TW201335184A - Human tissue factor antibody and uses thereof - Google Patents

Abstract

The invention relates to a humanized form of an antibody capable of preventing tissue factor (coagulation factor F3) signaling but which does not interfere with Factor VII binding or FX binding to tissue factor and does not prolong coagulation time. The antibody of the invention is useful in treating conditions, such as tumor progression, in which the associated cells express tissue factor and tissue factor signaling occurs.

Description

Human tissue factor antibody and use thereof

The present application claims priority to U.S. Patent Application Serial No. 61/452,674, filed on Mar.

The present invention relates to a human adaptive antibody that binds to human tissue factor and comprises an antigen present on the extravascular tissue of a tumor cell, and the antibody does not inhibit blood coagulation of tissue factor mediators. The invention also relates to methods of using such antibodies to treat cancer conditions such as the presence of human tissue factor and receptor function.

Tissue factor (TF), also known as factor III (F3), tissue thromboplastin or CD142, which is a penetrating membrane glycoprotein with a 219 amino acid extracellular region, and the extracellular region contains 2 A fibronectin type III region and a short intracellular region with a serine residue capable of phosphorylation. TF is a cellular receptor for FVII/FVIIa.

TF, in the form of cellular receptors, exhibits a higher level of tissue-specific distribution in the normal brain, lungs, and placenta, but lower levels in the spleen, thymus, skeletal muscle, and liver. It has also been found in cell-derived microparticles and as a soluble form of selective splicing. In addition to performance in normal organizations, TF has been reported in most masters To be overexpressed within tumor types and in many tumor-derived cell lines (RufW, J. Jhromb Haemost, 5: 1584 to 1587, 2007; Milsom et al., 2009, Aererioscler Thromb Vasc Biol., 29:2005-2014) .

Serum protein is a physiological response to injury that is caused by injury. Exposing blood to proteins containing collagen (intrinsic pathways) and tissue factor (external pathways) causes platelets and plasma protein fibrinogen, which is a coagulation factor, to begin to change. As the blood vessels are damaged, Factor VII (FVII) leaves the circulation pathway and then contacts with tissue factor (TF) expressed on tissue factor-supporting cells (interstitial fibroblasts and white blood cells) to form an activated TF- FVIIa complex. TF-FVIIa activates Factor IX (FIX) and Factor X (FX). FVII can be activated ectopically by TF and activated by thrombin, FXIa, cytosolic, FXII and FXa. TF-FVIIa forms a ternary complex with FXa.

Tissue factor (TF), expressed by non-vascular cells, plays a crucial role in hemostasis by activating blood clotting. The TF line is further involved in a process different from hemostasis and is directly related to function on the cell surface, and the TF line is expressed on the surface. The TF-dependent combination of clotting proteases on vascular and non-vascular cells activates protease-activated receptors (PARs), which are G-protein-linked receptors. Thus, the TF:VIIa complex is capable of inducing cellular signaling through PARs, primarily PAR2 for tumor formation, angiogenesis, tumor progression, and metastasis (Camerer et al., 2000, Proc. USA 97: 5255 to 5260; Riewald and Ruf, 2001, Proc. Natl. Acad. Sci. USA 98:7742 to 7747; 2003, Ruf et al., J Thromb Haemost, 1:1495. 4503; Chen et al., 2001, Thromb Haemost, 86: 334-45).

The ternary complex TF/FVIIa/FXa is formed directly by acting on the TF:VIIa complex of FX, or indirectly after TF:VIIa, which can split FX into FXa, ie FIX becomes FIXa. The TF/FVIIa/FXa complex produces a receptor that can cause a message or activate other receptors such as PAR1-4. TF/FVIIa/FXa complex formation leads to the induction of interleukin-8 (IL-8), which stimulates tumor cell migration (2004 Hjortor et al., Blood 103:3029) To 3037). Both PAR1 and PAR2 are involved in tumor metastasis (Shi et al., Mol Cancer Res., 2:395-402, 2004), whereas activated binary and ternary complexes TF-VIIa and TF-VIIa-FXa It is an activator of PAR2, which also leads to cellular signaling (Rao and Pendurthi, 2005, Aeterioscler Thromb. Vasc. Biol. Journal 25: 47-56). It is therefore of interest to determine whether the oncogenic role of tissue factor can be separated from the role of procoagulant, which has long been suspected of involving tumor movement, extravasation and metastasis mechanisms.

Such as described in Morrisy (published in Thromb Res Journal 52 (3): 247 to 261; US 5,223, 427) and Magdolen (published in Biol Chem, 379: 157 to 165, 1996), corresponding Individual antibodies to tissue factor, which have been used to explore functional and immunological views of ligand binding sites. A monoclonal antibody system capable of binding tissue factor can be used to block thrombotic events by interfering with TF to form or maintain the ability of the TF-VIIa complex or by blocking the ability of the complex to activate FX. Antibodies that bind to tissue factors and do not block blood clotting are also known. Factor VIIa initiates TF signaling blockade rather than coagulation blocking antibodies, such as antibody 10H10, has also been described (Ahamed et al., 2006, Proc Natl Acad Sci USA 103 (38): 13932 to 13937), and such antibodies Opportunities have been provided to understand the role and use of vectors with such activity in solid tumors (Versteeg et al., 2008, Blood 111(1): 190-199). The method and composition for inhibiting tissue factor signaling is disclosed in the published application WO2007056352A3 by Ruf et al., without interfering with patient hemostasis.

Because cancer development is a multifaceted process, it would be desirable to have a therapeutic candidate that is a TF-binding antibody that blocks carcinogenic, metastatic, angiogenic, and anti-apoptotic functions on tumor cells while not interfering with patient hemostasis.

The present invention provides a human adaptive anti-human tissue factor specific antibody for use as a human therapeutic for retaining the binding epitope of the murine antibody 10H10, which does not compete with FVIIa for binding to tissue factor, and thus does not substantially block TF Pre-coagulant of the -VIIa complex, Indoleamine hydrolyzes activity but blocks the signaling of TF-VIIa mediators as well as downstream carcinogenic effects such as interleukin IL-8 release.

The human adaptive anti-system of the invention consists of a human IgG variable region framework that is ligated to the CDR variant residues, which are determined according to the sequence of the CDR sequence by reference to the 10H10 murine antibody and according to the sequence identification number: 6- Representatives 11 and 27. Human frameworks FR1 and FR2 and FR3, which bind to CDRs and CDR variants with FR4, are provided which permit a combination of antibody binding domains with murine antibody 10H10 immunospecificity. In one embodiment of the invention, the six CDR sequences represented by the sequence identifiers: 6-11, or the groups represented by the sequence identifiers: 6, 8-11 and 27, are associated with the human germ cell line FRs. Binding, defined as the non-CDR position of the human antibody IgG variable region, is selected such that the binding affinity to human TF 10H10 is retained. In one aspect, human HC variable region FRs are derived from 1, 3 or 5 members of the human IGHV gene family as represented by the IMGT database. In one aspect, the human LC variable region FRs are derived from two or four members of the human IGKV gene family. In one embodiment, the antibody Fv (HC variable region paired with an LC variable region) comprises an HC variable region selected from SEQ ID NO: 12-21 and is selected from the group consisting of: An LC variable region.

In a specific embodiment, human FRs (HC variable regions that are paired with an LC variable region) that form an antibody Fv comprise IGHV5 and IGKV2 FRs. The antibody of the present invention comprises an HC variable region having the H-CDR3 of SEQ ID NO: 8; one having a sequence selected from the sequence number: 6 And H-CDR1 of a sequence of 62-83; an H-CDR2 having a sequence selected from one of sequence identification numbers: 7, 27 and 84-107; and a selectivity selected from IGVJ4 (SEQ ID NO: 60 ) or its variant HC FR4 region. The antibody of the present invention further comprises those having an LC variable region having an L-CDR1; L-CDR2; and an L-CDR3; and a selectivity selected from IGKJ2 (SEQ ID NO: 61 ) or its variant LC FR4 region. The L-CDR1 has a sequence selected from the group consisting of: SEQ ID NO: 9, 108-116; the L-CDR2 has a sequence selected from one of sequence identification numbers: 10 and 117-120; and the L-CDR3 has an option From the sequence identification number: one of the sequences of 11 and 121-128. In a specific embodiment, the human framework sequence is derived from IGHV5_a and the resulting variable region comprises a sequence selected from the group consisting of: SEQ ID NO: 19, 129-155. In another embodiment, the human framework sequence is derived from IGKV2D40_O1 and the resulting variable region comprises a sequence selected from the group consisting of: SEQ ID NO: 23, 156-163.

The anti-system of the invention may be expressed as a form of an antibody having a binding domain, an H-CDR3, which is derived from an IGHV5_a framework defined as a non-CDR position having the sequence SGYYGNSGFAY (sequence) Identification number: 8), wherein the sequence at the H-CDR-1 position can be represented by the following formula: H-CDR1 GYTFX ₁ X ₂ X ₃ WIE (I) (SEQ ID NO: 83) wherein X1 is selected from A, D, G, I, L, N, P, R, S, T, V and Y; X2 is selected from A, P, S and T and X3 is selected from F, H and Y; The sequence may be GFTFITYWIA (SEQ ID NO: 81); and the sequence at the H-CDR-2 position may be represented by the following formula: H-CDR2 DIX ₁ PGX ₂ GX ₃ TX ₄ (II) (SEQ ID NO: 107) Wherein X1 is selected from I and L, X2 is selected from S and T, X3 is selected from A, F, H and w; and X4 is selected from D, H, I, L and N; Except for H189 where H-CDR2 is DILPASSSTN (sequence identification number: 105).

An antibody of the invention is represented by an antibody having a binding domain derived from an IGKV2D40_O1 framework defined as a non-CDR position, wherein the L-CDR-1 and/or L-CDR-2 and L-CDR are The sequence of -3 has a sequence which can be represented by the following formula: L-CDR1 KSSQSLLX ₁ X ₂ X ₃ X ₄ QX ₅ NYLT ( III ) (SEQ ID NO: 116) wherein X1 is selected from F, P, S, T, W and Y; X2 is selected from F, S, T, R and V; X3 is selected from A, G, P, S, W, Y and V; X4 is selected from G, N and T And X5 are selected from K, R and S; L-CDR2 X ₁ ASTRX ₂ S ( IV ) (SEQ ID NO: 120) wherein X1 is selected from H and W; X2 is selected from D, E And S; L-CDR3 QNDX ₁ X ₂ X ₃ PX ₄ T ( V ) (SEQ ID NO: 128) wherein X1 is selected from D, F and L; X2 is selected from S, T and Y; X3 is selected from W and Y; and X4 is selected from L and M.

Thus, the heavy and light chain CDR residues of the antibody are substantially modified from the CDRs of murine 10H10. For example, according to the above description, the heavy chain of the antibody may be only 70% (3/10 residues in CDR1 have been altered) and 60% (4/10 residues in CDR2 have been altered) and the CDRs of murine 10H10 Similar (CDR3 unchanged). Only 71% (5/17 altered), (71%) (2/7 altered) or 55% (4/9 altered) of the light chain CDR residues were similar to the CDRs of murine 10H10.

The invention further provides human adaptive antibodies that compete for binding to human tissue factor and thus bind to an epitope that is substantially identical to the murine 10H10 antibody on human TF-ECD. The invention further provides a method of treating a human subject using such antibodies, wherein the human subject has a condition in which the TF manifestation and the local biological activity produced by the TF manifest are directly or indirectly related to the condition to be treated.

The invention further provides methods for preparing such antibodies, as well as pharmaceutically acceptable formulations of such antibodies, a container comprising the formulation, and a kit comprising the container, wherein the anti-system of the invention is made For the treatment of the use of human subjects.

abbreviation

TF is a tissue factor, huTF is a human tissue factor, muTF is a mouse tissue factor, cynoTF is a Malay monkey tissue factor, TF-FVIIa is a tissue factor-factor VIIa complex, and TF/FVIIa is a tissue factor-factor VIIa complex. HC is a heavy chain, LC is a light chain, v-region is a variable region, VH is a heavy chain variable region, VL is a light chain variable region, CCD is a charge coupled device, CDR is a complementarity determining region, and CHES is 2-(N -cyclohexylamino)-ethanesulfonic acid, EDTA is ethylenediaminetetraacetic acid, ECD is the extracellular region, and HEPES is N-(2-hydroxyethyl)-piper [mouth + well]-N'-2-B Sulfonic acid, HEK is human embryonic kidney cells, MES is 2-(N-morpholine)ethanesulfonic acid, PAR is protease activated receptor, PBMC is peripheral blood mononuclear cells, PBS phosphate buffer solution, PDB is protein database PEG is polyethylene glycol, SDS PAGE is sodium dodecyl sulfate polyacrylamide gel electrophoresis, SEC is particle size screening chromatography, MAb is monoclonal antibody, FR is frame antibody, and HFA is adapted to human framework. .

Definition and terminology

As used herein, "antibody" includes whole antibodies and any antigen-binding fragments thereof or single chains thereof. Thus, the antibody includes any protein or peptide comprising a molecule comprising at least a portion of an immunoglobulin molecule, such as, but not limited to, a complementarity determining region (CDR) of at least one heavy or light chain or a ligand binding portion thereof, A chain or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or a portion of at least one binding protein, which can be administered to an antibody of the invention. The term "antibody" is intended to further encompass antibodies, truncated fragments, specific portions and variants thereof, including antibody mimetics, or antibody portions comprising mimicking the structure and/or function of a particular antibody or portion or portion thereof, including Chain and single-chain domain antibodies and fragments. A functional fragment includes an antigen binding fragment to a preselected target. Examples of binding fragments encompassing the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH domains; (ii) F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) a Fd fragment consisting of VH and CH domains; (iv) Fv fragment consisting of the VL and VH domains of one arm of the antibody; (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546) consisting of a VH domain; and (vi) isolated Complementarity determining region (CDR). In addition, although the two domains, VL and VH of the Fv fragment are encoded by different genes, they can be joined using a recombinant method, through a synthetic linker, which can make them into a single protein chain. , where VL and VH regions Pairing forms a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, 1988, Bird et al., Science, 242: 423-426, and 1988, Huston et al., Proc. Natl. Acad Sci. USA, 85: 5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and these fragments are screened for utility in the same manner as intact antibodies. Conversely, a scFv construction library can be used to screen for antigen binding capacity and then spliced into other DNA encoding human germ cell line gene sequences using conventional techniques. An example of such a library is "HuCAL: Human Combinatorial Antibody Library" (Knappik, A. et al. J Mol Biol (2000) 296(1): 57-86).

The term "CDR" refers to the complementarity determining region or the hypervariable region amino acid residue of an antibody that is involved in or responsible for antigen binding. The hypervariable region or CDR of a human IgG subtype antibody comprises residues 24-34 (L-CDR1), 50-56 (L-CDR2) and 89-97 (L-CDR3) from the light chain variable region and Amino acid residues such as residues 31-35 (H-CDR1), 50-65 (H-CDR2) and 95-102 (H-CDR3) of the heavy chain variable region, as described by Kabat et al. (1991) National Institutes of Health in Bethesda, Maryland, fifth edition of Public Health Services, sequences of immunologically important proteins), and/or those residues from highly variable loops (ie, residues of the light chain variable region 26) -32 (L1), 50-52 (L2) and 91-96 (L3) and heavy chain variable regions 26-32 (H1), 52-56 (H2) and 95-101 (H3), as in 1987 Chothia and Lesk published in J. Mol. Biol. Journal 196: 901-917 Said). Chothia and Lesk refer to a structurally conservative high-variable loop as a "canonical structure." The framework or FR1-4 residues are those variable region residues that surround the hypervariable regions. The numbering system of Chothia and Lesk takes into account the difference in residue numbers in the ring by showing the extensions represented by lowercase symbols at the specified residues, such as 30a, 30b, 30c, and so on. More recently, a universal coding system has been developed and widely used, known as the International Immunogenetics Information System® (IMGT) (LaFranc et al., 2005, Nucl Acids Res. 33: D593-D597).

Here, the CDRs are referred to by sequential numbering depending on both the amino acid sequence and position within the light or heavy chain. Since the CDR "position" within the immunoglobulin variable region structure is retained between species and is present in a structure called a loop, the numbering system is used by aligning the variable region region sequence according to structural features, It is easy to identify CDRs and framework residues. This information is used to graft and replace CDR residues from a species of immunoglobulin into a receptor framework that is typically derived from a human antibody.

The term "Fc", "Fc-containing protein" or "Fc-containing molecule" as used herein refers to a monomeric, dimeric or heterodimeric protein having at least one immunoglobulin CH2 and CH3 domain. The CH2 and CH3 domains can form at least a portion (e.g., an antibody) of the dimer region of the protein/molecule.

The term "epitope" refers to a protein determinant that specifically binds to an antibody. The epitope is usually classified by the chemically active surface of the molecule, such as an amino acid or a sugar side chain. It is constructed and usually has specific three-dimensional structural features as well as specific charge characteristics. The difference between the conformational and non-structural epitopes is that the binding to the former is lost in the presence of a denaturing solvent, but the latter does not.

As used herein, refers to the dissociation constant K _D, certain words, for a predetermined antibody K _D antigen, and the magnitude of the affinity of a particular target for the antibody. High affinity antibody is an antigen having a predetermined K _D of 10 ^-8 M or less, is more preferably 10 ^-9 M or less, and further preferable is 10 ^-10 M or less. The reciprocal of K _D is K _A , which is the association constant. As used herein the term "k _dis" or "k _2" or "k _d", intended to refer to a particular antibody - antigen interaction dissociation rate. "K _D " is the dissociation rate (k ₂ ) (also known as the ratio of the exit rate (k _off ) to the association rate (k ₁ ) or the "connection rate (k _on )". Therefore, K _{D is} equivalent to k ₂ / k ₁ or k _off /k _on and expressed in molar concentration (M) _{. The} smaller the K _D , the stronger the binding. Therefore, a K _D of 10 ^-6 M (or 1 μM) means 10 ^-9 M ( Or 1 nM) weak binding.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of an antibody molecule consisting of a single molecule. The monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. The term also includes "recombinant antibodies" and "recombinant monoclonal antibodies", all of which are prepared, expressed, produced or isolated by recombinant methods, such as (a) animal or fusion antibody secreting animal cells and fusion partners (fusion partner) An antibody that is fused to the prepared fusion tumor; (b) an antibody that is isolated from a host cell that has been transformed to express the antibody (eg, from a transfectoma); (c) self-weight An antibody that binds to a human or other species antibody library; and (d) an antibody that is prepared, expressed, produced, or isolated by any other method involving splicing of the immunoglobulin gene sequence to other DNA sequences. As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies having different antigenic specificities. However, an isolated antibody that specifically binds to an epitope of one of the heterogeneous or variant human TFs may have a cross-reactivity to other related antigens, eg, from other species (eg, TF species homologs). Furthermore, the isolated antibodies can be substantially free of other cellular materials and/or chemicals. In one embodiment of the invention, combinations of "isolated" monoclonal antibodies having different specificities are combined into a well-defined composition.

As used herein, "specific binding", "immunospecific binding" and "immunospecific binding" mean the binding of an antibody to a predetermined antigen. Typically, the antibody binding has a dissociation constant (K _D ) of 10 ^-7 M or less, and the K _D bound to the predetermined antigen is at least less than its binding to a non-specific antigen (eg, BSA, casein or any other specific polypeptide) K _D Double. The idiom "an antibody that recognizes an antigen" and "a specific antibody to an antigen" are used interchangeably with the term "an antibody that specifically binds an antigen" herein. As used herein, "highly specific" antibody binding relative K _D means that a particular target epitope region than at least the antibody binds other ligands K _D is 10 times smaller.

"Isotype" as used herein means an antibody classification (eg, IgM or IgG) encoded by a heavy chain constant region gene. Some antibody classes further include subclasses, which are also encoded according to the heavy chain constant region. Moreover, this subclass is further decorated with oligosaccharides at specific residues in the constant region (such as IgG1, IgG2, IgG3, and IgG4), thereby further imparting biological functions to the antibody. For example, in the human antibody isotypes IgG1, IgG3, and to a lower range, IgG2 shows that effector functions are like mouse IgG2a antibodies.

By "effector" function or "effector positive" is meant that the antibody comprises a domain different from the specific binding domain of the antigen and is capable of interacting with a receptor or other blood component (eg, complement), resulting in, for example, recruitment of macrophages Cells, as well as events that result in destruction of cells that are bound by the antigen binding domain of the antibody. Antibodies have several effector functions mediated by binding to effector molecules. For example, the complement C1 component binds to an antibody to activate the complement system. Complement activation is important in phagocytosis and breakdown of cell bodies. Complement activation stimulates the inflammatory response and also involves autoimmune hypersensitivity. Furthermore, the antibody binds to the cell via the Fc region and binds to the Fc receptor (FcR) on the cell at the Fc receptor position of the antibody Fc region. There are a number of Fc receptors that are specific for different types of antibodies, including IgG antibodies (gamma receptors), IgE (n receptors), IgA (alpha receptors), and IgM (μg receptors). The binding of antibodies to the Fc receptor on the cell surface triggers many important and diverse biological reactions, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, and targeting of cells by killer cell antibodies (referred to as antibody-dependent cells). Decomposition of the antibody-dependent cell-mediated cytotoxicity, or ADCC, release of the mediator, placental transfer, and control of immunoglobulin production.

The terms "tissue factor protein", "tissue factor" and "TF" are used to refer to a polypeptide having an amino acid sequence, wherein the amino acid sequence corresponds to a naturally occurring human tissue factor or a recombination as described below. Organization factor. Naturally produced TFs contain tissue factors from humans and other animals such as rabbits, rats, pigs, non-human primates, horses, rats, and sheep (see, for example, Hartzell et al., 1989, Mol. Cell. Biol. : 2567-2573; Andrews et al., 1991, published in Gene Journal 98: 265-269, and 1991 by Takayenik et al., Biochem. Biophys. Res. Comm. 181: 1145-1150). The amino acid sequence of the human tissue factor was obtained from UniProt Record P13726 (SEQ ID NO: 1), Malay Monkey (SEQ ID NO: 2), and the mouse was from UniProt P20352 (SEQ ID NO: 3). Amino acid sequences of other mammalian tissue factor proteins are typically known or obtained by conventional techniques.

The anti-system of the present invention is used to administer a human subject or to contact a human tissue that is intended to block human TF function produced by a TF message and expressed on a cell, tissue or organ, and which is also expected It will substantially alter the TF coagulation function produced by the formation of the TF:FVIIa complex. Such uses are found in the treatment of tumors, particularly primary or secondary solid tumors of breast, prostate, lung, pancreatic and ovarian cancers.

The present invention also encompasses nucleic acids encoded by sequences of the antibodies of the invention that bind to those sequences known in the relevant art for construction and production by recombinant means or by transmitting information to the expression of antibodies in the environment. Production, and the environment means that it is intended to be formed in a medium such as a culture medium, a place and a body. Means for performing such nucleic acid manipulations which are intended to produce antibodies of the invention are known to those skilled in the art.

The invention further provides, for example, a pharmaceutically acceptable formulation, or a stable formulation for administration and storage of the isolated form of the antibody of the invention.

1. Composition of antibodies characteristic

The present invention is based on the surprising discovery that a non-clotting blocking murine antibody (Edgington et al., U.S. Patent No. 5,223,427), which binds to human TF, is known as 10H10, and is capable of abolishing TF signaling in specific cells (Ahmed et al., 2006) The above situation, WO2007/056352A2). Thus, the antibody of the present invention is one of the binding epitopes of the murine antibody 10H10, wherein the antibody does not compete with FVIIa for binding to tissue factor and does not substantially block the procoagulant and guanidine of the TF-VIIa complex. Amine hydrolysis activity, but will block the signaling of TF-VIIa media and downstream carcinogenic effects, such as the release of interleukin IL-8. The antibodies of the present invention are suitable for the human germ cell line IgG gene represented in the IMGT database and retain binding to human TF, while the presence of calcium in human plasma does not interfere with the ability of TF to initiate coagulation.

An antibody that retains the antigen binding region of murine antibody 10H10, which is generally assessed by the ability of the antibody to bind to TF and 10H10 competes for binding to human TF for evaluation, and at the same time, when present in a sample containing TF in the presence of human plasma, with a similar sample of human plasma in the absence of the antibody In comparison, it will not substantially extend the time required for TF to initiate coagulation for plasma. In another sense, the epitope of the antibody can be mapped according to the laws of the art using techniques known in the relevant art, including but not limited to deletion mutations, substitution mutations, restriction of the TF to which the antibody binds. Hydrolysis, followed by permeation of peptide fragment recognition and co-crystallization and X-ray diffraction analysis to map the adjacent regions of the atomic structure of the major structure of TF and the antibody binding domain, thereby defining the antibody and human TF The three-dimensional correlation between (Figure 1).

Thus, the epitope can be defined as non-overlapping where it binds to FVIIa (Figures 2 and 3). More specifically, the epitope determined by the antibody of the present invention may be in the N domain of TF (mature strand residues 1-104 represented by SEQ ID NO: 1), and FVII is not contacted, such as residue 65- 70. One or more of the residues are in contact without contact with residues K165 and K166 in the C domain, which is important for matrix binding (Kirchofer et al., 2000, Thromb Haemostat 84: 1072-1081) The presence of calcium in human plasma does not interfere with the ability of TF to initiate coagulation.

In one embodiment, the binding rate of the antibody (k _a at 1/M‧s) is greater than 1 x 10 ^-5 . In another embodiment, the antibody is directed against TF in off-rate (1 / k _d when s) is less than 1.0 × 10 ^-5, K _D thus produced is less than 1 × 10 ^-9 M (less than 1 nM). In a particular embodiment, the antibody is a human antibody germline gene adaptability which K _D of less than 0.5 × 10 ^-9 M. In one embodiment, the antibody has binding domains selected from those heavy and light chain pairs as shown in Table 11, such as M1639, M1645, M1647, M1652, M1641, M1644, M1587, M1604, M1593, M1606, M1584, M1611, M1596, M1601, M1588, M1594, M1607, M1612, M1595, M1599, M1589, M1592, M1583 and M1610.

The antibody composition may be further referred to as comprising a sequence of an amino acid residue located within the binding domain selected from one or more amino acid sequences which may be represented by SEQ ID NO: 6-166.

Antibody variants with altered Fc function

Since the use of therapeutic monoclonal antibodies produced by recombinant methods is expanding, the functions and properties of these complex compositions are being explored. Although immunospecific and antigenic target functions are typically within the variable regions and subdomains, such as the loop ends of the hypervariable regions, also referred to as CDRs, the complex is formed with a constant domain, such as the Fc portion of an IgG. The other receptors and serum components provided by the structure interact.

Antibodies and other Fc-containing protein lines can be functionally compared by several well-known in vitro assays. In particular, there is interest in the affinity of FcγRI, FcγRII and FcγRIII family members of the Fcγ receptor. These measurements can be made using recombinant soluble forms of the receptor or cell-associated forms of the receptor. In addition, it can be used, for example: recombinant solubility FcRn, which measures the affinity of FcRn through the BIA nucleus, and FcRn is the receptor responsible for the extended half-life of IgG. Cell functional assays, such as ADCC analysis and CDC analysis, provide insights into the possible functional outcomes of a particular variant structure. In one embodiment, the ADCC assay is configured to use NK cells as the primary effector cells, thereby reflecting the functional effects on the FcyRIII receptor. Phagocytosis assays can also be used to compare immune effector functions of different variants, as measured by cellular responses, such as superoxide or inflammatory mediators. Where the in vivo model is applicable, and, for example, a variant of the anti-CD3 antibody is used to measure the T cell activation status of the mouse, and the activity of binding to a specific ligand, such as an Fc gamma receptor, is determined depending on the Fc domain.

2. Tissue factor signaling blocks antibody production

An antibody having the antibody function and biological activity described in the present application, which may comprise or be derived from any mammal such as, but not limited to, human, mouse, rabbit, mouse, rodent, primate, goat or Any combination, and includes isolated human, primate, rodent, mammalian, chimeric, human or primate adaptive antibodies, immunoglobulins, cleavage products thereof, and other specific portions and variants . The monoclonal antibody system can be prepared by any method known in the art, such as fusion tumor technology (Kohler and Milstein, 1975, Nature 256:495-497) and related methods, using an undead fusion partner fused to B cells. . Resistance for use in the present invention The body can also be produced by culturing and expressing an immunoglobulin variable region cDNA which is produced from a single lymphocyte using a single lymphocyte antibody method, and the single lymphocyte system is produced. By, for example, Babcook, J., et al., 1996, published in Proc. Natl. Acad. Sci. USA Journal 93 (15): 7843-78481; WO 92/02551; WO 2004/051268 and International Patent Application WO 2004/106377 The method described is selected to be suitable for the manufacture of a particular antibody.

An antibody comprising a functional non-target binding domain of a target binding domain or subdomain, a constant domain, and an Fc domain as used herein, which can be derived by several methods known in the art. In one embodiment, sequences of naturally occurring antibody domains are conveniently obtained from published or online documents or databases, such as the V-Database (provided by the MRC Protein Engineering Center), and the National Center for Biological Products Information (NCBI). Ig embryo) or the ImMunoGeneTics® (IMGT) database provided by the International Immunogenetics Information System.

Human antibody

The invention also provides human immunoglobulins (or antibodies) that bind to human TF. These antibodies can also be characterized as engineered or adapted. The immunoglobulin has a variable region substantially from the human germ cell line immunoglobulin and includes related variants of the residues with which the antigen is recognized, such as Kabat or a high variable loop CDRs as defined by the structure. The constant region, if present, may also be substantially derived from a human immunoglobulin. Human TF antibodies exhibited K _D of at least about 10 ^-6 M (1 mM), about ^{10 -7 M (100 nM),} 10 -9 M (1 nM) or lower. In order to influence the change in affinity, such as improved affinity or K _D of reducing human TF antibody, alternative CDR residues or other residues be.

The production source of the human antibody for binding to TF is preferably a sequence as a variable region provided herein, the variable region comprising a sequence selected from the sequence identification numbers: 129-163, and a sequence selected from the sequence Identification number: one of the FRs and CDRs of 28-61, wherein the CDRs are selected from one or more of the sequence identifiers: 6-11, 27, 62-128, and the gene derived from the human-derived Fab presented on the filamentous phage particle. The spectrum is identified as being able to bind to human TF and interact with the Malay monkey TF.

Substitution of any non-human CDR into any human variable region FR may not allow the same spatial orientation to be formed between structures corresponding to the parent variable FR at the origin of the CDR. The heavy and light chain variable framework regions to be assigned to the final Mab can be derived from the same or different human antibody sequences. The human antibody sequence may be a sequence of a naturally occurring human antibody, may be a sequence derived from a human germline cell immunoglobulin sequence, or may be a consensus sequence of several human antibody and/or germ cell line sequences.

Suitable human antibody sequences are identified by computer comparison of the amino acid sequence of the mouse variable region with the sequence of a known human antibody. The comparisons are performed on heavy and light chains, respectively, but the principles are similar to each other.

With regard to empirical methods, it has been found to be particularly convenient to create variant sequences that can be used to screen for desired diversity, binding affinity or specificity. Library. One format for creating such variant libraries is a phage display vector. Further, the variant system can be produced using other methods for encoding the variegated nucleic acid sequence into a target residue within the variable region.

Another method of deciding whether further substitution is required, as well as the choice of amino acid residues for substitution, can be accomplished using a computer model. Computer hardware and software for making three-dimensional images of immunoglobulin molecules are widely available. In general, molecular models are initiated by the structure of the resolved immunoglobulin chain or its domain. The strand to be modeled is compared to the amino acid sequence similar to the chain or domain of the resolved three-dimensional structure, and the chain or domain showing the greatest similarity is selected as the starting point for constructing the molecular model. The resolved starting structure is modified such that the difference between the authentic amino acids of the immunoglobulin chain or domain is modeled and is equal to the starting structure. The modified structure is then assembled into a combined immunoglobulin. Ultimately, the model is refined by energy simulation and by confirming that all atoms are within a suitable distance from each other and that the combined length and angle are within chemically acceptable limits.

Due to the degeneracy of the coding, the variability of the nucleic acid sequence will encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence can be produced by de novo (re)solid phase DNA synthesis, or by PCR mutations of variants prepared early in the desired polynucleotide. All nucleic acids encoding the antibodies disclosed herein are encompassed by the present invention.

The variable segments of human antibodies produced as described herein are typically linked to at least a portion of a human antibody constant region. The antibody will contain There are both light chain and heavy chain constant regions. The heavy chain constant region typically includes a CH1 domain, a hinge domain, a CH2 domain, a CH3 domain, and sometimes a CH4 domain.

Human antibodies can comprise any type of constant domain from any antibody species, including IgM, IgG, IgD, IgA, and IgE, as well as any subtype (homotype), including IgGl, IgG2, IgG3, and IgG4. When it is desired human antibody visualized cytotoxic activity, the constant domain is usually a complement - fixing constant domain and the type typically IgG _1. When such cytotoxic activity is not desired, the constant domain can be of the IgG ₂ species. A humanized antibody can include sequences from more than one species or isotype.

A nucleic acid encoding a human light chain and a heavy chain variable region, which is linked to the constant region as needed, can be inserted into the expression vector. The light and heavy chains are optionally colonized on the same or different expression vectors. A DNA segment encoding an immunoglobulin is operably linked to a regulatory sequence of a performance vector to ensure expression of the immunoglobulin polypeptide. Such regulatory sequences include signal sequences, promoters, enhancers, and transcription termination sequences (see, in 1998, Queen et al., Proc. Natl. Acad. Sci. USA Journal 86, 10029; WO 90/07861; 1992 Co et al. Published in J. Immunol. Journal 148, 1149, incorporated herein by reference in its entirety for all its purposes.

The antibody or Fc, or a composition and domain thereof, and which may also be selected from such domains or constituent databases, such as phage databases. The phage library can be generated by insertion of a random oligonucleotide database or a library of polynucleotides of interest, such as B cells from immunized animals or humans (Hoogenboom et al., 2000, Immunol) .Today 21 (8) 371-8). The antibody phage library contains heavy (H) and light (L) chain variable region pairs in a phage that enables expression of a single Fv fragment or Fab fragment (Hoogenboom et al., supra , 2000). Differences in the phage library can be adjusted to increase and/or alter the immunospecificity of individual antibodies in the database, thereby producing additional, desired human monoclonal antibodies and subsequent recognition. For example, the coding lines of heavy (L) and light (L) immunoglobulin molecules can be randomly mixed (shuffled) to create new HL pairs in the combined immunoglobulin molecule. Furthermore, either or both of the H and L chain encoding genes can be mutagenized within the complementarity determining regions (CDRs) of the immunoglobulin peptide variable regions, followed by screening for the desired affinity and neutralizing ability. The antibody database can also be generated by selecting more than one human FR sequence, then introducing a CDR gate set derived from a human antibody gene profile or passing through a post-design variant (Kretzschmar and von Ruden, 2000, published in Current Opinion in Biotechnology 13: 598-602). The position of the difference is not limited to the CDRs, but may also comprise the FR segment of the variable region or may comprise an antibody variable region, such as a peptide.

Other libraries may be included that contain other target binding or non-target binding components in addition to the variable regions of the antibody, for ribosome presentation, yeast presentation, and bacterial presentation. Ribosome presentation is a method of translating mRNA into its cognate protein while maintaining the protein attached to the symptoms of RNA. The nucleic acid coding sequence was recovered by RT-PCR (Mattheakis, L.C. et al., 1994, Proc. Natl. Acad. Sci. USA Journal 91, 9022). Membrane-associated α-agglutination The yeast yeast adhesion receptor, part of the mating type system is based on the fusion protein structure of aga1 and aga2 (Broder et al., 1997, Nature Biotechnology 15: 553-7). Bacterial presentation is based on the fusion of the target to the cell membrane or cell wall-associated export bacterial protein (Chen and Georgiou, 2002, Biotechnol Bioeng. 79:496-503).

The present invention also provides a nucleic acid encoding composition of the present invention as an isolated polynucleotide, or as a mutagenic representation comprising expression, secretion, and/or composition of the prokaryotic, eukaryotic or filamentous phage, or the use thereof. The expression carrier portion of the carrier.

3. Method for producing antibody of the present invention

Once an antibody molecule of the invention has been identified according to the structural and functional characteristics described herein, the nucleic acid sequence encodes a desired portion or the entire antibody chain can be subjected to colonization, replication or chemical synthesis, and can be singled out It is isolated and used to express antibodies by conventional methods. Purification of the antibodies of the invention can be carried out by any method known in the relevant art for purification of immunoglobulin molecules, for example by chromatography (e.g., ion exchange, affinity and molecular sieve chromatography), centrifugation, differential Solubility, or by any other standard technique suitable for protein purification. Furthermore, antibodies or fragments thereof of the invention can be fused to other heterologous peptide sequences described herein or known in the related art to facilitate purification.

Host cell selection or host cell processing

As described herein, host cells selected for recombinant Fc-containing or monoclonal antibody expression are an important contributor to the final composition, including but not limited to oligosaccharides that decorate proteins within the immunoglobulin CH2 domain. Changes in part of the composition. Accordingly, an embodiment of the invention comprises selecting a host cell suitable for use in the production and/or development of a producer cell that is capable of expressing a desired therapeutic protein.

Furthermore, the host cell may be of mammalian origin or may be selected from the group consisting of COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, Lymphoma, yeast, insect or plant cell, or any derivative, apoptotic or transformed cell thereof.

Alternatively, the host cell line may be selected from a species or organism that is not capable of saccharifying the peptide, such as a prokaryotic cell or organism of the native or processed Escherichia, Klebsiella or Pseudomonas.

4. Method of using anti-TF antibody

Compositions (antibodies, antibody variants or fragments) produced by any of the above methods can be used to diagnose, treat, detect or modulate a human disease or a particular condition in a cell, tissue, organ, body fluid or, typically, a host. As taught herein, modification of the Fc portion, Fc fusion protein or Fc fragment of an antibody after target binding provides a more accurate range of effector functions, but wherein the antibody retains the original target characteristics, Antibody variants are produced that are suitable for the particular application and therapeutic indication.

A disease or condition that can be used for treatment using a composition provided by the present invention includes, but is not limited to, cancer; the cancer includes primary solid tumors and metastatic cancers; cancer, adenocarcinoma, melanoma, such as , lymphoma, leukemia and myeloma liquid tumors and invasive lumps with cancer development; soft tissue cancer; sarcoma, osteosarcoma, thymoma, lymphosarcoma, fibrosarcoma, leiomyosarcoma, lipoma, glial cells Tumor, astrocytoma sarcoma, prostate cancer, breast cancer, ovarian cancer, stomach cancer, pancreatic cancer, laryngeal cancer, esophageal cancer, testicular cancer, liver cancer, salivary gland cancer, biliary tract cancer, colon cancer, rectal cancer, cervical cancer, uterine cancer, Endometrial cancer, thyroid cancer, lung cancer, kidney cancer or bladder cancer.

Since the antibody of the present invention reduces the precarcinogenic environment in tissues by blocking the ability of TF to participate in the release of downstream interleukins, such as inflammatory interleukins, IL-8, etc., the anti-system of the present invention is considered as a prophylactic Use, or in combination, with other therapies for inhibiting tumor cell proliferation and angiogenesis. Most age-related cancers are derived from epithelial cells of regenerative tissues. An important element of epithelial tissue is the interstitial, extracellular matrix and epithelial layer composed of several types of cells including fibroblasts, macrophages and vascular endothelial cells. In cancerous tumors, interstitial is the key to tumor growth and development, while TF is expressed on mesenchymal cells as well as cancer epithelial cells. Therefore, the presence of TF leads to downstream factors, and the presence of downstream factors in the interstitial TF:VIIa message transmission may create a pre-carcinogenic tissue environment that synergizes with oncogenic mutations and drives the formation of tumor tissues.

Likewise, when the TF system is expressed in adipose tissue, it can modify tissue function in symptoms such as obesity, metabolic complications, and diabetes. Antibodies of the invention can be used in the treatment of these conditions by blocking TF:VIIa signaling. Downstream TF: Some of the factors produced by VIIa signaling, including IL-8 and IL-6, are potent inflammatory regulators. Other uses of the antibodies of the invention thus include treatment of inflammatory conditions such as, but not limited to, rheumatoid arthritis, inflammatory bowel disease, and asthma.

Since the antibodies of the invention inhibit TF:VIIa signaling and reduce downstream effects of promoting angiogenesis, the antibodies of the invention can be used in addition to cancer, involving the treatment of other diseases, disorders and/or conditions of angiogenesis. These diseases, disorders, and/or symptoms include, but are not limited to, benign tumors, such as: hemangioma, acoustic neuroma, neurofibromatosis, trachoma, pyogenic granuloma; arteriosclerotic plaque; ocular angiogenesis, eg diabetes Retinopathy, retinopathy of prematurity, degeneration of yellow spots, corneal graft rejection, neovascular glaucoma, post-crystal fibrous tissue hyperplasia, iris redness, retinoblastoma, ocular uveitis, and pterygium (abnormalities) Vascular growth); rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; angiogenesis; granulation; hypertrophic scar (kelsy); non-healing fracture; scleroderma; Angiogenesis; coronary collateral; cerebral collateral; arteriovenous malformation; ischemic limb angiogenesis; hereditary hemorrhagic vasodilation; plaque neovascularization; microvascular dilatation; hemophilic joint; angiofibroma; Dysplasia; wound granulation; Crohn's disease and atherosclerosis.

To date, because the antibodies of the invention inhibit TF:VIIa signaling, the anti-system can be used to treat and/or diagnose hyperproliferative diseases, disorders, and/or symptoms including, but not limited to, tumors. The antibody is capable of inhibiting the spread of the disorder by direct or indirect interaction. Examples of hyperproliferative diseases, disorders, and/or symptoms that can be treated and/or diagnosed by the antibodies of the invention include, but are not limited to, high gamma globulinemia, such as Castel's disease Lymphoid proliferative disorders, disorders and/or symptoms, atypical proteinemia, purpura, sarcoma-like, Sezary syndrome, Walden's giant globulinemia, Gaucher's disease, histiocytosis and any other Hyperproliferative diseases of body organs, tissues or fluid compartments.

Other means by which the antibodies of the invention may be used for treatment include, but are not limited to, direct cytotoxicity of the antibody, such as by mediation of complement (CDC) or effector cells (ADCC), or indirect cytotoxicity of the antibody, Such as when the immune linker.

While the invention has been described in detail, the embodiments of the invention are in the In the experimental description, certain reagents and procedures are used to make proteins or antibodies or designated fragments. The analytical methods routinely used to characterize the antibody are as follows.

Materials and Method Protein and antibody standards

The recombinant extracellular region (ECD) of human TF is constructed in two forms: for ELISA and Biacore based direct binding assays, the amino acid of the TF mature strand (eg, SEQ ID NO: 1) is a C-terminal The His6-labeled peptide pattern is expressed in mammalian systems; for co-crystallographic studies, the amino acid 5-213 line of sequence identification number: 1 is expressed in the bacterial system as a C-terminal His6-labeled peptide. NHS esters on human _TF1-219- based proteins are chemically labeled with amine residues for biotinylation. For coagulation assays, Innovin® (Dade Behring Company Code B4212), a lyophilized recombinant human tissue factor that binds to diagnostic phospholipids, calcium, buffers and stabilizers, is used.

The cyno TF-ECD (SEQ ID NO: 2) was selected for colonization using the PCR technique and cDNA obtained from the BioChain Institute (Hayward, Calif.) in a separate form of Malay test tissue.

Several anti-systems were used as reference antibodies: i) 10H10 from the original fusion tumor TF9.10H10-3.2.2 (US 7223427); ii) a 10H10 mouse-human chimera containing human IgG1 /Kappa constant region, specifically M1 sequence identifier: 4 and sequence identifier: 5; iii) M59, a human FR-adapted antibody, comprising 6 CDRs of 10H10 and acting as a master antibody for affinity maturation , comprising a sequence with human IgG1 and human Kappa constant region: 19 and SEQ ID NO: 23; iv) murine anti-human tissue factor antibody TF8-5G9 (US 7223427); v) from antibody 5G9, humanized , called CNTO 860, as human IgG1/Kappa (US7605235); and vi) An isotype control (human IgG1/kappa) antibody binds to an incoherent antigen (RSV) called B37.

Antibody performance and purification

Routine procedures are used to perform the performance and purification of the disclosed antibodies. For the initial screening, DNA was encoded for these molecules and transiently expressed in HEK 293E cells in 96-well plates, and then the supernatant was tested for activity (binding) 96 hours after transfection. The hitter is determined and then subjected to experimental scale performance and purification. Experimental scale performance was performed instantaneously in 750 ml volumes of HEK 293F cells or CHO-S. The supernatant from the harvest was purified by protein A chromatography, followed by evaluation of its affinity and functional activity against the purified protein. In addition, the purified protein was subjected to biophysical characterization by SDS-PAGE, SE-HPLC, and interaction chromatography (CIC). The theoretical isoelectric point (pI) of each variant is also calculated. From experimental scale characterization, a series of ultimately important candidates were transfected in a WAVE bioreactor and then purified by protein A.

Fab production and single Fab ELISA

The glycerol strain obtained from the phage screening round was subjected to a small amount of extraction, and the pIX gene was removed by digestion with NheI/SPEI. After rejoining, the DNA was allowed to plate on LB/agar plates overnight to convert to TG-1 cells and grow. The next day, colonize the colonies, grow overnight, and then The medium was used for (i) colony PCR and V-region sequencing, and (ii) production of Fab induction. For the production of Fab, the overnight medium is diluted 10 to 100 times in a new medium and then grown at 37 degrees Celsius for 5 to 6 hours. The Fab production was induced by the addition of fresh medium containing IPTG, and then the medium was grown overnight at 30 degrees Celsius. The next day, the medium was briefly centrifuged, and then the supernatant containing the soluble Fab protein was used in an ELISA.

For Fab ELISA, the Fab was grasped onto the plate by multiple anti-Fd (CH1) antibodies. After appropriate washing and blocking, biotinylated hTF was added at a concentration of 0.2 nM. This concentration allows the Fab variants to be ranked, depending on the percentage of parental binding, with the parental Fab line appearing as a control in all plates defined as a 100% binding. The biotinylated hTF line was detected using the chemiluminescent readings of HRP conjugated streptavidin and a plate reader.

TF-ECD binding to Mab substrate ELISA

A solution phase direct TF binding ELISA using chemiluminescence detection was used to sequence binders from the human framework adaptive database. The 96-well black enzyme-labeled reaction plate was coated with 100 μl at 4 ° C, diluted with a pH 9.4 carbonate-bicarbonate buffer to 4 μg/ml of goat anti-human IgG Fc overnight, and then washed. The buffer (PBS solution with 0.05% Tween-20 (trade name, polysorbate ester laurate 20)) was washed 3 times and blocked with 300 μl, 1% BSA/10 mM PBS solution. After 1 hour, It is then cleaned as previously described. The sample or standard was diluted to 50 Ng/ml in assay buffer (1% BSA in PBS + 0.05% Tween), followed by 100 μL of shaking into the assay plate for 1 hour at room temperature. The plate was washed 3 times, 100 μl of the assay buffer was diluted to 100 Ng/ml, and His-tagged human or male monkey TF-ECD was added to each plate, followed by incubation at room temperature for 2 hours. . After washing, 100 μl of Qiagen catalase conjugated Penta-His diluted in assay buffer 1:2000 was added to each plate, followed by shaking at room temperature for 1 hour. A fresh BM chemiluminescent matrix (BM Chemilum of Roche POD) was then prepared by dilution in a 1:100 buffer and 100 microliters was added to the plate after the last wash. After 10 minutes, the plate was read on a Perkin Elmer Envision Reader reader using the BM ChemiLum program.

MDA-MB-231 whole cell binding by FACS anti-tissue factor mAb

This assay is used to detect direct binding of antibodies to endogenous human TF expressed in breast cancer cells. Prepare test mAbs in 4 points titrated in FACS buffer (1% FBS in PBS) in duplicate. Start the titration at 1000 ng/ml with a 1:4 dilution. The maternal M1 line was used as a positive control group, while the anti-RSV mAb and B37 lines were used as a negative negative/isotype control group. Unstained cells and secondary antibodies, Cy-5 conjugated goat anti-human IgG Fc anti-system were used as a control group in FACS buffer 1:200 and prepared immediately before use.

The MDA-MB-231 cells attached to the culture flask were washed once with PBS (without Ca+2/Mg+2) using standard tissue culture techniques. The cells were lifted with Versene and the number of cells was counted, and then 200,000 cells were seeded in each of the polystyrene V-shaped bottom plates. FACS Analysis Protocol: Tissue Factor Binding. The pellet was pelleted at 4° C., and the cells in Allegra X-15R were resuspended in buffer FACS (2% FBS in PBS) by centrifugation at 450 × g for 3 minutes, and then 200,000 cells in 200 μl were plated. On each board. The pellet cells were centrifuged at 450 x g for 3 minutes at 4 °C. The supernatant was discarded and 100 microliters of test or control mAb was added to each plate to each plate and then incubated on ice or at 4 °C for 1 hour (+/- 10 minutes). The pellet cells were centrifuged at 450 x g for 3 minutes at 4 °C. The supernatant was discarded and the cells were washed once with FACS buffer. The cells were resuspended in 200 microliters of FACS buffer per plate, and the pellet cells were centrifuged at 450 x g for 3 minutes at 4 °C. The supernatant was discarded and 100 microliters of secondary antibody was added to each plate (titration) per plate and then incubated on ice for 1 hour (+/- 10 minutes). The pellet cells were centrifuged at 450 x g for 3 minutes at 4 °C. The supernatant was discarded and the cells were washed twice with FACS buffer and then resuspended in 200 microliters of FACS buffer per plate (titration). The pellet cells were centrifuged at 450 x g for 3 minutes at 4 °C. The supernatant was discarded and the cells were resuspended in 100 microliters of CytoFix buffer per plate. The reaction was analyzed by flow cytometry (BD FACSArray). The FlowJo soft system performs FACS data analysis by finding the primary cell distribution in the unstained control plate and applying the pathway to the entire data set. The data system Export to a geometric mean fluorescence intensity (MFI) chart in the red channel of the applied path.

Thermofluor analysis

The Thermofluor technology measures the dynamics of the reverse folding of a molecule as it is heated. Once the molecule is heated, the dye (ANS) is able to bind to the anti-folded molecule. Once the dye is bound to the molecule, it will fluoresce and then measure the fluorescence over time. In this analysis, antibody anti-folding at 37 to 95 ° C was measured and detected every 0.5 ° C. The Tm values of the parental molecules in both the murine and chimeric (10H10, M1, 5G9 and CNTO860) were also measured under the two mAbs with known Tm values as the control control (Emmp 4A5 and Emmp 5F6).

This analysis was used to predict the thermal stability of human framework adaptive database variants. The purified antibody was diluted to a concentration of 0.5 mg/ml in PBS, and then 2 μl of the sample was taken to each well, i.e., 1 μg of sample per well. Each sample was added in duplicate. Stock ANS was dissolved in DMSO at a concentration of 500 mM. Dilute stock ANS (40 mM) with DMSO; make dye/Tween solution by combining 20 μl of 40 mM ANS solution, 2.8 μl, 10% Tween and 1.98 ml PBS; add 2 μl Dye/Tween solution and 2 microliters of oil. Centrifuge the plate (450 rpm, 2 minutes). Thermofluor setting: Shutter set to manual, ramp temperature 0.5 C/sec, continuous ramp, temperature ramp: 50 to 95 °C. Choose to maintain at high temperature for 15 seconds, Exposure time 10 seconds / 1 round, get normal = 2, select "single SC picture / board".

Interaction chromatography (CID)

In order to determine the interaction of various antibodies with other human antibodies, a column of human IgG binding (Sigma Aldrich) was used for the chromatographic experiment. Briefly, 50 mgs of human IgG was combined with a 1 ml NHS-Sepharose column (GE Healthcare) following the manufacturer's instructions. Wash with 0.1 M Tris, pH 8, 0.5 M NaCl to remove unbound IgG and block the unreacted NHS functional groups with the same buffer. Pierce's Coomassie Plus Assay Kit (Thermo Pierce) was used to measure the concentration of protein remaining in the unreacted binding buffer and washing solution, and the amount of protein before fixation was subtracted, thereby determining the binding efficiency. A control column was also prepared, using the same protocol, but no protein was added to the resin.

The control column was equilibrated with PBS, pH 7, flow rate of 0.1 ml/min, and run for the first time on a Dionex UltiMate 3000 HPLC. A 20 liter reserve protein solution was first injected to ensure that the non-specific binding sites were blocked, followed by 20 liters of 10% acetone to check the integrity of the column.

The sample to be analyzed was diluted to dissolve in PBS, pH 7, 0.1 mg/ml. Twenty microliters of each sample was injected onto each column and allowed to run for 30 minutes at a flow rate of 0.1 ml/min. The residence time was recorded and the retention factor (K') of each variant was calculated.

The calculated value of K' is the difference between the residence time t _R on the protein-derived column (IgG combination column) and the residence time t ₀ on the column without protein binding. The calculated value also takes into account the residence time of acetone on the two columns in order to standardize the column. The acceptable value for K' is less than 0.3.

Solubility

In order to determine the solubility of various antibodies at room temperature, a concentration test was performed using a centrifugal filtration apparatus. Briefly, antibody preparations in PBS were added to a Vivaspin-15 (15 ml) centrifugal filter unit (Sartorius 30,000 MWCO, Göttingen, Germany) at room temperature. The filter was spun at 3000 xg for 20 minutes in a Beckman Allegra X15-R centrifuge using a spiral arm rotor. Once the volume was reduced to approximately 2 ml, the supernatant was transferred to a Vivaspin-4 (4 ml) filtration apparatus (30,000 MWCO) and then centrifuged at 4,000 x g for 20 minute intervals. Once the volume was reduced by 500 liters, the samples were transferred to a Vivaspin-500 liter filter and centrifuged at 15,000 xg for 15 minutes in an Eppendorf 5424 centrifuge. This is repeated until the protein concentration reaches 100 mg/ml or more. Protein concentration was determined by appropriate dilution on the BioTek Synergy HT spectrometer, absorbance at 280 nm and 310 nm. At this point, centrifugation was stopped and the sample was allowed to stand at room temperature overnight to reach equilibrium. The next morning, check the sample for signs of precipitation. If the concentration is greater than 100 mg/ml, the process is stopped.

Factor VIIa-induced IL-8 inhibition

This assay was used to test whether TF-binding antibodies neutralize FVIIa-induced IL-8 release from human cells expressing TF. Human breast cancer cells (MDA-MB-231) (ATCC HTB-26) grown in DMEM and 10% FBS (GIBCO: Class Number 11995 and Class Number 16140) using standard cell culture techniques at 20000 per well The density of cells (100,000 cells/ml) was plated on a 96-well cell culture plate (NUNC: Category No. 167008). The cells were allowed to recover for 2 days before starting antibody treatment and serial dilutions of 1:2 or 1:4 at 2 ug/ml in DMEM without FBS. One hour prior to the start of treatment with FVIIa (Innovative Research: Classification Number IHFVIIa, Lot No. 2824), the antibody was first added to a final concentration of 50 nm in DMEM without FBS. The cells were placed in the incubator for 24 hours. After treatment, the supernatant was collected and the amount of IL-8 was detected by ELISA according to the manufacturer's agreement (R&D Systems: Category No. D8000C). Briefly, the optical density values (OD) of the respective treatment samples were read at 450 nm and 540 nm. The reading at 540 nm is used to correct the optical defects in the analytical version, while at 450 nm, the corrected reading (OD 450 minus OD 540) is used to prepare the IL-8 according to the manufacturer's agreement. The standard curve is used to calculate the IL-8 content in the treated sample. Wells with cells but not treated with antibodies and FVIIa are used to define endogenous IL-8 levels, while wells with cells but only FVIIa are used to define "no inhibition" levels of IL-8, This defines the lowest and highest IL-8 levels, respectively. The mAb titration treatment samples were normalized according to the maximum and minimum IL-8 levels defined above and expressed as a percentage of inhibition. The normalized data is represented by a bar graph or with a four-parameter logistic curve that corresponds to the extracted EC ₅₀ value corresponding to each mAb.

Coagulation test

This assay is used to determine whether anti-human TF antibodies block coagulation in vitro in the presence of calcium and recombinant human TF preparation (Innovin from Dade Behring) in human plasma. The anti-human TF antibody was diluted into an antibody (Gibco, class number 14175) dissolved in HBSS at 2 mg/ml. Pooled human plasma with sodium citrate (George King Biomedical, Novi, Michigan) was spun down at 1000 rpm for 5 minutes and then the clear plasma was transferred to a new tube. In each well of a clean 96-well assay plate (NUNC, accession number 439454), 25 microliters of diluted antibody was added to 100 microliters of human plasma. 125 μl of Innovin (Dade Behring, Inc., part number B4212) diluted 1:500 into HBSS containing 22 mM calcium chloride was added to each well containing plasma with or without antibody, ie The reaction begins. Immediately after the start of the reaction, the clotting reaction was dynamically monitored at OD 405 for 30 minutes at 37 ° C using a SpectraMax M2E reader (Molecular Devices, Sunnyvale, Calif.). The Softmax Pro software was used to determine the T1⁄2 Max of each antibody, ie the number of seconds required to reach a maximum optical density of 50%. Will be suitable The time required for the sample is normalized to the reference value on each plate, but is statistically suitable for samples containing no antibody, suitable for 10H10 and the average time for all 10H10 derived and human adaptive variants, That is, there is no difference between the samples between 150 seconds and 200 seconds.

Example 1: Sequence of 10H10

A murine antibody, 10H10, produced by the Scripps Research Institute in La Jolla, California (US 5,223,427, published by Morrisey et al., Thromb Res., 52(3): 247-261, 1998) by fusion tumor TF9 Produced by .10H10-3.2.2. Antibody sequences from 10H10 fusion tumor strains have not been reported in the past.

These sequences were identified using the 5' RACE method (published in Focus Journal 25(2): 25-27, 2003; Maruyama, 1994, Gene Journal 138, 171-174, 1994), in which the two antibody chains VH and VL are Amplification was performed using 5'GeneRacer ^(TM) (InVitrogen) primers and 3' consensus primers, which are complementary to one of the mouse IgGl constant region and the mouse Kappa constant region, respectively. Nested PCR amplification using the 5'GeneRacer nested primer and the 3' consensus primer was used to generate a VL product that is more suitable for sequence analysis.

At least 16 colonies were selected for identification of the variable regions of each strand. The primers are used to sequence through the unknown interval of the insert. The original sequence data was downloaded from the ABI DNA Sequencer to Vector NTI (Invitrogen Informax) for sequence analysis. Identify a function VH and one Function VL. Both the VH and VL genes were further analyzed to find their natural message sequences, FR, CDR and J segments.

The 10H10 FR and CDRs other than the corresponding regions of CDR-1 of VH were numbered sequentially and segmented according to the Kabat definition (published in Kabat et al., 1991, in the fifth edition of the Public Health Service of the National Institutes of Health). For this region, it is used in conjunction with the Kabat and Chothia definitions (Raghunathan, G., published in US 2009/0118127 A1; Chothia and Lesk, 1987, J Jol Biol, 196 J(4): 901-17).

The selected V region is engineered in the human IgG1/Kappa constant region and cloned into mammalian expression vectors suitable for recombinant expression in HEK293 and CHO cell lines, which are used in analytical development. A mouse-human chimeric antibody specifically designated as M1 as a reference antibody. The V-region of HC was also engineered with only the human IgG1 CH1 domain and the C-terminal 6 histidine to produce the 10H10 Fab used in the crystal structure analysis.

Example 2: Mapping of epitopes of non-anticoagulant tissue factor antibodies

The antigenic determinant map alignment for 10H10 was carried out by measuring the crystal structure of the complex between the human TF ECD and the corresponding Fab fragment. His-tagged human TF ECD (SEQ ID NO: 1 residue 5-213) is expressed in E. coli and affinity is performed using a HisTrap HP column (GE Healthcare) and a Q HP column (GE Healthcare), respectively. It was purified by ion exchange chromatography. The His-tagged chimeric version of the 10H10 Fab (mouse V region, human constant domain) is expressed in HEK cells using affinity (GEON TALON column) and molecular sieve (GE Healthcare's HiLoad Superdex 200 column) Purification by chromatography.

The Fab and human TF ECD were mixed by molar ratio of 1:1.2 (excess TF) to prepare a complex. The mixture was incubated at room temperature for 20 minutes and then loaded onto a Superdex 200 column (GE Healthcare) equilibrated with 20 mM HEPES, pH 7.5 and 0.1 M sodium chloride. The fraction corresponding to the main peak was collected, concentrated to 10 mg/ml, and then used as a crystal. The composite system was crystallized by a vapor diffusion method at 20 °C. The 10H10:TF complex was crystallized from a solution containing 0.1 M CHES, 18% PEG 8000, pH 9.5. For X-ray data collection, the composite crystals were immersed in a mother liquor supplemented with 20% glycerol for a few seconds and then frozen instantaneously under a 100 K nitrogen stream. Using X-ray diffraction intensity based detector with Saturn 944 CCD X-STREAM ^TM 2000 and a refrigerated cooling system (a Rigaku) of Rigaku MicroMaxTM-007HF polyethylene micro X-ray generator was measured. This structure was determined by molecular replacement using a CCP4 kit suitable for macromolecular crystallography (Collaborative Computational Project, 1994, Acta Cryst. D50, 760-763).

The TF ECD is composed of two topologically identical domains with immunoglobulin folds. The N-terminal domain extends to residues 1-103 and the C-terminal domain extends to residues 104-210 (of ECD, sequence identifier: 1). The 10H10 epitope was found to be in the center of the residue K149-D150 of the ECD, reaching the deep small block region of the 10H10 heavy and light chain variable regions. The interface between 10H10 and TF is very broad and contains all six CDR cycles (Figure 1).

A notable finding is that the TF epitope of 10H10 does not overlap with the binding positions of FVII and FX (Figures 2 and 3). In addition, the epitopes of 10H10 and 5G9 (another mouse human-TF-binding antibody capable of blocking coagulation, and the antigenic determinant was previously published by Huang et al in 1998) The table, in J Mol Biol Journal 275:873-94, does overlap partially, explaining the competitive binding of human antibodies to human TF (Figures 2 and 3).

Human TF ECD: 10H10 interface

The position of 10H10 binding TF is at the interface between the N- and C-terminal domains of the ECD. The convex surface of the TF conforms to the concave surface of the antibody CDR. The total area buried by the formation of the complex exceeds 1,100 Å 2 on each boundary molecule. All six CDR lines are involved in direct contact with TF (contact is defined as the 4-A interatomic distance). There are a total of 24 epitope residues and 25 antibody binding site residues. L1, H1 and H3 of the CDR form the major part of the contact. The residues forming the epitope of the 10H10:TF complex and the antibody binding site are as shown in Figure 1.

The 10H10 epitope comprises two fragments from the N-domain of TF ECD and three fragments from the C-domain. Two fragments from the N-domain will interact with the antibody: residues 65-70 interact with H-CDR1 and H-CDR3, while residue 104 interacts with H-CDR1. Three fragments in the C-domain interact with antibodies: residues 195 and 197 interact with H-CDR1 and H-CDR2, residues 171-174 interact with L-CDR1 and L-CDR3, residue 129- 150 interacts with L-CDR1, L-CDR3, H-CDR1 and H-CDR3; TF residue K149-D150 is located in the center of the antigen-determining region; reaching a deep small block region formed between the VL and VH domains, wherein The main partners were D97 (sequence identification number: 5) of the LC variable region of 10H10 and W33 (sequence identification number: 4) of the HC variable region.

B. Antibody specificity

The amino acid sequence of human, cyno monkey (SEQ ID NO: 2) and mouse TF ECD (SEQ ID NO: 3) are arranged in Figure 2. There is a high degree of similarity between human and male monkey TF ECD sequences, which differ in only one residue and are located at 10H10 contact residues: SEQ ID NO: 1 position 197, which is R (Arg) in the Malay monkey sequence. ). Once T197 in the human sequence is exposed to a single H-CDR2 residue, the high degree of reactivity of the current 10H10-derived antibody group can be explained.

By arranging the human and mouse TF sequences, it was demonstrated that the epitope of the 10H10 species-specific epitope is a significant amino acid difference that occurs in the epitope of the epitope: 24 residues in human TF 10H10 contact The human and mouse sequences differ at positions 68, 69, 70 and 104; in the N-domain, and the sequence identifier: 1 pair of positions 136, 142, 145 and 197 in the C-domain of sequence identification number: 3. This difference is consistent with the post-impaired binding affinity of 10H10 suitable for mouse TF.

Figure 2 also shows the interaction position on human TF suitable for FVII and FX based on the theoretical three-dimensional model (Fig. 3), describing its relationship with the ternary complex (2003 Nordled et al., published in the Proteins Journal 53: 640). -648). The antibody 5G9 and TF bind to an epitope determined to partially overlap with the FX binding position. Therefore, 5G9 will compete with FX, which will lead to blockade of coagulation. 10H10 and 5G9 differ in that they do not block coagulation, while effectively cutting off the transmission of messages via TF-associated PAR. According to the ternary TF/FVII/FX complex model It is understood that the 10H10 epitope will be expected to be on the free surface of the TF and at the center around the residues K149-D150. Early evidence of this is provided by mapping the 10H10 binding position and the peptide epitope determination map by mutagenesis.

The complex crystal structure currently between TF ECD and 10H10 Fab provides a spatial map alignment in which the antibody binds to TF but does not interfere with FVII and FX and TF, or their interaction with each other. The 10H10 epitope as shown by this structure contains the free surface of TF that is theoretically present in the ternary complex. In addition, the 10H10 epitope will partially overlap to the coagulation-blocking MAb 5G9 epitope (published in supra in 1998), and the common residues are K149 and N171. Neither 10H10 nor 5G9 would block the binding of FVII to TF. The epitopes of 10H10 and FX also do not overlap, but in the current model, stereoscopic conflicts occur between the constant domain of Fab and the protease (globular) domain of FX. However, it should be noted that the FX orientation may actually be different from the model, and the relationship between FX and TF may give some flexibility in the protease domain. There is also considerable flexibility at the corners between the variable and constant domains of the Fab, which may cause 10H10 to bind to the ternary complex without conflict.

Example 3: Binding domain modified for human use

The efficacy of therapeutic proteins may be limited by unwanted immune responses. Non-human monoclonal antibodies may have linear amino acid sequences and local structures that may cause substantial extensibility of the human immune response Conformation. Transfer of immunospecific residues responsible for the binding of non-human mAb targets to human antibodies tends to support results that do not cause substantial loss of binding affinity for the target antigen. Therefore, the use of rational design principles to create antibody molecules is very valuable, with minimal immune response when injected into the human body, while retaining the binding and biophysical appearance of maternal non-human molecules.

As described previously in US 20090118127 A1 and as described in Folsson et al., J Mol Biol, 398: 214-231, in 2010, a two-step process is used to humanize and restore or enhance binding affinity to produce binding to humans. The antibody species of the invention exhibiting the targeted effect of murine antibody 10H10 at TF. This two-step process is called Human Framework Adaptability (HFA) and consists of 1) human framework selection and 2) affinity maturation steps.

In the HFA process, binding position residue (CDR) lines are selected for human germ cell line genes selected based on sequence similarity and structural considerations. Two CDR assignment systems suitable for antibodies are: Kabat definition based on antibody sequence variability and Chothia definition based on antibody three-dimensional structure analysis. Among the six CDRs, one or other systems can be used when there are differences. In the case of light chain CDRs, the Kabat definition is used.

In the case of the heavy chain CDR3, the definitions of Kabat and Chothia are identical. In the case of the heavy chain CDR1, the definition of Chothia is used to define the start and the definition of Kabat is the termination (W, followed by the pattern defined by the hydrophobic amino acid such as V, I or A). In the case of VH-CDR2, the Kabat definition is used. However, in most antibody structures In this sequence definition, the FR3 part is assigned to belong to CDR2. Thus, a shorter version of this CDR can also be used to terminate 7 residues on the C-terminal region of this CDR, as referred to herein as Kabat-7.

Human FR selection

A human FR defined as a region within the V region that does not contain an antigen binding site is selected from the gene profiles of the functional human germ cell lines IGHV and IGHJ genes. The gene lineage of the human germ cell line gene sequence was obtained by searching the IMGT database (Lefranc 2005) and compiling all the "01" alleles. From this compilation, redundant genes (amino acid levels of 100% identical) and unpaired cysteine residues were removed from the compilation. The gene compilation was recently updated on October 1, 2007.

Preliminary selection of human sequences suitable for HH of VH, based on sequence similarity of human VH germ cell line genes corresponding to the total length of mouse VH regions, including FR-1 to 3 and H-CDR-1 and H-CDR- 2. In the next stage, the selected human sequences are ranked using a score that takes into account both the CDR length, the sequence similarity between the CDRs of the mouse and the human sequence. Standard mutation matrices, such as the BLOSUM 62 substitution matrix (1992, Henikoff, Proc Natl Acad Sci USA Journal 15; 89(22): 10915-9), used for scoring of CDR sequences in mouse and human sequences, and if When there are insertions and/or deletions in the CDR cycle, it causes a large loss. people The FR-4 class is selected based on the sequence similarity between the IGHJ germ cell line gene (Lefranc 2005) and the mouse 10H10 sequence and IGHJ4 (eg, SEQ ID NO: 60).

A similar procedure was used to select the human FR of VL. In this case, the IGVK, germ cell line genes selected by the same procedure except for the use of the IGHV gene were used as genes suitable for selection of 1-3 of FR and L-CDRs 1-3. The human IGJ-K2 gene (SEQ ID NO: 61) was selected as FR4 of all variants.

Eleven VH and seven VL germ cell line chains were selected. The selected VH genes are mainly from the IGVH-1 gene family: 6 sequences from IGVH1 with IGVH1-69 and IGVH1-f with longer and shorter H-CDR2, 2 from IGVH3, and one used The IGVH5 gene CDR2 of the long cum short H-CDR2. The VL gene represents 6 IGVK2 and 1 IGVK4 gene family.

Thus, VH variants H15, H19 and H21 with longer H-CDR2 (SEQ ID NO: 7) correspond to H22, H23 and H24, respectively, using shorter mouse CDR-H2 (eg SEQ ID NO: 27). . The prefix "S" indicates that the test variant carries fewer mouse residues in the beta strand region with more human residues. The V-region designations used and the gene sequences used are shown in Tables 2 and 3 below.

Human MA FR variant representing 11 heavy and 7 light chains, plus 96 MAb databases of the murine 10H10 chimeric strand, expressed in HEK 293E cells in 96-well format to provide primary screening The supernatant. For initial screening, DNA encoding the selected variable region was recombined using standard recombinant methods to form intact MAbs transiently expressed in 96 well plates in HEK 293E cells. 96 small after transfection The activity test activity (binding) was carried out against the supernatant fluid from the culture medium.

The 19 variants were selected for experimental scale performance in HEK 293-F cells and then purified based on the results of the primary screening. Experimental scale performance was performed instantaneously in 750 ml volumes of HEK 293F cells or CHO-S. The supernatant from the harvest was purified by protein A chromatography, followed by evaluation of its affinity and functional activity against the purified protein. In addition, the purified protein was subjected to biophysical characterization by SDS-PAGE, SE-HPLC, and interaction chromatography (CIC). The theoretical isoelectric point (pI) of each variant is also calculated. From experimental scale characterization, a series of ultimately important candidates were transfected in a WAVE bioreactor and then purified by protein A.

Combined assessment

Binding of the chimeric chimeric antibody, Ml and HFA variants to human and male monkey TF was performed using chemiluminescence detection according to the direct ELISA format. In order to initially screen the library variants in the original supernatant, the samples or control groups were normalized to 50 Ng/ml in FreeStyle 293 HEK medium (Gibco) and analyzed under a single concentration assay. In this experiment, the antibody concentration was 5 ng (using 0.1 ml), and the TF ECD and antigen were His6-TF-ECD _1-219 , and the final concentration was 10 ng/well.

The screening results for the entire pooled database indicated that all other VHs were bound to hTF with different intensities except H14. Several HFA variants provide a higher binding signal than the parental 10H10 (H13, L1), especially some L3 and L5 combinations. H18 and H21 do not bind to human antigens, nor bind to other VHs, indicating insufficient binding to the male monkey antigen. Between VL, L6 and L8 do not bind to either antigen when other bindings are at detectable levels. H14 and L8 also produced lower performance when combined with any VL. Fifty of the 77 antibodies (VH, VL combination) exhibited TF binding as shown in Table 4.

Ten variants were selected for large scale performance and purification using ELISA, based on the relative binding affinities suitable for TF. K _D s by BIAcore measured summary, ELISA data analysis, whole cell binding by 50 nM FVIIa, 2 [mu] g / ml of Mab inhibition of IL-8 and MDA-MB231 cells from induced and analysis in accordance Thermoflour The measured TMS is shown in Table 5.

Several new human mAb variants exhibited higher affinity for TF, relative to M1 (with 10H10 variable chains: H13 and L1) and some lower. The K _D value for M61 (0.21 nM) is 2.5 times lower than the mouse mother MAb (K _D = 0.56 nM). The data in Table 5 includes four Mabs containing H15 and four with H22, both of which are constructed from the same germline lineage gene (IGHV5-a). Those with H22 and shorter H-CDR2 usually have higher binding affinity than the corresponding molecule with H15. Although many are new variants that are bound by Malay TF, the ordering of male monkey binding affinity is different from that suitable for human TF binding.

The Tm of the mouse 10H10 mAb was 74.2 °C. The Tm of the selected molecule ranges from 75 to 82.2 °C. Thus, the HFA process creates an antibody construct with a new Fd region suitable for increased binding affinity of human and non-human primate TF, and also produces stable intact antibody variants with human domains.

Characterization of additional new antibody constructs verified that the antibody recognizes native TF on cells derived from human tumor tissue (MDA-MB231 breast cancer-derived cells) and by inhibiting IL-8 induction from MDA-MB231 The presence of VIIa is measured to reduce TF message transmission.

Additional biophysical characterization (solubility and interaction chromatography) and analytical results led to the selection of M59 consisting of variable regions H22 and L3 suitable for affinity maturation.

Example 4: Antibody maturation

The Fab database is constructed in the pIX phage display system as described in the applicant's co-pending application WO2009/085462, which is incorporated herein by reference.

Based on the experimental structure of 10H10 in the hTF complex, two databases were designed to start with M59 paired H116 (SEQ ID NO: 19) and L3 (SEQ ID NO: 23) to achieve V _L and V. _H The diversity of both. The database differs in the target location due to diversity and is also different in the amino acids used to diversify the target location. A database is diversified into a total of 8 locations representing the CDRs, which were previously shown to be in contact with the TF. This design focuses on L1, L3, H1 and H2. The position of contact in L2 is not diversified, and most of the positions in H3 are also the same. Avoid, for example, cysteine and methionine, unstable or reactive amino acids.

The second set of databases was designed to diversify the amino acids located around the antigen binding site, and the antigen binding sites were determined by calculating the solvent accessibility of the bound and unbound Fab crystal structures. Residues that are buried in contact with solvent molecules are also targeted because of their diversity. A total of 12 residues (6 in V _L and 6 in V _H ) were determined using this method, which was diversified into groups with 8 amino acid reductions, including: Arg(R), Asn (N), Asp (D), Gly (G), His (H), Ser (S), Trp (W), and Tyr (Y). The combined database size is estimated to be 8 ₁₂ or 10 ₁₀ variants, which can be applied using standard databases to limit selection techniques.

For the CDR-contacting residue library, this position is diversified into 15 amino acids using nucleotide dimer (N-dimer) synthesis (except for Met, Cys, Lys, Gln, and Glu). ).

The Fab library presented on phage coated protein IX is screened for biotinylated hT-ECD according to screening schemes known in the relevant art, by selecting a lower exit rate (increased exit rate value) or faster binding. The rate (the rate of binding rate is reduced), or both, leads to an increase in affinity. Screening was used in human and male monkey TF as a target antigen. Phage systems are produced by helper phage infection. The binding agent is recovered by the addition of SA-beads to form a bead/antigen/phage complex. After the last wash, the phage system was rescued by infection with multiplied TG-1 E. coli cells. The phage system is regenerated and then an additional screening round is performed.

The pIX gene was digested with NheI/SpeI and removed from selected strains. After self-adhesion, the DNA was transformed into TG-1 cells and grown overnight on LB/agar plates. The overnight medium was used for (i) colony PCR and sequencing against the V-region, and (ii) soluble Fab production. Soluble Fab proteins are captured on the plate by multiple anti-Fd (CH1) antibodies. After appropriate washing and blocking, biotinylation at a concentration of 0.2 nM was added. hTF was then used to detect biotinylated hTF using HRP conjugated streptavidin and previous chemiluminescence readings. This concentration of hTF allowed the Fab variants to be ranked, depending on the percentage of parental binding, with the parental Fab line appearing as a control in all plates defined as a 100% binding.

According to this criterion, 381 Fabs that bind to more than 100% of the M59 Fab were selected to bind human TF.

Analysis of selected strains successfully showed Y2, I5, T6 and Y7 at the 27 and 30-32 heavy chain CDR1 (SEQ ID NO: 6) corresponding to the V-region (SEQ ID NO: 4 or 19) There have been significant changes. Although no residues were contacted, only one aromatic amino acid (Tyr and Phe) was allowed at position 27. Position 30 is relatively loose for positions 30-32 in direct contact with K68, T101, Y103, and L104 (Fig. 1) of human TF sequence identification number: 1 in 10H10 Fab, but positions 31 and 32 are restricted. (Table 6).

In the L-CDR2 variation, L3, S6, and S8 (sequence identification number: 7) corresponding to positions L52, S55, and S57 of sequence identification number: 4 or 19, which are associated with human TF sequence recognition in 10H10 Fab No.: P194, S195 and T197 (Fig. 1) are directly contacted with residues, and the permissible substitutions are limited.

In H-CDR3, position N6 (sequence identification number: 8) corresponding to N104 of sequence identification number: 4 or 19, which directly corresponds to F147, G148 and K149 of human TF-ED (sequence identification number: 1) Contact is limited.

The permissible amino acid changes in the various database locations selected by screening for compatible binding affinities by phage screening and solid phase capture assays using 0.2 nM human TF-ECD1-219 are shown in Table 6.

S9, G10 of L-CDR1 (SEQ ID NO: 9) corresponding to positions 32, 33, 34 and 36 of the LC variable region (SEQ ID NO: 5 or 23) for the VL in the selected strain Changes in N11 and K13, which are aligned to the human TF sequence identification number by epitope mapping: 1 The contacts E174, D129, S142, R144, and D145 (Fig. 1) are relatively loose as shown in Table 7. For L-CDR2, the residue W1 in the sequence identifier: 5 or 23, position 56 (Kabat residue number 50) is a contact residue, and residue E61 shows that the substitution tolerance is limited. In the light chain CDR3 (SEQ ID NO: 9) corresponding to the residue position of the sequence identification number: 5 or 23, the sequence has the human TF sequence identification number: 1 K149, D150, N171 and T172 directly Contact (Fig. 1), position D97 (Kabat position 91) is limited. The selection based on the affinity for binding to the amino acid use of the human TF-ECD1-219 strain is summarized in Table 7.

In summary, the identification of a range of affinity-improved Fab variants is through the construction of a phage library based on the appropriate variable region H22 (SEQ ID NO: 19) and L3 (SEQ ID NO: :23) The direct variegation of the CDR position of the amino acid in the contact position of the human TF, followed by screening and selection by screening for compatibility or better binding in the alignment with the starting sequence A species with affinity.

Overall, a modest increase in affinity was obtained for the VH and VL pairs selected from the designed HFA library. However, the parent amino acid was reselected after strict phage screening from the VH library exposed to the variegated residue. From the structural analysis explanation, only two antibody binding sites were significantly changed in position. These two amino acid substitutions are involved in the CDR during affinity maturation, and T31P and S57F are the contact residues involved. An improvement in affinity by 5 fold is attributable to an increase in the S195 contact interface between F57 and TF. The interaction of VL with TF appears to be susceptible, with many variations in contact residues and peripheral residues.

Forty-three of the 381 VH and VL pairs were selected for further characterization. The selected 43 MAbs represent 27 different VHs and 8 VLs (see heavy and light chain sequence pairings in Table 8) and can be classified into 3 subgroups: Group 1 variants have the same light chain (L3) , sequence identification number: 23), groups 2 and 3 are paired with 2 different heavy chains (minutes 8 light chains for H116 and H171, sequence identification numbers: 131 and 67)

A group belonging to 27 antibodies having the same light chain as M59 (L3, SEQ ID NO: 23), which are in H-CDR1 (GYTFX ₁ X ₂ X ₃ WIE (SEQ ID NO: 83) The three positions are different, wherein X1 is selected from A, D, G, I, L, N, P, R, S, T, V, Y, and X2 is selected from A, P, S, T And X3 are selected from F, H and Y); except for H189, wherein H-CDR1 is GFTFITYWIA (SEQ ID NO: 81), and in H-CDR2 (DIX ₁ PGX ₂ GX ₃ TX ₄ (sequence) There are four positions in the identification number: 107), wherein X1 is selected from I and L, X2 is selected from S and T, X3 is selected from A, F, H and w; and X4 is selected from D. , H, I, L, and N; except for H189, where H-CDR2 is DILPASSSTN (SEQ ID NO: 105), and H-CDR3 and FRs are identical to H22 sequence (SEQ ID NO: 19) and are SGYYGNSGFAY (SEQ ID NO: 8) The unique composition of the heavy chains for these 27 Mabs is as follows (Table 9).

H171 (SEQ ID NO: 139), in contrast to H116, contains additional changes in H-CDR1 and H-CDR2, namely I31A and S55T.

The two Mab cohorts are represented by 8 LCs (Table 10) paired with one of two different HC:H22 (SEQ ID NO: 19) or heavy chain H171 (SEQ ID NO: 139). These 8 light chains each have an FR of L3 (derived from IGKV240_O1) and have 5 positions in L-CDR1 (KSSQSLLX ₁ X ₂ X ₃ X ₄ QX ₅ NYLT (SEQ ID NO: 116), among which X1 is selected From F, P, S, T, W and Y; X2 is selected from F, S, T, R and V; X3 is selected from A, G, P, S, W, Y and V; X4 Selected from G, N and T; X5 is selected from K, R and S), and two positions in L-CDR 2 (X ₁ ASTRX ₂ S (SEQ ID NO: 120), wherein X1 is selected from In H and W; X2 is selected from D, E and S) and in L-CDR3 (QNDX ₁ X ₂ X ₃ PX ₄ T (SEQ ID NO: 128), wherein X1 is selected from D, F and L; X2 is selected from the group consisting of S, T and Y; wherein X3 is selected from W and Y; and X4 is selected from the sequence changes of L and M). The composition of these eight LCs is shown in Table 10.

Some of these Mabs were further characterized and tested in an in vivo xenograft mode (Example 5).

Example 5: MAB characterization

Compatible with a M59-based variant library containing a single LC variable region (L3, SEQ ID NO: 23) and a single HC variable region (H22, SEQ ID NO: 19), with partial changes in CDR residues The adaptability and re-selection of the human framework of the residue allows the novel mAb to undergo biophysical and biological activity analysis, while a pair of M1587 lines with altered antibody binding site residues are used to re-examine the original suitable for 10H10Fab Whether there is a change in the epitope determined by binding to the TF-ECD (Example 2) property.

Human TF ECD: M1587 interface

Co-crystallisation of human adaptive and affinity matured antibodies based on 10H10 CDRs, M1587 (L3 and H116) with TF ECD The procedure was carried out in the same manner as for 10H10 (Example 3), except that the M1587-Fab:TF complex system was crystallized from a solution containing 16% PEG 3350, 0.2 M ammonium acetate, 0.1 M sodium acetate, and pH 4.5.

Comparison of the eutectic structure of human TF ECD with M1587Fab with affinity maturation indicated that human adaptation and affinity maturation of 10H10 did not change the footprint of the antibody epitope, as shown in Figure 2, and there was no altered conformation of the CDR. . Three amino acid substitutions (T31P, S57F and N59T) were introduced into H-CDR1 and H-CDR2 during human framework adaptability and affinity maturation (SEQ ID NO: 6 and 27, respectively, using the CDRs described in Example 2) Definitions) (SEQ ID NO: 6 and 7 are replaced by SEQ ID NO: 63 and 86), and the contact residues at residues 31 and 57 containing H116 (SEQ ID NO: 133) are T31P and S57F. A 5 fold improvement in affinity is attributable to an increased interface of F57 with S195 of TF. The human TF ECD structure with M1587 Fab confirmed that the epitope was preserved even during the HFA and affinity maturation, even if the H-CDR1 and H-CDR2 antibody binding site residues were altered.

Biophysical and biological assay results

The pooled data for these antibodies are as follows: KD analysis by Biacore (Table 11), human plasma clotting time caused by human TF (Table 12), IL-from MD-MB-231 cells stimulated by FVIIa 8 Release the applicable EC50 (Tables 13, 14 and 5).

43 for a selected affinity mature mAb (M1) the K _D value of variant human adaptation and frame with a selected murine 10H10, CDR chimeric Mab version has not changed (the number M is less than 100) is generated from 10H10 The data is shown in Table 11. Thus, selecting the human framework and CDR residues replaced compositions which produce human antibody K _D between 80 to 950pM, off-rate (Koff) of between 2.2 × 10-5 to 2.6 × 10-3 sec-1 sec -1 The bonding rate is between 10 ⁴ M-1 sec-1 and 2.3×10 ⁵ M-1 sec-1. Comparing the original mouse 10H10 or chimeric structure values, the equilibrium dissociation constant (K _D ) of the novel mAb was as much as 10 times lower, from 0.77 nM to 0.08 nM; showing a faster binding rate (K _on >10 ⁵ M) -1 second -1) or slower exit rate (Koff = 10 ⁵ sec -1). These characteristics can be used to select a Mab for a particular application that wants to stay or penetrate tissue.

Coagulation

The novel mAb is characterized by the ability to bind to human TF in the presence of calcium and exogenously added human TF in vitro, but does not block coagulation in human plasma (Table 12). Seventeen HFA (M number less than 100) variants and 38 affinity mature variants (M1583 and above) were analyzed and then T _1⁄2 Max (seconds required to reach a maximum optical density of 50%) was reported.

All showed a reaction similar to that observed with 10H10, T _1⁄2 Max was less than 205 seconds (Table 12), indicating that these antibodies were compared to the vehicle control group with 159 ± 17 (n = 14) without antibody. Down, will not prolong the clotting time. The human TF-binding antibody (US7605235 B2) as previously described and derived from CNTO860, which blocks FX binding to the mouse antibody 5G9 of TF, prolonged coagulation in the same assay, but never reached coagulation within 1800 seconds. Of the 43 MAbs with altered CDRs as described in Example 4, were not tested in the coagulation assay because their initial concentration was below 2 mg/ml. The M1, M59 and CNTO860 values of the multiple tests were averaged.

Signal blocking activity

The novel mAb is also capable of blocking the transmission of signals through the TF/FVIIa complex as described. TF/VIIa/PAR2 signaling in breast cancer cells induces extensively directed angiogenic factors such as VEGF25, Cyr61, VEGF-C, CTGF, CXCL1 and IL-8. It has previously been reported that FVIIa induces MDA-MB-231, a human breast cancer cell that exhibits detectable IL-8 in TF (Albrektsen et al., J Thromb Haemost, 5: 1588-1597, 2007). Therefore, this assay was used as a bioassay to assess the activity of variant antibodies against TF/VIIa-induced IL-8 production.

Analytical details as provided herein above, and testing the CDRs of 19 Example 3 HFA (M10-M68) variants and 29 Example 4 The variant results were tested for inhibition of IL-8 production activity against a single concentration of TF (0.5 mg per ml). The primary antibody-RSV antibody (B37) that did not bind to tissue factor was used as a negative control group. At this concentration, many mAbs of HBA were able to block IL-8 induction by more than 67% (Table 13). Figure 5 shows the comparison of 10H10 (SEQ ID NO: 6 and 7 respectively) due to 27 shared L3 light chains (SEQ ID NO: 23) and MAbs with substitutions in H-CDR1 or H-CDR2 Relative inhibition of IL-8 release. In addition, four of them: M1584, M1611, F7M1612, and TF7M1607 were placed together with M in a complete titration IL-8 induction assay. The calculated relative IC50 further supports a more potent argument that the affinity-improved variant is smaller than the 10H10-small mouse-human chimera M1 (Table 14). Other affinity maturation cohorts of the affinity matured antibodies described in Example 4 produced similar results.

Example 6: Antibody antitumor activity Mouse xenograft model with MDA-MB-231

MDA-MB-231 human breast cancer cells were cultured in DMEM medium containing 10% FBS and 1% LNN, harvested by trypsin digestion at logarithmic growth phase, and were at a concentration of 5 × 10 ⁷ cells/ml. Resuspend in sterile serum free DMEM medium. Twenty female SCID Beige (CB-17/IcrCrl-scid-bgBR) mice were obtained from Charles River Laboratories and acclimated for 14 days prior to the experiment. At about 8 weeks old, 2.5 × 10 ⁶ th implanted MDA-MB-231 cells in the right axillary mammary fat pad of the mice. When the tumor was approximately 100 cubic millimeters, the mice were stratified into tumor groups according to tumor size (N=10 per group) in Dulbecco's phosphate buffered saline (DPBS) or M1593 at 10 mg/kg kg. The intraperitoneal treatment was started from the stratified day and then continued once a week for a total of 6 times. Tumors and body weight were recorded weekly. The study was terminated when the mean tumor volume of each group reached 1500 cubic millimeters. The statistical test applied was a two-factor repeated measurement of variance analysis ANOVA (GraphPad 4.0 PRIZM).

In the MDA-MB-231 xenograft mode, M1593 significantly inhibited tumor growth from day 22 (* P < 0.01) and continued until day 29 (** P < 0.001), at which time the control group (DPBS) Processed) euthanasia. The M1593 treatment group was euthanized on the 36th day. M1593 is at On day 29, tumor growth was inhibited by approximately 49%. Tumors were delayed for approximately 11 days in the M1593 treated group relative to the DPBS treated control group (Figure 6).

Mouse xenograft mode with A431

A431 human squamous carcinoma cells were cultured in DMEM medium containing 10% FBS and 1% LNN, harvested by trypsinization at logarithmic growth phase, and resuspended at a concentration of 1 × 10 ⁷ cells/ml. In sterile HBSS. Twenty female SCID Beige (CB-17/IcrCrl-scid-bgBR) mice were obtained from Charles River Laboratories and acclimated for 14 days prior to the experiment. Approximately 8 weeks of age, 2 × 10 ⁶ th implanted A431 cells in the right armpit mouse mammary fat pad at. When the tumor was approximately 118 cubic millimeters, the mice were stratified into treatment groups according to tumor size (N=10 per group). Intraperitoneal treatment was started from stratified days with DPBS or M1593 at 10 mg/kg kg, and then continued once a week for a total of 6 times. Secondary tumors and body weight were recorded weekly. The study was terminated when the mean tumor volume of each group reached 1000 mm3. The statistical test applied was a two-factor repeated measurement of variance analysis ANOVA (GraphPad 4.0 PRIZM).

M1593 significantly inhibited tumor growth from day 22 (* p = 0.0067), at which time the control group (treated with DPBS) was euthanized. The CNTO592 treatment group was euthanized on the 39th. CNTO592 inhibited tumor growth by approximately 54% on day 22. Processed relative to DPBS In the control group, tumors were delayed in growth for approximately 17 days in the M1593 treated group (Figure 7).

Example 7: Antibody composition with altered Fc

Naturally occurring human Fc receptor variants have substantially different affinities from the human antibody FC-portion. In addition, clinical studies have demonstrated improved response rates and survival in patients with tightly binding Fc genotypes following treatment with Fc-engineered mAbs (Musolino et al., J Clin Oncol, 26: 1789-1796, 2008) 2008); Bibeau et al., 2009, J Clin Oncol, 27: 1122-1129).

Although inhibition of TF signaling is expected to reduce cellular responses, leading to tumor cell proliferation, migration, and metastatic spread, it has been shown that TF antigens are present on tumor cells, providing an Fc receptor-associated mechanism by which antibody Fc binds. A means of selectively killing target cells. It is known that the surface characteristics of FC-domain antibodies are affected by the glycan composition as well as the primary sequence of the heavy chain, and modifications of either or both can alter FC-receptor binding.

The MAb line identified as M1593 is produced as a low-trehalose-modified IgG1, and may also be an IgG1-1CH2 domain variant (S239D, I332E, wherein the number belongs to the Kabat EU system).

MAb composition and production method

Preparation of antibody with low trehalose content (M1593-LF), as shown below, encoding the M1593 (IgG1/Kappa) chain by electroporation vector to encode the signal peptide to the protein algae from the CHO host cell line Among the CHO host cell sub-strains selected for rock glycation. Sequence ID: 165 represents the complete light chain comprising the variable region, residues 1-113 (SEQ ID NO: 23 plus FR4, SEQ ID NO: 61, bottom line) and the human Kappa constant light domain. a heavy chain comprising variable region residues 1-120 with wild-type human antibody isoforms CH1, CH2 and CH3 (comprising SEQ ID NO: 139 and FR4 SEQ ID NO: 60, bottom line), wherein Kabat positions 239 and 332 (SEQ ID NO: 167 and 335 of 167) were modified from wild type residues S and D, to D and E, respectively, to form variant M1593-DE.

M1593-light chain

DIVMTQTPLSLPVTPGEPASISCKSSQSLLSSGNQKNYLTWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQNDYTYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID: 165)

M1593-heavy chain, where Kabat position S239 is D and I332 is E

EVQLVQSGAEVKKPGESLRISCKGSGYTFAPYWIEWVRQMPGKGLEWMGDILPGTGFTTYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARSGYYGNSGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGGLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP D VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP E EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 167)

After 4 rounds of negative lectin selection (selection does not bind to trehalose-binding lectin) and FACS sequencing, CHO cells are produced by sub-selection to isolate a pool as a host cell. The use of naturally occurring low trehalose cells. This cell line is derived from the same host cell used to make M1593, so the cultivation and processing of the cells is carried out in the same manner. Screening of transfected cells was performed by methylcellulose panelization using G proteins suitable for detection and sorting into cell lines in 96 well plates. The medium was spread to a shaker flask for titration. The top parental strain produced M1593-LF up to 708 mg/liter in standard shake flask medium (in standard media).

LC-MS glycopeptide map alignments were performed on two M1593-LF production lines (C2452B and C2452D) to determine the percentage of fucosylation and to evaluate the stability of the glycation profile over time and production process (Table 15). Samples were collected from the first generation feed batch to the tenth batch of medium during the stability study and then purified. The purified samples evaluated from the bioreactor were also analyzed. This glycosylation pattern is advantageous when the glycopeptide map alignment shows a low percentage of total fucosylation from C2452B and C2452D. Importantly, the percentage of fucosylation does not increase significantly over time, indicating that the fucosylation of the host cell is stable. Thus the trehalose content for M1593-LF is less than 10%, and is typically less than 5%, and in some formulations less than 2%. A Mab produced in a non-exogenous lectin-selected host CHO cell that contains more than 80% of the hyperglycosylated glycan groups.

For the mutation of the FC variant (M1593-DE) of M1593, the plasmid was shown to be M1593 subjected to site-directed mutagenesis.

Biological activity

The three anti-human TF FC variants (M1593, M1593-LF and M1593-DE) tended to be both human and male monkey Fc receptors (FcyRI, FcyRIIa, FcyRIIIa). These analyses were carried out as described in the applicant's patent application (US No. 61/426,619) or by a combined analysis of the surface plasmon resonance (Biacore) basis.

These analyses show that the two Fc-modified anti-TF antibodies bind to the recombinant human FcγIIIa receptor more tightly, compared to the parental unmodified IgG1 M1593 antibody, which is 18-fold (M1593-LF) and 40 times (M1593-DE) (Table 16).

ADCC is stimulated by FcyRIIIa junction. ADCC analysis was performed as previously described (Scallon et al., 2007, Mol Immunol, 44: 1524-1534).

Using human PBMC as an effector cell and a human breast cancer cell line as a target cell, in vitro ADCC analysis can reflect the improved Fc receptor binding function (Fig. 8).

Figure 1 shows the epitope determined by X-ray diffraction analysis of a 10H10 Fab (antigen-binding fragment) or a co-crystal of a human adaptive variant (M1593 Fab) and human TF-ECD residue 5-208. The two contact residues that have been altered in M1593 H-CDR1 (T31P) and HCDR-2 (S57F) are shown.

Figure 2 is an amine of human (SEQ ID NO: 1, 1-219), cyno (SEQ ID NO: 2, 1-220) and mouse TF-ECD (SEQ ID NO: 3, 1-221) residues Base acid arrangement, the figure shows the position of the residue contacted by the murine antibody TF8-5G9 (published in 1998 by Huang et al., J Mol Biol 275: 873 to 94) and 10H10, and those known to be associated with the factor FVII /VIIa and FX contact residues.

Figure 3 shows a three-dimensional projection of human TF-EC with regions labeled by the antibody binding sites of 5G9 and 10H10, as well as the clotting factors FVII and FX, of which only residues L104 and T197 are via 10H10 and FX. Being contacted.

Figure 4 shows an amino acid sequence arrangement of the mouse antibody 10H10 (SEQ ID NO: 4 and 5) heavy chain (upper layer arrangement) and light chain (lower layer arrangement) variable region, antibody M59 (SEQ ID NO: The human framework adaptable sequences of 19 and 23, respectively, and the two selected affinity matured variable region sequences H116 (SEQ ID NO: 133) and H171 (SEQ ID NO: 139).

Figure 5 shows the relative percentage inhibition, which is based on the same type of control B37, 27 affinity matured monoclonal antibodies (mAbs) against FVIIa-induced from MDB-MB-231 breast cancer cells at a concentration of 0.24 μg/ml. IL-8 is released.

Figure 6 shows a graph of tumor volume after implantation of MDA-MB231 tumor cells into immunocompromised mice for several days, wherein the group given M1593 showed a slowing of tumor growth.

Figure 7 shows a graph of tumor volume after implantation of A431 human squamous tumor cells into immunocompromised mice for several days, wherein the group given M1593 showed a slowing of tumor growth.

Figure 8 is a graph showing the percentage of target cell breakdown (MDA-MB231 cells) consisting of human PBMC corresponding to MAb concentration, wherein MAb concentrations are suitable for murine variable region-human IgG1 (M1), positions 234 and 235, respectively. Alanine-substituted murine variable region-human IgG4, M1593 as wild-type IgG1 produced in uncorrected cholesterol (CHO), produced in CHO strain selected to produce glycans with low trehalose content M1593-LF and M1593-DE substituted with Kabat at S239D and I332E.

SEQUENCE LISTING

<110> Jiansheng Biotechnology Company

<120> Human tissue factor antibodies and uses thereof

<130> CEN5309WOPCT

<140> 101108699

<141> 2012-03-14

<150> 61/452,674

<151> 2011-03-15

<160> 167

<170> PatentIn version 3.5

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<400> 108

<210> 109

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 109

<210> 110

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 110

<210> 111

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 111

<210> 112

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 112

<210> 113

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 113

<210> 114

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 114

<210> 115

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 115

<210> 116

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<220>

<221> Mutagenesis

<223> Xaa is selected from F, P, S, T, W and Y

<222> (8)..(8)

<220>

<221> Mutagenesis

<223> Xaa is selected from F, S, T, R and V

<222> (9)..(9)

<220>

<221> Mutagenesis

<223> Xaa is selected from A, G, P, S, W, Y and V

<222> (10)..(10)

<220>

<221> Mutagenesis

<223> Xaa is selected from G, N and T

<222> (11)..(11)

<220>

<221> Mutagenesis

<223> Xaa is selected from K, R and S

<222> (13)..(13)

<400> 116

<210> 117

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 117

<210> 118

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 118

<210> 119

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 119

<210> 120

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<220>

<221> Mutagenesis

<222> (1)..(1)

<223> Xaa is selected from H and W

<220>

<221> Mutagenesis

<222> (6)..(6)

<223> Xaa is selected from D, E and S

<400> 120

<210> 121

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 121

<210> 122

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 122

<210> 123

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 123

<210> 124

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 124

<210> 125

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 125

<210> 126

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 126

<210> 127

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 127

<210> 128

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<220>

<221> Mutagenesis

<222> (4)..(4)

<223> Xaa is selected from D, F and L

<220>

<221> Mutagenesis

<222> (5)..(5)

<223> Xaa is selected from S, T and Y

<220>

<221> Mutagenesis

<222> (6)..(6)

<223> Xaa is selected from W and Y

<220>

<221> Mutagenesis

<222> (8)..(8)

<223> Xaa is selected from L and M

<400> 128

<210> 129

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 129

<210> 130

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 130

<210> 131

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 131

<210> 132

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 132

<210> 133

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 133

<210> 134

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 134

<210> 135

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 135

<210> 136

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 136

<210> 137

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 137

<210> 138

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 138

<210> 139

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 139

<210> 140

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 140

<210> 141

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 141

<210> 142

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 142

<210> 143

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 143

<210> 144

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 144

<210> 145

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 145

<210> 146

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 146

<210> 147

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 147

<210> 148

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 148

<210> 149

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 149

<210> 150

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 150

<210> 151

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 151

<210> 152

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 152

<210> 153

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 153

<210> 154

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 154

<210> 155

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 155

<210> 156

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 156

<210> 157

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 157

<210> 158

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 158

<210> 159

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 159

<210> 160

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 160

<210> 161

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 161

<210> 162

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 162

<210> 163

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 163

<210> 164

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 164

<210> 165

<211> 220

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 165

<210> 166

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 166

<210> 167

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Murine CDRs or their mutagenesis in the human IgG variable domain framework

<400> 167

Claims (31)

An isolated antibody that competes with the murine antibody 10H10 for binding to human tissue factor, wherein the antibody binding domain is suitable for a human framework (FR) region of the structure FR1-CDR1-FR2-CDR2-FR3-CDR3, wherein The FR amino acid sequence is not altered by the amino acid sequence encoded by the human germ cell line gene sequence, and the germ cell line is recognized by the IMGT database and the CDR sequence carries the sequence identification number: 6-11 The mouse 10H10 CDR represented by 27 is not less than 50% identical in sequence. The antibody of claim 1, wherein the antibody does not compete with FVIIa for binding to tissue factor, and does not substantially block the procoagulant and guanamine hydrolyzing activity of the TF-VIIa complex, but blocks MDA TF-VIIa mediates signal transduction detected by the release of interleukin IL-8 from MB-231 cells. An antibody according to claim 2, wherein one or more of the CDR sequences are selected from the sequence consisting of the sequence numbers: 6-11 and 27. The antibody of claim 3, wherein the three light chain CDR sequences L-CDR1, L-CDR2 and L-CDR3 are represented by SEQ ID NO: 9-11, respectively. The antibody of claim 3, wherein the three heavy chain CDR sequences H-CDR1, H-CDR2 and H-CDR3 are respectively identified by sequence number: 6-8 or by sequence identifiers: 6, 27 and 8 representatives. An antibody according to claim 3, wherein the L-CDR1, L-CDR2 and L-CDR3 are represented by SEQ ID NO: 9-11, respectively, and the three heavy CDR sequences are H-CDR1, H-CDR2 and H-CDR3 is represented by sequence identifiers: 6-8 or by sequence identifiers: 6, 27 and 8, respectively. The antibody of claim 1, wherein the human HC variable region framework is derived from an IGHV family 1, 3 or 5 member represented by the IMGT database. An antibody according to claim 7 which comprises an HC variable region selected from the group consisting of SEQ ID NO: 12-21. The antibody of claim 1, wherein the human LC variable region framework is derived from a human IGKV family 2 or 4 member. An antibody according to claim 9 which comprises an LC variable region selected from the group consisting of SEQ ID NO: 22-26. The antibody of claim 1, wherein the human HC variable region framework is derived from a human germline cell gene family selected from the group consisting of IGHV5 and IGKV2. An antibody according to claim 1, wherein the antibody comprises an HC variable region selected from the group consisting of: SEQ ID NO: 12-21 and a human LC variable region selected from the group consisting of SEQ ID NO: 22-26. An antibody according to claim 1, wherein the antibody comprises an HC variable region having the H-CDR3 of SEQ ID NO: 8; and an H-selected from a sequence of SEQ ID NO: 6, 62-83 CDR1; an H-CDR2 having a sequence selected from one of SEQ ID NO: 7, 27, and 84-107; and a HC FR4 region selected from one of IGVJ4 (SEQ ID NO: 60), or one of Variant. An antibody according to claim 1, which comprises an LC variable region having an L-CDR1 selected from the sequence of SEQ ID NO: 9, 108-116; selected from the sequence identification number L-CDR2 of one of 10 and 117-120; L-CDR3 selected from one of sequence identification numbers: 11 and 121-128; and one selected from IGKJ2 (SEQ ID NO: 61) LC FR4 region, or a variant thereof. An antibody according to claim 1, which comprises an HC variable region and an LC variable region and a selective LC FR4 region, the HC variable region The H-CDR3 having the sequence identifier: 8; an H-CDR1 having a sequence selected from one of the sequence identifiers: 6, 62-83; and one having a sequence number selected from: 7, 27 and 84-107 a sequence of H-CDR2; and a variant selected from the HC FR4 region of IGVJ4 (SEQ ID NO: 60), or a variant thereof, and the LC variable region having one having a sequence identification number selected from : L-CDR1 of one of sequence 108-116; L-CDR2 having a sequence selected from one of sequence identification numbers: 10 and 117-120; and one having a sequence number selected from: 11 and 121- L-CDR3 of one of 128 sequences; and the LC FR4 region is selectively selected from IGKJ2 (SEQ ID NO: 61), or variants thereof. The antibody of claim 1, wherein the HC human framework sequence is derived from IGHV5_a and the HC variable region has the sequence selected from the group consisting of: SEQ ID NO: 19, 129-155. The antibody of claim 1, wherein the LC human FR sequence is derived from IGKV2D40_O1, and the LC variable region has a sequence selected from the group consisting of SEQ ID NO: 23, 157-164. The antibody of claim 1, wherein the HC human framework sequence is derived from IGHV5_a, and the HC variable region has the sequence selected from SEQ ID NO: 19, 129-155, the LC human FR sequence The line is derived from IGKV2D40_O1 and the LC variable region has a sequence selected from the group consisting of SEQ ID NO: 23, 157-164. An antibody according to claim 1, wherein the antibody has a binding domain derived from the IGHV5_a framework defined as a non-CDR position, and an H-CDR3 having the sequence SGYYGNSGFAY (SEQ ID NO: 8) and wherein the H- The sequence of the CDR-1 and/or H-CDR-2 position can be represented by the following formula: H-CDR1 GYTFX ₁ X ₂ X ₃ WIE (I) (SEQ ID NO: 83) wherein X1 is selected from A, D , G, I, L, N, P, R, S, T, V and Y; X2 is selected from A, P, S and T; and X3 is selected from F, H and Y; or the sequence is GFTFITYWIA (SEQ ID NO: 81); and H-CDR2 DIX ₁ PGX ₂ GX ₃ TX ₄ (II) (SEQ ID NO: 107) wherein X1 is selected from I and L, and X2 is selected from S and T, X3 is selected from A, F, H and w; and X4 is selected from D, H, I, L and N; or wherein H-CDR2 is DILPASSSTN (SEQ ID NO: 105). An antibody according to claim 1, wherein the antibody has a binding domain, wherein the non-CDR position is derived from the IGKV2D40_O1 framework, wherein the allele is in L-CDR-1 and/or L-CDR-2 and L-CDR The sequence of -3 has a sequence represented by the following formula: L-CDR1 KSSQSLLX ₁ X ₂ X ₃ X ₄ QX ₅ NYLT ( III ) (SEQ ID NO: 116) wherein X1 is selected from F, P, S, T , W and Y; X2 is selected from the group consisting of F, S, T, R and V; X3 is selected from the group consisting of A, G, P, S, W, Y and V; X4 is selected from G, N and T; And X5 are selected from K, R and S; L-CDR2 X ₁ ASTRX ₂ S ( IV ) (SEQ ID NO: 120) wherein X1 is selected from H and W; X2 is selected from D, E and S; L-CDR3 QNDX ₁ X ₂ X ₃ PX ₄ T ( V ) (SEQ ID NO: 128) wherein X1 is selected from D, F and L; X2 is selected from S, T and Y; X3 is selected from W and Y; and X4 is selected from L and M. A method of treating a human subject suffering from a condition in which TF manifestation and local biological activity produced by the TF manifestation are directly or indirectly related to the symptom to be treated, the method comprising An antibody as claimed in claim 1, 3, 19 or 20 is administered to a subject in need of such treatment. The method of claim 21, wherein the symptom is cancer. The method of claim 22, wherein the cancer is selected from the group consisting of a primary solid tumor, a metastatic cancer; a cancer, an adenocarcinoma, a melanoma, a liquid tumor, a lymphoma, a leukemia, a myeloma, a soft tissue cancer, Sarcoma, osteosarcoma, thymoma, lymphosarcoma, fibrosarcoma, leiomyosarcoma, lipoma, glioblastoma, astrocytoma, prostate cancer, breast cancer, ovarian cancer, stomach cancer, pancreatic cancer, laryngeal cancer, esophageal cancer, testis Cancer, liver cancer, salivary gland cancer, biliary tract cancer, colon cancer, rectal cancer, cervical cancer, uterine cancer, endometrial cancer, thyroid cancer, lung cancer, kidney cancer or bladder cancer. The method of claim 21, wherein the symptom is selected from the group consisting of a benign tumor, a hemangioma, an acoustic neuroma, a neurofibroma, a trachoma, and a purulent granuloma; an arteriosclerotic plaque; an ocular neovascular disease, a diabetic retinopathy Retinopathy of prematurity, degeneration of yellow spots, corneal graft rejection, neovascular glaucoma, post-lens fibrous tissue hyperplasia, iris redness, retinoblastoma, ocular uveitis and pterygium (abnormal vascular growth) Rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; angiogenesis; granulation; hypertrophic scar (kelsy); non-healing fracture; scleroderma; Myocardial angiogenesis; coronary collateral; cerebral collateral; arteriovenous malformation; ischemic limb angiogenesis; hereditary hemorrhagic vasodilatation (Osler-Webber Syndrome); plaque neovascularization; microvascular dilatation; hemophilia Sexual joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease and atherosclerosis. A pharmaceutical composition for treating a subject comprising an antibody such as in Patent Application No. 1, 3, 19 or 20 in a pharmaceutically acceptable formulation. A kit comprising antibodies and instructions for use in a stable form, as in claim 1, 3, 19 or 20. An isolated nucleic acid encoding one or more antibody binding domains of an antibody as in claim 1, claim 3, 19 or 20. A vector comprising at least one polynucleotide as in claim 27 of the patent application. A host cell comprising a vector as in claim 28 of the patent application. An isolated antibody or fragment of claim 1, 3, 19 or 20 which has an IgGl or IgG4 isotype. An isolated antibody or fragment according to claim 30, wherein the Fc domain comprises a human IgG1 isotype, wherein the Kabat positions 239 and 332 (SEQ ID NO: 167 of 242 and 335) are in the Fc region, Modifications from wild type residues S and D to D and E mutations.