HK1191960A - Human tissue factor antibody and uses thereof - Google Patents

Description

Human tissue factor antibodies and uses thereof

Priority application

Priority of U.S. application No. 61/452,674, filed on 3/15/2011, is claimed and is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to human adapted antibodies that bind human tissue factor, an antigen present on extravascular tissue including tumor cells, which do not inhibit tissue factor mediated blood coagulation. The invention also relates to methods of using the antibodies to treat disorders associated with the presence of human tissue factor and receptor function, such as malignancies.

Background

Tissue Factor (TF), also known as coagulation factor III (F3), tissue thromboplastin, or CD142, is a transmembrane glycoprotein having an ectodomain of 219 amino acids, which comprises two fibronectin type III domains, and a short endodomain with one serine residue that can be phosphorylated. TF is a cellular receptor for FVII/FVIIa.

TF exhibits a tissue-specific distribution in the form of cellular receptors of high levels in the normal brain, lungs and placenta and low levels in the spleen, thymus, skeletal muscle and liver. It is also present in microparticles of cellular origin and in another spliced soluble form. Besides expression in normal tissues, TF has been reported to be overexpressed in most major tumor types and in many tumor-derived cell lines (Ruf W JThromb Haemost.5: 1584-1587, 2007; Milsom et al, Arterioscler Thromb Vasc biol.29: 2005-2014, 2009).

Coagulation of serum proteins in response to injury is an important physiological response to injury. Exposure of blood to proteins including collagen (intrinsic pathway) and tissue factor (extrinsic pathway) initiates changes in platelets and plasma proteins fibrinogen, coagulation factors. Following vascular injury, factor vii (fvii) leaves the circulation and begins to contact Tissue Factor (TF) expressed on tissue factor-bearing cells (mesenchymal fibroblasts and leukocytes), thereby forming an activated TF-FVIIa complex. TF-FVIIa activates Factor IX (FIX) and Factor X (FX). FVII is allosterically activated by TF, as well as by thrombin, FXIa, plasmin, FXII and FXa. TF-FVIIa forms a ternary complex with FXa.

Tissue Factor (TF) expression by non-vascular cells plays an essential role in hemostasis to stimulate blood clotting. TF is also associated with a process different from hemostasis and is directly associated with a function at the surface of the cell expressing TF. TF-dependent assembly of coagulation proteases on vascular and non-vascular cells activates the Protease Activated Receptor (PAR), which is a G protein coupled receptor. Thus, by PAR (mainly PAR2), TF: the VIIa complex is capable of inducing cell signaling (Camerer et al, Proc. Natl. Acad. Sci. USA 97: 5255-5260, 2000; Riewald and Ruf, Proc. Natl. Acad. Sci. USA 98: 7742-.

The ternary complex TF/FVIIa/FXa consists of TF acting on FX: VIIa complex is formed directly or in TF: VIIa cleavage is followed indirectly by the TF: cleavage with VIIa cleaves FX to FXa. The formation of the TF/FVIIa/FXa complex may lead to signaling or activation of other receptors such as PAR 1-4. The formation of the TF/FVIIa/FXa complex leads to the induction of interleukin-8 (IL-8), which IL-8 stimulates tumor cell migration (Hjortor et al, Blood 103: 3029-3037, 2004). Both PAR1 and PAR2 are involved in tumor metastasis (Shi et al, Mol Cancer Res.2: 395-402, 2004), however, the activated binary and ternary complexes (TF-VIIa and TF-VIIa-FXa) are activators of PAR2, and the PAR2 also leads to cell signaling (Rao and Pendurti, Arterioscler.Thromb.Vasc.biol.25: 47-56, 2005). Therefore, there is interest in determining whether tissue factor carcinogenesis can be separated from procoagulant effects, and there is a long-standing doubt that this involves tumor migration, extravasation, and the metastatic mechanism.

The relationship of monoclonal antibodies to tissue factor, such as those described by Morrisey (1988, Thromb Res52 (3): 247-. Monoclonal antibodies capable of binding tissue factor can be used to block thrombotic events by interfering with the ability of TF to form or retain the TF-VIIa complex, or by blocking the ability of said complex to activate FX. Antibodies that bind to tissue factor and do not block coagulation are also known. TF-elicitor factor VIIa signaling blocking but not coagulation blocking antibodies (such as antibody 10H10) have also been described (Ahamed et al, 2006ProcNatl Acad Sci USA103 (38): 13932-. Ruf et al, published application WO2007056352A3, disclose methods and compositions for inhibiting tissue factor signaling without interfering with hemostasis in patients.

Since malignant tumor progression is a multifaceted process, therapeutic candidates for TF-binding antibodies that can block tumor cell carcinogenesis, metastasis, angiogenesis, and anti-apoptotic functions without interfering with hemostasis in patients are desired.

Disclosure of Invention

The present invention provides human adapted anti-human tissue factor specific antibodies for human therapy that retain the binding epitope of murine antibody 10H10, which antibody does not compete with tissue factor for FVIIa binding and therefore does not substantially block procoagulant, amidolytic activity of the TF-VIIa complex but blocks TF-VIIa mediated signaling and downstream oncogenic effects (such as cytokine IL-8 release).

The human adaptive antibodies of the invention are composed of human IgG variable domain framework combined CDR variant residues represented by the reference 10H10 murine antibody CDR sequences and represented by SEQ ID NO: 6-11 and 27. The combination of human frameworks FR1 and FR2 and FR3 with CDRs and CDR variants, with FR4 is provided, which allows the assembly of antibody binding domains with the immunospecificity of the murine antibody 10H 10. In one embodiment of the invention, the polypeptide represented by SEQ ID NO: 6-11 or the six CDR sequences represented by SEQ ID NOs: 6. the groups represented by 8-11 and 27 were combined with human germline FRs, defined as the non-CDR positions of the human IgG variable domains, selected such that the binding affinity of 10H10 to human TF was preserved. In one aspect, the human HC variable region FR is derived from a member of IGHV gene family 1, 3 or 5 as represented by the IMGT database. In one aspect, the human LC variable region FR is derived from a human IGKV gene family 2 or 4 member. In one embodiment, the antibody Fv (HC variable region paired with LC variable region) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-21 and a HC variable domain selected from SEQ ID NOs: 22-26.

In particular embodiments, human FRs that form antibody Fv (HC variable region paired with LC variable region) include IGHV5 and IGKV2 FR. The antibody of the invention comprises an HC variable domain having: SEQ ID NO: 8, H-CDR 3; has a sequence selected from SEQ ID NO: 6 and 62-83, H-CDR 1; has a sequence selected from SEQ ID NO: 7. 27 and 84-107, H-CDR 2; and optionally, an HC FR4 region selected from IGVJ4(SEQ ID NO: 60) or a variant thereof. Antibodies of the invention also include those having an LC variable domain having: has a sequence selected from SEQ ID NO: 9. 108-116 of the sequence L-CDR 1; has a sequence selected from SEQ ID NO: 10 and 117-120 of the sequence L-CDR 2; and has an amino acid sequence selected from SEQ ID NO: 11 and 121-128 of the sequence L-CDR 3; and optionally, an LC FR4 region selected from IGKJ2(SEQ ID NO: 61) or a variant thereof. In particular embodiments, the human framework sequence is derived from IGHV5_ a, and the created variable domain comprises a sequence selected from SEQ ID NOs: 19. 129-155. In another embodiment, the human framework sequence is derived from IGKV2D40_ O1, and the created variable domain comprises a sequence selected from SEQ ID NOs: 23. 156-163.

One form of the antibody of the invention may be represented as an antibody having: a H-CDR3 derived from the binding domain of the IGHV5_ a framework, defined as a non-CDR position, having the sequence SGYYGNSGFAY (SEQ ID NO: 8), wherein the sequence at the H-CDR-1 position is given by:

H-CDR1 GYTFX₁X₂X₃WIE(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 may be GFTFITYWIA (SEQ ID NO: 81); and the sequence at position H-CDR2 is given by:

H-CDR2 DIX₁PGX₂GX₃TX₄(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 in H189, where H-CDR2 is DILPASSSTN (SEQ ID NO: 105).

The antibodies of the invention are represented as antibodies having binding domains derived from the IGKV2D40 — O1 framework defined as non-CDR positions, and wherein the sequences at L-CDR-1 and/or LCDR-2 and L-CDR3 have sequences given by the formula:

L-CDR1 KSSQSLLX₁X₂X₃X₄QX₅NYLT(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; x5 is selected from K, R and S;

L-CDR2 X₁ASTRX₂S(IV) (SEQ ID NO：120)

wherein X1 is selected from H and W; x2 is selected from D, E and S;

L-CDR3 QNDX₁X₂X₃PX₄T (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; x4 is selected from L and M.

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

The invention also provides human adaptive antibodies that compete for binding to human tissue factor and thus to substantially the same epitope of human TF-ECD as murine 10H10 antibody. The invention also provides methods of using such antibodies to treat a human subject having a disorder in which TF expression and local biological activity resulting from TF expression are directly or indirectly associated with the disorder being treated.

The invention also provides methods of making antibodies and pharmaceutically acceptable formulations of the antibodies, containers comprising the formulations, and kits comprising the containers, wherein the antibodies of the invention can be made against methods of use to treat human subjects.

Drawings

FIG. 1 shows the epitope revealed by X-ray diffraction analysis of 10H10Fab or co-crystallized with human adapted variant (M1593Fab) and human TF-ECD residues 5-208, showing two contact residues that were varied in M1593H-CDR1(T31P) and HCDR-2 (S57F).

FIG. 2 is an alignment of the amino acid residues 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), showing the residue positions of the contact murine antibodies TF8-5G9(Huang et al, 1998J Mol Biol 275: 873-94) and 10H10 and those residues known to contact the coagulation factors FVII/VIIa and FX.

Fig. 3 shows a three-dimensional projection of human TF-ECD with paratopes contacting 5G9 and 10H10 and the regions indicated for coagulation factors FVII and FX, where only residues L104 and T197 contact both 10H10 and FX.

FIG. 4 shows an amino acid sequence alignment of the heavy (upper alignment) and light (lower alignment) variable domains of murine antibody 10H10 (SEQ ID NOS: 4 and 5, respectively), the human framework-adapted sequences of antibody M59 (SEQ ID NOS: 19 and 23, respectively), and two selected affinity matured variable domain sequences H116(SEQ ID NO: 133) and H171(SEQ ID NO: 139).

Figure 5 shows the relative inhibition percentage of 27 affinity matured mabs against IL-8 release from FVIIa-induced MDB-MB-231 breast malignant cells at 0.24 ug/ml compared to isotype control B37.

FIG. 6 shows a graph of tumor volume over a certain number of days following MDA-MB231 tumor cell engraftment in immunocompromised mice, where the group administered M1593 reduced established tumor growth.

Fig. 7 shows a graph of tumor volume over days following implantation of a431 human squamous tumor cells into immunocompromised mice, in which the group administered M1593 reduced established tumor growth.

Figure 8 shows a plot of the target percent cytolysis of human PBMC (MDA-MB231 cells) versus MAb concentration for murine variable domain-human IgG1(M1), murine variable domain-human IgG4 with alanine substitutions at positions 234 and 235, wild type IgG1 produced M1593 in unmodified CHO, M1593-LF produced in CHO lines selected for production of glycans with low fucose content, and M1593-DE with Kabat position substitutions at S239D and 1332E.

Sequence listing description

Detailed Description

Abbreviations

TF-tissue factor, huTF-human tissue factor, muTF-murine tissue factor, cynoTF-cynomolgus monkey tissue factor, TF-FVIIa-tissue factor-factor VIIa complex, TF/FVIIa-tissue factor-factor VIIa complex, HC-heavy chain, LC-light chain, v-variable domain, VH-heavy chain variable domain, VL-light chain variable domain, CCD-charge coupling device, CDR-complementarity determining domain, CHES-2- (N-cyclohexylamino) -ethanesulfonic acid, EDTA-ethylenediaminetetraacetic acid, ECD-ectodomain, HEPES-N- (2-hydroxyethyl) -piperazine-N' -2-ethanesulfonic acid, HEK-human kidney cells, MES-2- (N-morpholino) ethanesulfonic acid, PBMC-protease activated receptor, PBMC-peripheral mononuclear cells, PBS-phosphate buffered saline (PDB) buffer protein data, PEG ═ polyethylene glycol, SDS PAGE ═ sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SEC ═ size exclusion chromatography, MAb ═ monoclonal antibody, FR ═ antibody framework, HFA ═ human framework adaptation.

Definitions and explanations of terms

As used herein, "antibody" includes whole antibodies and any antigen-binding fragment or single chain thereof. Thus, an antibody includes any protein or peptide comprising a molecule that comprises at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one Complementarity Determining Region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a Framework (FR) region, or any portion thereof, or at least a portion of a binding protein that can be incorporated into an antibody of the invention. The term "antibody" is also intended to encompass antibodies, digested fragments thereof, specified portions and variants, including antibody mimetics or antibody portions that comprise structures and/or functions that mimic antibodies or specified fragments or portions thereof, including single chain antibodies and single domain antibodies and fragments thereof. Functional fragments include antigen-binding fragments directed against a preselected target. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL and CH domains; (ii) a F (ab') 2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bond; (iii) an Fd fragment consisting of VH and CH domains; (iv) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of VH domains (Ward et al, (1989) Nature 341: 544-546); and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by synthetic linking peptides that allow them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for utility in the same manner as intact antibodies. Conversely, scFv construction libraries can be used to screen for antigen binding and then spliced together with other DNA encoding human germline gene sequences using conventional techniques. An example of such a library is "HuCAL: human combinatorial antibody libraries "(Knappik, A. et al, J MolBiol (2000)296 (1): 57-86).

The term "CDR" refers to the complementarity determining region or hypervariable region amino acid residues of an antibody which are involved in or responsible for antigen binding. The hypervariable regions or CDRs of the human IgG antibody subclass comprise residues 24-34(L-CDR1), 50-56(L-CDR2) and 89-97(L-CDR3) from the light chain variable domain and residues 31-35(H-CDR1) from the heavy chain variable domain, amino acid residues from 50-65(H-CDR2) and 95-102(H-CDR3) (as described by Kabat et al (1991Sequences of Proteins of immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md.) and/or residues from the hypervariable loops of the light chain variable domain (i.e., residues 26-32(L1), 50-52(L2) and 91-96(L3)) and the hypervariable loops of the heavy chain variable domain 26-32(H1), 52-56(H2) and 95-101(H3) (as described by Chothia and Lesk, 1987 J.mol.biol.196: 901-917)). Chothia and Lesk refer to structurally conserved hypervariable loops as "canonical structures". Framework or FR1-4 residues are those variable domain residues other than and excluding the hypervariable region. The numbering system of Chothia and Lesk takes into account the numbering differences of the residues in the loops by showing extensions at the designated residues in lower case notation, e.g., 30a, 30b, 30c, etc. Recently, a universal numbering system, i.e., the International immunogenetics information System, has been developed and widely adopted(intemational ImMunoGeneTics information) (IMGT) (LaFranc et al, 2005.Nucl Acids Res.33: D593-D597).

Herein, CDRs are represented by sequence numbering in terms of amino acid sequence and position in the light or heavy chain. Since the "position" of a CDR in the structure of an immunoglobulin variable domain is conserved across species and is present in a structure called a loop, the CDR and framework residues are readily identified by using a numbering system that aligns the variable domain sequences according to structural features. This information is used to graft and replace CDR residues from an immunoglobulin of one species into the acceptor framework from a typically 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 may form at least a portion of a dimer region of a protein/molecule (e.g., an antibody).

The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groups of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

As used herein, K_DRefers to the dissociation constant, specifically, the K of the antibody to the predetermined antigen_DAnd is a measure of the affinity of the antibody for a particular target. K of high affinity antibodies to predetermined antigens_DIs 10^-8M or less, more preferably 10^-9M or less, and more preferably 10^-10M or less. K_DIs reciprocal of K_AAnd is the association constant. As used herein, the term "k_dis"or" k₂"or" k_d"means a specific antibody-antiOff-rate of the original interaction. ' K_D"is the dissociation rate (k)₂Also known as the "off-rate" (k)_off) ") and association rate (k)₁) Or "on-rate" (k)_on) "is used in the following description. Thus, K_DIs equal to k₂／k₁Or k_off／k_onAnd is expressed in molar concentration (M). It follows the principle that K_DThe smaller the binding, the stronger. Thus, and 10^-9M (or 1nM) comparison, 10^-6K of M (or 1. mu. mol)_DIndicating a weak binding.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. The term also includes "recombinant antibodies" and "recombinant monoclonal antibodies" because all antibodies are made, expressed, produced or isolated by recombinant means, e.g., (a) antibodies isolated from an animal or hybridoma, which are made by antibody secreting animal cells and fusion partners; (b) antibodies isolated from host cells transformed to express the antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant, combinatorial human or other species antibody libraries and (d) antibodies prepared, expressed, produced or isolated by any other method that involves splicing immunoglobulin gene sequences to other DNA sequences. As used herein, "isolated antibody" means an antibody that: substantially free of other antibodies having different antigen specificities. However, an isolated antibody that specifically binds to a human TF epitope, isoform or variant may be cross-reactive to other related antigens, e.g., from other species (e.g., TF species homologs). Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, combinations of "isolated" monoclonal antibodies with different specificities are mixed in a well-defined composition.

As used herein, "specific binding," immunospecific binding, "and" immunizationSpecifically binding "refers to the binding of an antibody to a predetermined antigen. Typically, the antibody is present in 10^-7Dissociation constant (K) of M or less_D) K binding to and binding to a predetermined antigen_DK for its binding to a non-specific antigen other than the predetermined antigen (e.g. BSA, casein or any other specific polypeptide)_DAt least 1/2. The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen". As used herein, "highly specific" binding refers to the relative K of an antibody to a particular target epitope_DK for binding of the antibody to other ligands_DAt least 1/10.

As used herein, "isotype" refers to the class of antibodies (e.g., IgM or IgG) encoded by the heavy chain constant region gene. Some antibody classes also include subclasses that are also encoded by heavy chain constant regions and that are also modified with oligosaccharides at specific residues within constant region domains (e.g., IgG1, IgG2, IgG3, and IgG4) that also impart biological function to the antibody. For example, among human antibody isotypes, IgG1, IgG3, and the lesser existing IgG2 exhibit the same effector functions as the murine IgG2a antibody.

By "effector" function or "effector positive" is meant that the antibody comprises a domain that is distinguishable from the antigen-specific binding domain, which is capable of interacting with a receptor or other blood component (e.g., complement), resulting in, for example, macrophage recruitment, and causing the destruction of cells bound by the antigen-binding domain of the antibody. Antibodies have several effector functions mediated by the binding of effector molecules. For example, binding of the C1 component of complement to antibodies activates the complement system. Activation of complement is important for the opsonization and lysis of cellular pathogens. Activation of complement stimulates inflammatory responses and may also involve autoimmune hypersensitivity. In addition, the antibody binds to the cell via the Fc region, i.e., the Fc receptor site on the Fc region of the antibody binds to an Fc receptor (FcR) on the cell. There are many Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptor), IgE (eta receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of antibodies to Fc receptors on cell surfaces triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-encapsulated particles, clearance of immune complexes, lysis of antibody-encapsulated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and modulation of immunoglobulin production.

The terms "tissue factor protein", "tissue factor" and "TF" are used to refer to a polypeptide having an amino acid sequence corresponding to naturally occurring human tissue factor or recombinant tissue factor as described below. Naturally occurring TF includes tissue factors of the human species as well as other species such as rabbits, rats, pigs, non-human primates, horses, mice and sheep (see, e.g., Hartzell et al, (1989) mol. cell. biol., 9: 2567-. The amino acid sequence of human tissue factor is given by UniProt records P13726(SEQ ID NO: 1), cynomolgus monkey (SEQ ID NO: 2) and UniProt P20352 in mice (SEQ ID NO: 3). The amino acid sequences of other mammalian tissue factor proteins are well known or can be obtained by conventional techniques.

The antibodies of the invention are useful for administration to a human subject or for contacting human tissue, wherein blocking of human TF function expressed on a cell, tissue, or organ caused by TF signaling is desired, and wherein also substantially no change in the expression of TF: the formation of the FVIIa complex leads to a TF procoagulant function. Such uses may be found in the treatment of tumors, in particular, primary or secondary solid tumors of the breast, prostate, lung, pancreas and ovary.

The invention also includes nucleic acids encoding the antibody sequences of the invention, which can be combined with those known in the art for use in the construction and manufacture of antibodies expressing information by recombinant methods or in the surrounding environment where it is desired to form them in such cultures, in situ and in vivo. Methods for manipulating such nucleic acids to produce antibodies of the invention are well known to those skilled in the art.

The invention also provides stable formulations such as pharmaceutically acceptable formulations or for administration and storage of the antibodies of the invention in isolated form.

1. Antibody compositions

Properties of

The present invention is based on the unexpected discovery of a non-clotting blocking murine antibody, designated 10H10, that binds human TF (Edgington et al, US5,223,427), which is capable of abrogating TF signaling in certain cells (Ahmed et al, 2006, cited above, WO 2007/056352 a 2). Thus, the antibodies of the invention are antibodies that retain the binding epitope of murine antibody 10H10, which does not compete with tissue factor for FVIIa binding and does not substantially block procoagulant, amidolytic activity of the TF-VIIa complex but blocks TF-VIIa-mediated signaling and downstream oncogenic effects (such as cytokine IL-8 release). The antibodies of the invention are suitable for human germline IgG genes represented in the IMGT database and retain the ability of TF to bind human TF without interfering with the initiation of coagulation by TF in the presence of calcium in human plasma.

Generally, antibodies that retain the epitope to which murine antibody 10H10 binds can be evaluated by evaluating the following abilities: the antibody binds TF and competes with 10H10 for binding to human TF while the time required for TF to initiate plasma coagulation when present in a sample comprising TF in the presence of human plasma will not be substantially prolonged compared to a similar human plasma sample in the absence of the antibody. In another aspect, the epitope of an antibody can be physically mapped using techniques known in the art, including but not limited to deletion mutagenesis, substitution mutagenesis, limited proteolysis of antibody-bound TF following identification of peptide fragments, and co-crystallization and X-ray diffraction methods to map the atomic structure of the TF major structure and the proximity of the antibody binding domain, thereby defining a three-dimensional association between the antibody and human TF (fig. 1).

Thus, epitopes can be defined as non-overlapping with the FVIIa binding site (fig. 2 and 3). More specifically, the epitope to which the antibody of the invention binds may contact one or more residues in the N domain of TF that is not in contact with FVII (residues 1-104 of the mature chain represented by SEQ ID NO: 1), such as residues 65-70, and not contact residues K165 and K166 in the C domain, which is important for matrix binding (Kirchofer et al, 2000Thromb Haemostat 84: 1072-81) without interfering with the ability of TF to initiate coagulation in the presence of calcium in human plasma.

In one embodiment, the binding rate (k) of the antibody_a1/M.s) is greater than 1X 10^-5. In another embodiment, the dissociation rate (k) of the antibody to TF_d1/s) less than 1.0X 10^-5And K is obtained_DLess than 1 x 10^-9M (less than 1 nM). In specific embodiments, the antibody is of less than 0.5 x 10^-9K of M_DThe human germline gene adaptive antibody of (1). In one embodiment, the antibody has binding domains selected from those of heavy and light chain pairings 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 can also be characterized as comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-166, or a sequence of amino acid residues in the binding domain of one or more of the amino acid sequences set forth.

Antibody variants with altered Fc function

The characteristics and properties of these complex compositions were explored when the therapeutic monoclonal antibodies produced were amplified by recombinant methods. While immune specificity and antigen targeting characteristics are generally in the variable domains and subdomains, such as the loop ends of hypervariable regions (also known as CDRs), the complex interacts with other receptors and serocomponents provided by structures formed by constant domains, such as the Fc portion of IgG.

The function of antibodies and other Fc-containing proteins can be compared by several well-known in vitro assays. In particular, of interest are the affinities of the Fc γ RI, Fc γ RII and members of the Fc γ RIII family of Fc γ receptors. These measurements can be made using recombinant soluble forms of the receptor or cell associated forms of the receptor. Furthermore, the affinity of FcRn, a receptor that acts to prolong the circulating half-life of lgG, can be measured, for example, by BIAcore using recombinant soluble FcRn. The potential functional consequences of a particular variant structure can be understood in depth using cell-based functional assays (e.g., ADCC and CDC assays). In one embodiment, the ADCC assay is configured to use NK cells as the primary effector cells, reflecting the functional effects on Fc γ RIIIA receptors. Phagocytosis assays can also be used to compare immune effector function of different variants, while assays that measure cellular responses (e.g., peroxide or inflammatory mediator release) can also be used to make comparisons. For example, in the case of using variants of anti-CD 3 antibodies, an in vivo model can also be used to measure T cell activation in mice, which activity depends on the Fc domain engaging a particular ligand (e.g., an Fc γ receptor).

2. Production of tissue factor signal-blocking antibodies

Antibodies having the characteristics and biological activities of the antibodies described herein can include or be derived from any mammal, such as, but not limited to, a human, a mouse, a rabbit, a rat, a rodent, a primate, a goat, or any combination thereof, and include isolated human, primate, rodent, mammalian, chimeric, human or primate adapted antibodies, immunoglobulins, lysates, and other specified portions and variants thereof. Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler and Milstein, 1975, Nature, 256: 495-497) and related methods of fusion with B cells using immortal fusion partners. The antibodies used in the present invention are produced by cloning and expressing immunoglobulin variable region cDNA produced by a single lymphocyte selected for the production of specific antibodies, and may also be produced using a single lymphocyte antibody method, as described, for example, in Babcook, j. et al, 1996, proc.natl.acad.sci.usa93 (15): 7843-78481; WO 92/02551; WO 2004/051268 and International patent application No. WO 2004/106377.

As herein describedThe antibody comprising a target binding domain or subdomain, a constant domain and a functional non-target binding domain (such as an Fc domain) may be derived from a variety of methods well known in the art. In one aspect, the sequence of the naturally occurring antibody domain can be conveniently obtained from published or online literature or databases (such as V-base (provided by MRC Centre for Protein Engineering), the national center for biological information (NCBI Igblast), or by the International immunogenetics information System (International immunogenetics information)) The immunogenetics (imgt) database provided).

Human antibodies

The invention also provides human immunoglobulins (or antibodies) that bind human TF. These antibodies may also be characterized as engineered or adapted. The immunoglobulins have one or more variable regions substantially from human germline immunoglobulins and include directed variations of residues known to be involved in antigen recognition, such as the CDRs of Kabat or residues of structurally defined hypervariable loops. The one or more constant regions (if present) are also substantially from a human immunoglobulin. Human antibodies exhibit at least about 10 against TF^-6M (1. mu.M), about 10^-7M(100nM)、10^-9M (1nM), or less K_D. To influence the change in affinity, e.g., to improve the affinity of a human antibody for TF or to reduce the K of a human antibody for TF_DCDR residues or other residues may be substituted.

The source for generating human antibodies that bind TF is preferably a sequence provided herein, such sequences are sequences comprising a sequence selected from the group consisting of SEQ ID NOs: the variable region of the 129-163 sequence selected from the group consisting of SEQ ID NO: 28-61, and CDRs, wherein the CDRs are selected from the group consisting of SEQ ID NOs: 6-11, 27, 62-128.

The replacement of any non-human CDR to any human variable domain FR may not allow the same spatial orientation provided by the parent variable FR conformation from which the CDR originates. The heavy and light chain variable framework regions that will be paired in the final Mab may be derived from the same or different human antibody sequences. The human antibody sequences can be naturally occurring human antibody sequences, derived from human germline immunoglobulin sequences, or can be a consensus sequence of several human antibody and/or germline sequences.

Suitable human antibody sequences can be identified by computer comparison of the amino acid sequences of the mouse variable regions to the sequences of known human antibodies. Comparisons of heavy and light chains were made separately, but the principle of each comparison was the same.

In terms of experimental methods, it has been found particularly convenient to create libraries of variant sequences which can be screened for a desired activity, binding affinity or specificity. One form for creating such variant libraries is a phage display vector. Alternatively, variants may be generated using other methods of variation of the nucleic acid sequence encoding the target residue in the variable domain.

Another method of determining whether further substitutions are required and selecting amino acid residues for substitution can be accomplished using computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are widely available. Typically, the generation of molecular models begins with the solved structure of an immunoglobulin chain or domain thereof. The chains to be modeled are compared for amino acid sequence similarity to chains or domains of resolved three-dimensional structures, and the chain or domain exhibiting the highest sequence similarity is selected as the starting point for molecular model construction. The solved starting structures are modified to allow for differences between the actual amino acids in the immunoglobulin chains or domains being modeled and those in the starting structures. The modified structures are then assembled into complex immunoglobulins. Finally, the model was refined by energy minimization and by verifying that all atoms are at the proper distance from each other and that the bond length and bond angle are within chemically acceptable limits.

Due to the degeneracy of the code, multiple nucleic acid sequences will encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence may be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of an earlier prepared variant of the desired polynucleotide. All nucleic acids encoding the antibody described in the present application are specifically included in the present invention.

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

The human antibody may comprise any type of constant domain from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and from any subclass (isotype), including IgG1, IgG2, IgG3, and IgG 4. When it is desired that the humanized antibody exhibit cytotoxic activity, the constant domain is typically a complement-binding constant domain, and the species is typically an IgG₁. When such cytotoxic activity may not be required, the constant domain may be an IgG₂And (4) class. Humanized antibodies may comprise sequences from more than one species or isotype.

Nucleic acids encoding the humanized light chain variable region and the heavy chain variable region, optionally linked to a constant region, are inserted into an expression vector. The light and heavy chains may be cloned in the same or different expression vectors. The DNA segment encoding the immunoglobulin chain is operably linked to control sequences in one or more expression vectors that ensure expression of the immunoglobulin polypeptide. Such control sequences include signal sequences, promoters, enhancers and transcription termination sequences (see Queen et al, Proc. Natl. Acad. Sci. USA86, 10029 (1989); WO 90/07861; Co et al, J.Immunol.148, 1149(1992), which are incorporated herein by reference in their entirety for all purposes).

Antibodies or Fc or components and domains thereof may also be obtained by library (e.g., phage library) selection of such domains or components. Phage libraries, such as B-cells from immunized animals or humans (Hoogenboom et al, 2000, immunol. today21(8)371-8), can be created by inserting random oligonucleotide libraries or polynucleotide libraries comprising sequences of interest. Antibody phage libraries contain one phage heavy (H) and light (L) chain variable region pair that allows for the expression of single-chain Fv fragments or Fab fragments (Hoogenboom et al, 2000 supra). The diversity of the phagemid library can be manipulated to increase and/or alter the immunospecificity of the monoclonal antibodies in the library to generate and subsequently identify additional desired human monoclonal antibodies. For example, heavy (H) chain and light (L) chain immunoglobulin molecules encoding genes can be randomly mixed (shuffled) to create new HL pairs in the assembled immunoglobulin molecules. In addition, one or both of the H and L chains of the encoding gene can be mutagenized in the Complementarity Determining Regions (CDRs) of the variable region of an immunoglobulin polypeptide, and subsequently screened for the desired affinity and neutralizing capacity. Antibody libraries can also be created by the following syntheses: one or more human FR sequences are selected and a collection of CDR cassettes derived from a human antibody repertoire is introduced or by designed variants (Kretzschmar and von Ruden2000, Current Opinion in Biotechnology, 13: 598-. The position of diversity is not limited to the CDR, but may also include FR fragments of variable regions or may include fragments other than antibody variable regions, such as peptides.

Other libraries that may include target-binding or non-target-binding components in addition to antibody variable regions are ribosome display, yeast display, and bacterial display. Ribosome display is a method of translating mrnas into their cognate proteins while keeping the proteins attached to the RNA. The nucleic acid coding sequence was recovered by RT-PCR (Mattheakis, L.C. et al, 1994.Proc. Natl. Acad. Sci. USA91, 9022). Yeast display is the construction of a fusion protein based on the membrane-associated alpha-lectin yeast adhesion receptors aga1 and aga2, which is part of a hybrid-type system (Broder et al, 1997.Nature Biotechnology, 15: 553-7). Bacterial display is based on the fusion of a target to exported bacterial proteins associated with cell membranes or cell walls (Chen and Georgiou2002.Biotechnol Bioeng, 79: 496-503).

The invention also provides nucleic acids encoding the compositions of the invention, as isolated polynucleotides or as part of expression vectors, including vectors compatible with prokaryotic, eukaryotic, or filamentous phage expression, secretion, and/or display of the compositions or targeted mutagens thereof.

3. Methods of producing antibodies of the invention

Once the antibody molecules of the invention have been identified based on the structural and functional properties described herein, the nucleic acid sequences encoding the desired portion of the antibody chain, or the entire antibody chain, can be cloned, replicated, or chemically synthesized and can be isolated and used to express the antibody by conventional methods. The antibodies of the invention may be purified by any method of immunoglobulin molecule purification known in the art, for example, by chromatography (e.g., ion exchange, affinity and size column chromatography), centrifugation, differential solubility, or by any other standard technique of protein purification. In addition, the antibodies or fragments thereof of the present invention can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

Host cell selection or host cell engineering

As described herein, the choice of host cell for expression of recombinant Fc-containing proteins or monoclonal antibodies is critical to the final composition, including but not limited to changes in the composition of the oligosaccharide portion of the decorative protein in the immunoglobulin CH2 domain. Accordingly, one aspect of the present invention relates to the selection of appropriate host cells for use in and/or development of producer cells 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, SP 2/0, 293, Hela cells, myeloma, lymphoma, yeast, insect or plant cells, or any derived, immortalized or transformed cell thereof.

Alternatively, the host cell may be selected from a species or organism that is not capable of glycosylating a polypeptide, for example a prokaryotic cell or organism, such as a prokaryotic cell or organism belonging to a natural or engineered escherichia species (e.coli spp.), Klebsiella spp, or Pseudomonas species (Pseudomonas spp.).

4. Methods of using anti-TF antibodies

The compositions (antibodies, antibody variants, or fragments) produced by any of the above methods are useful for diagnosing, treating, detecting, or modulating a human disease or a particular pathology of a cell, tissue, organ, bodily fluid, or generally a host. As taught herein, the Fc portion, Fc fusion protein, or Fc fragment of an antibody is modified to provide a range of effector functions with suitable specificity upon binding of more targets, but wherein retention of the original target properties in the antibody will result in antibody variants for specific applications and therapeutic indications.

Diseases or pathologies that may be treated using the compositions provided herein include, but are not limited to: a malignant tumor; including primary solid tumors and metastases; carcinomas, adenocarcinomas, melanomas, liquid tumors such as lymphomas, leukemias and myelomas, and aggressive masses formed by the progression of malignant tumors; soft tissue malignancies; sarcoma, osteosarcoma, thymoma, lymphosarcoma, fibrosarcoma, leiomyosarcoma, lipoma, glioblastoma, astrosarcoma, malignancy of the prostate, breast, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, thyroid, lung, kidney, or bladder.

As regards the ability of the antibodies of the invention to reduce the pre-oncogenic surrounding environment in tissues by blocking the ability of TF to participate in the release of downstream cytokines (such as inflammatory cytokines, IL-8), the antibodies of the invention can be used prophylactically or in combination with other therapies directed at inhibiting tumor proliferation and angiogenesis. Most age-related malignancies are derived from epithelial cells of regenerable tissue. An important component of epithelial tissue is the stroma, the subepithelial layer composed of extracellular matrix and a number of cell types, including fibroblasts, macrophages and endothelial cells. The malignant tumor stroma is critical for tumor growth and progression, and TF can be expressed on stromal cells as well as malignant epithelial cells. Thus, the ratio of TF in the stroma: the presence of downstream factors due to VIIa signaling may create a pro-oncogenic tissue environment that synergizes oncogenic mutations to drive the formation of neoplastic tissue.

Similarly, when TF is expressed in adipose tissue, it may alter tissue function in disorders such as obesity, metabolic syndrome and diabetes. The antibodies of the invention are produced by blocking TF: VIIa signaling may be useful in treating these conditions. Some TF: factors produced downstream of FVIIa signaling (including IL-8 and IL-6) are powerful inflammatory mediators. Thus, additional uses of the antibodies of the invention include the treatment of inflammatory disorders such as, but not limited to, rheumatoid arthritis, inflammatory bowel disease and asthma.

Since the antibodies of the invention inhibit TF: VIIa signaling and reduced downstream effects that promote angiogenesis, the antibodies of the invention are useful for treating other diseases, disorders, and/or conditions involving angiogenesis, in addition to malignancies. Such diseases, disorders, and/or conditions include, but are not limited to: benign tumors such as hemangioma, acoustic neuroma, neurofibroma, trachoma, and pyogenic granuloma; atherosclerotic plaques; ocular angiogenic diseases of the eye, such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uveitis and pterygium (abnormal blood vessel growth); rheumatoid arthritis; psoriasis; delay of wound healing; endometriosis; (ii) angiogenesis; granulation occurs; hypertrophic scars (keloids); nonunion of fracture; scleroderma; sand holes; vascular adhesion; myocardial angiogenesis; coronary artery collateral; a collateral cerebral branch; arteriovenous malformation; ischemic limb angiogenesis; Osler-Webber syndrome; plaque neovascularization; expanding the capillary; hemophilic joints; angiofibroma; fibromuscular dysplasia; wound granulation occurs; crohn's disease; and atherosclerosis.

With respect to the antibodies of the invention inhibiting TF: VIIa signaling, the antibodies can be used to treat and/or diagnose hyperproliferative diseases, disorders, and/or conditions, including but not limited to neoplasms. The antibodies may inhibit the proliferation of the disorder through direct or indirect interaction. Examples of hyperproliferative diseases, disorders, and/or conditions that can be treated, and/or diagnosed by the antibodies of the invention include hyperproliferative diseases, disorders, and/or conditions (including but not limited to hypergammaglobulinemia, lymphoproliferative diseases, disorders such as castleman's disease, and/or pathological proteinemia, purpura, sarcoidosis, Sezary syndrome, waldenstrom's macroglobulinemia, gaucher's disease, histiocytosis) and any other hyperproliferative disease in an organ, tissue, or fluid compartment.

Additional aspects of the antibodies of the invention may be used in therapy including, but not limited to, direct cytotoxicity of the antibody (e.g., mediated by complementary sequences (CDC) or by effector cells (ADCC)), or indirect cytotoxicity of the antibody (e.g., immunoconjugate).

While the invention has been described in general terms, embodiments of the invention will also be disclosed in the following examples, which should not be construed as limiting the scope of the claims. In experimental descriptions, certain reagents and procedures are used to produce proteins or antibodies or specific fragments. The following describes conventional analytical methods for characterizing antibodies.

Materials and methods

Protein and antibody standards

The human TF recombinant extracellular domain (ECD) was constructed in two forms: ELISA and Biacore based directed binding assays, amino acids 1-219 of the mature chain of TF (SEQ ID NO: 1) expressed in a mammalian system with a C-terminal His 6-tag peptide; cocrystallogical studies of bacterial systems with a C-terminal His 6-tag peptide expressing SEQ ID NO: 1, amino acids 5-213. Use of NHS esters on proteins to chemically target amine residues_TF1-219And (4) biotinylation. For blood coagulation assays, use is made of(Dade Behring inc. cat # B4212), lyophilized recombinant human tissue factor in combination with phospholipids, calcium, buffers and stabilizers for diagnostic use.

Macaca fascicularis (cyno) TF-ECD (SEQ ID NO: 2) was cloned using PCR from cDNA isolated from cynomolgus monkey testis tissue (available from BioChain Institute (Hayward, Calif.)).

Several antibodies were used as reference antibodies: i)10H10 cloned from primary hybridoma TF9.10H10-3.2.2(US 7223427); ii) a 10H10 mouse-human chimera comprising SEQ ID NO: 4 and SEQ ID NO: 5, named M1; iii) M59, a human FR-adapted antibody comprising six CDRs of 10H10 and having the function of an affinity matured parent antibody, comprising the amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 23; iv) the murine anti-human tissue factor antibody TF8-5G9(US 7223427); v) a humanized form of antibody 5G9, designated CNTO860, human IgG 1/κ (US 7605235); and vi) an isotype control (human IgG 1/kappa) antibody that binds to an unrelated antigen (RSV), designated B37.

Expression and purification of antibodies

The disclosed antibodies are expressed and purified using conventional procedures. For primary screening, DNA encoding these molecules was transiently expressed in 96-well plate HEK293E cells, and supernatant activity (binding) was tested 96 hours after transfection. Hits were identified and selected for pilot scale expression and purification. Pilot scale expression was transiently performed in 750ml volumes of HEK293F cells or CHO-S. The harvested supernatants were purified by Protein a chromatography and the purified proteins were evaluated for their affinity and functional activity. In addition, the purified protein was biophysically characterized by SDS-PAGE, SE-HPLC and cross-interaction chromatography (CIC). The theoretical isoelectric point (pI) was also calculated for each variant. From pilot scale characterization, a panel of final major candidates was transfected in a WAVE bioreactor and purified by Protein a chromatography.

Fab production andmonoclonal Fab ELISA

Glycerol stocks from phage panning rounds were miniprepped and the ex vivo pIX gene was digested by NheI/SpeI. After religation, the DNA was transformed into TG-1 cells and allowed to grow overnight on LB/agar plates. The next day, colonies were picked, allowed to grow overnight, and the cultures were used for (i) colony PCR and V-region sequencing; and (ii) inducing Fab production. For Fab production, overnight cultures were diluted 10-100 fold in fresh medium and allowed to grow for 5-6 hours at 37 degrees celsius, Fab production was induced by addition of fresh medium containing IPTG, and cultures were grown overnight at 30 degrees celsius. The next day, the spinner culture was stopped and the supernatant (containing soluble Fab protein) was used for Fab ELISA.

For Fab ELISA, Fab were captured on plates by polyclonal anti-Fd (CH1) antibody. After appropriate washing and capping biotinylated hTF was added at a concentration of 0.2 nM. This concentration enables grading of Fab variants, defined as the percent binding of the parent, wherein the parent Fab present as a control in all plates is defined as 100% binding. Biotinylated hTF was detected by HR cross-linked streptavidin and chemiluminescence read in a plate reader.

TF-ECD binding Mab based ELISA

Solution phase-directed TF binding ELISA using chemiluminescence detection was used to rank the first binders from the human framework-adapted library. A96-well black maxisorp plate was coated with 100uL of 4 ug/ml goat anti-human IgG FC diluted in carbonate-bicarbonate buffer at pH9.4, overnight at 4 ℃, and then washed three times with buffer (PBS with 0.05% Tween-20 solution) and blocked with 300 μ l of 1% BSA/10mM PBS solution for 1 hour, followed by washing as before. Samples or standards were diluted to 50 ng/ml (1% BSA in PBS + 05% Tween) in assay buffer and 100ul was added to the assay plate, shaking for 1 hour at room temperature. The plates were washed three times and 100 ul/well His-tagged human or cynomolgus monkey TF-ECD was added at 100 ng/ml diluted in assay buffer and incubated for 2 hours at room temperature. After washing, 100 ul/well was cross-linked to five his Qiagen peroxidase in assay buffer at 1: 2000 dilutions were added and incubated at room temperature for 1 hour with shaking. BM ChemiLum Substrate (BM ChemiLum, POD, Roche) was treated with a 1: 100 dilutions were freshly formulated in buffer and 100ul was added to the plates after the final wash. After 10 minutes, the plates were read on a Perkin Elmer Envision Reader, BM Chemilum program.

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

This assay is used to detect the directional binding of antibodies to endogenous human TF expressed by breast malignant cells. Four-point titers of test mabs were prepared in parallel in FACS buffer (1% FBS in PBS). To have a 1: titer 1,000 ng/ml for the 4 dilutions started. M1 (parent molecule) was used as a positive control, while B37 (anti-RSV mAb) was used as a negative/isotype control. Non-disrupted cells and secondary antibody, FACS buffer 1: a Cy-5 cross-linked goat anti-human IgG Fc antibody in 200 was used as a control and prepared immediately prior to use.

Adherent MDA-MB-231 cells in the flask were rinsed once with PBS (w/o Ca + 2/Mg +2) using standard tissue culture techniques. Cells were lifted with verene and cells and seeds were counted at 200,000 cells/well in a polystyrene V-plate. FACS analysis protocol: tissue factor binding. Cells were pelleted at 450 Xg 3 min at 4 ℃ in an Allegra X-15R centrifuge, resuspended in FACS buffer (2% FBS in PBS) and placed at 200uL at 200,000 cells/well. Cells were pelleted at 450 Xg 3 min at 4 ℃. The supernatant was discarded and 100 uL/well of test or control mAb was added to the indicated wells and incubated on ice or at 4 ℃ for 1 hour (+/-10 minutes). Cells were pelleted at 450 Xg 3 min at 4 ℃. The supernatant was discarded and the cells were washed once in FACS buffer. Cells were resuspended in 200 uL/well FACS buffer and pelleted at 450 Xg 3 min at 4 ℃. The supernatant was discarded and 100 uL/well of secondary antibody was added to the designated wells (triterate) and incubated on ice for 1 hour (+/-10 minutes). Cells were pelleted at 450 Xg 3 min at 4 ℃. The supernatant was discarded and the cells were washed 2 times in FACS buffer before resuspending the cells in 200 uL/well FACS buffer (triterate). Cells were pelleted at 450 Xg 3 min at 4 ℃. The supernatant was discarded and the cells were resuspended in 100 uL/well CytoFix buffer. The reaction was analyzed by flow cytometry (BD FACSArray). FlowJo software was used for FACS data analysis by accessing the major cell population in an undamaged control well and applying this access to the entire dataset. Data were derived as a table of geometric Mean Fluorescence Intensity (MFI) in the red channel of the applied inlet.

Thermo fluorescence (thermolfluor) assay

The thermal fluorescence technique is a kinetic measurement of unfolded molecules as they are heated. When the molecule is heated, the dye (ANS) is able to bind to the molecule (due to its unfolding). When the dye binds to the molecule, the dye will fluoresce and this fluorescence is measured over time. In this assay, unfolded antibodies were measured from 37-95 ℃ and detected every 0.5 ℃. Tm was also measured for both the parental molecules, both murine and chimeric forms (10H10, M1, 5G9 and CNTO860), as well as 2 mabs (Emmp4a5 and Emmp5F6) with known Tm used as assay controls.

This assay was used to predict the thermostability of human framework-adapted library variants. The purified antibody was diluted to 0.5 mg/mL in PBS and 2ul of sample was added to each well for a total of 1ug of sample per well. Each sample was added in parallel. Stock ANS was 500mM in DMSO. Mixing stock solution ANS 1: 12 dilution into DMSO (to 40 mM); Dye/Tween solutions were prepared by combining 20ul of 40mM ANS solution, 2.8ul of 10% Tween and 1.98mL of PBS; 2uL Dye/Tween solution and 2uL oil were added. Centrifuge plate (450rpm2 min). Thermal fluorescence setting: the Shutter is set to manual, RampTemperature0.5C/s, Continuous Ramp, Temperatureramp: 50-95 ℃. Hold 15 is selected under "Single SC Image/plate" selection at high t, Exposure time10 s/1 rep, Gain normal 2.

Cross-interaction Chromatography (CID)

To determine the interaction of various antibodies with other antibodies, chromatography experiments were performed using a column (Sigma Aldrich) coupled to human IgG. Briefly, 50mg of human IgG was coupled to a 1ml NHS-Sepharose column (GE Healthcare) according to the manufacturer's instructions. Unconjugated IgG was removed by washing with 0.1M Tris, pH8, 0.5M NaCl, and unreacted NHS groups were capped with the same buffer. The coupling efficiency was determined by measuring the protein concentration remaining in the unreacted coupling buffer and washing with Pierce's coomassie Plus Assay Kit (Thermo Pierce) and subtracting from the amount of protein previously immobilized. Control columns were also prepared using the same protocol with no protein added to the resin.

The control column was first run on a Dionex UltMate 3000HPLC after equilibration with PBS, pH7, 0.1 ml/min flow. The 201 stock solution of protein was injected first to ensure capping of non-specific binding sites, and then 20110% acetone was injected to check the integrity of the column.

The sample to be analyzed was diluted to 0.1 mg/ml in PBS, pH7. 20 μ L of each sample was injected into each column and allowed to run at 0.1 ml/min for 30 min. The retention time was recorded and the retention factor (k') was calculated for each variant.

k' is calculated as the difference between the retention time tR on the protein-derivatized column (IgG-coupled column) and the retention time t0 of the column to which no protein is coupled. This calculation also takes into account the acetone retention time on both columns to normalize the columns. Acceptable k' values are less than 0.3.

Solubility in water

To determine the solubility of various antibodies at room temperature, concentration experiments were performed using a centrifugal filtration device. Briefly, antibody preparations in PBS were added to a Vivaspin-15(15m1) centrifugal filtration device (30,000MWCO, Sartorius, Goettingen, Germany) at room temperature. The filtrate was spun at 3000 Xg in a Beckman Allegra X15-R centrifuge using a float bowl rotor over a 20 minute interval. Once the volume was reduced to about 2ml, the supernatant was transferred to a Vivaspin-4(4ml) filtration unit (30,000MWCO) and centrifuged at 4,000 Xg over a 20min interval. Once the volume had been reduced to 500l, the sample was transferred to a Vivaspin-500 filtration unit and centrifuged at 15,000 Xg for 15 minutes in an Eppendorf5424 centrifuge. This procedure was repeated until the protein concentration reached 100 mg/ml or more. Protein concentration was determined by absorbance at 280nm and 310nm of appropriate dilutions on a BioTek synergy ht (TM) spectrophotometer. At this point, centrifugation was stopped and the sample was kept overnight at room temperature to reach equilibrium. The next morning, samples were examined for signs of sedimentation. If the concentration is greater than 100 mg/ml, the process is terminated.

Factor VIIA-induced IL-8 inhibition assay

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) (adapted to be grown in DMEM and 10% FBS (Gibco: cat #11995 and cat # 16140)) were placed at a density of 20000 cells/well (100,000 cells/mL) in 96-well cell culture plates (Nunc: cat #167008) using standard cell culture techniques. Two days before the start of antibody treatment 1: 2 or 1: cells were recovered at 4 serial dilutions. Antibodies were added at a final concentration of 50nM in DMEM without FBS one hour prior to treatment with human FVIIa (Innovative Research: cat # IHFVIIa, lot: 2824). The cells were placed in an incubator for 24 hours. After treatment, the supernatant was collected and according to the manufacturing protocol (R)&DSystems: cat # D8000C) was used to determine the amount of IL-8 by ELISA. Briefly, the Optical Density (OD) of each treatment sample was read at 450nm and 540 nm. Using an IL-8 standard curve drawn according to the manufacturing protocol, the reading at 540nm was used to correct for optical imperfections in the assay plate, while the reading corrected at 450nm (OD450 minus OD540) was used to calculate the IL-8 content. Wells of cells not receiving antibody and FVIIa treatment were used to define endogenous IL-8 levels, whereas wells of cells receiving FVIIa only were used to define "no inhibition" IL-8 levels, thus defining the minimum and maximum IL-8 levels, respectively. MAb titer treatment samples normalized to maximum and minimum IL-8 levels were expressed as defined above and percent inhibition. Normalized data is represented in a histogramIn or adapted to the EC for the extraction of each MAb₅₀Four parameter log plot of values.

Blood coagulation assay

This assay was used to determine whether the anti-human TF antibody blocks coagulation in vitro using human plasma in the presence of calcium and the addition of a recombinant human TF preparation (Innovin, Dade Behring Inc). Anti-human TF antibody was diluted to 2 mg/ml antibody in HBSS (Gibco, cat # 14175). Human plasma combined with sodium citrate (George King biological, Novi, MI) was spun slowly at 1000rpm for 5min and the clarified plasma was transferred to a new tube. In each well of a clean 96-well assay plate (NUNC, cat #439454), 25ul of diluted antibody was added to 100ul of human plasma. The reaction was performed by mixing HBSS with 22mM CaCl2 at 1: addition of 125 μ l Innovin (DadeBehring inc., cat # B4212) diluted at 500 was initiated to each well containing plasma (with or with antibodies). The clotting reaction was monitored kinetically at OD405 using a SpectraMax M2e reader (Molecular Devices, Sunnyvale, Calif.), initiating the subsequent reaction immediately and for 30min at 37 ℃. For antibodies, T1/2Max was determined using Softmax Pro Software, which takes time (seconds) to reach 50% of the maximum optical density. The time of the samples (in seconds) was normalized to the reference on each plate, however, there was statistically no difference between the mean times of 150s and 200s for the sample list without antibody, 10H10, and all 10H10 derived and human adapted variants.

Example 1: sequencing of 10H10

A murine antibody designated 10H10 generated in The Scripps Research Institute, La Jolla, Calif. (US5223427, Morrisey et al, 1988Thromb Res.52 (3): 247-261) was produced by hybridoma TF9.10H10-3.2.2. Antibody sequences from the 10H10 hybridoma clone have not been reported previously.

The sequence was identified using the 5 'RACE method (Focus25 (2): 25-27, 2003; Mamyama1994Gene138, 171-174) using 5' GeneRacer complementary to the sequences of the mouse IgG1 constant region and mouse k constant region, respectively^TMThe (InVitrogen) primers and the 3' consensus primer amplified two antibody chains (VH and VL). Nested PCR amplification using 5 'GeneRacer nested primers and 3' consensus primers was used to generate VL products more suitable for sequence analysis.

At least 16 clones were selected to identify the variable region of each strand. Sequencing by unknown insert region using primers. Raw sequence data was downloaded from ABI DNA sequence to Vector NTI (Invitrogen informamax) for sequence analysis. One functional VH and one functional VL were identified. Genes for both VH and VL were also analyzed to find their native signal sequences, FR, CDR and J segments.

The 10H10FR and CDRs were numbered and segmented sequentially, except for the region corresponding to VH CDR-1, according to the Kabat definition (Kabat et al, 5 th edition, Public Health Service, NIH, Washington, DC, 1991). For this region, a combination of Kabat and Chothia definitions was used (Raghunnathan, G., US 2009/0118127A 1; Chothia and Lesk, J Mol Biol196 (4): 901-17, 1987).

Table 1: 10H10 variable region sequence and its sequence structure

The cloned V regions were engineered with human IgG 1/kappa constant regions and cloned into mammalian expression vectors for recombinant expression in HEK293 or CHO cell lines, creating a mouse-human chimeric antibody designated M1 for analytical studies as a reference antibody. The HC V region was also engineered with only the human IgG1CH1 domain and the C-terminal hexa-histidine for the purpose of generating 10H10Fab for use in crystal structure analysis.

Example 2: epitope mapping of non-anticoagulant tissue factor antibodies

Epitope mapping of 10H10 was performed by crystal structure determination of the complex between the human TF ECD and the corresponding Fab fragment. His-tagged human TFECD (residues 5-213 of SEQ ID NO: 1) was expressed in E.coli (Escherichia coli) and purified by affinity and ion exchange chromatography using HisTrap HP column (GEHealthcare) and Q HP column (GE Healthcare), respectively. The 10H10Fab of a His-tagged chimeric version (mouse V-region, human constant domain) was expressed in HEK cells and purified by affinity (talen column, GE Healthcare) and size exclusion (HiLoad Superdex200 column, GE Healthcare) chromatography.

The complex was prepared by mixing Fab with human TF ECD in a molar ratio of 1: 1.2(TF excess). The mixture was incubated at room temperature for 20min and loaded onto a Superdex200 column (GE Healthcare) equilibrated with 20mM HEPES, pH7.5 and 0.1M NaCl. Fractions corresponding to the main peak were combined, adjusted to a concentration of 10 mg/mL and used for crystallization. The composite was crystallized by a vapor diffusion method at 20 ℃. Crystallization of 10H10 from a solution containing 18% PEG8000 (in 0.1M CHES, ph 9.5): the TF complex. For X-ray data acquisition, one crystal of the complex was soaked for a few seconds in a mother liquor supplemented with 20% glycerol and flash frozen with a 100K nitrogen stream. Using a detector equipped with Satum944CCD and an X-stream^TMThe X-ray diffraction intensity was measured with a RigakuMicroMaxTM-007HF microfocus X-ray generator of a 2000cryo cooling system (Rigaku). The structure was determined by molecular replacement using the package of macromolecular crystallography CCP4 (macromolecular computerized procedure ect, number4.1994.acta Crystal. D50, 760-.

The TF ECD consists of two topologically identical domains with immunoglobulin folds. The N-terminal domain spans residues 1-103, and the C-terminal domain spans residues 104-210 (belonging to ECD, SEQID NO: 1). The 10H10 epitope was found to be centered at ECD residues K149-D150, which resulted in a deep pocket between the 10H10 light chain and the light chain variable domain. The interface between 10H10 and TF is extensive and involves all six CDR loops (FIG. 1).

It was notably found that the TF epitope of 10H10 does not overlap with the FVII and FX binding sites (fig. 2 and 3). In addition, the partial overlap of the epitopes of 10H10 and 5G9 (another murine human TF-binding antibody with the ability to block blood coagulation and the epitopes previously published, Huang et al, 1998J Mol Biol 275: 873-94) indicates competition between the two antibodies for binding to human TF (FIGS. 2 and 3).

Human TF ECD: 10H10 contact surface

10H10 incorporates TF at the interface between the N and C terminal domains of the ECD. The convex surface of the TF fits into the concave CDR surface of the antibody. The total area of each interacting molecule embedded during complex formation exceeds. Including direct contact with TF (contact is defined asAtomic distance) of all six CDRs. There are a total of 24 epitope residues and 25 paratope residues. CDRL1, H1, and H3 form the majority of the contacts. Formation of 10H 10: the residues of the epitope and paratope of the TF complex are schematically shown in fig. 1.

The 10H10 epitope includes two fragments from the N domain and three fragments from the C domain of the TF ECD. Two fragments from the N domain interact with the antibody: residues 65-70 interact with the H-CDR1 and H-CDR3, and residue 104 interacts with the H-CDR 1. Three fragments in the C domain interact with the antibody: residues 195 and 197 interact with H-CDR1 and H-CDR2, residues 171 and 174 interact with L-CDR1 and L-CDR3, and residues 129 and 150 interact with L-CDR1, L-CDR3, H-CDR1 and H-CDR 3; TF residues K149-D150 are centered on the epitope; the deep pocket formed between the VL and VH domains was obtained with their major partners being D97 for the LC variable region (SEQ ID NO: 5) and W33 for the HC variable region (SEQ ID NO: 4), respectively, of 10H 10.

B. Specificity of antibodies

The amino acid sequences of human, cynomolgus monkey (cyno) (SEQ ID NO: 2) and mouse TF ECD (SEQ ID NO: 3) are aligned in FIG. 2. The human and cynomolgus monkey TF ECD sequences share high similarity and differ from each other in only one residue in the 10H10 contact residue: SEQ ID NO: 1, which is r (arg) in the cyno sequence, position 197. The high level of cross-reactivity observed in the 10H 10-derived antibody panel herein is interpretable due to the single H-CDR2 residue contacting T197 in the human sequence.

By aligning human and mouse TF sequences, it was determined that the 10H10 species-specific epitope residues clearly differ significantly in the amino acid residues: of the 24 residues of 10H10 that were exposed to human TF, the human and mouse sequence differences are set forth in SEQ ID NO: 1 to SEQ ID NO: 3 at positions 68, 69, 70 and 104 in the N domain and positions 136, 142, 145 and 197 in the C domain. These differences correspond to the reduced binding affinity of 10H10 for mouse TF.

FIG. 2 also shows the human TF interaction sites for FVII and FX based on a theoretical 3D model (FIG. 3) which describes their association as a ternary complex (Norridge et al, Proteins, 53: 640-. Antibody 5G9 binds TF at an epitope that partially overlaps with the FX binding site. Thus, 5G9 competes with FX, and this causes a blockade of the coagulation cascade. 10H10 differs from 5G9 in that it does not block coagulation, but it effectively shuts off signaling of PAR associated through TF. Based on the ternary TF/FVII/FX complex model, one would expect that the 10H10 epitope could be centered on the TF free surface and around residues K149-D150. Peptide epitope mapping was provided by mutagenesis and early indication of this to map the binding of 10H 10.

The crystal structure of the complex between TF ECD and 10H10Fab herein provides a spatial mapping means in which the antibody can bind TF without preventing FVII and FX from interacting with TF or each other. The 10H10 epitope shown by the structure covers the TF free surface, although it is theoretically present in a ternary complex. Furthermore, the 10H10 epitope partially overlaps with the common residues of the coagulation-blocking MAb, the 5G9 epitope (Huang1998 supra), K149 and N171. Neither 10H10 nor 5G9 blocked FVII binding to TF. The epitope of 10H10 and FX do not overlap, but steric hindrance occurs between the Fab constant domain and the protease (globular) domain of FX in the current model. However, it should be noted that FX orientation may in fact differ from the model, and that the association between FX and TF may allow some flexibility in the protease domain. There is also considerable flexibility in the corner angle between the Fab variable and constant domains, which may allow to avoid steric hindrance when 10H10 is bound to the ternary complex.

Example 3: modulation of binding domains for use in humans

The efficacy of therapeutic proteins may be limited to unwanted immune responses. The non-human monoclonal antibodies can have substantial extensions of the linear amino acid sequence and local structural conformations that elicit human immune responses. The transfer of residues responsible for the immunospecificity of target binding of the non-human MAb to the human antibody scaffold is less likely to cause substantial loss of binding affinity for the target antigen. Thus, this is of great value for using design principles to create antibody molecules that elicit small immunogenic responses while retaining the binding and biophysical profile of the parent non-human molecule when injected into humans.

As previously described in US20090118127a1 and Fransson et al, 2010J Mol Biol 398: 214-231, humanization and restoration or enhancement of binding affinity to generate the antibody species of the invention all used a two-step approach that exhibited the target effect of murine antibody 10H10 when conjugated to human TF. A two-step process, known as Human Frame Adaptation (HFA), consisting of: 1) human framework selection and 2) affinity maturation step.

In the HFA approach, binding site residues (CDRs) are combined with human germline genes selected based on sequence similarity and structural considerations. Two systems of CDR assignment for antibodies are: kabat definition, which is based on the variability of antibody sequences; and Chothia definition, which is based on analysis of the three-dimensional structure of antibodies. Of the six CDRs, one or the other system can be used where they are separated. For light chain CDRs, the Kabat definition is used.

For the heavy chain CDR3, Kabat and Chothia definition is the same. For heavy chain CDR1, the Chothia definition is used to define the beginning and the Kabat definition is used to define the end (pattern defined by W followed by hydrophobic amino acids such as V, I or a). For the VH-CDR2, the Kabat definition was used. However, in most antibody structures, this sequence-based definition assigns a portion of FR3 as belonging to CDR 2. Thus, shorter versions of this CDR that end seven (7) residues early in the C-terminal region of this CDR, referred to herein as Kabat-7, may also be used.

Human FR selection

Human FRs (defined as regions in the V regions not included in the antigen binding site) are selected from all of the functional human germline IGHV and IGHJ genes. All of the human germline gene sequences were obtained by searching the IMGT database (Lefranc2005) and compiling all "01" alleles. From this compilation, abundant genes (100% identical at amino acid level) and those with unpaired cysteine residues were removed from the compilation. The last upgrade of gene compilation was completed in 2007 on 1/10.

The initial selection of human sequences for HFAs of VH is based on the sequence similarity of human VH germline genes to the entire length of the mouse VH region including FR-1 to 3 and H-CDR-1 and H-CDR-2. In the next stage, the selected human sequences are ranked in order using a score that takes into account both the CDR length and the sequence similarity between the mouse CDR and the human sequence. Standard mutation matrices, such as the BLOSUM62 substitution matrix (Henikoff and Henikoff1992Proc Natl Acad Sci U S A15; 89 (22): 10915-9) are used to score mouse CDR and human sequence alignments and apply large penalties if there are insertions and/or deletions in the CDR loops. Human FR-4 was selected based on the sequence similarity of the IGHJ germline gene (Lefranc2005) to the mouse 10H10 sequence, IGHJ4(SEQ ID NO: 60).

Human FR was selected for VL using a similar procedure. In this case, the IGVK germline gene selected using the same procedure was used for the IGHV gene, selecting for gene action for FR1-3 and L-CDR 1-3. The human IGJ-K2 gene (SEQ ID NO: 61) was selected as FR4 for all variants.

Eleven VH and seven VL species tethers were selected. The VH genes selected were mainly from the IGVH-1 gene family: 6 sequences from IGVH1 with IGVH1-69 and IGVH1-f used with longer and shorter H-CDR2, 2 from IGVH3, and an IGVH5 gene used with long and short H-CDR 2. The VL genes represent six IGVK2 and one IGVK 4gene family.

Thus, the VH variants H15, H19 and H21 have longer H-CDR2(SEQ ID NO: 7), which correspond to H22, H23 and H24, respectively, where the shorter mouse CDR-H2(SEQ ID NO: 27) is used. The prefix "s" represents that the test variant has fewer mouse residues and more human residues in the beta-chain region. The V region names used and the gene sequences used are shown in tables 2 and 3 below.

TABLE 2：

Polypeptide ID	IMGT gene used
		H13	VH-10H10
H14	IGHV1-2
		H15	IGHV5-a

H16	IGHV1-46
		H17	IGHV1-3
H18	IGHV3-74
		H19	IGHV1-69
H20	IGHV1-18
		H21	IGHV1-f
H22	s1_IGHV5-a
		H23	s1_IGHV1-69
H24	s1_IGHV1-f

TABLE 3：

Peptide ID	IMGT gene name
		L1	VL-10H10
L2	IGKV4-1_B3
		L3	IGKV2D40_O1
L4	IGKV2D-28_A3
		L5	IGKV2D-29_A2
L6	IGKV2-30_A17
		L7	IGKV2-24_A232
L8	IGKV2D-26_A21

A library of 96 mabs (representing the 11 heavy and 7 light chain human FR variants plus the murine 10H10 chimeric chain) was expressed in 96-well HEK293E cells to provide supernatants for primary screening. For primary screening, the DNA encoding the selected variable domains was recombined to form intact mabs, which were transiently expressed in 96-well plate HEK293E cells, using standard recombination methods. At 96 hours post transfection, supernatants from cultures were tested for fluid activity (binding).

Nineteen variants were selected for pilot scale expression in HEK293-F cells and purified based on preliminary screening results. Pilot scale expression was transiently performed in 750ml volumes of HEK293F cells or CHO-S. The harvested supernatants were purified by Protein a chromatography and the purified proteins were evaluated for their affinity and functional activity. In addition, the purified protein was biophysically characterized by SDS-PAGE, SE-HPLC and cross-interaction chromatography (CIC). The theoretical isoelectric point (pI) of each variant was also calculated. From pilot scale characterization, a set of final major candidates were transfected in a WAVE bioreactor and purified by Protein a chromatography.

Binding evaluation

The binding of the parent chimeric antibody, M1 and HFA variants to both human and cyno TF was performed in a directed ELISA format using chemiluminescent label detection. Initial screens for library variants, samples or controls in crude supernatants were normalized to 50 ng/ml in the specific FreeStyle293HEK medium (Gibco) and the assay was determined at a single concentration. In this assay, the concentration of antibody was 5ng (0.1 ml was used) and the TF ECD and antigen were His6-TF-ECD_1-219(used at a final concentration of 10 ng/well).

The results of the entire combinatorial library screen showed that all other VH's had different strength binding to hTF, except H14. Several HFA variants gave higher binding signals than the parent 10H10(H13, L1), especially some L3 and L5 combinations. H18 and H21 did not bind to human antigens as well as other VH and showed insignificant binding to cyno antigen. VL, L6 and L8 did not bind antigen to each other, while others bound at the level of detection. H14 and L8 also gave low expression when combined with any VL. 50 of the 77 antibodies (VH, VL combination) showed TF binding as shown in Table 4.

Table 4: vH and vL sequence IDs of 50 human TF-binding human FR variants

Antibody ID	Light chain peptide ID	Light chain SEQ ID NO:	heavy chain peptide ID	Heavy chain SEQ ID NO:
					M1	L1	5	H13	4
M9	L2	22	H15	12
					M10	L3	23	H15	12
M11	L4	24	H15	12
					M12	L5	25	H15	12
M14	L7	26	H15	12
					M16	L2	22	H16	13
M17	L3	23	H16	13
					M18	L4	24	H16	13
M19	L5	25	H16	13
					M21	L7	26	H16	13
M23	L2	22	H17	14
					M24	L3	23	H17	14
M25	L4	24	H17	14
					M26	L5	25	H17	14
M28	L7	26	H17	14
					M30	L2	22	H18	15
M31	L3	23	H18	15
					M32	L4	24	H18	15
M33	L5	25	H18	15
					M35	L7	26	H18	15
M37	L2	22	H19	16
					M38	L3	23	H19	16
M39	L4	24	H19	16
					M40	L5	25	H19	16
M42	L7	26	H19	16
					M44	L2	22	H20	17
M45	L3	23	H20	17
					M46	L4	24	H20	17
M47	L5	25	H20	17
					M49	L7	26	H20	17

antibody ID	Light chain peptide ID	Light chain SEQ ID NO:	heavy chain peptide ID	Heavy chain SEQ ID NO:
					M51	L2	22	H21	18
M52	L3	23	H21	18
					M53	L4	24	H21	18
M54	L5	25	H21	18
					M56	L7	26	H21	18
M58	L2	22	H22	19
					M59	L3	23	H22	19
M60	L4	24	H22	19
					M61	L5	25	H22	19
M63	L7	26	H22	19
					M65	L2	22	H23	20
M66	L3	23	H23	20
					M67	L4	24	H23	20
M68	L5	25	H23	20
					M70	L7	26	H23	20
M72	L2	22	H24	21
					M73	L3	23	H24	21
M74	L4	24	H24	21
					M75	L5	25	H24	21
M77	L7	26	H24	21

using ELISA, ten variants were selected for scale-up of expression and purification based on relative binding affinity to TF. K measured by BIAcore_DThe summary, ELISA assay data, whole cell binding, IL-8 induced inhibition by 50nM FVIIa, 2 ug/ml Mab from MDA-MB231 cells, and Tm measured by the Thermofour assay are shown in Table 5.

TABLE 5：

Several novel human MAb variants showed higher TF affinity than M1 (with 10H10 variable chains: H13 and L1), and some were lower. Of M61K_D(0.21nM) lower than the murine parent MAb (K)_D0.56nM)2.5 times. The data in table 5 includes four mabs with H15 and four mabs with H22, both constructed from the same germline gene (IGHV 5-a). Those with H22 and shorter H-CDR2 generally exhibited higher binding affinity than the corresponding molecule with H15. Although many new variants bind cyno TF, the classification of cyno binding affinity differs from that of binding to human TF.

The Tm of the mouse 10H10MAb is 74.2 ℃. The Tm range of the selected molecules is 75 to 82.2 ℃. Thus, the HFA approach leads to antibody construction with novel Fd regions with improved binding affinity for human and non-human primate TF, and also results in stable intact antibody variants with human domains.

Additional characterization of the novel antibody construct demonstrated that the antibody was able to recognize self-TF originating on human tumor tissue (MDA-MB231 breast malignancy-derived cells) cells and reduce TF signaling measured in the presence of VIIa induced by IL-8 inhibition of MDA-MB 231.

Additional biophysical characterization (solubility and cross-interaction chromatography) and assay results involved the selection of M59, consisting of variable regions H22 and L3 for affinity maturation.

Example 4: antibody maturation

Fab libraries were constructed in a pIX phage display system described in U.S. patent No.6,472,147(Scripps) and in the applicants co-pending application published as WO 2009/085462 with small modifications to restriction enzyme sites.

Based on the experimental structure of 10H10 in complexes with hTF, two M59-initiated libraries with H116(SEQ ID NO: 19) and L3(SEQ ID NO: 23) pairings were designed for diversity of both VL and VH. The libraries differ in the targeted diversity position, as well as in the amino acids used for the diversity targeted position. A library diversifies the total of eight positions representing each CDR, previously shown to be exposed to TF. Design emphasis was placed on L1, L3, H1 and H2. The position of contact L2 was not diversified from the position in most H3. Avoiding unstable or reactive amino acids (such as Cys and Met).

The second set of libraries was designed to allow the diversity of amino acids around the antigen binding site to be determined by calculating the solvent available for binding and unbinding Fab crystal structures. Targeting buried residues for diversity upon binding and contact with solvent molecules. A total of 12 residues (6 in VL and 6 in VH) were identified using this method, diversified with a reduced set of eight amino acids, including: arg (R), Asn (N), Asp (D), Gly (G), His (H), Ser (S), Trp (W) and Tyr (Y). Estimation of combinatorial library size of 8¹²Or 10¹⁰Variants, which can be overlaid using standard library restriction cloning techniques.

For the CDR-contact residue library, position diversity was performed with 15 amino acids (all but Met, Cys, Lys, Gln and Glu) using a nucleotide dimer (N-dimer) synthesis method.

The Fab library displayed on phage coat protein IX was panned for biotin acylation of hT-ECD by selecting either slower dissociation rate (increasing dissociation rate value) or faster association rate (decreasing association rate value), or using both to direct increased affinity, according to panning protocols known in the art. Panning used both human and cyno TF as target antigens. Phage are generated by helper phage infection. The binders were retrieved by adding SA beads to form bead/antigen/phage complexes. After the final wash, phages were rescued by infecting exponentially growing TG-1 E.coli cells. Phage were again generated and subjected to additional rounds of panning.

The pIX gene was isolated by NheI/SpeI digestion from selected clones, religated, DNA transformed into TG-1 cells and grown overnight on LB/Agar plates. Overnight cultures were used for (i) colony PCR and sequencing for V-regions; and (ii) soluble Fab production. The soluble Fab protein was captured onto the plate by polyclonal anti-Fd (CH1) antibody. After appropriate washing and capping, biotinylated hTF was added at a concentration of 0.2nM and detected by HRP-crosslinked streptavidin and chemiluminescence read before. At this concentration of hTF, a grading of Fab variants (defined as the percentage of binding of the parent, where the parent Fab is defined as 100% binding in all plates present as a control) is possible.

By this criterion 381 fabs were selected that bind human TF 100% or more relative to the M59 Fab.

Analysis of the selected clones succeeded in indicating some changes at Y2, I5, T6 and Y7 corresponding to the heavy chain CDR1(SEQ ID NO: 6) at positions 27 and 30-32 of the V region (SEQ ID NO: 4 or 19). Although there are no contact residues, only aromatic amino acids (Tyr and Phe) are allowed at position 27. For positions 30-32, it is identical to the human TF SEQ ID NO: k68, T101, Y103 and L104 of 1 are in direct contact (fig. 1), position 30 is relatively loose, but 31 and 32 are strict (table 6).

Changes in L3, S6 and S8 (corresponding to positions L52, S55 and S57 of SEQ ID NO: 4 or 19) in L-CDR2(SEQ ID NO: 7), which are residues in direct contact with P194, S195 and T197 of human TF SEQ ID NO: 1 in 10H10Fab (FIG. 1), somewhat limited the set of permissible substitutions.

In H-CDR3, position N6 (SEQ ID NO: 8) (corresponding to N104 of SEQ ID NO: 4 or 19) was brought into direct contact with F147, G148 and K149 of human TF-ED (SEQ ID NO: 1) shown to be restricted.

Allowed amino acid changes in each library position selected by phage panning and screening for comparable binding affinity determined using solid phase capture of 0.2nM human TF-ECD1-219 are shown in Table 6.

TABLE 6：

For VL in selected clones, the changes in S9, G10, N11 and K13 (corresponding to positions 32, 33, 34 and 36 of the LC variable region (SEQ ID NO: 5 or 23)) of L-CDR1(SEQ ID NO: 9), which are shown by mapping the E174, D129, S142, R144 and D145 (FIG. 1) epitopes of SEQ ID NO: 1 exposed to human TF, are relatively loose in Table 7. For the L-CDR2, residue W1 (position 56 in SEQ ID NO: 5 or 23(Kabat residue number 50)) is the contact residue, although residue E61 was shown to be of limited tolerance to substitution. The light chain CDR3(SEQ ID NO: 9) (corresponding to residue positions 97-100 of SEQ ID NO: 5 or 23), which is in direct contact with K149, D150, N171 and T172 of human TF SEQ ID NO: 1 (FIG. 1), position D97(Kabat position 91) is restricted. The selection based on the affinity binding to the human TF-ECD1-219 clone using the lenient amino acids is summarized in Table 7.

TABLE 7：

In summary, a series of affinity-improved Fab variants were identified by constructing phage libraries based on directed variation of the CDR positions of the amino acids in the contact positions with the antigen human TF variable domains H22(SEQ ID NO: 19) and L3(SEQ ID NO: 23), followed by panning and selection of species with affinity in a binding screening assay comparable or better than the starting sequence.

Overall, modest increases in VH and VL pairing affinities selected from the designed HFA library can be obtained. However, from VH libraries of diverse contact residues, the parental amino acids were reselected after stringent phage panning. Only the two paratope positions are significantly changed as explained by structural analysis. These two amino acid substitutions (T31P and S57F) that introduce CDRs during affinity maturation comprise contact residues. The 5-fold improvement in affinity is attributable to an increase in the F57 interface with S195 of TF. The interaction of VL with TF appears to be plastic, allowing for many changes in the touching residues as well as adjacent residues.

For the 381VH and VL pairings, 43 were selected for further characterization. The 43 mabs selected represented 27 different VH and 8 VL (see table 8 heavy and light chain sequence pairings) and could be classified into three subgroups: group 1 variants have identical light chains (L3, SEQ ID NO: 23), and groups 2 and 3 are represented by eight light chains paired with two different heavy chains (H116 and H171, SEQ ID NO: 131 and 67, respectively).

TABLE 8：

For the group of 27 antibodies with the same light chain (L3, SEQ ID NO: 23) as M59, the 27 heavy chains differ: at three positions in H-CDR1 (GYTFX)₁X₂X₃WIE (SEQ ID NO: 83), 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 and T; and X3 is selected from F, H and Y); in addition to H189, where the H-CDR1 is GFTFITYWIA (SEQ ID NO: 81), and at four positions in H-CDR2 (DIX)₁PGX₂GX₃TX₄(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 (SEQ ID NO: 105)), and H-CDR3 and FR are represented by the sequence H22(SEQ ID NO: 19) unchanged and SGYYGNSGFAY (seq id NO: 8). The unique heavy chain composition for these 27 mabs is given below (table 9).

TABLE 9：

H171(SEQ ID NO: 139) contained additional changes in H-CDR1 and H-CDR2 (I31A and S55T) compared to H116.

Two sets of mabs were represented by eight LCs (table 10) paired with one or both of two different HCs (H22(SEQ ID NO: 19) or heavy chain H171(SEQ ID NO: 139)). The eight light chains all have the FR of L3 (from IGKV240_ O1) and have the following sequence variations: five positions in L-CDR1 (KSSQSLLX)₁X₂X₃X₄QX₅NYLT (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; x5 is selected from K, R and S), two of L-CDR2 (X)₁ASTRX₂S (SEQ ID NO: 120), wherein X1 is selected from H and W; x2 is selected from D, E and S), and four of L-CDR3 (QNDX)₁X₂X₃PX₄T (SEQ ID NO: 128), wherein X1 is selected from D, F and L; x2 is selected from S, T and Y; wherein X3 is selected from W and Y; x4 is selected from L and M). The composition of the eight LCs is shown in table 10.

Watch 10：

Some of these mabs were further characterized and tested in an in vivo graft tumor model (example 5).

Example 5: characterization of MAB

Following human framework adaptation and re-selection of a library of variants based on M59 (with altered residues in certain CDR residues) comprising a single LC variable region (L3, SEQ ID NO.23) and a single HC variable region (H22, SEQ ID NO: 19), the novel mabs were used for biophysical and biological activity assays, and a comparison M1587 (with altered paratope residues) was used to recheck whether the epitope originally characterizing the 10H10Fab binding to TF-ECD (example 2) was altered.

Human TF ECD: m1587 contact surface

Human adapted and 10H10 CDR-based affinity matured antibody, cocrystallization of M1587 with TF ECD (L3 and H116) was performed in the same way as 10H10 (example 3), except that M1587-Fab: the TF complex was crystallized from a solution containing 16% PEG3350, 0.2M ammonium acetate, 0.1M sodium acetate, ph 4.5.

Comparison of the cocrystal structures of human TF ECD and M1587Fab with affinity maturation showed that human adapted and affinity matured 10H10 did not have a changed antibody epitope footprint, nor did it have a change in CDR conformation, as shown in figure 2. Introduction of three amino acid substitutions (T31P, S57F and N59T) into H-CDR1 and H-CDR2(SEQ ID NOS: 6 and 27, respectively, using the CDR definitions described in example 2), the contact residues at residues 31 and 57, which are T31P and S57F, were included during human framework adaptation and affinity maturation (SEQ ID NOS: 6 and 7 were replaced with SEQ ID NOS: 63 and 86) at residues 31 and 57 of H116(SEQ ID NO: 133). The five-fold improvement in affinity is attributable to an increase in the F57 interface with S195 of TF. Despite the changes in the H-CDR1 and H-CDR2 paratope residues, the structure of the human TF ECD with M1587Fab confirmed that epitope and affinity maturation was preserved during HFA.

Biophysical and biometric results

Summary data for these antibodies are shown below in the KD analysis of Biacore (table 11), clotting time of human plasma of human TF (table 12) and EC50 released from IL8 from MD-MB-231 cells after FVIIa stimulation (tables 13, 14 and fig. 5).

KD and data including murine 10H10 and selected human framework adapted variant Mab (M1) chimeric versions (M numbering less than 100) with unmodified CDRs from 10H10 for 43 selected affinity matured mabs are generated and shown in table 11. Thus, the combination of human framework selection and CDR residue substitutions resulted in a peptide having a K in the range of 80 to 950pM_DThe human antibody of (a), the K_DComprising: an off-rate (Koff) in the range of 2.2X 10-5s-1 to 2.6X 10-3 s-1; and10⁴m-1s-1 to 2.3X 10⁵Binding Rate (Kon) in the range of M-1 s-1. The novel Mab had a lower than equilibrium dissociation constant (K) compared to the values of the original murine 10H10 or chimeric construct_D) Up to 10 fold, from 0.77 to 0.08 nM; shows a faster binding rate (K)_on＞10⁵M-1s-1), or have a slow dissociation rate (Koff ═ 10)⁵s-1). These properties can be used to advantage in selecting MAbs for a particular application where residence time or ability to penetrate tissue is desired.

TABLE 11：

Blood coagulation

The novel Mab was characterized by the ability to bind human TF without blocking the clotting of human plasma measured in vitro in the presence of calcium and with exogenous addition of human TF (table 12). Seventeen HFA (M numbering less than 100) variants and 38 affinity matured variants were determined (M1583 and above) and T was reported_1/2Max (time (sec) to reach 50% of maximum optical density).

All responses shown are similar to T with less than 205 seconds_1/2Observed for 10H10 (table 12) at Max values, indicating that these antibodies do not prolong clotting time when compared to the vehicle control without the 159+17 (n-14) antibody. CNTO860, previously described (US7605235B2) and derived from the murine antibody 5G9, a human TF binding antibody (which blocks FX binding to TF) prolonged platelet aggregation and never coagulated blood within the same assay 1800 s. Five of the 43 mabs described in example 4, although having altered CDRs, were not subjected to coagulation assays because their starting concentration was less than 2 mg/ml. M1, M59, and CNTO860 values in several testsThe averaging is performed.

TABLE 12：

Signal blocking Activity

Novel mabs can also be described in terms of their ability to block signaling by the TF/FVIIa complex. TF/VIIa/PAR 2 signaling in breast malignant cells induces a wide array of angiogenic promoting factors (such as VEGF25, Cyr61, VEGF-C, CTGF, CXCL1, and IL-8). FVIIa has previously been reported to induce IL-8 in detectable MDA-MB-231, TF-expressing human breast malignant cell lines (Albrektsen et al, J Thromb Haemost 5: 1588-1597, 2007). Thus, the assay is used as a bioassay to evaluate the TF/VIIa-inducing ability of variant antibodies to inhibit IL-8 production.

Details of this assay are given herein above, and test 19 results for the H-FA (M10-M68) variant of example 3 and the 29 CDR variants of example 4 were tested for the ability to inhibit IL-8 production at a single concentration of TF (0.5. mu.g/ml). An anti-RSV antibody (B37) that does not bind tissue factor was used as a negative control. At this concentration, many HFA mabs were able to block more than 67% of IL-8 induction (table 13). FIG. 5 shows the relative inhibition of IL 8-release by 27 MAbs sharing a L3 light chain (SEQ ID NO: 23) and having either H-CDR1 or H-CDR2 substitutions, compared to those of 10H10 (SEQ ID NOS: 6 and 7, respectively). Furthermore; four of these: m1584, M1611, TF7M1612 and TF7M1607 were placed in the full titer IL-8 induction assay as well as M. The calculated relative IC50 values further support the observation that the improved affinity variants have greater potential compared to the M1, 10H10 mouse-human chimeras (table 14). Other affinity maturation groups of affinity matured antibodies are described in example 4, where similar results were generated.

Watch 13：

Variant ID	% IL-8 inhibition	SD
			10H10	93.9	8.0
M1	96.1	7.6
			M9	102.7	4.5
M10	90.7	0.0
			M11	87.9	9.4
M12	98.6	3.1
			M16	98.3	6.2
M19	87.3	5.8
			M26	79.1	1.3
M37	71.2	11.6
			M42	86.0	20.1
M46	82.5	10.7
			M51	71.5	9.4
M52	67.7	4.0
			M58	88.5	8.5
M59	83.8	8.0
			M60	99.6	4.5
M61	106.8	4.9
			M68	89.8	11.1

TABLE 14：

MAb ID	IC50(ug／ml)
		M1	0.527
M59	0.382
		M1584	0.332
M1607	0.395
		M1611	0.398
M1612	0.413

Example 6: anti-tumor activity of antibody

Mouse transplantation tumor model using MDA-MB-231

MDA-MB-231 human breast malignant cells were cultured in DMEM medium with 10% FBS and 1% LNN, harvested in log phase by trypsinization, and harvested at 5X 10⁷cells/mL were resuspended in sterile serum free DMEM medium. Twenty female SCID Beige (c.b-17/IcrCrl-SCID-bgBR) mice were obtained from Charles River Laboratories and acclimated for 14 days prior to the experiment. By 2.5X 10⁶MDA-MB-231 cells were implanted into the fat pad of the right axillary breast of mice approximately eight weeks of age. When the tumor is about 100mm³In size, mice were stratified by tumor size into treatment groups (N ═ 10/group). Treatment with Dulbecco's Phosphate Buffered Saline (DPBS) or intraperitoneal injection of M1593 at 10 mg/kg body weight, started on the day of stratification and continued once a week for a total of six doses. Tumors and body weights were recorded once a week. When the mean tumor volume per group reached 15001mn³When this is the case, the study is ended. Statistical testing ANOVA (PRIZM4.0, GraphPad) was measured repeatedly using two methods.

In the MDA-MB-231 transplant tumor model, M1593 significantly inhibited tumor growth on the first 22 days (. P < 0.01) and subsequently until day 29 (. P < 0.001), the control (DPBS-treated) group was euthanized at this point. The M1593 treatment group was euthanized on day 36. M1593 inhibited tumor growth by approximately 49% on day 29. The M1593 treated group had approximately 11 days of tumor growth delay relative to the DPBS treated control group (fig. 6).

Mouse graft tumor model using a431

A431 human squamous cell carcinoma cells were cultured in DMEM medium with 10% FBS and 1% LNN, harvested in log phase by trypsinization, and harvested at 1X 10⁷cells/mL were resuspended in sterile HBSS. From Charles RiverTwenty female SCID Beige (c.b-17/IcrCrl-SCID-bgBR) mice were obtained by Laboratories and acclimated for 14 days prior to the experiment. By 2X 10⁶A431 cells were implanted into the right side of mice at about eight weeks of age. When the tumor is about 118mn³In size, mice were stratified by tumor size into treatment groups (N ═ 10/group). Treatment with DPBS or M1593 intraperitoneal injections at 10 mg/kg body weight, starting the day of stratification and once a week for a total of six doses. Tumors and body weights were recorded twice a week. When the average tumor volume of each group reaches 1000mm³When this is the case, the study is ended. Statistical testing ANOVA (PRIZM4.0, GraphPad) was measured repeatedly using two methods.

M1593 significantly inhibited tumor growth on day 22(═ P ═ 0.0067), at which point the control (DPBS treated) group was euthanized. The CNTO592 treated group was euthanized on day 39. CNT0592 inhibited tumor growth by approximately 54% on day 22. The M1593 treated group had approximately 17 days of tumor growth delay relative to the DPBS treated control group (fig. 7).

Example 7: with altered F _C Antibody composition of (1)

Naturally occurring human Fc receptor variants have substantially different affinities for the Fc portion of human antibodies. In addition, clinical studies have shown improved response rates and patient survival of tightly bound Fc genotypes following treatment with Fc-engineered mAbs (Musolino et al, 2008J Clin Oncol 26: 1789-.

While inhibition of TF signaling is expected to reduce the cellular response leading to tumor proliferation, migration, and metastatic spread, the fact that the TF antigen is displayed on tumor cells provides a means to selectively kill target cells through a mechanism associated with the engagement of the antibody Fc with Fc receptors. It is known that glycan compositions and the primary sequence of the heavy chain affect the surface characteristics of the antibody Fc domain, and modifications to one or both of the glycan composition and the primary sequence of the heavy chain can alter Fc receptor binding.

The MAb identified as M1593 was produced as low fucose glycan modified IgG1 and also as an IgG1-CH2 domain variant (S239D, 1332E, where the numbering belongs to the Kabat EU system).

MAb compositions and methods of preparation

Antibodies with low fucose content (M1593-LF) were produced by electroporation of vectors encoding M1593(IgG 1/k) chains with signal peptide (as shown below) into CHO host cell sublines selected for low fucosylation of proteins from the CHO host cell line. SEQ ID NO: 165 denotes the complete light chain comprising variable domain residues 1-113(SEQ ID NO: 23 plus FR4, SEQ ID NO: 61, underlined) and the human kappa constant light domain. The heavy chain with wild-type human IgG isotype 1 comprises variable domain residues 1-120 (which includes SEQ ID NO: 139 and FR4, SEQ ID NO: 60, underlined), CH1, CH2, and CH3, wherein Kabat positions 239 and 332 (which are 242 and 335 of SEQ ID NO: 167) are modifications from wild-type residues S and D, respectively, to D and E to form variant M1593-DE.

M1593-light chain

DIVMTQTPLSLPVTPGEPASISCKSSQSLLSSGNQKNYLTWYLQKPGQSPQL LIYWASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQNDYTYPLT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：165)

M1593-heavy chain, wherein Kabat position S239 is D and 1332 is E

EVQLVQSGAEVKKPGESLRISCKGSGYTFAPYWIEWVRQMPGKGLEWMG DILPGTGFTTYSPSFQGHVTISADKSISTAYLQWS SLKASDTAMYYCARSG YYGNSGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：167)

CHO cells were generated by subcloning and FACS sorting after four rounds of negative lectin selection (selection without binding to lectin-binding fucose) to isolate a pool of naturally occurring low fucose cells used as host cell lines. This line is derived from the same host cell used to produce M1593 and, therefore, the cells are strictly cultured and processed in the same manner. Transfected cells were screened by methylcellulose plates using protein G detection, and colonies were picked into 96-well plates. The cultures were expanded to shake flasks for titer. In this batch of shake flask cultures, the first parental clone produced up to 708 mg/L of M1593-LF (in standard medium).

LC-MS glycopeptide mapping was performed on two M1593-LF generating clones (C2452B and C2452D) to determine percent fucosylation and to evaluate glycosylation stability profile and generation method over time (table 15). Samples were collected and purified from fed-batch channel 1 and channel 10 batch cultures during the stability study. Purified samples from the bioreactor were also analyzed. Glycopeptide mapping showed an advantageous glycosylation pattern with low percent total fucosylation from C2452B and C2452D. Importantly, the percent fucosylation did not increase significantly over time, indicating that the fucosylation of the host cell is stable. Thus, the fucose content of M1593-LF is less than 10% and typically less than 5%, and in some formulations less than 2%. Mabs produced in non-lectin-selected host CHO cells contained polyglycosyl groups (of which more than 80% were fucosylated).

Watch 15：

Cloning	% fucosylation	Sample analysis
			C2452B	2.81	P1 Shake flask fed batch
C2452B	1.83	Batch p10 stability study
			C2452B	3.67	Bioreactor
C2452D	2.20	P1 Shake flask fed batch
			C2452D	2.25	Batch p10 stability study
C2452D	7.31	Bioreactor

For the mutant Fc variant of M1593 (M1593-DE), the plasmid expressing M1593 was provided with site directed mutagenesis.

Biological activity

Three anti-human TF Fc variants (M1593, M1593-LF, and M1593-DE) have affinity for the Fc receptors (Fc γ RI, Fc γ RIIa, Fc γ RIIIa) of both human and cynomolgus monkeys. These assays were either as described in the applicant's co-pending application (us serial No. 61/426619) or by using plasmon resonance (Biacore) based binding assays (.

The results of these assays showed that the Fc modified anti-TF antibodies all bound more tightly to the recombinant human Fc γ IIIa receptor (18 fold (M1593-LF) and 40 fold (M1593-DE)) compared to the parent, unmodified IgG1M1593 antibody (table 16).

Table 16: affinity of anti-TF antibodies to human Fc γ IIIa receptor

ADCC is stimulated by Fc γ RIIIa conjugation. ADCC assays were performed as described previously (Scallon et al, Mol Immunol 44: 1524-.

The in vitro ADCC assay using human PBMC as effector cells and human breast malignant cell line MDA-MB-231 as target cells reflected improved Fc receptor binding in function (fig. 8).

Claims (31)

1. An isolated antibody that competes with murine antibody 10H10 for binding to human tissue factor, wherein the antibody binding domain is adapted for the human Framework (FR) region, and the antibody has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3, wherein the FR amino acid sequence is unaltered from the amino acid sequence encoded by the human germline gene sequence, wherein the germline is recognized in the IMGT database and the CDR sequences have NO less than 50% sequence identity with the murine 10H10 CDRs represented by SEQ ID NOS: 6-11 and 27.

2. The isolated antibody of claim 1, wherein the antibody does not compete with FVIIa for tissue factor binding and does not substantially block procoagulant, amidolytic activity of TF-VIIa complex but does block TF-VIIa mediated signaling as measured by cytokine IL-8 release from MDA-MB-231 cells.

3. The antibody of claim 2, wherein one or more of the CDR sequences is selected from the sequences of SEQ ID NOs 6-11 and 27.

4. The antibody of claim 3, wherein the three light chain CDR sequences, L-CDR1, L-CDR2, and L-CDR3 are represented by SEQ ID NOS: 9-11, respectively.

5. The antibody of claim 3, wherein the three heavy chain CDR sequences, H-CDR1, H-CDR2, and H-CDR3 are represented by SEQ ID NOS 6-8, respectively, or SEQ ID NOS 6, 27, and 8, respectively.

6. The antibody of claim 3, wherein L-CDR1, L-CDR2, and L-CDR3 are represented by SEQ ID NOs 9-11, respectively; and the three heavy chain CDR sequences, H-CDR1, H-CDR2 and H-CDR3 are represented by SEQ ID NOS: 6-8, respectively, or by SEQ ID NOS: 6, 27 and 8, respectively.

7. 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.

8. The antibody of claim 7, comprising an HC variable region selected from SEQ ID NOS 12-21.

9. The antibody of claim 1, wherein the human LC variable region framework is derived from a human IGKV family two or four member.

10. The antibody of claim 9, comprising an LC variable region selected from SEQ ID NOS 22-26.

11. The antibody of claim 1, wherein the human HC variable region framework is derived from a human germline gene family selected from IGHV5 and IGKV 2.

12. The antibody of claim 1, comprising an HC variable region selected from SEQ ID NOs 12-21 and a human LC variable region selected from SEQ ID NOs 22-26.

13. The antibody of claim 1, comprising an HC variable region having: H-CDR3 of SEQ ID NO. 8; H-CDR1 having a sequence selected from SEQ ID NO 6, 62-83; H-CDR2 having a sequence selected from SEQ ID NOs 7, 27 and 84-107; and optionally, an HC FR4 region selected from IGVJ4(SEQ ID NO: 60) or a variant thereof.

14. The antibody of claim 1, comprising an LC variable region having: L-CDR1 having a sequence selected from the group consisting of SEQ ID NOS 9, 108-116; L-CDR2 having a sequence selected from the group consisting of SEQ ID NOs 10 and 117-120; and L-CDR3 having a sequence selected from the group consisting of SEQ ID NOS 11 and 121-128; and optionally, an LC FR4 region selected from IGKJ2(SEQ ID NO: 61) or a variant thereof.

15. The antibody of claim 1, comprising an HC variable region and an LC variable domain, the HC variable region having: H-CDR3 of SEQ ID NO. 8; H-CDR1 having a sequence selected from SEQ ID NO 6, 62-83; H-CDR2 having a sequence selected from SEQ ID NOs 7, 27 and 84-107; and optionally, an HC FR4 region selected from IGVJ4(SEQ ID NO: 60) or a variant thereof, and said LC variable domain has: L-CDR1 having a sequence selected from the group consisting of SEQ ID NOS 9, 108-116; L-CDR2 having a sequence selected from the group consisting of SEQ ID NOs 10 and 117-120; and L-CDR3 having a sequence selected from the group consisting of SEQ ID NOS 11 and 121-128; and optionally, an LC FR4 region selected from IGKJ2(SEQ ID NO: 61) or a variant thereof.

16. The antibody of claim 1, wherein the HC human framework sequence is derived from IGHV5_ a, and the HC variable region has a sequence selected from the group consisting of SEQ ID NO 19, 129-155.

17. 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 SEQ ID NOS 23, 157-164.

18. The antibody of claim 1, wherein the HC human framework sequence is derived from IGHV5_ a, and the HC variable region has a sequence selected from the group consisting of SEQ ID NO 19, 129-155; the LC human FR sequence is derived from IGKV2D40_ O1, and the LC variable domain has a sequence selected from SEQ ID NO 23, 157-164.

19. The antibody of claim 1, having: a binding domain derived from the IGHV5_ a framework, defined as a non-CDR position, a H-CDR3 having the sequence SGYYGNSGFAY (SEQ ID NO: 8), and wherein the sequence at the H-CDR-1 and/or H-CDR-2 position is given by:

H-CDR1 GYTFX₁X₂X₃WIE （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 may be GFTFITYWIA (SEQ ID NO: 81); and is

H-CDR2 DIX₁PGX₂GX₃TX₄ （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; or wherein the H-CDR2 is DILPASSSTN (SEQ ID NO: 105).

20. The antibody of claim 1, having a binding domain wherein the non-CDR positions are derived from the IGKV2D40_ O1 framework and wherein the sequences at L-CDR-1 and/or LCDR-2 and L-CDR3 have sequences given by the formula:

L-CDR1 KSSQSLLX₁X₂X₃X₄QX₅NYLT （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 is selected from K, R and S;

L-CDR2 X₁ASTRX₂S （IV）（SEQ ID NO: 120）

wherein X1 is selected from H and W; x2 is selected from D, E and S;

L-CDR3 QNDX₁X₂X₃PX₄T （V）（SEQ ID NO: 128）

wherein X1 is selected from D, F and L; x2 is selected from S, T and Y; wherein X3 is selected from W and Y; and X4 is selected from L and M.

21. A method of treating a human subject suffering from a condition wherein TF expression and local biological activity resulting from said TF expression are directly or indirectly associated with the condition being treated, comprising administering to such subject in need of such treatment an antibody according to claim 1, 3, 19 or 20.

22. The method of claim 21, wherein the disorder is a malignancy.

23. The method of claim 22, wherein the malignancy is selected from: primary solid tumors, metastases, carcinomas, adenocarcinomas, melanomas, liquid tumors, lymphomas, leukemias, myelomas, soft tissue malignancies, sarcomas, osteosarcomas, thymomas, lymphosarcomas, fibrosarcomas, leiomyosarcomas, lipomas, glioblastoma, astrosarcoma, malignancies of the prostate, breast, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid, biliary tree, colon, rectum, cervix, uterus, endometrium, thyroid, lung, kidney or bladder.

24. The method of claim 21, wherein the disorder is selected from: benign tumors, hemangiomas, acoustic neuromas, neurofibromas, trachomas and pyogenic granulomas; atherosclerotic plaques; ocular angiogenic diseases of the eye, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uveitis and pterygium (abnormal blood vessel growth); rheumatoid arthritis; psoriasis; delay of wound healing; endometriosis; (ii) angiogenesis; granulation occurs; hypertrophic scars (keloids); nonunion of fracture; scleroderma; sand holes; vascular adhesion; myocardial angiogenesis; coronary artery collateral; a collateral cerebral branch; arteriovenous malformation; ischemic limb angiogenesis; Osler-Webber syndrome; plaque neovascularization; expanding the capillary; hemophilic joints; angiofibroma; fibromuscular dysplasia; wound granulation occurs; crohn's disease; and atherosclerosis.

25. A pharmaceutical composition useful in treating a subject comprising the antibody of claim 1, 3, 19 or 20 in a pharmaceutically acceptable formulation.

26. A kit comprising an antibody according to claim 1, 3, 19 or 20 in a stable form and instructions for use.

27. An isolated nucleic acid encoding one or more of the antibody binding domains of the antibody of claim 1, 3, 19, or 20.

28. A vector comprising at least one polynucleotide of claim 27.

29. A host cell comprising the vector of claim 28.

30. The isolated antibody or fragment of claim 1, 3, 19, or 20, having an IgG1 or IgG4 isotype.

31. The isolated antibody or fragment of claim 30, wherein the Fc region comprises a human IgG1 isotype, with Kabat positions 239 and 332 (which are 242 and 335 of SEQ ID NO: 167) modified in the Fc region from wild type residues S and D to mutations of D and E.