Light chain KSSPSLLD I DGKTYLN 138 LVSKLDS 114 WQGTHFPF 115 18 Heavy chain H YAMT 142 TITTGGS H TYYADSV D G 128 QDGNFLMDY 112 Light chain KSSPSLLD I DGKTYLN 138 LVSKLDS 114 WQGTHFPF 115 19 Heavy chain Q YAMN 144 TIT KK GS F TYYADSV D G 145 QDG E FLMDY 146 Light chain KSSPSLLDSDGKTYLN 137 LVSKLDS 114 WQGTHFPF 115 20 Heavy chain Q YAMN 144 TI KK GGS F TYYADSVDG 147 QDG E FLMDY 146 Light chain KSSPSLLDSDGKTYLN 137 LVSKLDS 114 WQGTHFPF 115 21 Heavy chain SYAMN 110 TIT K GGSYTYYADSVDG 148 QDGNFLMDY 112 Light chain KSSPSLLDSDGKTYLN 137 LVSKLDS 114 WQGTHFPF 115 22 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDG H FLMDY 117 Light chain KSSPSLLD V DGKTYLN 139 LVSKLDS 114 WQGTYFPF 140 23 Heavy chain H YAMN 149 TITTGGSYTYYADSVEG 123 QDG H FLMDY 117 Light chain KSSPSLLD I DGKTYLN 138 LVSKLDS 114 WQGTH L PF 125

2-4: Cloning of IgG gene of clone 308-4 mutant antibody, which is an anti-TFPI antibody obtained from yeast display

Each of the light chain variable region genes of the 308-4 mutant antibody, anti-TFPI antibody obtained in Example 2-2 and Example 2-3, was amplified using PrimeSTAR HS DNA polymerase (CAT.NO.R040B, Takara) PCR was performed with the included forward primer (Table 6: SEQ ID NO: 150) and reverse primer (Table 6: SEQ ID NO: 151). The kappa constant light region of the human antibody was also subjected to PCR using a forward primer (Table 6: SEQ ID NO: 152) and a reverse primer (Table 6: SEQ ID NO: 153). The conditions were 10 minutes of exposure at 94 ° C, 30 seconds of exposure at 94 ° C for 15 seconds, 56 ° C for 30 seconds, and 72 ° C for 90 seconds, followed by reaction at 72 ° C for 10 minutes. The amplified genes were identified with DNA bands of the expected size in 1% agarose gel and separated using a gel extraction kit. Then, each light chain variable region and the light chain constant region are mixed at a ratio of 1: 1. Using the forward primer (Table 5: SEQ ID NO: 150) and the reverse primer (Table 5: SEQ ID NO: 153), conditions were 94 ° C. for 10 minutes, 94 ° C. for 15 seconds, 56 ° C. for 30 seconds, Exposure was repeated 30 times, followed by reaction at 72 ° C for 10 minutes to perform overlapping PCR. The amplified genes were identified with DNA bands of the expected size in 1% agarose gel and separated using a gel extraction kit. The isolated gene was reacted with KpnI (CAT.NO.R0142L, NEB), HindIII (CAT.NO.R0104L, NEB) restriction enzyme at 37 ° C for 12 hours or longer, and then the restriction enzyme-treated gene was further amplified with 1% agarose And separated from the gel. The pcIW plasmid vector was also digested in the same manner and isolated on agarose gel. The isolated light chain region gene was inserted into the NotI and HindIII sites of the linear pcIW vector using T4 DNA ligase (Cat. No. M0203S, NEB). The ligation reaction was transformed into XL1-Blue bacteria (Electroporation-Competent Cells (Cat. No. 2002228, Stratagene), and then LB plates containing carbenicillin (Cat.NO.LN004CA, Narae Biotech), cultured at 37 ° C for 12 hours or more, single colonies were selected, cultured, and plasmids were isolated using plasmid minikits (Cat. No. 27405, QIAGEN) DNA sequencing.

The heavy chain variable region was constructed by using a 308-4 mutant antibody heavy chain variable region gene as a template and using a PrimeSTAR HS DNA polymerase (Takara) as a reverse primer containing KpnI (Table 6: SEQ ID NO: 154) and ApaI-containing reverse primer (Table 6: SEQ ID NO: 155). The PCR conditions were 98 ° C for 2 minutes, 98 ° C for 10 seconds, 58 ° C for 10 seconds, and 72 ° C for 30 seconds, followed by 30 cycles of reaction at 72 ° C for 5 minutes. The amplified genes were identified with DNA bands of the expected size in 1% agarose gel and separated using a gel extraction kit. Three different genes were then reacted with KpnI and ApaI restriction enzyme at 37 ℃ for 4 hours. The gene that had been reacted with the restriction enzyme was further resolved on 1% agarose gel. The pCIW plasmid vector was also digested in the same manner and isolated on agarose gel. Using the T4 DNA ligase, the isolated gene was inserted into the KpnI (CAT.NO.R0142L, NEB), ApaI (CAT.NO.R0114L, NEB) region of the linear pcIw vector containing the human heavy chain constant region . The ligation reaction product was transformed into XL1-Blue bacteria (Electroporation-Competent Cells; Stratagene), plated on an LB plate (Cat.NO.LN004CA, Narae Biotech) containing carbenicillin, cultured at 37 ° C for 12 hours or more Single colonies were selected and cultured. Plasmids were isolated using a plasmid mini kit (Cat. No. 27405, QIAGEN), and DNA sequencing of the isolated plasmids was performed.

Table 6 shows the primers for the cloning of the IgG gene of the clone 308-4 mutant antibody which is the anti-TFPI antibody obtained in the yeast display.

name Base sequence SEQ ID NO: VH Fo TGCTGTGGGTGAGTGGTACCTGTGGG GAA GTC CAG CTG GTG GAG TCT GGA GGT 150 VH Re AGT GGG AAC ACG GAG GGC CCC TTG GTG CTG GCG GAG CTC ACA GTC ACC AGC GTG CC 151 VL Fo TGCTGTGGGTGAGTGGTACCTGTGGG GAC GTG GTG ATG ACA CAG ACG CCG CTG 152 VL Re_CL overlap GAT GAA CAC AGA AGG GGC AGC CAC CGT GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA CGT AAA 153 Ck Fo ACG GTG GCT GCC CCT TCT GTG TTC ATC 154 Ck Re GAT TGG ATC CAA GCT TAC TAG CAC TCA CCC CTG TTG AAA GAC TTA 155

2-5: IgG production and purification of anti-TFPI 308-4 clone mutant antibodies

In order to produce and purify the anti-TFPI 308-4 clone mutant antibody cloned in Example 2-4, Expi293F ™ cells were inoculated at a concentration of 2.5 × 10 ⁶ cells / mL the day before transfection. In a 24 hour incubation (37 ℃, 8% CO2, 125rpm) after Expi293 ™ Expression medium was added to cells (Cat.No.A1435101, Gibco) 2.5 X 10 6 cells / mL concentrations (survival rate (viability) ≥95%) Were prepared. Twenty micrograms of DNA (15 μg pcIw-anti-TFPI heavy chain and 15 μg pcIw-anti-TFPI light chain) were diluted in OptiPro ™ MS medium (Cat. No. 12309019, Gibco) to a total volume of 1.5 mL, Respectively. 80 μL of ExpiFectamine ™ 293 reagent (Cat. No. A14524, Gibco) was mixed with 1.5 mL of OptiPro ™ MS medium (Cat. No. 12309019, Gibco) to a total volume of 1.5 mL, followed by reaction at room temperature for 5 minutes. After 5 minutes of reaction, each diluted DNA and 1.5 mL of ExpiFectamine ™ 293 reagent were mixed well and reacted at room temperature for 20 to 30 minutes. Expi293F ™ cells were treated with DNA and 3 mL of ExpiFectamine ™ 293 reagent mixture. 150 μL of ExpiFectamine ™ 293 Enhancer 1 (Cat. No. A14524, Gibco) and 1.5 mL of ExpiFectamine ™ 293 Enhancer 2 (Cat. No. A14524, Gibco) were added for 16-18 hours at 37 ° C. and 8% Followed by suspension culture. After the culture was completed, the cell debris was removed by centrifugation at 4000 rpm for 20 minutes, and the supernatant was passed through a 0.22 μm filter. 100 μL of Protein A resin MabSelect Xtra (Cat. No. 17-5269-02, GE Healthcare) was prepared for each 30 mL of the culture solution, centrifuged at 1000 rpm for 2 minutes to remove the storage solution, and Protein A Binding buffer (Cat. No. 21007, Pierce) was washed three times with 400 μL each. Protein A resin was added to the prepared culture medium, and the mixture was subjected to a rotation reaction at room temperature for 30 minutes. The culture and resin mixture was placed in a Pierce spin column-snap cap (Cat. No. 69725, Thermo) and the resin was left in the column using a QIAvac 24 Plus (Cat. No. 19413, QIAGEN) vacuum manifold . The resin was washed with 5 mL of Protein A binding buffer, resuspensioned with 200 μL of Protein A elution buffer (Cat. No.21009, Pierce), and reacted at room temperature for 2 minutes And then eluted by centrifugation at 1000 rpm for 1 minute. Each eluate was neutralized by the addition of 2.5 μL of 1.5 M Tris-HCl (pH 9.0). Elution was carried out 4 to 6 times, and each fraction was quantified using Nanodrop 200C (Thermo scientific). Protein-detected fractions were pooled and resuspended in PBS (Phosphate-Buffered Saline) buffer using Zeba Spin Desalting Columns, 7K MWCO, 5 mL (Cat. No. 0889892, Pierce) Protein electrophoresis (SDS-PAGE) was performed under reducing conditions, and finally, the quantification of the concentration and the state of the antibody were verified and stored at 4 ° C

As a result, the purified anti-TFPI 308-4 clone mutant antibody in good condition was confirmed on protein electrophoresis (SDS-PAGE).

Example 3: Preparation of anti-TFPI 308-4 clone affinity maturation antibody using phage display Fab library

3-1: Construction of phage display Fab library

In order to construct the Fab library, the light chain variable region library was first constructed after the construction of the heavy chain variable region library. First, heavy chain variable region fragment 1 was prepared by adding a forward primer (Table 3: SEQ ID NO: 1) and a reverse primer (SEQ ID NO: 2 to 9) to the heavy chain variable region gene sequence of anti-TFPI 308-4 clone as a template, The heavy chain variable region fragment 2 was prepared by adding a forward primer (Table 3: SEQ ID NO: 10) and a reverse primer (Table 3: SEQ ID NOS: 11 to 22) as a template with the heavy chain variable region gene sequence of anti-TFPI 308-4 clone, The variable region fragment 3 was prepared by adding the forward primer (SEQ ID NO: 23) and the reverse primer (SEQ ID NO: 24 to 30) to the heavy chain variable region gene sequence of the anti-TFPI 308-4 clone as a template Were subjected to PCR using AccuPower Pfu PCR PreMix (CAT.NO.K-2015, Bioneer). The PCR conditions were 95 ° C for 30 minutes, 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 60 seconds, 30 cycles, and 72 ° C for 10 minutes. The amplified genes were identified by DNA bands of the size expected from 1% agarose gel and separated using a gel extraction kit (QIAquick Gel Extraction Kit, QIAGEN). The forward primer (SEQ ID NO: 31) and the reverse primer (SEQ ID NO: 32) were added to the obtained heavy chain variable region fragment in a molar ratio of 1: 1: PCR was performed using a PCR premix (Takara). The PCR conditions were as follows: 95 ° C for 2 minutes, 95 ° C for 10 seconds, 55 ° C for 20 seconds, 72 ° C for 30 seconds, 20 cycles, and 72 ° C for 5 minutes. The amplified gene was confirmed to have a DNA band of a size expected from 1% agarose gel and separated using a QIAquick Gel Extraction Kit (QIAGEN) to obtain a heavy chain variable region gene. The obtained gene was treated with XhoI (CAT.No.R0146L, NEB) and ApaI (Cat.No.R0114L, NEB) restriction enzyme at 37 ° C for 4 hours. The restriction enzyme treated gene was further resolved on 1% agarose gel. Using the T4 DNA ligase (Cat. No. M0203S, NEB), the isolated gene was inserted into the XhoI and ApaI sites of the linear pComb3x vector containing the 308-4 light chain variable-constant region. The ligation reaction product was transformed into XL1-Blue bacteria (Electroporation-competent cells: Cat. No. 2002228, Stratagene) and then cultured in 300 ml of LB medium for 1 hour at 37 ° C and 220 rpm. 150 μl of Carbenicillin , and tetracycline (300 μL), followed by suspension culture at 37 ° C and 220 rpm for 12 hours or more. Next, a heavy chain variable region plasmid constructed using the Midi prep kit (CAT.No.12143, QIAGEN) was obtained. One hour after the transformation, 100 μl of the culture solution was sampled and plated on an LB plate (Cat.NO.LN004CA, Narae Biotech) containing carbenicillin by a stepwise dilution method, followed by incubation at 37 ° C for 12 hours or more , Followed by colony counting.

The light chain variable region fragment 1 was prepared by adding a forward primer (SEQ ID NO: 33) and a reverse primer (SEQ ID NO: 34 to SEQ ID NO: 43) using a light chain variable region gene sequence of an anti-TFPI 308-4 clone as a template , The light chain variable region fragment 2 was obtained by adding a forward primer (Table 3: SEQ ID NO: 44) and a reverse primer (Table 3: SEQ ID NOS: 45 to 48) to the light chain variable region gene sequence of the anti-TFPI 308-4 clone as a template Each fragment was subjected to PCR using AccuPower Pfu PCR PreMix (CAT NO. K-2015, Bioneer). The PCR conditions were 95 ° C for 30 minutes, 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 60 seconds, 30 cycles, and 72 ° C for 10 minutes. The amplified genes were identified by DNA bands of the size expected from 1% agarose gel and separated using a gel extraction kit (QIAquick Gel Extraction Kit, QIAGEN). The light chain variable region was obtained by adding a forward primer (SEQ ID NO: 52), a reverse primer (Table 3: SEQ ID NO: 53) to a Takara primer star PCR premix using a template at a 1: Takara) was used for PCR. The PCR conditions were 95 ° C for 10 minutes, 55 ° C for 30 seconds, 72 ° C for 40 seconds, 20 cycles at 95 ° C for 2 minutes, followed by 72 ° C for 5 minutes. The amplified gene was confirmed to have a DNA band of a size expected from 1% agarose gel, and the DNA band was isolated using a QIAquick Gel Extraction Kit (QIAGEN) to obtain a light chain variable region gene. The obtained gene was reacted with NruI (CAT.No.R0192L, NEB) and XbaI (Cat.No.R0145L, NEB) restriction enzyme at 37 ° C for 4 hours. The gene that had been reacted with the restriction enzyme was separated again on 1% agarose gel. Using the T4 DNA ligase (Cat. No. M0203S, NEB), the isolated gene was inserted into the NruI, XbaI site of the linear pComb3x vector containing the 308-4 heavy chain variable region library. The ligation reaction product was transformed into XL1-Blue bacteria (Electroporation-competent cells: Cat. No. 2002228, Stratagene) and then cultured in 300 ml of LB medium for 1 hour at 37 ° C and 220 rpm. Then, 150 μl of carbencillin , 300 μL of tetracycline, and cultured at 37 ° C and 220 rpm for 1 hour. After treatment with 4.5 mL (10 ¹¹ pfu) of VCS M13 helper phage, the cells were shake-cultured at 37 ° C and 220 rpm for 1 hour, treated with 300 μL of kanamycin and 300 μL of carbenicillin and cultured overnight at 37 ° C. and 220 rpm . The next day, the cultured cells were centrifuged at 4000 rpm for 20 minutes and the supernatant was transferred to a new container. To precipitate the phage, 5X PEG / NaCl was added to the supernatant to 1X and left on ice for at least 30 minutes. The precipitated phage was centrifuged at 8000 rpm for 30 minutes. The supernatant was discarded and the precipitated phage was resuspended in 10 mL of PBS. To remove cell debris, the phage dissolved in 10 mL of PBS was centrifuged at 14,000 rpm for 10 minutes, and the supernatant was separated and stored at 4 ° C. One hour after the transformation, 100 μl of the culture was sampled and plated on an LB plate (Narae Biotech) containing carbenicillin by a stepwise dilution method, followed by culturing at 37 ° C. for 12 hours or more, followed by colony counting colony counting).

3-2: Selection of anti-TFPI mutant antibodies

1 mL of the recombinant human protein TFPI was added to the solid phase polystyrene tube (Cat. No. 444202, Nunc) and the tube coated at 4 ° C for 12 hours or more was washed three times with 5 mL of 0.05% PBST. To the Immuno tube coated with TFPI, 5 mL of 1% BSA / PBS was added, and the mixture was blocked at room temperature for 2 hours. After removing the blocking buffer of the immuno tube, the phage library was treated with tubes and allowed to react at room temperature for 2 hours. And then washed 4 times with 5 mL of PBST. Immuno tubes were treated with 1 mL of Glycine (pH 2.0) elution buffer and reacted at room temperature for 10 minutes to obtain supernatant. Then, 100 μl of 1.5 M Tris-Cl (pH 8.8) was added to the eluted phages to neutralize them. Neutralized phage was treated with 10 mL of XLI-Blue Electroporation-Competent Cell cultured for about 2 hours (OD600 = 0.8-1.0). 10 μl of SB, 10 μl of tetracycline (50 mg / ml) and 10 μl of carbenicillin (100 mg / ml) were added to 10 ml of infected XLI-Blue Electroporation Competent Cell for 30 minutes at room temperature. Shake culture (200 rpm). 1 mL of VCSM13 helper phage (> 10 ¹¹ pfu / mL) was treated and incubated at 37 ° C for 1 hour (200 rpm). After incubation for 1 hour, 100 μl of SB 80ml, kanamycin, and carbenicillin (100mg / ml) were treated and cultured at 200 rpm at 37 ° C overnight. After incubation for more than 12 hours, the supernatant was centrifuged at 4000 rpm for 15 minutes. The supernatant was removed, mixed with 5X PEG / NaCl buffer 1X, and left on ice for 30 minutes. The supernatant was removed by centrifugation at 8000 rpm for 30 minutes and the pellet was resuspended in 2 mL of 1% BSA / PBS and centrifuged at 12000 rpm for 10 minutes to take the supernatant and use for panning of the next order. The above procedure was repeated four times.

3-3: ELISA method securing anti-TFPI individual clone antibody

After collecting a single colony of the final amplification population of the library, the cells were cultured in 1.5 ml of SB / carbenicillin at an OD600 of 0.8-1.0 at 37 ° C and 220 rpm, followed by incubation at 1 mM IPTG, 30 ° C, 200 rpm for 12 hours or more . The reaction mixture was centrifuged at 5,500 rpm for 5 minutes. Each supernatant was added to an ELISA plate coated with TFPI antigen, reacted at room temperature for 2 hours, washed 4 times with PBST (1XPBS, 0.05% tween 20) HRP / anti-hFab-HRP conjugate (CAT.No.A0293, Sigma) diluted 1/5000 in 1% BSA / 1XPBS was added and reacted at room temperature for 1 hour. PBST (1XPBS, 0.05% tween 20), added with TMB solution, treated for 5-10 minutes, added with TMB stop solution, read the value at 450 nm wavelength using TECAN sunrise, and clone having high OD value was cloned into individual clone Respectively.

As a result, as shown in Table 7, clones that specifically bind to human TFPI could be selected and their amino acid sequences were confirmed. Among the antibodies described in Korean Patent Application No. 10-2015-0026555, the antibody used in the present invention is described as '2015-26555_ (Priority Sequence No.)'.

Table 8 shows the CDR amino acid sequences of the clone antibodies of Table 7 on the basis of Kabat numbering.

Clone Variable area Amino acid sequence SEQ ID NO: A24 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTF H SYAMNWVRQAPGKGLEWVSTITT R GSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 156 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 A25 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 58 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD R DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 157 A52 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGS H TYYADSV D GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 158 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57 A63 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 159 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD V DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 64 A67 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 166 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD L DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 62 A71 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTITTGGSYTYYADSV H GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 160 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD T DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 91 A74 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAM H WVRQAPGKGLEWVSTITTGGSYTYYADSV Q GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWGQGTLVTVSS 161 Light chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLD I DGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR 57

Clone Variable area CDR1 amino acid sequence SEQ ID NO: CDR2 amino acid sequence SEQ ID NO: CDR3 amino acid sequence SEQ ID NO: A24 Heavy chain SYAMN 110 TITT R GSYTYYADSVEG 161 QDGNFLMDY 112 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTHFPF 115 A25 Heavy chain SYAMN 110 TITTGGS H TYYADSVEG 116 QDGNFLMDY 112 Light chain KSSQSLLD R DGKTYLN 162 LVSKLDS 114 WQGTHFPF 115 A52 Heavy chain SYAMN 110 TITTGGS H TYYADSV D G 128 QDGNFLMDY 112 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115 A63 Heavy chain SYAMN 110 TITTGGSYTYYADSV Q G 126 QDGNFLMDY 112 Light chain KSSQSLLD V DGKTYLN 121 LVSKLDS 114 WQGTHFPF 115 A67 Heavy chain SYAMN 110 TITTGGSYTYYADSVEG 123 QDGNFLMDY 112 Light chain KSSQSLLD L DGKTYLN 119 LVSKLDS 114 WQGTHFPF 115 A71 Heavy chain SYAMN 110 TITTGGSYTYYADSV H G 163 QDGNFLMDY 112 Light chain KSSQSLLD T DGKTYLN 132 LVSKLDS 114 WQGTHFPF 115 A74 Heavy chain SYAM H 164 TITTGGSYTYYADSV Q G 126 QDGNFLMDY 112 Light chain KSSQSLLD I DGKTYLN 113 LVSKLDS 114 WQGTHFPF 115

3-4: Cloning IgG Gene of Anti-TFPI 308-4 Clone Mutant Antibody

Using the obtained anti-TFPI 308-4 clone mutant antibody light chain variable region gene as a template, a forward primer containing KpnI (Table 6: SEQ ID NO: 150) and a reverse primer containing a primer Hp DNA polymerase (Takara) (Table 6: SEQ ID NO: 151). The kappa constant light region of the human antibody was also subjected to PCR using a forward primer (Table 6: SEQ ID NO: 152) and a reverse primer (Table 6: SEQ ID NO: 153). The conditions were 10 minutes of exposure at 94 ° C, 30 seconds of exposure at 94 ° C for 15 seconds, 56 ° C for 30 seconds, and 72 ° C for 90 seconds, followed by reaction at 72 ° C for 10 minutes. The amplified genes were identified with DNA bands of the expected size in 1% agarose gel, and were separated using a gel extraction kit. Then, each light chain variable region and the light chain constant region were mixed at a ratio of 1: 1. Using the forward primer (Table 5: SEQ ID NO: 150) and the reverse primer (Table 5: SEQ ID NO: 153), conditions were 94 ° C. for 10 minutes, 94 ° C. for 15 seconds, 56 ° C. for 30 seconds, Exposure was repeated 30 times, followed by reaction at 72 ° C for 10 minutes to perform overlapping PCR. The amplified genes were identified with DNA bands of the expected size in 1% agarose gel, and were separated using a gel extraction kit. The isolated gene was reacted with KpnI (CAT.NO.R0142L, NEB), HindIII (CAT.NO.R0104L, NEB) restriction enzyme at 37 ° C for 12 hours or longer, and the gene reacted with the restriction enzyme was further reacted with 1% agarose And separated from the gel. The pcIW plasmid vector was also cut in the same manner and isolated on agarose gel. The isolated light chain region gene was inserted into the NotI and HindIII sites of the linear pcIW vector using T4 DNA ligase (Cat. No. M0203S, NEB). The ligation reaction product was transformed into XL1-Blue bacteria (Electroporation-Competent Cells; Stratagene, Cat. No. 2002228) and then ligated to an LB plate (Cat.NO.LN004CA, Narae Biotech) containing carbenicillin After culturing, the cells were cultured at 37 ° C. for 12 hours or more. Then, single colonies were picked and cultured, and plasmids were isolated using a plasmid mini kit (Cat. No. 27405, QIAGEN) .

The heavy chain variable region was constructed by using a 308-4 mutant antibody heavy chain variable region gene as a template and using a PrimeSTAR HS DNA polymerase (Takara) as a reverse primer containing KpnI (Table 6: SEQ ID NO: 154) and ApaI-containing reverse primer (Table 6: SEQ ID NO: 155). The PCR conditions were 98 ° C for 2 minutes, 98 ° C for 10 seconds, 58 ° C for 10 seconds, and 72 ° C for 30 seconds, followed by 30 cycles of reaction at 72 ° C for 5 minutes. The amplified genes were identified with DNA bands of the expected size in 1% agarose gel, and were separated using a gel extraction kit. Three different genes were then reacted with KpnI and ApaI restriction enzyme at 37 ℃ for 4 hours. The gene that had been reacted with the restriction enzyme was further resolved on 1% agarose gel. The pCIW plasmid vector was also digested in the same manner and isolated on agarose gel. Using the T4 DNA ligase, the isolated gene was inserted into the KpnI (CAT.NO.R0142L, NEB) and ApaI (NEB, CAT.No.R0114L) sites of the linear pcIw vector containing the human heavy chain constant region . The ligation reaction product was transformed into XL1-Blue bacteria (Electroporation-Competent Cells; Stratagene, Cat. No. 2002228), and then ligated to an LB plate (Narae Biotech, Cat. No. LN004CA) containing carbenicillin After culturing, the cells were cultured at 37 ° C. for 12 hours or more. Then, single colonies were picked and cultured, and plasmids were isolated using a plasmid mini kit (Cat. No. 27405, QIAGEN) .

3-5: IgG production and purification of anti-TFPI 308-4 clone mutant antibody

In order to produce and purify the anti-TFPI 308-4 clone mutant antibody cloned in Example 3-4, Expi293F ™ cells were inoculated at a concentration of 2.5 × 10 ⁶ cells / mL the day before transfection. In a 24 hour incubation (37 ℃, 8% CO2, 125rpm) after Expi293 ™ Expression medium was added to cells (Cat.No.A1435101, Gibco) 2.5 X 10 6 cells / mL concentrations (survival rate (viability) ≥95%) Were prepared. Twenty micrograms of DNA (15 μg pcIw-anti-TFPI heavy chain and 15 μg pcIw-anti-TFPI light chain) were diluted in OptiPro ™ MS medium (Cat. No. 12309019, Gibco) to a total volume of 1.5 mL, Respectively. 80 μL of ExpiFectamine ™ 293 reagent (Cat. No. A14524, Gibco) was mixed with 1.5 mL of OptiPro ™ MS medium (Cat. No. 12309019, Gibco) to a total volume of 1.5 mL, followed by reaction at room temperature for 5 minutes. After 5 minutes of reaction, each diluted DNA and 1.5 mL of ExpiFectamine ™ 293 reagent were mixed well and reacted at room temperature for 20 to 30 minutes. Expi293F ™ cells were treated with DNA and 3 mL of ExpiFectamine ™ 293 reagent mixture. 150 μL of ExpiFectamine ™ 293 Enhancer 1 (Cat. No. A14524, Gibco) and 1.5 mL of ExpiFectamine ™ 293 Enhancer 2 (Cat. No. A14524, Gibco) were added for 16-18 hours at 37 ° C. and 8% Followed by suspension culture. After the culture was completed, the cell debris was removed by centrifugation at 4000 rpm for 20 minutes, and the supernatant was passed through a 0.22 μm filter. 100 μL of Protein A resin MabSelect Xtra (Cat. No. 17-5269-02, GE Healthcare) was prepared for each 30 mL of the culture solution, centrifuged at 1000 rpm for 2 minutes to remove the storage solution, and Protein A Binding buffer (Cat. No. 21007, Pierce) was washed three times with 400 μL each. Protein A resin was added to the prepared culture medium, and the mixture was subjected to a rotation reaction at room temperature for 30 minutes. The culture and resin mixture was placed in a Pierce spin column-snap cap (Cat. No. 69725, Thermo) and the resin was left in the column using a QIAvac 24 Plus (Cat. No. 19413, QIAGEN) vacuum manifold . The resin was washed with 5 mL of Protein A binding buffer, resuspensioned with 200 μL of Protein A elution buffer (Cat. No.21009, Pierce), and reacted at room temperature for 2 minutes And then eluted by centrifugation at 1000 rpm for 1 minute. Each eluate was neutralized by the addition of 2.5 μL of 1.5 M Tris-HCl (pH 9.0). Elution was carried out 4 to 6 times, and each fraction was quantified using Nanodrop 200C (Thermo scientific). Protein-detected fractions were pooled and resuspended in PBS (Phosphate-Buffered Saline) buffer using Zeba Spin Desalting Columns, 7K MWCO, 5 mL (Cat. No. 0889892, Pierce) Protein electrophoresis (SDS-PAGE) was performed under reducing conditions, and finally, the quantification of the concentration and the state of the antibody were verified and stored at 4 ° C

As a result, the purified anti-TFPI 308-4 clone mutant antibody in good condition was confirmed on protein electrophoresis (SDS-PAGE).

Example 4 Quantitative Determination of Binding Capacity of 308-4 Mutant Antibody to TFPI Antigen

The quantitative binding affinity (affinity) of the 308-4 clone heavy chain variable region antibody 12, 1023, 1202, and 3241 purified in Example 2 and Example 3 to the recombinant human TFPI was measured using Biacore T -200 (GE Healthcare) biosensor. Protein A was immobilized on a CM5 chip (CAT.No. BR-1005-30, GE Healthcare) using an amine-carboxyl reaction so as to have a 200 Rmax, and each of purified 12, 1023, 1202 and 3241 clones was incubated for 1 minute After binding to the immobilized Protein A, recombinant human TFPI, which was serially diluted in HBS-EP buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20), was added at a concentration range of 0.078 nM to 5 nM Association for 120 seconds, dissociation for 3600 seconds at a flow rate of 30 μL / min. The dissociation of TFPI bound to the antibody was induced by flowing 10 mM Glycine-HCl pH 1.5 at a flow rate of 30 μL / min for 30 seconds. Affinities were obtained with kinetic rate constants (K _on and K _off ) and equilibrium dissociation constants (K _D ) as shown in Table 9 using Biacore T-200 evaluation software

Table 9 shows the affinity of the anti-TFPI antibody for the recombinant human TFPI protein as kinetic rate constants (K _on and K _off ) and equilibrium dissociation constant (K _D ).

K _on K _off K _D 12 4.87X10 ⁶ 3.99X10 ^-5 8.19X10 ^-12 1023 4.91X10 ⁶ 1.5X10 ^-4 3.01X10 ^-11 1202 7.56X10 ⁶ 7.16X10 ^-5 9.47X10 ^-12 3241 1.91X10 ⁶ 1.4X10 ^-4 7.4X10 ^-11

Example  5: Fxa  Active measurement

Blood coagulation is induced by the intrinsic pathway and the extrinsic pathway, and both pathways activate thrombin through a common pathway that activates Factor X in common, which ultimately forms fibrin, Thereby inducing solidification. In addition, TFPI is composed of Kunitz 1 (K1), Kunitz 2 (K2) and Kunitz 3 (K3) domains. The K1 domain binds to FVIIa and the K2 domain binds to FXa. It is known that TFPI binds to blood clotting factors and inhibits blood clotting. Therefore, the degree of FXa activity was evaluated to confirm the effect of MG1113 (anti-TFPI antibody) on the whole blood coagulation process.

In order to minimize the influence of various factors, FXa activity is shown by constructing the test system with only FXa, TFPI and candidate antibody, and when the candidate substance binds with TFPI, it does not inhibit the function of FXa. On the other hand, if the candidate substance fails to bind effectively with TFPI, TFPI binds to FXa and inhibits the function, thereby decreasing the degree of color development. Therefore, residual activity of FXa, which is not inhibited by TFPI, is measured by the degree of substrate decomposition. The substrate used here is S-2765, an FXa-specific substrate, which degrades and generates measurable chromophoric pNA at 405 nm. The assay is based on an amidolytic assay.

Dilution using the respective FXa, TFPI, mAb2021, refer to Table 10 S-2765 solution in assay buffer (20mM HEPES, 150mM NaCl, 1mg / mL of BSA, 0.02% NaN 3, 5mM CaCl 2, pH7.4) One solution was prepared in a 1.5 ml tube.

Materials Preliminary dilution concentration
(NM) Working concentration
(Working conc.) (NM) Remarks FXa 2nM 0.5 nM TFPI 40 nM 10 nM S-2765 2 mM 0.5 mM Standard curve
(Standard curve) 10 nM 0.02, 0.1, 0.5, 2.5 nM FXa mAb2021 160 nM 2.5, 5, 10, 20 nM Positive control group

50 μL of mAb2021 antibody (anti-TFPI Ab, Novo Nordisk) and candidate antibodies were added to each well to give 20, 10, 5, and 2.5 nM. 50 niL of 40 nM TFPI solution was placed in each well and allowed to stand at room temperature for 30 minutes. To obtain a standard curve, 50 μL of FXa solution was added to each well at a concentration of 50 μL, and 2 μM of FXa solution was added to each well at 50 μL, followed by reaction at 37 ° C. for 10 minutes. After adding 50 μL of 2 mM S-2765 solution to each well, the reaction was carried out at 37 ° C. for 30 minutes, and the results were analyzed in an endpoint mode at a wavelength of 405 nm using a microplate reader.

As a result, as shown in Fig. 1, the affinity maturation antibody of MG1113 candidate antibody, anti-TFPI antibody, was detected. 1015, 1021, 1023, 3007, 3016 and 3024 antibodies. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced. Among them, No. In the case of antibody 1015, the positive control TFPI showed an effect of suppressing TFPI by about 83% in the 20 nM treated sample compared with the untreated sample and an effect of suppressing about 71% in the 10 nM treated sample. In addition, In the case of 1023 antibody, the effect of inhibiting TFPI by about 86% in the 20 nM treated sample versus the positive control sample and the effect of suppressing TFPI by about 84% in the 10 nM treated sample were shown. Considering that the amount of TFPI treated was 10 nM, No. 1015 antibody. 1023 antibody showed excellent TFPI inhibitory ability.

In addition, as shown in Fig. 2, the affinity maturation antibody of the MG1113 candidate antibody, anti-TFPI antibody, 3036, 3115, 3120, 3131, 4017, 4141 antibodies. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced. Among them, No. For the 4017 antibody, the positive control TFPI showed an effect of inhibiting TFPI by about 90% in the 20 nM treated sample versus about 70% in the 10 nM treated sample compared to the untreated sample

In addition, as shown in Fig. 3, the affinity maturation antibody of the MG1113 candidate antibody, which is an anti-TFPI antibody, 1001, 1024, 1104, and 1123 antibodies. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced. Among them, No. In the case of antibody 1123, the positive control TFPI showed an effect of inhibiting TFPI by about 88% in the 20 nM treated sample compared with the untreated sample, and by about 69% in the 10 nM treated sample.

In addition, as shown in Fig. 4, the affinity maturation antibody A24, A25, A51, A52, A63 and A67 antibodies of the MG1113 candidate antibody as an anti-TFPI antibody was confirmed. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced. In the case of the A67 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 79% in the 20 nM treated sample compared with the untreated sample, and by about 67% in the case of the 10 nM treated sample.

Further, as shown in Fig. 5, the affinity maturation antibody of the MG1113 candidate antibody, anti-TFPI antibody, 3203, 3241, 4206, 4208 antibodies. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced. Among them, No. For the 3241 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 82% in the 20 nM treated sample compared with the untreated sample, and about 83% inhibition in the 10 nM treated sample

In addition, as shown in Fig. 6, the affinity maturation antibody of the MG1113 candidate antibody, anti-TFPI antibody, 1, 2, 3, 7, 8, and 10, respectively. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced. Among them, No. 2 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 76% in the 20 nM treated sample compared with the untreated sample, and an effect of suppressing about 79% in the 10 nM treated sample. No. 3 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 81% in the 20 nM treated sample and a 70% inhibition effect in the 10 nM treated sample compared to the untreated sample. No. 8 antibody showed a TFPI inhibitory effect of about 80% in the 20 nM treated sample and a 69% inhibitory effect in the 10 nM treated sample compared to the untreated sample of the positive control TFPI.

In addition, as shown in Fig. 7, the affinity maturation antibody of MG1113 candidate antibody, anti-TFPI antibody, 1214, 1216, 1224, 1234, 1238, 4287 antibodies. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced. Among them, No. For the 1214 antibody, the positive control TFPI showed an effect of inhibiting TFPI by about 77% in the 20 nM treated sample versus the untreated sample, and by about 63% in the 10 nM treated sample.

In addition, as shown in Fig. 8, the affinity maturation antibody of the MG1113 candidate antibody, anti-TFPI antibody, 16, 19, 20, 21, and 23 antibodies. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced. Among them, No. 16 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 55% in the 20 nM treated sample compared with the untreated sample and a suppressive effect of about 34% in the 10 nM treated sample.

In addition, as shown in Fig. 9, the affinity maturation antibody of MG1113 candidate antibody, anti-TFPI antibody, 11, 12, 13, and 1202 antibodies. All of the above antibodies confirmed that the absorbance was increased depending on the antibody treatment concentration, and thus it was confirmed that the effect of inhibiting TFPI in a concentration dependent manner was enhanced. Among them, No. 11 antibody, the positive control TFPI showed an effect of inhibiting TFPI by about 89% in the 20 nM treated sample compared with the untreated sample, and a suppressive effect of about 81% in the 10 nM treated sample. No. 12 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 82% in the 20 nM treated sample compared with the untreated sample and a suppression effect of about 82% at the treated sample of 10 nM. No. 13 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 85% in the 20 nM treated sample compared with the untreated sample and an effect of suppressing about 76% in the 10 nM treated sample. No. In the case of 1202 antibody, the positive control TFPI showed an effect of inhibiting TFPI by about 87% in the 20 nM treated sample compared to the untreated sample, and about 82% inhibition in the 10 nM treated sample.

Example 6: Measurement of TF / FVIIa / FX complex

The most important factors in the exogenous mechanism of blood clotting are Tissue Factor (TF), Factor VII (FVII), and Factor X (FX). If TF and FVIIa are complexed by an external signal, FX is activated by FXa. Then, FXa activates prothrombin with thrombin, which converts fibrinogen into fibrin and acts on blood clotting. However, the Tissue factor pathway inhibitor (TFPI) binds to FXa and inhibits its function, thereby interfering with blood coagulation. The TF / FVIIa / FXa complex test was performed to evaluate the effect of MG1113, an anti-TFPI antibody, in the pathway. The degree to which FXa is produced and inhibited by the TF / FVIIa complex in the presence of TFPI with or without the anti-TFPI antibody MG1113 is determined by the degree of color development of the substrate (S2765) degraded by FXa, The effect was confirmed. That is, the greater the effect of MG1113, an anti-TFPI antibody, on the inhibition of TFPI, the greater the production of FXa and the greater the amount of substrate degradation, thereby increasing the absorbance.

Briefly, TFA (4500 L / B, Sekisui diagnostics), FVIIa (pH 7.4) was performed in a 1.5 mL tube using an assay buffer (20 mM HEPES, 150 mM NaCl, 1 mg / mL BSA, 0.02% NaN ₃ , 5 mM CaCl ₂ , Novo Nordisk, Novo Seven), and FX (PP008A, Hyphen biomed) were prepared so as to have the concentrations as shown in Table 11.

matter TF FVIIa FX density 0.6 ng / mL 1 nM 17nM - > 5nM

The mixed solution was dispensed in a 96-well plate (70 μL / well). 70 [mu] L of Assay buffer was dispensed into the blank well. After reaction at 37 ° C for 15 minutes, 30 μL of TFPI was added to each well to give 50 nM. However, 30 μL of assay buffer was dispensed into blank wells and positive control wells (anti-TFPI antibody MG1113 and TFPI untreated samples). 30 占 퐇 aliquots were added to each well so that the anti-TFPI antibody MG1113 was 12.5, 25, 50 and 100 nM. 30 μL of assay buffer was dispensed into blank wells, positive control wells (anti-TFPI antibody and TFPI untreated samples), negative control wells (anti-TFPI antibody MG1113 untreated samples) and reacted at 37 ° C. for 15 minutes. EDTA (E7889, Sigma-Aldrich) was added to all wells in an amount of 20 μL to 50 mM. Subsequently, 50 μL of S2765 (Chromogenix, S-2765) was added to all wells to 200 μM, and the mixture was incubated at 37 ° C. for 10 minutes using a microplate reader. Absorbance was measured at 405 nm.

Table 12 is a numerical representation of the MG1113 efficacy test result of the affinity maturation anti-TFPI antibody through the TF / FVIIa / FX complex test.

Antibody concentration Normalization
(Normalized) TFPI (50 nM) mAb2021 T417 308-4 1015 1023 4017 100 nM 0.918 0.119 0.937 0.949 0.938 0.944 0.951 0.943 50 nM 0.929 0.945 0.926 0.919 0.947 0.919 25 nM 0.918 0.873 0.664 0.269 0.795 0.307 12.5 nM 0.218 0.242 0.223 0.179 0.228 0.181 6.25 nM 0.168 0.179 0.177 0.158 0.168 0.150 3.13 nM 0.145 0.147 0.155 0.148 0.152 0.138 1.56 nM 0.125 0.134 0.135 0.141 0.143 0.134

Table 13 is a numerical representation of the affinity maturation anti-TFPI antibody MG1113 potency test result through TF / FVIIa / FX complex test.

Antibody concentration Normalization
(Normalized) TFPI (50 nM) mAb2021 T417 308-4 1015 1023 4017 100 nM 100.0% 13.0% 102.0% 103.3% 102.1% 102.8% 103.6% 102.7% 50 nM 101.2% 102.9% 100.8% 100.1% 103.1% 100.1% 25 nM 100.0% 95.0% 72.3% 29.3% 86.5% 33.4% 12.5 nM 23.7% 26.3% 24.2% 19.4% 24.8% 19.7% 6.25 nM 18.2% 19.4% 19.2% 17.2% 18.2% 16.3% 3.13 nM 15.7% 16.0% 16.8% 16.1% 16.5% 15.0% 1.56 nM 13.6% 14.5% 14.7% 15.4% 15.6% 14.5%

As a result, as shown in Fig. 10, Table 12, and Table 13, the affinity maturation antibody of the MG1113 candidate antibody, anti-TFPI antibody, 1015, 1023, and 4017 antibodies. It was confirmed that the absorbance was increased depending on the antibody treatment concentration, so that all of the candidate antibodies showed an effect of inhibiting TFPI in a concentration dependent manner. No. For the 1015 antibody, the positive control TFPI showed a 100% inhibition of TFPI in the 50 nM treated sample versus a 29.3% inhibition in the 25 nM treated sample compared to the untreated sample. No. In the case of 1023 antibody, the effect of inhibiting TFPI by 50 nM in the treated sample compared to the positive control sample was suppressed by about 100%, and that by the treated sample was about 86.5%. No. 4017 antibody showed a 100% inhibition of TFPI in the 50 nM treated sample versus a positive control sample, and a 33.4% inhibition effect in the 25 nM treated sample. Therefore, 1023 antibody showed the best ability to inhibit TFPI.

Table 14 is a numerical representation of the affinity maturation anti-TFPI antibody MG1113 efficacy test result through TF / FVIIa / FX complex test.

Antibody concentration Normalization
(Normalized) TFPI (50 nM) mAb2021 T417 308-4 1023 1123 A67 100 nM 0.955 0.143 0.966 0.945 0.926 0.935 0.905 0.907 50 nM 0.951 0.908 0.909 0.905 0.770 0.895 25 nM 0.955 0.880 0.716 0.923 0.272 0.914 12.5 nM 0.233 0.251 0.222 0.259 0.157 0.290 6.25 nM 0.180 0.186 0.185 0.190 0.150 0.196 3.13 nM 0.171 0.160 0.164 0.167 0.151 0.177 1.56 nM 0.151 0.145 0.154 0.153 0.140 0.154

Table 15 is a numerical representation of the MG1113 efficacy test result of the affinity maturation anti-TFPI antibody through the TF / FVIIa / FX complex test.

Antibody concentration Normalization
(Normalized) TFPI (50 nM) mAb2021 T417 308-4 1023 1123 A67 100 nM 100.0% 15.0% 101.2% 99.0% 97.0% 98.0% 94.8% 95.0% 50 nM 99.6% 95.1% 95.2% 94.8% 80.7% 93.7% 25 nM 100.1% 92.2% 75.0% 96.6% 28.5% 95.8% 12.5 nM 24.4% 26.3% 23.2% 27.1% 16.4% 30.3% 6.25 nM 18.8% 19.5% 19.4% 19.9% 15.7% 20.5% 3.13 nM 17.9% 16.8% 17.2% 17.5% 15.8% 18.5% 1.56 nM 15.8% 15.2% 16.1% 16.0% 14.7% 16.1%

As shown in Fig. 11, Table 14, and Table 15, the results of the above tests show that the most excellent effect was obtained. 1023 and another affinity maturation antibody (No. 1123 and A67 antibodies were evaluated. It was confirmed that the absorbance was increased depending on the treatment concentration of the antibody, and thus it was confirmed that the effect of suppressing TFPI was increased in all of the candidate antibodies in a concentration-dependent manner. No. In the case of the 1023 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 94.8% in the 50 nM treated sample compared with the untreated sample, and a 96.6% inhibitory effect in the 25 nM treated sample. No. In the case of antibody 1123, the effect of suppressing TFPI by about 80.7% in the 50 nM treated sample versus the positive control sample and the effect of suppressing the 25 nM treated sample by 28.5% were shown. In the case of the A67 antibody, the effect of inhibiting TFPI by 93.7% in the 50 nM treated sample versus the positive control sample and the effect of inhibiting by 95.8% in the 25 nM treated sample were shown. Therefore, 1023 antibody and A67 antibody showed similar TFPI inhibitory abilities.

Table 16 is a numerical representation of the affinity maturation anti-TFPI antibody MG1113 potency assay results through TF / FVIIa / FX conjugate test.

Antibody concentration Normalization
(Normalized) TFPI (50 nM) mAb2021 T417 1023 A67 3203 3241 100 nM 0.915 0.115 0.952 0.931 0.939 0.947 0.932 0.937 50 nM 0.953 0.938 0.938 0.938 0.934 0.935 25 nM 0.932 0.894 0.914 0.908 0.424 0.911 12.5 nM 0.241 0.253 0.290 0.330 0.208 0.305 6.25 nM 0.156 0.183 0.185 0.195 0.173 0.197 3.13 nM 0.143 0.158 0.160 0.172 0.162 0.171 1.56 nM 0.137 0.160 0.135 0.149 0.147 0.157

Table 17 quantifies the MG1113 efficacy results of the affinity maturation anti-TFPI antibody through the TF / FVIIa / FX conjugate test.

Antibody concentration Normalization
(Normalized) TFPI (50 nM) mAb2021 T417 1023 A67 3203 3241 100 nM 100.0% 12.6% 104.0% 101.7% 102.6% 103.5% 101.8% 102.3% 50 nM 104.1% 102.5% 102.5% 102.5% 102.1% 102.1% 25 nM 101.9% 97.7% 99.8% 99.2% 46.3% 99.6% 12.5 nM 26.3% 27.7% 31.6% 36.1% 22.7% 33.3% 6.25 nM 17.0% 20.0% 20.2% 21.3% 18.9% 21.5% 3.13 nM 15.6% 17.2% 17.5% 18.7% 17.7% 18.7% 1.56 nM 15.0% 17.5% 14.7% 16.3% 16.1% 17.2%

Also, as shown in Fig. 12, Table 16, and Table 17, the results of the above tests show that the most excellent effect was obtained. 1023 and A67 and affinity maturation. 3203 and No. 3241 antibody was evaluated. It was confirmed that the absorbance was increased depending on the treatment concentration of the antibody, and thus it was confirmed that the effect of suppressing TFPI was increased in all of the candidate antibodies in a concentration-dependent manner. No. For the 1023 antibody, the positive control TFPI showed a 100% inhibition of TFPI in the 50 nM treated sample versus a 99.8% inhibition in the 25 nM treated sample compared to the untreated sample. The A67 antibody showed a 100% inhibition of TFPI in the 50 nM treated sample versus a 99.2% inhibition in the 25 nM treated sample compared to the positive control sample. No. In the case of the 3203 antibody, the effect of inhibiting TFPI by 50% in the 50 nM treated sample versus the positive control sample was inhibited by about 46.3% in the 25 nM treated sample. No. In the case of the 3241 antibody, the effect of inhibiting TFPI by 50 nM in the treated sample compared with the positive control sample was suppressed by about 99.6%, and the effect by the 25 nM treated sample was suppressed by about 99.6%. Therefore, 1023 antibody and A67 antibody and No. 3241 antibody showed similar TFPI inhibitory abilities.

Table 18 is a numerical representation of the affinity maturation anti-TFPI antibody MG1113 potency assay results through TF / FVIIa / FX conjugate test.

Antibody concentration Normalization
(Normalized) TFPI (50 nM) mAb2021 T417 1023 2 3 8 100 nM 0.809 0.04 0.805 0.745 0.810 0.835 0.842 0.834 50 nM 0.733 0.509 0.652 0.735 0.743 0.673 25 nM 0.344 0.154 0.216 0.416 0.527 0.391 12.5 nM 0.082 0.072 0.079 0.083 0.141 0.088 6.25 nM 0.050 0.050 0.056 0.052 0.059 0.052 3.13 nM 0.047 0.049 0.048 0.045 0.046 0.050 1.56 nM 0.043 0.045 0.050 0.045 0.045 0.041

Table 19 is a numerical representation of the affinity maturation anti-TFPI antibody MG1113 potency assay results through TF / FVIIa / FX complex test.

Antibody concentration Normalization
(Normalized) TFPI (50 M) mAb2021 T417 1023 2 3 8 100 nM 100.0% 4.9% 99.4% 92.0% 100.1% 103.2% 104.1% 103.0% 50 nM 90.6% 62.9% 80.5% 90.9% 91.8% 83.2% 25 nM 42.5% 19.0% 26.6% 51.4% 65.1% 48.3% 12.5 nM 10.1% 8.9% 9.7% 10.3% 17.4% 10.8% 6.25 nM 6.1% 6.2% 6.9% 6.4% 7.2% 6.4% 3.13 nM 5.7% 6.0% 5.9% 5.6% 5.7% 6.1% 1.56 nM 5.3% 5.5% 6.2% 5.5% 5.6% 5.1%

In addition, as shown in FIG. 13, Table 18 and Table 19, the FX treatment concentration was changed from 17 nM to 5 nM level in order to decrease the reaction rate and increase the resolution for confirming the effect between the candidate antibodies. Through the above test, 1023 antibody and affinity maturation. 2, 3, and 8 antibodies were evaluated. It was confirmed that the absorbance was increased depending on the treatment concentration of the antibody, and thus it was confirmed that the effect of suppressing TFPI was increased in all of the candidate antibodies in a concentration-dependent manner. No. In the case of 1023 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 80.5% in the 50 nM treated sample compared with the untreated sample, and an effect of suppressing about 26.6% in the 25 nM treated sample. No. 2 antibody showed an effect of inhibiting TFPI by about 90.9% in the 50 nM treated sample versus about 51.4% in the 25 nM treated sample compared to the positive control sample. No. 3 antibody showed an effect of inhibiting TFPI by about 91.8% in the 50 nM treated sample versus about 65.1% in the 25 nM treated sample compared to the positive control sample. No. 8 antibody showed an effect of suppressing TFPI by about 83.2% in the 50 nM treated sample compared to the positive control sample and by 48.3% in the case of the 25 nM treated sample.

Table 20 is a numerical representation of the affinity maturation anti-TFPI antibody MG1113 potency assay results through TF / FVIIa / FX conjugate test.

Antibody concentration Normalization
(Normalized) TFPI (50 nM) mAb2021 T417 1023 12 13 1202 100 nM 0.848 0.035 0.852 0.810 0.846 0.847 0.859 0.859 50 nM 0.730 0.600 0.681 0.803 0.818 0.843 25 nM 0.462 0.273 0.371 0.489 0.528 0.509 12.5 nM 0.105 0.074 0.088 0.097 0.101 0.091 6.25 nM 0.062 0.050 0.071 0.067 0.075 0.059 3.13 nM 0.046 0.047 0.051 0.048 0.054 0.051 1.56 nM 0.044 0.045 0.041 0.041 0.046 0.043

Table 21 is a numerical representation of the affinity maturation anti-TFPI antibody MG1113 potency test result through TF / FVIIa / FX complex test.

Antibody concentration Normalization
(Normalized) TFPI (50 nM) mAb2021 T417 1023 12 13 1202 100 nM 100.0% 4.1% 100.4% 95.5% 99.8% 99.9% 101.3% 101.2% 50 nM 86.1% 70.7% 80.3% 94.6% 96.5% 99.4% 25 nM 54.4% 32.1% 43.7% 57.7% 62.2% 60.0% 12.5 nM 12.4% 8.7% 10.3% 11.4% 11.9% 10.7% 6.25 nM 7.3% 5.8% 8.3% 7.8% 8.8% 6.9% 3.13 nM 5.4% 5.5% 6.0% 5.6% 6.4% 6.0% 1.56 nM 5.2% 5.2% 4.8% 4.8% 5.4% 5.1%

As shown in Fig. 14, Table 20 and Table 21, no. 1023 antibody and affinity maturation. 12, 13, 1202 antibodies were evaluated. It was confirmed that the absorbance was increased depending on the treatment concentration of the antibody, and thus it was confirmed that the effect of suppressing TFPI was increased in all of the candidate antibodies in a concentration-dependent manner. No. In the case of 1023 antibody, the positive control TFPI showed an effect of suppressing TFPI by about 80.3% in the 50 nM treated sample compared with the untreated sample, and an effect of suppressing about 43.7% in the 25 nM treated sample. No. 12 antibody showed an effect of inhibiting TFPI by about 94.6% in the 50 nM treated sample versus about 57.7% in the 25 nM treated sample compared to the positive control sample. No. 13 antibody showed an effect of inhibiting TFPI by about 96.5% in a 50 nM treated sample versus about 62.2% in a 25 nM treated sample compared to a positive control sample. No. In the case of the 1202 antibody, the effect of inhibiting TFPI by about 99.4% in the 50 nM treated sample compared with the positive control sample and the effect of suppressing about 60.0% in the 25 nM treated sample were shown.

Example 7 Measurement of Thrombin Production

The blood coagulation mechanism is divided into an intrinsic pathway and an extrinsic pathway. It is known that the most important role of the exogenous pathway by TF (Tissue factor) is the explosive generation of thrombin which is very important as an active feedback function in the blood coagulation mechanism and is generated very rapidly. The most important factors in this mechanism are TF, FVII, and FX. If TF and FVIIa are complexed by an external signal, FX is activated by FXa. Then, FXa activates prothrombin to thrombin, which acts on blood clotting by transforming fibrinogen into fibrin. However, TFPI binds FXa and inhibits FXa, thereby interfering with blood coagulation. The thrombin production test was performed by treating a sample to be evaluated with plasma and then converting the fluorogenic substrate into a fluorescent substance in the presence of an active agent (PPP reagent, PPP-Reagent Low reagent) Is a method of verifying the amount of thrombin produced by the amount of fluorescent material, and measuring the amount actually produced by correcting it with the amount of thrombin already known as a calibrator.

Add 20 μL of the PPP reagent (PPP-Reagent Low solution) to the sample loading well and 20 μL of the calibrator solution in the well of the 96-well plate (round bottom immulon 2HB 96 well plate). Pre-dissolve the anti-TFPI candidate antibody Diluted at a concentration of 0.3125, 0.625, 1.25, or 2.5 nM with a diluted FVIII-deficient plasma, and then treated at room temperature for 10 minutes so that TFPI and the anti-TFPI candidate antibody can bind.

Add a sample dilution (FVIII-deficient plasma) to the calibrator and blank wells and 80 μL of the diluted test solution in the remaining wells. If you press the start button at the bottom of the software screen, the cleaning is executed first. Place the inlet tube in distilled water warmed to 37 ° C in a water bath and place the outlet tube in an empty container for cleaning. When cleaning is finished, press next button and empty process. Place the injection tube in the FluCa solution warmed to 37 ℃ and prime it to fill the solution. After attaching the drain tube to the M-hole in the dispenser and pressing the next button, 20 μL of FluCa solution is automatically dispensed into each well, followed by shaking and analysis.

As a result, as shown in FIG. 15, among the affinity maturation antibody candidates selected through the Fxa activity measurement test and the TF / FVIIa / FXa complex test, For the 1023 antibody, a comparative test for measurement of thrombin production using a T417 chimeric antibody was performed. Compared to the blank treated with the sample diluent alone, the T417 antibody at about 2.5 nM contained about 335%, 1023 antibody showed about thrombin peak value of about 401%. In the case of ETP showing the total production amount of thrombin, about 293% in the case of the T417 antibody in the 2.5 nM treated sample compared with the negative control (antibody member) And that of 1023 antibody was increased to about 309%. Comparison of the two antibodies revealed that the antibody derived from the affinity maturation. 1023 antibody was more effective than the T417 antibody.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Mogam Biotechnology Institute <120> Novel Antibody Binding to TFPI, and Composition Comprising the          Same <130> P15-B263 <160> 167 <170> Kopatentin 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> VH FR1 Fo <400> 1 gaagtccagc tggtggagtc tggaggt 27 <210> 2 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VH FR1 Re_S <400> 2 cggggcctga cgaacccagt tcatggcata gctgctgaag gtgaagccgc tcgctgc 57 <210> 3 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VH FR1 Re_H <400> 3 cggggcctga cgaacccagt tcatggcata atggctgaag gtgaagccgc tcgctgc 57 <210> 4 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VH FR1 Re_K <400> 4 cggggcctga cgaacccagt tcatggcata tttgctgaag gtgaagccgc tcgctgc 57 <210> 5 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VH FR1 Re_R <400> 5 cggggcctga cgaacccagt tcatggcata tctgctgaag gtgaagccgc tcgctgc 57 <210> 6 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VH FR1 Re_T <400> 6 cggggcctga cgaacccagt tcatggcata agtgctgaag gtgaagccgc tcgctgc 57 <210> 7 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VH FR1 Re_Y <400> 7 cggggcctga cgaacccagt tcatggcata atagctgaag gtgaagccgc tcgctgc 57 <210> 8 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VH FR1 Re_I <400> 8 cggggcctga cgaacccagt tcatggcata aatgctgaag gtgaagccgc tcgctgc 57 <210> 9 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VH FR1 Re_L <400> 9 cggggcctga cgaacccagt tcatggcata aaggctgaag gtgaagccgc tcgctgc 57 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Fo <400> 10 tatgccatga actgggttcg tcaggcc 27 <210> 11 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Re_T-YH-EQDH <400> 11 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg tgtttgaccc 60 accggttgtg atggtgctga cccattccaa gcc 93 <210> 12 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Re_T-RK-EQDH <400> 12 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg ttyttgaccc 60 accggttgtg atggtgctga cccattccaa gcc 93 <210> 13 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Re_FYLH-YH-EQDH <400> 13 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg tgtttgaccc 60 acctwrtgtg atggtgctga cccattccaa gcc 93 <210> 14 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Re_FYLH-RK-EQDH <400> 14 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg ttyttgaccc 60 acctwrtgtg atggtgctga cccattccaa gcc 93 <210> 15 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Re_KRI-YH-EQDH <400> 15 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg tgtttgaccc 60 accthttgtg atggtgctga cccattccaa gcc 93 <210> 16 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Re_KRI-RK-EQDH <400> 16 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg ttyttgaccc 60 accthttgtg atggtgctga cccattccaa gcc 93 <210> 17 <211> 93 <212> DNA <213> Artificial Sequence <220> VH FR2 Re_T-YH-EQDH_ # 2 <400> 17 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg tatrtgaccc 60 accggttgtg atggtgctga cccattccaa gcc 93 <210> 18 <211> 93 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > VH FR2 Re_T-RK-EQDH_ # 2 <400> 18 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg ttyttgaccc 60 accggttgtg atggtgctga cccattccaa gcc 93 <210> 19 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Re_FYLH-YH-EQDH_ # 2 <400> 19 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg tatrtgaccc 60 acctwrtgtg atggtgctga cccattccaa gcc 93 <210> 20 <211> 93 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > VH FR2 Re_FYLH-RK-EQDH_ # 2 <400> 20 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg ttyttgaccc 60 acctwrtgtg atggtgctga cccattccaa gcc 93 <210> 21 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Re_KRI-YH-EQDH_ # 2 <400> 21 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg tatrtgaccc 60 accthttgtg atggtgctga cccattccaa gcc 93 <210> 22 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> VH FR2 Re_KRI-RK-EQDH_ # 2 <400> 22 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg ttyttgaccc 60 accthttgtg atggtgctga cccattccaa gcc 93 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> VH FR3 Fo <400> 23 gggcgcttca ccatttcccg cgataac 27 <210> 24 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VH FR3 Re_N <400> 24 gccctggccc caataatcca tcagaaaatt gccatcctgg cgcgcgcaat aatataccgc 60                                                                           60 <210> 25 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VH FR3 Re_F <400> 25 gccctggccc caataatcca tcagaaaaaa gccatcctgg cgcgcgcaat aatataccgc 60                                                                           60 <210> 26 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VH FR3 Re_H <400> 26 gccctggccc caataatcca tcagaaaatg gccatcctgg cgcgcgcaat aatataccgc 60                                                                           60 <210> 27 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VH FR3 Re_K <400> 27 gccctggccc caataatcca tcagaaattt gccatcctgg cgcgcgcaat aatataccgc 60                                                                           60 <210> 28 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VH FR3 Re_Q <400> 28 gccctggccc caataatcca tcagaaattg gccatcctgg cgcgcgcaat aatataccgc 60                                                                           60 <210> 29 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VH FR3 Re_R <400> 29 gccctggccc caataatcca tcagaaatct gccatcctgg cgcgcgcaat aatataccgc 60                                                                           60 <210> 30 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VH FR3 Re_Y <400> 30 gccctggccc caataatcca tcagaaaata gccatcctgg cgcgcgcaat aatataccgc 60                                                                           60 <210> 31 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> VH Final Fo <400> 31 ggttctggtg gtggtggttc tgctagcgac gtggtgatga cacagacgcc gctg 54 <210> 32 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> VH Final Re <400> 32 ggagctcaca gtcaccagcg tgccctggcc ccaataatcc atcagaaa 48 <210> 33 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Fo <400> 33 gacgtggtga tgacacagac gccgctg 27 <210> 34 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_S <400> 34 gagccaattc agatacgtct tgccgtcgga gtccagcagc gactggcttg atttgca 57 <210> 35 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_I <400> 35 gagccaattc agatacgtct tgccgtcaat gtccagcagc gactggcttg atttgca 57 <210> 36 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_L <400> 36 gagccaattc agatacgtct tgccgtcaag gtccagcagc gactggcttg atttgca 57 <210> 37 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_N <400> 37 gagccaattc agatacgtct tgccgtcagc gtccagcagc gactggcttg atttgca 57 <210> 38 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_Q <400> 38 gagccaattc agatacgtct tgccgtcttg gtccagcagc gactggcttg atttgca 57 <210> 39 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_R <400> 39 gagccaattc agatacgtct tgccgtctct gtccagcagc gactggcttg atttgca 57 <210> 40 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_F <400> 40 gagccaattc agatacgtct tgccgtcaaa gtccagcagc gactggcttg atttgca 57 <210> 41 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_K <400> 41 gagccaattc agatacgtct tgccgtcttt gtccagcagc gactggcttg atttgca 57 <210> 42 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_T <400> 42 gagccaattc agatacgtct tgccgtcagt gtccagcagc gactggcttg atttgca 57 <210> 43 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> VL FR1 Re_V <400> 43 gagccaattc agatacgtct tgccgtcaac gtccagcagc gactggcttg atttgca 57 <210> 44 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> VL FR2 Fo <400> 44 gacggcaaga cgtatctgaa ttggctccag 30 <210> 45 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> VL FR2 Re_T-YH <400> 45 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaac ggaaagtrgg tgccctgcca 60 gcaatagtag acgcc 75 <210> 46 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> VL FR2 Re_T-LIHQNK <400> 46 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaac ggaaawwkgg tgccctgcca 60 gcaatagtag acgcc 75 <210> 47 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> VL FR2 Re_FYIN-YH <400> 47 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaac ggaaagtraw wgccctgcca 60 gcaatagtag acgcc 75 <210> 48 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> VL FR2 Re_FYIN-LIHQNK <400> 48 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaac ggaaawwkaw wgccctgcca 60 gcaatagtag acgcc 75 <210> 49 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> VL Final Re <400> 49 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaa 39 <210> 50 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> VL Final Fo SfiI <400> 50 cgtggcccag gcggccgacg tggtgatgac acagacgccg ctg 43 <210> 51 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> VL Final Fo NruI <400> 51 ctatcgcgat tgcagtggca ctggctggtt tcg 33 <210> 52 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> VL Overlapping Fo <400> 52 ggcacgctgg tgactgtgag ctccggaggc ggcggaagtg gcggaggagg cagcggcgga 60 ggcgggagtg acgtggtgat gacacagacg ccgctg 96 <210> 53 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> VL Final Re <400> 53 gtcctcttca gaaataagct tttgttcgga tccgcgttta atttcaacct tagtgccttg 60 gccgaacgta aa 72 <210> 54 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> VH Homologous recombination <400> 54 gctctgcagg ctagtggtgg tggtggttct ggtggtggtg gttctggtgg tggtggttct 60 gctagc 66 <210> 55 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VL Homologous recombination <400> 55 ttgttatcag atctcgagct attacaagtc ctcttcagaa ataagctttt gttcggatcc 60                                                                           60 <210> 56 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1001_Variable heavy chain <400> 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 57 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 1001_Variable light chain <400> 57 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 58 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1015_Variable heavy chain <400> 58 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 59 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1021_Variable heavy chain <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 60 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1023_Variable heavy chain <400> 60 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 61 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1024_Variable heavy chain <400> 61 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 62 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 1024_Variable light chain <400> 62 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Leu              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 63 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1104_Variable heavy chain <400> 63 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 64 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 1104_Variable light chain <400> 64 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Val              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 65 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1123_Variable heavy chain <400> 65 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 66 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1202_Variable heavy chain <400> 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr  65 70 75 80 Leu Lys Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 67 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 1208_Variable light chain <400> 67 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Val              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr Tyr Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 68 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1216_Variable heavy chain <400> 68 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr              20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 69 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 1216_Variable light chain <400> 69 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 70 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1223_Variable heavy chain <400> 70 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 71 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1234_Variable heavy chain <400> 71 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 72 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 1234_Variable light chain <400> 72 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Glu Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 73 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1243_Variable heavy chain <400> 73 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 His Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 74 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 1248_Variable heavy chain <400> 74 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 75 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 3007_Variable heavy chain <400> 75 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr              20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Leu Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 76 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 3016_Variable heavy chain <400> 76 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 77 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 3016_Variable light chain <400> 77 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 78 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 3024_Variable heavy chain <400> 78 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 79 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 3120_Variable heavy chain <400> 79 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 80 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 3131_Variable heavy chain <400> 80 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Gln Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 81 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 3203_Variable heavy chain <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 82 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 4011_Variable heavy chain <400> 82 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Tyr Ser Tyr              20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 83 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 4011_Variable light chain <400> 83 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Val              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 84 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 4017_Variable heavy chain <400> 84 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 85 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 4034_Variable heavy chain <400> 85 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 86 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 4041_Variable heavy chain <400> 86 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 87 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 4141_Variable heavy chain <400> 87 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 88 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 4146_Variable heavy chain <400> 88 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 89 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 4206_Variable heavy chain <400> 89 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr  65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 90 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 4208_Variable heavy chain <400> 90 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Ser 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 91 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 4208_Variable light chain <400> 91 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Thr              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 92 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Clone 4278_Variable heavy chain <400> 92 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr              20 25 30 Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Leu Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Tyr Leu Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 93 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Clone 4287_Variable heavy chain <400> 93 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr              20 25 30 Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln His Pro Tyr Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 94 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 4_Variable heavy chain <400> 94 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 95 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 6_Variable heavy chain <400> 95 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 96 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 9_Variable heavy chain <400> 96 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 97 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 11_Variable heavy chain <400> 97 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 98 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 12_Variable heavy chain <400> 98 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 99 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 13_Variable heavy chain <400> 99 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 100 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 14_Variable light chain <400> 100 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Ser Ser Leu Leu Asp Ser              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr Tyr Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 101 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 15_Variable light chain <400> 101 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Ser Ser Leu Leu Asp Ser              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Phe Tyr Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 102 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 16_Variable heavy chain <400> 102 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr              20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 103 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 17_Variable heavy chain <400> 103 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 104 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 18_Variable heavy chain <400> 104 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr              20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 105 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 19_Variable heavy chain <400> 105 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr              20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Lys Lys Gly Ser Phe Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Glu Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 106 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 20_Variable heavy chain <400> 106 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr              20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Lys Lys Gly Gly Ser Phe Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Glu Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 107 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 21_Variable heavy chain <400> 107 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Lys Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 108 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 22_Variable light chain <400> 108 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Ser Ser Leu Leu Asp Val              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr Tyr Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 109 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 23_Variable heavy chain <400> 109 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr              20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 110 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Clone 1001_Variable heavy chain CDR 1 <400> 110 Ser Tyr Ala Met Asn   1 5 <210> 111 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 1001_Variable heavy chain CDR 2 <400> 111 Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Asp   1 5 10 15 Gly     <210> 112 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Clone 1001_Variable heavy chain CDR 3 <400> 112 Gln Asp Gly Asn Phe Leu Met Asp Tyr   1 5 <210> 113 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Clone 1001_Variable light chain CDR 1 <400> 113 Lys Ser Ser Gln Ser Leu Leu Asp Ile Asp Gly Lys Thr Tyr Leu Asn   1 5 10 15 <210> 114 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Clone 1001_Variable light chain CDR 2 <400> 114 Leu Val Ser Lys Leu Asp Ser   1 5 <210> 115 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Clone 1001_Variable light chain CDR 3 <400> 115 Trp Gln Gly Thr His Phe Pro Phe   1 5 <210> 116 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 1015_Variable heavy chain CDR 2 <400> 116 Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val Glu   1 5 10 15 Gly     <210> 117 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Clone 1021_Variable heavy chain CDR 3 <400> 117 Gln Asp Gly His Phe Leu Met Asp Tyr   1 5 <210> 118 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Clone 1024_Variable heavy chain CDR 1 <400> 118 Ser Tyr Ala Met Ser   1 5 <210> 119 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Clone 1024_Variable light chain CDR 1 <400> 119 Lys Ser Ser Gln Ser Leu Leu Asp Leu Asp Gly Lys Thr Tyr Leu Asn   1 5 10 15 <210> 120 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 1104_Variable heavy chain CDR 2 <400> 120 Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val Gln   1 5 10 15 Gly     <210> 121 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Clone 1104_Variable light chain CDR 1 <400> 121 Lys Ser Ser Gln Ser Leu Leu Asp Val Asp Gly Lys Thr Tyr Leu Asn   1 5 10 15 <210> 122 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Clone 1208_Variable light chain CDR 3 <400> 122 Trp Gln Gly Thr Tyr Leu Pro Phe   1 5 <210> 123 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 1214_Variable heavy chain CDR 2 <400> 123 Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Glu   1 5 10 15 Gly     <210> 124 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Clone 1216_Variable heavy chain CDR 1 <400> 124 His Tyr Ala Met Asn   1 5 <210> 125 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Clone 1216_Variable light chain CDR 3 <400> 125 Trp Gln Gly Thr His Leu Pro Phe   1 5 <210> 126 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 1234_Variable heavy chain CDR 2 <400> 126 Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Gln   1 5 10 15 Gly     <210> 127 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 1243_Variable heavy chain CDR 2 <400> 127 Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val His   1 5 10 15 Gly     <210> 128 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 1248_Variable heavy chain CDR 2 <400> 128 Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val Asp   1 5 10 15 Gly     <210> 129 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 3007_Variable heavy chain CDR 2 <400> 129 Thr Ile Thr Leu Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Gln   1 5 10 15 Gly     <210> 130 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Clone 3016_Variable light chain CDR 1 <400> 130 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn   1 5 10 15 <210> 131 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Clone 3120_Variable heavy chain CDR 3 <400> 131 Gln Asp Gly Tyr Phe Leu Met Asp Tyr   1 5 <210> 132 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Clone 3131_Variable heavy chain CDR 3 <400> 132 Gln Asp Gly Gln Phe Leu Met Asp Tyr   1 5 <210> 133 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Clone 4278_Variable heavy chain CDR 1 <400> 133 Lys Tyr Ala Met Asn   1 5 <210> 134 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 4278_Variable heavy chain CDR 2 <400> 134 Thr Ile Thr Leu Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Asp   1 5 10 15 Gly     <210> 135 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Clone 4278_Variable heavy chain CDR 3 <400> 135 Gln Tyr Leu Asp Gly Asn Phe Leu Met Asp Tyr   1 5 10 <210> 136 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Clone 4287_Variable heavy chain CDR 3 <400> 136 Gln His Pro Tyr Gly Asn Phe Leu Met Asp Tyr   1 5 10 <210> 137 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Clone 1_Variable light chain CDR 1 <400> 137 Lys Ser Ser Pro Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn   1 5 10 15 <210> 138 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Clone 2_Variable light chain CDR 1 <400> 138 Lys Ser Ser Ser Leu Leu Asp Ile Asp Gly Lys Thr Tyr Leu Asn   1 5 10 15 <210> 139 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Clone 3_Variable light chain CDR 1 <400> 139 Lys Ser Ser Ser Leu Leu Asp Val Asp Gly Lys Thr Tyr Leu Asn   1 5 10 15 <210> 140 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Clone 14_Variable light chain CDR 3 <400> 140 Trp Gln Gly Thr Tyr Phe Pro Phe   1 5 <210> 141 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Clone 15_Variable light chain CDR 3 <400> 141 Trp Gln Gly Phe Tyr Phe Pro Phe   1 5 <210> 142 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Clone 16_Variable heavy chain CDR 1 <400> 142 His Tyr Ala Met Thr   1 5 <210> 143 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Clone 17_Variable heavy chain CDR 1 <400> 143 Ser Tyr Ala Met Thr   1 5 <210> 144 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Clone 19_Variable heavy chain CDR 1 <400> 144 Gln Tyr Ala Met Asn   1 5 <210> 145 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 19_Variable heavy chain CDR 2 <400> 145 Thr Ile Thr Lys Lys Gly Ser Phe Thr Tyr Tyr Ala Asp Ser Val Asp   1 5 10 15 Gly     <210> 146 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Clone 19_Variable heavy chain CDR 3 <400> 146 Gln Asp Gly Glu Phe Leu Met Asp Tyr   1 5 <210> 147 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 20_Variable heavy chain CDR 2 <400> 147 Thr Ile Lys Lys Gly Gly Ser Phe Thr Tyr Tyr Ala Asp Ser Val Asp   1 5 10 15 Gly     <210> 148 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 21_Variable heavy chain CDR 2 <400> 148 Thr Ile Thr Lys Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Asp   1 5 10 15 Gly     <210> 149 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Clone 23_Variable heavy chain CDR 1 <400> 149 His Tyr Ala Met Asn   1 5 <210> 150 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> VH Fo <400> 150 tgctgtgggt gagtggtacc tgtggggaag tccagctggt ggagtctgga ggt 53 <210> 151 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> VH Re <400> 151 agtgggaaca cggagggccc cttggtgctg gcggagctca cagtcaccag cgtgcc 56 <210> 152 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> VL Fo <400> 152 tgctgtgggt gagtggtacc tgtggggacg tggtgatgac acagacgccg ctg 53 <210> 153 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> VL Re_CL overlap <400> 153 gatgaacaca gaaggggcag ccaccgtgcg tttaatttca accttagtgc cttggccgaa 60 cgtaaa 66 <210> 154 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Ck Fo <400> 154 acggtggctg ccccttctgt gttcatc 27 <210> 155 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Ck Re <400> 155 gattggatcc aagcttacta gcactcaccc ctgttgaaag actta 45 <210> 156 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone A24_Variable heavy chain <400> 156 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Ser Tyr              20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Arg Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 157 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone A25_Variable light chain <400> 157 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Arg              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 158 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone A52_Variable heavy chain <400> 158 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 159 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone A63_Variable heavy chain <400> 159 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 160 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone A71_Variable heavy chain <400> 160 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 His Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 161 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone A74_Variable heavy chain <400> 161 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 162 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Clone A25_Variable light chain CDR 1 <400> 162 Lys Ser Ser Gln Ser Leu Leu Asp Arg Asp Gly Lys Thr Tyr Leu Asn   1 5 10 15 <210> 163 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone A71_Variable heavy chain CDR 2 <400> 163 Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val His   1 5 10 15 Gly     <210> 164 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Clone A74_Variable heavy chain CDR 1 <400> 164 Ser Tyr Ala Met His   1 5 <210> 165 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> HUMAN Tissue factor pathway inhibitor <400> 165 Met Ile Tyr Thr Met Lys Lys Val His Ala Leu Trp Ala Ser Val Cys   1 5 10 15 Leu Leu Leu Asn Leu Pro Ala Pro Leu Asn Ala Asp Ser Glu Glu              20 25 30 Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu Pro Pro Leu Lys          35 40 45 Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys      50 55 60 Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu  65 70 75 80 Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser                  85 90 95 Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile             100 105 110 Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys Phe Leu Glu         115 120 125 Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn     130 135 140 Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly Gly Cys Leu Gly 145 150 155 160 Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys Asn Ile Cys Glu                 165 170 175 Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn             180 185 190 Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys Val Ser Seru         195 200 205 Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp Arg Gly     210 215 220 Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val Ile Gly 225 230 235 240 Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly Asn Glu Asn Asn                 245 250 255 Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala Cys Lys Lys Gly Phe Ile             260 265 270 Gln Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys         275 280 285 Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met     290 295 300 <210> 166 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Clone 3241_Variable heavy chain <400> 166 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 167 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Clone 1_Variable light chain <400> 167 Asp Val Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Ser Ser Leu Leu Asp Ser              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg

Claims (11)

An antibody specifically binding to a TFPI (Tissue Factor Pathway Inhibitor) represented by SEQ ID NO: 165, comprising a heavy chain variable region comprising a heavy chain CDR and a light chain variable region comprising a light chain CDR:
i) a heavy chain variable region comprising the heavy chain CDR1 of the amino acid sequence of SEQ ID NO: 110, the heavy chain CDR2 of the amino acid sequence of SEQ ID NO: 111, and the heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 112; A light chain variable region comprising the light chain CDR1 of the amino acid sequence of SEQ ID NO: 113, the light chain CDR2 of the amino acid sequence of SEQ ID NO: 114, and the light chain CDR3 of the amino acid sequence of SEQ ID NO: 115;
ii) a heavy chain variable region comprising the heavy chain CDR1 of the amino acid sequence of SEQ ID NO: 110, the heavy chain CDR2 of the amino acid sequence of SEQ ID NO: 116, and the heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 117; A light chain variable region comprising the light chain CDR1 of the amino acid sequence of SEQ ID NO: 113, the light chain CDR2 of the amino acid sequence of SEQ ID NO: 114, and the light chain CDR3 of the amino acid sequence of SEQ ID NO: 115;
iii) a heavy chain variable region comprising the heavy chain CDR1 of the amino acid sequence of SEQ ID NO: 110, the heavy chain CDR2 of the amino acid sequence of SEQ ID NO: 123, and the heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 112; A light chain variable region comprising the light chain CDR1 of the amino acid sequence of SEQ ID NO: 113, the light chain CDR2 of the amino acid sequence of SEQ ID NO: 114, and the light chain CDR3 of the amino acid sequence of SEQ ID NO: 115; or
iv) a heavy chain variable region comprising the heavy chain CDR1 of the amino acid sequence of SEQ ID NO: 110, the heavy chain CDR2 of the amino acid sequence of SEQ ID NO: 123, and the heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 112; And a light chain variable region comprising the light chain CDR1 of the amino acid sequence of SEQ ID NO: 138, the light chain CDR2 of the amino acid sequence of SEQ ID NO: 114, and the light chain CDR3 of the amino acid sequence of SEQ ID NO:
delete delete delete delete The method according to claim 1,
i) a heavy chain variable region of the amino acid sequence of SEQ ID NO: 60 and a light chain variable region of the amino acid sequence of SEQ ID NO: 57;
ii) a heavy chain variable region of the amino acid sequence of SEQ ID NO: 65 and a light chain variable region of the amino acid sequence of SEQ ID NO: 57;
iii) a heavy chain variable region of the amino acid sequence of SEQ ID NO: 66 and a light chain variable region of the amino acid sequence of SEQ ID NO: 57; or
iv) a heavy chain variable region of the amino acid sequence of SEQ ID NO: 166 and a light chain variable region of SEQ ID NO: 57.
7. A nucleic acid encoding an antibody according to claim 1 or 6.
A vector comprising the nucleic acid of claim 7.
9. A host cell comprising the vector of claim 8.
A method for producing an antibody, comprising culturing the host cell of claim 9 to express the antibody.
A pharmaceutical composition for the treatment of hemophilia comprising the antibody according to any one of claims 1 to 6 as an active ingredient.

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