EA041176B1 - HUMAN ANTIBODIES AGAINST TISSUE FACTOR - Google Patents

Description

The field of technology to which the invention belongs

This invention relates to antibodies directed to tissue factor, in particular human tissue factor, and uses of such antibodies, in particular their use in the treatment of cancer, inflammation and vascular disease.

State of the art

Tissue factor (TF), also called thromboplastin, factor III, or CD142, is a protein present in subendothelial tissue, platelets, and leukocytes necessary to initiate the formation of thrombin from the prothrombin zymogen. The formation of thrombin ultimately leads to coagulation of the blood. Tissue factor allows cells to initiate blood coagulation cascades and it functions as a high affinity receptor for coagulation factor VII. The resulting complex provides a catalytic event that is responsible for the initiation of protease coagulation cascades through specific limited proteolysis. Unlike the other cofactors of these protease cascades, which circulate as non-functional precursors, this factor is a potent initiator that is fully functional when expressed on cell surfaces.

Tissue factor is a cell surface receptor for the serine protease factor VIIa (FVIIa). FVIIa binding to tissue factor has been found to trigger signal transduction processes in that cell, with this signal transduction function playing a role in angiogenesis. While angiogenesis is a normal process in growth and development, as well as in wound healing, it is also a fundamental step in the transition of tumors from a dormant state to a malignant state: when cancer cells acquire the ability to produce proteins that are involved in angiogenesis, the so-called angiogenic growth factors, these proteins are released by the tumor into neighboring tissues and stimulate new blood vessels to sprout from existing healthy blood vessels towards the tumor and into the tumor. Once new blood vessels enter the tumor, it can rapidly increase in size and invade local tissue and organs. Through these new blood vessels, cancer cells can also leak into the bloodstream and localize to other organs to form new tumors (metastases).

In addition, TF plays a role in inflammation. The role of TF is thought to be mediated by blood coagulation (A.J. Chu: Tissue factor mediates inflammation in Archives of biochemistry and biophysics, 2005, vol. 440, No. 2, pp. 123-132). Thus, inhibition of TF, for example by monoclonal anti-TF antibodies, is important in interrupting the coagulation-inflammation cycle to promote not only anti-inflammation but also the treatment of vascular disease.

TF expression is observed in many types of cancer and is associated with more aggressive disease. In addition, human TF also exists in a soluble alternatively spliced form, asHTF. Recently, asHTF has been found to stimulate tumor growth (Hobbs et al., 2007 Thrombosis Res. 120(2) S13-S21).

The binding of antibodies to TF has been disclosed in the prior art:

WO 98/40408 describes antibodies that can bind native human TF, either alone or present in a TF:VIIa complex, effectively preventing factor X from binding to TF or the complex and thereby reducing blood coagulation. It is disclosed that these antibodies can be used to alleviate thrombosis after an invasive medical procedure, such as arterial or cardiac surgery, or to eliminate blood coagulation resulting from the use of a medical program. In addition, it is described that antibodies can be used in in vivo diagnostic methods, including in vivo diagnostic imaging of native human TF.

WO 04/094475 provides antibodies capable of binding to human tissue factor that do not inhibit factor-mediated blood coagulation compared to normal plasma as a control. Human antibodies have not been described. It is assumed that this antibody can be used to treat cancer.

WO 03/093422 relates to antibodies that bind with higher affinity to a TF:VIIa complex than to TF alone. These antibodies are proposed to be used as an anticoagulant in the treatment of certain diseases such as sepsis, disseminated intravascular coagulation, ischemic stroke, thrombosis, acute coronary syndromes, and coagulopathy in advanced cancer.

WO 01/27079 describes compositions and methods for inhibiting abnormal cell proliferation, in particular endothelial cell proliferation, for example in the case of cancer, abnormal embryonic development, impaired function of immune responses, and angiogenesis associated with neovascularization and tumor growth. Many active substances are proposed, including antibodies, but no specific antibodies are described.

WO 03/037361 relates to the use of a TF agonist or antagonist for treatment associated with apoptosis.

WO 03/029295 relates to isolated human antibodies that immunoreact with human TF to inhibit coagulation factor VIIa binding. However, this application does not disclose any

- 1 041176 an example of an antibody having these properties.

A number of monoclonal antibody therapies have been approved for various types of tumors, including bevacizumab (Avastin®), cetuximab (Erbitux®), panitumumab (Vectibix™) and trastuzumab (Herceptin®).

The essence of the invention

While much progress has been made, there remains a need for improved treatments for serious diseases, such as improved cancer treatments, based on therapeutic antibodies.

The aim of this invention is to provide new highly specific and effective human anti-TF antibodies for medical use. The antibodies of this invention exhibit TF binding characteristics that differ from those of antibodies described in the art. In preferred embodiments, the antibodies of this invention have high affinity for human tissue factor, mediate antibody-dependent cellular cytotoxicity (ADCC), inhibit FVIIa-induced ERK phosphorylation and IL-8 release, and do not or weakly inhibit coagulation.

Brief description of the figures

Fig. 1: Comparison of sequences of antibodies of the present invention. CDR1, CDR2 and CDR3 are highlighted according to IMGT: sequences in italics represent the CDR1 region, sequences not underlined represent the CDR2 region, sequences in bold represent the CDR3 region.

Fig. 2: IgG4 sequences (SEQ ID NOs: 113-114).

SEQ ID NO: 113: Amino acid sequence of the wild-type CH region of human IgG4. The sequence in italics represents the CH1 region, the highlighted sequence represents the hinge region, the normal sequence represents the CH2 region, and the ununderlined sequence represents the CH3 region;

SEQ ID NO: 114: Amino acid sequence of the hingeless CH region of human IgG4.

Fig. 3: Binding of anti-TF-HuMab to the extracellular domain of TF.

Fig. 4: Binding of anti-TF-HuMab to membrane-bound TF.

Fig. 5: Inhibition of FVIIa binding to TF.

Fig. 6: Inhibition of FVIIa-induced ERK phosphorylation.

Fig. 6a: Inhibition of FVIIa-induced ERK phosphorylation.

Fig. 7: Inhibition of FVIIa-induced release of IL-8.

Fig. 8: Inhibition of FXa generation.

Fig. 9: Inhibition of blood coagulation.

Fig. 10: TF-HuMab induces lysis of Bx-PC3 cells via ADCC.

Fig. 11: Deposition of complement components C3c and C4c on target cells.

Fig. 12: Immunohistochemical analysis of TF-HuMab binding to glomeruli (glomeruli).

Fig. 13: Immunohistochemical analysis of TF-HuMab binding to pancreatic tumors.

Fig. 14: In vivo performance of TF-HuMab in an established MDA-MB231 tumor xenograft.

Fig. 15: Bleeding time determined in cynomolgus monkeys after intravenous injections of TF-specific HuMab 011. This antibody was administered on days 1 (0 mg/kg), 8 (1 mg/kg), 15 (10 mg/kg) and 22 (100 mg/kg).

Fig. 16: In vivo efficacy of TF-HuMab in prophylactic and established Bx-PC3 tumor xenograft.

Fig. 17: Shuffled construction and TF domains.

Fig. 18: Binding of anti-TF antibodies to shuffled TF constructs.

Fig. 19: Binding of HuMab-TF Fab fragments to the extracellular domain of TF, ELISA.

Fig. 20: Binding of HuMab-TF Fab fragments to the extracellular domain of TF, FACS.

Fig. 21: Anti-TF-HuMab binding profile versus number of expressed TF molecules.

Information confirming the possibility of carrying out the invention

Definitions

The terms tissue factor, TF, CD142, tissue factor antigen, TF antigen, and CD142 antigen are used interchangeably herein and, unless otherwise indicated, include any variants, isoforms, and species homologues of human tissue factor that are naturally expressed by cells or expressed on cells, transfected with the tissue factor gene.

The term immunoglobulin refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains, and one pair of heavy (H) chains, which can, all four, be linked by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W.,

- 2 041176 ed., ^2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated here as VH or VH) and a heavy chain constant region. This heavy chain constant region typically consists of three domains, C _H 1 , CH2 and CH3. Each light chain typically consists of a light chain variable region (abbreviated here as V _L or VL) and a light chain constant region. This light chain constant region typically contains one domain, C _L . The VH and V _L regions can be further subdivided into regions of hypervariability (or hypervariable regions, which may be hypervariable in sequence and/or form structurally defined loops), also called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called frame regions (FR). Each of VH and V _L usually consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol Biol 196, 901-917 (1987)). Typically, the numbering of amino acid residues in this region is according to IMGT, Sequences of Proteins of Immunological Interest, 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) (here, phrases such as Kabat or Kabat variable domain numbering refer to this heavy chain or light chain variable domain numbering). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer amino acids or additional amino acids corresponding to a FR or CDR shortening or insertion into a variable domain FR or CDR. For example, a heavy chain variable domain may include a single amino acid insert (Kabat residue 52a) after VH CDR2 residue 52, and inserted residues (e.g. Kabat residues 82a, 82b and 82c, etc.) after heavy chain FR residue 82 . The Kabat numbering of residues can be determined for a particular antibody by matching regions of homology of that antibody sequence to a standard Kabat numbered sequence.

The term antibody (Ab) in the context of this invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule or a derivative of any of them, which has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 h or more, approximately 3, 4, 5, 6, 7 or more days, etc., or any other suitable functionally defined period (such as time sufficient to induce, stimulate, enhance and/or modulate a physiological response associated with the binding of the antibody to the antigen, and/or the time sufficient for this antibody to recruit effector a activities). The heavy and light chain variable regions of this immunoglobulin molecule contain a binding domain that interacts with the antigen. The constant regions of these antibodies (Ab) can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component in the classical complement activation pathway. The anti-TF antibody can also be a bispecific antibody, a diabody, or the like (see, for example, PNAS USA 90(14), 6444-8 (1993) for a description of diabodies). Indeed, the bispecific antibodies, diabodies, and the like provided by the present invention may bind any suitable target, along with the tissue factor portion or tissue factor-FVIIa complex. As indicated above, the term antibody, unless otherwise indicated, includes antibody fragments that retain the ability to specifically bind to that antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments included in the term antibody include (i) a Fab or Fab fragment, a monovalent fragment consisting of V _L , V _H , C _L and _CH 1 domains, or a monovalent antibody described in WO 2007059782 (Genmab );

(ii) F(ab') ₂ fragments, divalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region;

(iii) an Fd fragment consisting essentially of V _h u CH1 domains;

(iv) an Fv fragment consisting essentially of V _L and V _H domains, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a V _H domain, and also referred to as domain-consisting antibodies (Holt et al., Trends Biotechnol. 2003 Nov; 21(11):484-90);

(vi) camelids or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1):111-24) and (vii) a distinct complementarity determining region (CDR).

In addition, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they can be connected, using recombinant methods, with a synthetic linker that allows them to be prepared as a single protein chain in which the V _L and V regions _H pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv),

- 3 041176 see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are included in the term antibody unless otherwise indicated or otherwise clearly indicated by the context). Although such fragments are usually included in the concept of antibodies, they, together or each independently, are unique features of the present invention, exhibiting various biological properties and applicability. These and other applicable antibody fragments in the context of this invention are discussed further here. It should be understood that the term antibody, unless otherwise indicated, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to that antigen. (antigen binding fragments) provided by any known method such as enzymatic digestion, peptide synthesis and recombinant methods. The generated antibody may be of any isotype.

An anti-TF antibody is an antibody as described above that specifically binds to an antigenic tissue factor.

The term human antibody, as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of this invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or during gene rearrangement or somatic mutation in vivo). However, the term human antibody, in this context, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

In one preferred embodiment, the antibody of this invention is isolated. An isolated antibody refers in this context to an antibody that is substantially free of other antibodies having differing antigenic specificities (e.g., an isolated antibody that specifically binds to tissue factor is substantially free of antibodies that specifically bind antigens other than tissue factor ). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of human tissue factor may be cross-reactive with other related antigens, eg, from other species (such as tissue factor species homologues). In addition, the selected antibody may be essentially. free from other cellular material and/or chemicals. In one embodiment of the present invention, two or more isolated monoclonal antibodies having different antigen-binding specificities are combined in a well-defined composition.

When used here in the context of two or more antibodies, the term competes with or cross-competes with indicates that the two or more antibodies compete for TF binding, e.g. compete for TF binding in the assay described in Example 6 here. For some pairs of antibodies, competition in this assay of Example 6 occurs only when one antibody is coated on the plate and the other is used to compete, and not vice versa. The term rivals as used herein also encompasses such combinations of antibodies.

The terms monoclonal antibody or monoclonal antibody composition refers in this context to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term human monoclonal antibody refers to antibodies exhibiting a single binding specificity that have variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be generated by a hybridoma that includes a B cell derived from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome containing a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

As used herein, the term binding in the context of antibody binding to a predetermined antigen generally means binding with an affinity corresponding to a K _D of about ^10-7 M or less, such as about ^10-8 M or less, such as about 10-9 M or less , about ^10-10 M or less, or about ^10-11 M or even less, as determined, for example, by surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using this antigen as a ligand and this antibody as an analyte, and this antibody binds a predetermined antigen with an affinity corresponding to a KD that is at least ten times lower, such as at least 100 times lower, such as at least 1000 times lower, such as at least 10,000 times lower, e.g., at least 100,000 times lower, than its affinity for a non-specific antigen (e.g., BSA, casein), another, than a predetermined antigen or a closely related antigen. The amount by which this affinity is lower depends on the K _D of that antibody, so that when the K _D of an antibody is very low (i.e., the antibody is highly specific), the amount by which the affinity for that antibody is antigen is lower than the affinity for non-specific antigen, may be at least 10,000-fold.

The term kd (c ^-1 ) refers in this context to the dissociation rate constant of a particular antibody-antigen interaction. This value is also called the kf value.

The term _ka (M-c' ¹ ) refers in this context to the association rate constant of a particular antibody-antigen interaction.

The term K _D (M) refers in this context to the dissociation equilibrium constant of a particular antibody-antigen interaction.

The term KA (M'1) refers in this context to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing _ka by kd.

The invention also provides antibodies containing functional variants of the V _L region, the VH region, or one or more CDRs of the antibodies of these examples. A functional V _L , VH or CDR variant used in the context of an anti-TF antibody still allows that antibody to retain at least a substantial proportion (at least about 50, 60, 70, 80, 90, 95% or more ) the affinity/avidity and/or specificity/selectivity of the parent antibody, and in some cases, such an anti-TF antibody may be associated with higher affinity, selectivity and/or specificity than the parent antibody.

Such functional variants typically retain significant sequence identity relative to the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by those sequences (i.e. %homology=number of identical positions/total number of positions x100), given the number of gaps and the length of each gap that must be entered to optimally match the two. sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm as described in the non-limiting examples below.

Percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988)), which was included in the ALIGN program (version 2.0), using the PAM120 subtraction table for weight, gap length penalty (penalty) 12, and gap penalty 4. In addition, percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970)), which was included in the GAP program in the GCG software package (available at http://www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14 , 12, 10, 8, 6 or 4 and length weight 1, 2, 3, 4, 5 or 6.

The sequence of the CDR variants may differ from the CDR sequences of the parent antibody by the most conservative substitutions; for example, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more , about 95% or more (eg, about 65-99%, eg, about 96, 97, or 98%) of the substitutions in a variant are conservative amino acid residue substitutions.

The sequence of the CDR variants may differ from the sequence of the CDR sequences of the parent antibody by the most conservative substitutions; for example at least 10, eg at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of these substitutions in a variant are conservative amino acid residue substitutions.

In the context of this invention, conservative substitutions may be defined by substitutions in the classes of amino acids shown in one or more of the following three tables:

Amino acid residue classes for conservative substitutions

- 5 041176

Acid Residue Asp (D) and Glu (E) Basic remains Lys (S), Arg (R) and His (H) Hydrophilic uncharged residues Ser (K), Thr (T), Asn (N) and Leu(L) Aliphatic uncharged residues Gly (G), (Ala (A), Vai (V), Leu (L) and He (I) Non-polar uncharged residues Cys (C), Met (M) and Pro (P) aromatic residues Phe (F), Tyr (Y) and Trp (W)

Alternative classes of conservative amino acid substitutions i A S T

2D E 3 N Q 4 R TO 5 I L M 6F Y W

Alternative physical and functional classifications of amino acid residues

Residues S and T containing an alcohol group

Aliphatic residues I, L, V and M

Cycloalkenyl-associated residues F, H, W and Y

Hydrophobic residues A, C, F, G, Η, I, L, M,

R, T, V, W and Y

Negatively charged residues D and E

Polar residues C, D. E, H. K, N, Q, R, S and T

Positively charged residues H, K and R

Small residues A, C, D, G, N, P, S, T and

Very small residues of A, G and S

Residues involved in the formation of A, C, D, E, G, H, K, N, Q, R, turn S, P and T

Flexible residues Q, T, K, S, G, P, D, E and R

Groupings of more conservative substitutions include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

Additional groups of amino acids can also be prepared using the principles described in, for example, Creighton (1984) Proteins: Structure and Molecular Properties (2d Ed. 1993), W. H. Freeman and Company.

In one embodiment of this invention, conservatism in terms of hydrophobic/hydrophilic properties and weight/residue size is also substantially preserved in the CDR variant compared to the antibody CDR of these examples (e.g., weight class, hydrophobicity score, or both of these sequences are at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more (eg, about 65-99%) preserved). For example, conservative substitutions of residues may also or alternatively be based on the substitution of strong or weak weight-based conservative groups, which are known in the art.

Retention of similar residues can also or alternatively be measured by a similarity score determined using the BLAST program (for example, BLAST 2.2.8 is available through the NCBI, using standard settings BLOSUM62, open gap=11 and extended gap=1). Suitable variants typically exhibit at least about 45%, e.g., at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least at least about 95% or greater (eg, about 70-99%) similarity relative to the parent peptide.

As used herein, an isotype refers to a class of immunoglobulins (eg, IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that are encoded by heavy chain constant region genes.

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The term epitope means a protein determinant capable of specifically binding to an antibody. Epitopes usually consist of groupings of surface 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 differ in that the binding of the former, but not the latter, is lost in the presence of denaturing solvents. An epitope may contain amino acid residues directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide (in other words, that amino acid residue is located in within the specific antigen-binding peptide footprint).

In this context, a human antibody is derived from a particular germline sequence if the antibody is derived from a system using human immunoglobulin sequences, for example, by immunization of a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library, and where the V domain sequence of the selected human antibody is at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or, such as at least at least 99% identical in the amino acid sequence of the V domain to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, outside of the heavy chain CDR3, a human antibody derived from a particular human germline sequence will exhibit no more than 20 amino acid differences, e.g. no more than 10 amino acid differences, e.g. no more than 9, 8, 7, 6, or 5, e.g. no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

In this context, the term growth inhibition (for example, when referring to cells such as tumor cells) includes any measurable reduction in cell growth when contacted with an anti-TF antibody compared to the growth of the same cells without contact with anti-TF. -antibody, for example, inhibition of cell culture growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. This decrease in cell growth can occur through various mechanisms such as effector cell phagocytosis, ADCC, CDC and/or apoptosis.

The term bispecific molecule is intended to include any agent, such as a protein, peptide, or protein or peptide complex, that has two different binding specificities. For example, this molecule can bind or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term bispecific antibodies is intended to include any anti-TF antibody that is a bispecific molecule. The term bispecific antibodies also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and V _L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, causing these domains to pair with complementary domains of the other chain and creating two antigen-binding sites (see, for example, Holliger, P. et al., PNAS USA 90, 6444-6448 (1993), Poljak, RJ. et al., Structure 2, 1121-1123 (1994) ).

An antibody deficient in effector function or deficient in effector function refers to an antibody that has a significantly reduced ability or lack of ability to activate one or more effector mechanisms, such as complement activation or Fc receptor binding. Thus, effector-deficient antibodies have a significantly reduced ability or lack of ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). An example of such an antibody is IgG4.

The term monovalent antibody means in the context of this invention that an antibody molecule is capable of binding a single antigen molecule and therefore not capable of cross-linking antigens.

The term stabilized IgG4 antibody refers to an IgG4 antibody that has been modified to reduce the exchange of half molecules (see van der Neut Kolfschoten M et al. (2007) Science 14; 317(5844) and references in this article, as well as Labrijn et al (2009) Nature Biotechnology, 27, 767-771.

In this context, the term effector cell refers to an immune cell that participates in the effector phase of the immune response, as opposed to the recognition phase and the activation phase of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (such as B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells and basophils. Some effector cells express specific Fc receptors and carry out specific immune functions. In some embodiments, the effector cell is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), such as a natural killer cell, method

- 7 041176 to induce ADCC. For example, monocytes, macrophages that express FcRs, are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system or the binding of cells that present antigens. In some embodiments, the effector cell may phagocytize the target antigen or target cell. Expression of a particular FcR on an effector cell may be regulated by humoral factors such as cytokines. For example, FcyRI expression has been found to be upregulated by interferon-γ (IFN-γ) and/or G-CSF. This increased expression increases the cytotoxic activity of FcyRI-bearing cells against targets. The effector cell may phagocytize or lyse the target antigen or target cell.

The term vector refers in this context to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a plasmid, which is a circular double-stranded loop into which additional DNA segments can be ligated. Another type of vector is the viral vector, where additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (such as non-episomal mammalian vectors) can be integrated into the genome of a host cell after introduction into that host cell, and thereby replicate along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). Typically, expression vectors used in recombinant DNA methods are often in the form of plasmids. In this description, the plasmid and vector can be used interchangeably, since the plasmid is the most commonly used form of the vector. However, the present invention includes such other forms of expression vectors as viral vectors (such as replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

The term recombinant host cell (or simply host cell) refers in this context to a cell into which an expression vector has been introduced. It should be understood that such terms refer not only to the particular cell in question, but also to the progeny of such a cell. Since certain modifications may occur in surviving generations due to either mutation or environmental influences, such progeny may not actually be identical to the original cell, but it is still within the scope of the term host cell in this context. Recombinant host cells include, for example, transfectomes such as CHO cells, HEK293 cells, NS/0 cells, and lymphocytes.

The term transfectome includes, as used herein, recombinant eukaryotic host cells expressing the antibody, such as CHO cells, NS/0 cells, HEK293 cells, plant cells, or fungi, including yeast cells.

The term non-human transgenic animal refers to a non-human animal having a genome containing one or more human heavy and/or light chain transgenes or transchromosomes (either integrated or not integrated into the natural genomic DNA of that animal) that is capable of expressing fully human antibodies. For example, a transgenic mouse may have a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome such that the mouse produces anti-human TF antibodies when immunized with TF antigen and/or cells expressing TF. The human heavy chain transgene may be integrated into the chromosomal DNA of the mouse, or the human heavy chain transgene may be maintained extrachromosomally, as in the case of the transchromosomal KM mouse described in WO02/43478. Such transgenic and transchromosomal mice (collectively referred to herein as transgenic mice) are capable of producing multiple isotypes of human monoclonal antibodies to a particular antigen (such as IgG, IgA, IgM, IgD, and/or IgE) by undergoing V-D-J recombination and isotype switching. A transgenic non-human animal may also be used to generate antibodies against a specific antigen by introducing genes encoding that specific antibody, for example by operably linking those genes to a gene that is expressed in the animal's milk.

Treatment refers to the administration of an effective amount of a therapeutically active compound of this invention for the purpose of alleviating, reducing symptoms, delaying or eliminating (curing) the symptoms or conditions of a disease.

An effective amount is an amount effective, at suitable doses and for the necessary periods of time, to achieve the desired therapeutic result. The therapeutically effective amount of the anti-TF antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the anti-TF antibody to induce the desired response in that individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or portion

- 8 041176 antibodies are outweighed by therapeutically beneficial effects.

An anti-idiotypic (Id) antibody is an antibody that recognizes unique determinants normally associated with an antibody's antigen-binding site.

Additional aspects and variants of this invention

As described above, in a first aspect, this invention relates to a human antibody that binds human tissue factor.

In one embodiment, the antibody binds to the extracellular domain of tissue factor with an apparent affinity (EC ₅₀ ) of 3 nM or less, such as 0.50 nM or less, such as 0.35 nM or less, such as 0.20 nM or less, e.g., 0.1 nM or less, when determined as described in the assay in Example 13.

In another embodiment, the antibody binds to mammalian cells expressing tissue factor, such as A431 cells transfected with a construct encoding tissue factor, preferably with an apparent affinity (EC ₅₀ ) of 10 nM or less, such as 8 nM or less, such as 5 nM or less, such as 2 nM or less, such as 1 nM or less, such as 0.5 nM or less, such as 0.3 nM or less, when determined as described in the assay in Example 14.

In another embodiment, the antibody is capable of inducing antibody-dependent cellular cytotoxicity in A431 cells, preferably with an EC ₅₀ value of 2 nM or less, such as 1 nM or less, such as 0.7 nM or less, or 0.3 nM or less, such as 0.2 nM or less or 0.1 nM or less or 0.05 nM or less when determined as described in the assay in Example 20.

In another embodiment, the antibody is effective in inhibiting the growth of established MDA-MB-231 tumors as determined by the method described in Example 24 and/or in inhibiting the growth of established BxPC3 tumors as measured by the method described in Example 26.

In another embodiment, the antibody inhibits tissue factor-induced blood coagulation, preferably with a median inhibition concentration of less than 10 nM, such as less than 5 nM, such as less than 2 nM, such as less than 1 nM, as determined , as described in the analysis in Example 19.

In another embodiment, the antibody does not inhibit coagulation. In one embodiment, coagulation is inhibited by at most 30%, eg 25%, eg 20%, eg 15%, eg 10% or eg 5% compared to natural levels.

In a further embodiment, the antibody inhibits FVIIa binding to tissue factor, preferably with a maximum inhibition value of greater than 80%, such as greater than 90%, when determined as described in the assay in Example 15.

In a further embodiment, the antibody inhibits FVIIa-induced release of IL-8 by MDA-MB-231 cells, preferably with a maximum inhibition value of greater than 40%, such as greater than 50%, such as greater than 60%, when determined as described in the assay in example 17.

In a further embodiment, the antibody inhibits the conversion of FX to FXa by the TF/FVIIa complex, preferably by less than 50%, such as less than 40%, such as in the range of 1-30%, when determined as described in the assay in example 18.

In a further embodiment, the antibody competes for tissue factor binding with an antibody comprising a VH region containing the sequence of SEQ ID NO: 9 and a VL region containing the sequence of SEQ ID NO: 65.

In a further embodiment, the binding of an antibody of this invention to tissue factor does not include all three of the following residues: W at position 45, K at position 46, or Y at position 94 of the tissue factor. In the following embodiment, this binding does not include any of the following residues: W at position 45, K at position 46, or Y at position 94 (these numbers refer to mature TF, the equivalent positions in Genbank entry NP_001984 are 77, 78 and 126 ).

In another embodiment, the antibody competes for tissue factor binding with an antibody comprising a VH region containing the sequence of SEQ ID NO: 37 and a VL region containing the sequence of SEQ ID NO: 93.

In a further embodiment, the antibody inhibits FVIIa-induced ERK phosphorylation, preferably at a median inhibition concentration of less than 10 nM, such as less than 5 nM, e.g., less than 2 nM, when determined as described in the assay in Example 16.

In a further embodiment, the antibody inhibits ERK phosphorylation, preferably at a median inhibition concentration of less than 10 nM, such as less than 5 nM, such as less than 2 nM, when determined as described in the assay in Example 16, and does not inhibit FVII-induced release of IL-8, as described in the analysis in example 17, by more than (at most) 10%.

In a further embodiment, the antibody is capable of inducing C3c and C4c deposition, preferably when the antibody is capable of inducing C3c and C4c deposition as defined in

- 9 041176 example 21.

In a further embodiment, the Fab fragments of this antibody bind to the extracellular domain of tissue factor as described in Example 28 with an _EC50 value below 0.1 μg/ml, such as below 0.05 μg/ml, such as below 0.04 μg/ml as measured by ELISA.

In a further embodiment, the Fab fragments of this antibody bind to the extracellular domain of tissue factor, as described in Example 28, with an EC ₅₀ value greater than 1.0 μg/ml, as measured by ELISA.

In a further embodiment, the Fab fragments of this antibody bind to the extracellular domain of tissue factor as described in Example 28 with an EC ₅₀ value below 10 μg/ml, such as below 1 μg/ml, such as below 0.5 μg/ml or below 0.2 µg/ml.

In a further embodiment, the antibody binds to human tissue factor but murine tissue factor and exhibits reduced binding compared to binding to human TF with a 42-84 mm shuffled construct containing the human TF sequence, except for amino acids 42-84, which were replaced by the mouse sequence as described in Example 27.

In a further embodiment, the antibody binds to human tissue factor, but not to mouse tissue factor, and exhibits reduced binding compared to human TF binding with a 85-122 mm shuffled construct containing the human TF sequence, except for amino acids 85-122, which were replaced by the mouse sequence as described in Example 27.

In a further embodiment, the antibody binds to human tissue factor, but not to mouse tissue factor, and exhibits reduced binding compared to human TF binding with a 123-137 mm shuffled construct containing the human TF sequence, except for amino acids 123-137, which were replaced by the mouse sequence as described in Example 27.

In a further embodiment, the antibody binds to human tissue factor, but not to mouse tissue factor, and exhibits reduced binding compared to human TF binding with a 185-225 mm shuffled construct containing the human TF sequence, except for amino acids 185-225, which were replaced by the mouse sequence as described in Example 27.

In a further embodiment, the antibody binds to human tissue factor, but not to mouse tissue factor, and exhibits reduced binding compared to human TF binding with a 226-250 mm shuffled construct containing the human TF sequence, except for amino acids 226-250, which were replaced by the mouse sequence as described in Example 27.

In a further embodiment, the antibody exhibits reduced binding compared to binding to human TF with more than one shuffled construct. In a further embodiment, this antibody exhibits reduced binding to the 4284 mm construct as well as to the 85-122 mm construct.

In a further embodiment, this antibody exhibits reduced binding to the 123-137 mm construct as well as the 185-225 mm construct. In a further embodiment, this antibody exhibits reduced binding to the 123-137 mm construct as well as to the 185-225 mm construct and additionally to the 226-250 mm construct.

In a further embodiment, the antibody is capable of inducing C3c and C4c deposition, preferably when the antibody is capable of inducing C3c and C4c deposition as defined in Example 21.

In one embodiment of an antibody of this invention, said antibody competes for tissue factor binding with an antibody comprising a VH region containing the sequence of SEQ ID NO: 9 and a VL region containing the sequence of SEQ ID NO: 65, and does not compete for tissue factor binding with the antibody , containing a VH region containing the sequence of SEQ ID NO: 37, and a VL region containing the sequence of SEQ ID NO: 93.

In a further embodiment, this antibody contains a VH CDR3 region having

a) the sequence shown in SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, or

b) a variant of any of these sequences, such as a variant having at most 1, 2, 3, 4 or 5 amino acid modifications, preferably substitutions such as conservative substitutions.

In a further embodiment, the antibody comprises a VH CDR3 region having the sequence shown in SEQ ID NO: 12, or a variant thereof, wherein the variant contains a modification at one or more of positions 2, 3, 6, 9 and 11, preferably where this the modification is a substitution, more preferably where the substitution is selected from the group consisting of

a) R replaced by K when it is in position 2,

b) S replaced by A or T when it is in position 3,

- 10 041176

c) G replaced by T when it is in position 6,

d) L replaced by F when it is in position 9, and

e) S replaced by Y when it is in position 11.

In another embodiment, this antibody contains:

a) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 10, 11 and 12, and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 66, 67 and 68,

b) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 14, 15 and 16 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 70, 71 and 72,

c) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 18, 19, 20 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 74, 75 and 76,

d) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 22, 23 and 24, and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 78, 79 and 80,

e) a VH region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 26, 27 and 28, and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 82, 83 and 84, or

f) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2, or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in said sequences.

In the following embodiment, this antibody contains a VH having:

a) at least 80% identity, such as at least 90%, at least 95%, or at least 98 or 100% sequence identity of a VH region selected from the group consisting of SEQ ID NOs: 9, 13, 17, 21 and 25, or

b) at most 20, such as 15, or 10, or 5, 4, 3, 2, or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to a V _H region sequence selected from the group consisting of from SEQ ID NOs: 9, 13, 17, 21, 21 and 25.

In the following embodiment, this antibody contains a VL having:

a) at least 80% identity, such as at least 90%, at least 95%, or at least 98 or 100% sequence identity of a VL region selected from the group consisting of: SeQ ID NOs: 65, 69 , 73, 77 and 81, or

b) at most 20, e.g. 15 or 10 or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions such as conservative amino acid substitutions, compared to a VL region sequence selected from the group consisting of from SEQ ID NOs: 65, 69, 73, 77 and 81.

In the following embodiment, this antibody contains:

a) a VH region containing the sequence of SEQ ID NO: 9 and a VL region containing the sequence of SEQ ID NO: 65,

b) a VH region containing the sequence of SEQ ID NO: 13 and a VL region containing the sequence of SEQ ID NO: 69,

c) a VH region containing the sequence of SEQ ID NO: 17 and a VL region containing the sequence of SEQ ID NO: 73,

d) a VH region containing the sequence of SEQ GO N0:21 and a VL region containing the sequence of SEQ ID NO: 77,

e) a VH region containing the sequence of SEQ ID NO: 25 and a VL region containing the sequence of SEQ ID NO: 81, or

f) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2, or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in said sequences.

In a further embodiment, the antibody competes for tissue factor binding with an antibody comprising a VH region containing the sequence of SEQ ID NO: 9 and a VL region containing the sequence of SEQ ID NO: 65, and competes for tissue factor binding with an antibody, containing a VH region containing the sequence of SEQ ID NO: 37, and a VL region containing the sequence of SEQ ID NO: 93.

In the following embodiment, this antibody contains a VH CDR3 region having:

a) the sequence shown in SEQ ID NO: 8, SEQ ID NO: 52, or

b) a variant of any of these sequences, such as a variant having at most 1, 2 or 3 amino acid modifications, preferably substitutions such as conservative substitutions.

In the following embodiment, this antibody contains:

a) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 6, 7 and 8 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 62, 63 and 64,

b) a VH region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 50, 51 and 52, and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 106, 107 and 108, or

c) a variant of any of said antibodies, wherein said variant preferably has the most

- 11 041176 greater than 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in the indicated sequences.

In the following embodiment, this antibody contains a VH having:

a) at least 80% identity, such as at least 90%, at least 95%, or at least 98 or 100% identity, relative to a VH region sequence selected from the group consisting of SEQ ID NO: 5, and 49, or

b) at most 20, such as 15, or 10, or 5, 4, 3, 2, or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to a VH region sequence selected from the group consisting of SEQ ID NOs: 5 and 49.

In the following embodiment, this antibody contains a VL having:

a) at least 80% identity, such as at least 90%, at least 95%, or at least 98 or 100% identity, with respect to a VL region sequence selected from the group consisting of SEQ ID NO: 61, and 105 or

b) at most 20, such as 15, or 10, or 5, 4, 3, 2, or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to a VH region sequence selected from the group consisting of : SEQ ID NO: 61 and 105.

In the following embodiment, this antibody contains:

a) a VH region containing the sequence of SEQ ID NO: 5 and a VL region containing the sequence of SEQ ID NO: 61,

b) a VH region containing the sequence of SEQ ID NO: 49 and a VL region containing the sequence of SEQ ID NO: 105, or

c) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in said sequences.

In the following embodiment, the antibody does not compete for tissue factor binding with an antibody containing a VH region containing the sequence of SEQ ID NO: 9 and a VL region containing the sequence of SEQ ID NO: 65, and competes for tissue factor binding with the antibody , containing a VH region containing the sequence of SEQ ID NO: 37, and a VL region containing the sequence of SEQ ID NO: 93.

In the following embodiment, this antibody contains a VH CDR3 region having:

a) the sequence shown in SEQ ID NO: 32, SeQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 56, or

b) a variant of any of these sequences, such as a variant having at most 1, 2 or 3 amino acid modifications, preferably substitutions such as conservative substitutions.

In the following embodiment, this antibody contains:

a) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 30, 31 and 32 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 86, 87 and 88,

b) a VH region containing CDR1, 2 and 3 of SEQ ID NOs: 34, 35 and 36 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 90, 91 and 92,

c) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 38, 39 and 40 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 94, 95 and 96,

d) a VH region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 54, 55 and 56 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 110, 11 and 112, or

e) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in said sequences.

In the following embodiment, this antibody contains a VH having:

a) at least 80% identity, such as at least 90%, at least 95%, or at least 98 or 100% identity, with respect to a VH region sequence selected from the group consisting of SEQ ID NO: 29, 33, 37 and 53, or

b) at most 20, such as 15, or 10, or 5, 4, 3, 2, or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to a VH region sequence selected from the group consisting of SEQ ID NOs: 29, 33, 37 and 53.

In the following embodiment, this antibody contains a VL having:

a) at least 80% identity, such as at least 90%, at least 95%, or at least 98 or 100% identity, relative to the sequence of the VL region selected from the group consisting of SEQ ID NO: 85, 89, 93 and 109, or

d) at most 20, e.g. 15 or 10 or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions such as conservative amino acid substitutions, compared to a VH region sequence selected from the group consisting of from SEQ ID NOs: 85, 89, 93 and 109.

In the following embodiment, this antibody contains:

a) a VH region containing the sequence of SEQ ID NO: 29, and a VL region containing the sequence

- 12 041176 SEQ ID NO: 85,

b) a VH region containing the sequence of SEQ GO NO: 33 and a VL region containing the sequence of SEQ ID NO: 89,

c) a VH region containing the sequence of SEQ ID NO: 37 and a VL region containing the sequence of SEQ ID NO: 93,

d) a VH region containing the sequences of SEQ GO NO: 53 and a VL region containing the sequence of SEQ ID NO: 109,

e) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in said sequences.

In a further embodiment, the antibody comprises an antibody that competes for tissue factor binding with an antibody comprising a VH region containing the sequence of SEQ ID NO: 41 and a VL region containing the sequence of SEQ ID NO: 97.

In the following embodiment, this antibody contains a VH CDR3 region having:

a) the sequence shown in SEQ ID NO: 4, SEQ ID NO: 44, SEQ ID NO: 48, or

b) a variant of any of these sequences, such as a variant having at most 1, 2 or 3 amino acid modifications, preferably substitutions such as conservative substitutions.

In the following embodiment, this antibody contains:

a) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 2, 3 and 4 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 58, 59 and 60,

b) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 42, 43 and 44 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 98, 99 and 100,

c) a VH region containing the sequences CDR1, 2 and 3 of SEQ ID NOs: 46, 47 and 48 and a VL region containing the sequences of CDR1, 2 and 3 of SEQ ID NOs: 102, 103 and 104,

d) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2, or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in said sequences.

In the following embodiment, this antibody contains a VH having:

a) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity, relative to the sequence of the VH region selected from the group consisting of SEQ ID NO: 1 , 41 and 45, or

b) at most 20, such as 15, or 10, or 5, 4, 3, 2, or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to a VH region sequence selected from the group consisting of : SEQ ID NO: 1, 41 and 45.

In a further embodiment, the antibody comprises a VL having a) at least 80% identity, such as at least 90%, at least 95%, or at least 98 or 100% identity, relative to the VL region sequence selected from the group consisting of SEQ ID NOs: 57, 97 and 101, or

b) at most 20, e.g. 15 or 10 or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions such as conservative amino acid substitutions, compared to a VH region sequence selected from the group consisting of from SEQ ID NOs: 57, 97 and 101.

In the following embodiment, this antibody contains:

a) a VH region containing the sequence of SEQ ID NO: 1 and a VL region containing the sequence of SEQ ID NO: 57,

b) a VH region containing the sequence of SEQ ID NO: 41 and a VL region containing the sequence of SEQ ID NO: 97,

c) a VH region containing the sequence of SEQ ID NO: 45 and a VL region containing the sequence of SEQ ID NO: 101, or

d) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2, or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in said sequences.

In yet another embodiment, an antibody of this invention has an affinity for tissue factor that is less than 5 nM, eg less than 3.5 nM, eg less than 2 nM, as determined by the method described in Example 22 here.

A particularly interesting group of antibodies of this invention have tissue factor binding that is characterized by normal or high avidity and a high rate of dissociation (k _d ). As demonstrated here, such antibodies can exhibit tumor-specific binding in that they bind cancer tissue but do not, or less so, bind healthy tissue. Without wishing to be bound by any particular theory, the inventors hypothesize that this group of antibodies only binds well to cells that express high levels of TF, since this binding is only effective if it is divalent. Examples of these antibodies include antibody 044, 098 and 111 described here.

- 13 041176

Thus, in one embodiment, an antibody of this invention has a k _d greater than 10 ^-3 s ^-1 as determined by the affinity determination method described in Example 22 herein, and an avidity of less than 5 nM, such as less than 1 nM, such as less than 0.2 nM, when determined by the avidity determination method described in Example 22 here.

In another embodiment, an antibody of this invention has a kd greater than 10 ^-3 s ^-1 , as determined by the affinity determination method described in Example 22 here, and/or a k _a greater than 5x10 ⁴ , MoF-c, when determined by the affinity determination method described in example 22 here.

In the following embodiment, this antibody does not bind to healthy tissue, in particular, does not bind to human glomeruli (glomeruli), for example, when determined in the assay described in example 23, but does show binding to pancreatic tumors, for example, when determined in the assay described in Example 23 here.

In yet another embodiment, the antibody is effective in inhibiting the growth of established Bx-PC3 tumors as determined by the method described in Example 26 here.

In another embodiment, an antibody of this invention has one or more of the following properties: inhibition of tumor cells, inhibition of tumor angiogenesis, induction of apoptosis of tumor cells, binding to alternatively spliced tissue factor.

In a further embodiment, an antibody of this invention competes for tissue factor binding with an antibody comprising:

a) a VH region containing the sequence of SEQ ID NO: 9 and a VL region containing the sequence of SEQ ID NO: 65,

b) a VH region containing the sequence of SEQ ID NO: 1 and a VL region containing the sequence of SEQ ID NO: 57,

c) a VH region containing the sequence of SEQ ID NO: 5 and a VL region containing the sequence of SEQ ID NO: 61,

d) a VH region containing the sequence of SEQ GO N0:13 and a VL region containing the sequence of SEQ ID NO: 69,

e) a VH region containing the sequence of SEQ ID NO: 17 and a VL region containing the sequence of SEQ ID NO: 73,

f) a VH region containing the sequence of SEQ ID NO: 21 and a VL region containing the sequence of SEQ ID NO: 77,

g) a VL region containing the sequence of SEQ ID NO: 25 and a VL region containing the sequence of SEQ ID NO: 81,

h) a VL region containing the sequence of SEQ ID NO: 29 and a VL region containing the sequence of SEQ ID NO: 85,

i) a VH region containing the sequence of SEQ ID NO: 33 and a VL region containing the sequence of SEQ ID NO: 89,

j) a VH region containing the sequence of SEQ ID NO: 37 and a VL region containing the sequence of SEQ ID NO: 93,

k) a VH region containing the sequence of SEQ ID NO: 41 and a VL region containing the sequence of SEQ ID NO: 97,

l) a VH region containing the sequence of SEQ ID NO: 45 and a VL region containing the sequence of SEQ ID NO: 101,

m) a VH region containing the sequence of SEQ ID NO: 49 and a VL region containing the sequence of SEQ ID NO: 105, or

n) a VH region containing the sequence of SEQ ID NO: 53 and a VL region containing the sequence of SEQ ID NO: 109.

In a further embodiment, an antibody of this invention binds to the same epitope on tissue factor as an antibody having:

a) a VH region containing the sequence of SEQ ID NO: 9 and a VL region containing the sequence of SEQ ID NO: 65,

b) a VH region containing the sequence of SEQ ID NO: 1 and a VL region containing the sequence of SEQ ID NO: 57,

c) a VH region containing the sequence of SEQ ID NO: 5 and a VL region containing the sequence of SEQ ID NO: 61,

d) a VH region containing the sequence of SEQ ID NO: 13 and a VL region containing the sequence of SEQ ID NO: 69,

e) a VH region containing the sequence of SEQ ID NO: 17 and a VL region containing the sequence of SEQ ID NO: 73,

f) a VH region containing the sequence of SEQ ID NO: 21 and a VL region containing the sequence of SEQ ID NO: 77,

g) a VH region containing the sequence of SEQ ID NO: 25 and a VL region containing the sequence of SEQ ID NO: 81,

- 14 041176

h) a VH region containing the sequence of SEQ ID NO: 29 and a VL region containing the sequence of SEQ ID NO: 85,

i) a VH region containing the sequence of SEQ ID NO: 33 and a VL region containing the sequence of SEQ ID NO: 89,

j) a VH region containing the sequence of SEQ ID NO: 37 and a VL region containing the sequence of SEQ ID NO: 93,

k) a VL region containing the sequence of SEQ ID NO: 41 and a VL region containing the sequence of SEQ ID NO: 97,

l) a VH region containing the sequence of SEQ ID NO: 45 and a VL region containing the sequence of SEQ ID NO: 101,

m) a VH region containing the sequence of SEQ ID NO: 49 and a VL region containing the sequence of SEQ ID NO: 105, or

n) a VH region containing the sequence of SEQ ID NO: 53 and a VL region containing the sequence of SEQ ID NO: 109.

In a further embodiment, an antibody of this invention comprises a heavy chain variable region derived from a human germline VH sequence selected from the group consisting of IGHV1-18*01, IGHV3-23*01, IGHV3-30*01, IGHV3-33* 01, IGHV3-33*03, IGHV1-69*02, IGHV1-69*04 and IGHV5-51*01 and/or a light chain variable region derived from a human germline Vk sequence selected from the group consisting of IGKV3- 20*01, IGKV1-13*02, IGKV3-11*01 and IGKV1D-16*01.

In a further aspect, this invention relates to a monoclonal anti-TF antibody comprising a VH region having the sequence shown in SEQ ID NOs: 9, 1, 5, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, or 53, or a variant of any of these sequences, such as a variant having at most 25 amino acid modifications, such as 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7 , 6, 5, 4, 3, 2 or 1 amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions.

A variant of the sequence shown in SEQ ID NO: 9, 1, 5, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, or 53 may have at least 80% identity to any of the indicated sequences , such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 98% identity or 99% identity.

In one aspect of this invention, an isolated anti-TF monoclonal antibody comprises the VL sequence shown in SEQ ID NOs: 65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101, or 105, or a variant of any of said sequences, such as a variant having at most 25 amino acid modifications, e.g. 20, e.g. at most 15, 14, 13, 12 or 11 amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions.

A variant of the sequence shown in SEQ ID NOs: 65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101 or 105 may have at least 80% identity to any of the indicated sequences, for example, at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity.

In another embodiment, this antibody contains:

a) a VL region having a sequence selected from the group consisting of SEQ ID NOs: 65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101 or 105, and a VH region, having a sequence selected from the group consisting of SEQ ID NO: 9, 1, 5, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, or 53,

b) a variant of any of the above sequences, wherein said variant preferably has only conservative substitutions in said sequences.

In one preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 65 and a VH region having the sequence shown in SEQ ID NO: 9, or a variant of either of these two sequences, which variants have or

a) at most 25 amino acid modifications, for example 20, for example at most 15, 14, 13, 12 or 11 amino acid modifications, for example 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the amino acid modifications, such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 9 or SEQ ID NO: 65, respectively, such as at least 85% identity or 90% identity or 95% identity, e.g. 96% identity or 97% identity or 98% identity identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 57 and a VH region having the sequence shown in SEQ ID NO: 1, or a variant of either of these two sequences, wherein these options are either

- 15 041176 (a) at most 25 amino acid modifications, for example 20, for example at most 15, 14,

13, 12 or 11 amino acid modifications, for example 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 57, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 61 and a VH region having the sequence shown in SEQ ID NO: 5, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, such as 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 5 or SEQ ID NO: 61, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 69 and a VH region having the sequence shown in SEQ ID NO: 13, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, such as 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 13 or SEQ ID NO: 61, respectively, such as at least 85% identity or 90% identity or 95% identity, e.g. 96% identity or 97% identity or 98% identity identity or 99% identity.

In another preferred embodiment, this antibody contains a VL region having the sequence shown in SEQ ID NO: 73 and a VH region having the sequence shown in SEQ ID NO: 17, or a variant of either of these two sequences, and these variants have either (a) at most 25 amino acid modifications, e.g. 20, e.g. at most 15, 14, 13, 12 or 11 amino acid modifications, e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 17 or SEQ ID NO: 73, respectively, such as at least 85% identity or 90% identity or 95% identity, e.g. 96% identity or 97% identity or 98% identity identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 77 and a VH region having the sequence shown in SEQ ID NO: 21, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, such as 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 21 or SEQ ID NO: 77, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 81 and a VH region having the sequence shown in SEQ ID NO: 25, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, e.g. 20, e.g. at most 15, 14, 13, 12 or 11 amino acid modifications, e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 25 or SEQ ID NO: 81, respectively, such as at least 85% identity or 90% identity or 95% identity, e.g. 96% identity or 97% identity or 98% identity identity or 99% identity.

- 16 041176

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 85 and a VH region having the sequence shown in SEQ ID NO: 29, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, e.g. 20, e.g. at most 15, 14, 13, 12 or 11 amino acid modifications, e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 29 or SEQ ID NO: 85, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98 % identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 89 and a VH region having the sequence shown in SEQ ID NO: 33, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, such as 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 33 or SEQ ID NO: 89, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98 % identity or 99% identity.

In another preferred embodiment, this antibody contains a VL region having the sequence shown in SEQ ID NO: 93 and a VH region having the sequence shown in SEQ ID NO: 37, or a variant of either of these two sequences, and these variants have either (a) at most 25 amino acid modifications, such as 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 37 or SEQ ID NO: 93, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 97 and a VH region having the sequence shown in SEQ ID NO: 41, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, e.g. 20, e.g. at most 15, 14, 13, 12 or 11 amino acid modifications, e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 41 or SEQ ID NO: 97, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 101 and a VH region having the sequence shown in SEQ ID NO: 45, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, such as 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 45 or SEQ ID NO: 101, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 105 and a VH region having the sequence shown in SEQ ID NO: 49, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, such as 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative

- 17 041176 exchange, or

b) at least 80% identity to SEQ ID NO: 49 or SEQ ID NO: 105, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity.

In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID NO: 109 and a VH region having the sequence shown in SEQ ID NO: 53, or a variant of either of these two sequences, which variants have either (a) at most 25 amino acid modifications, such as 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or

b) at least 80% identity to SEQ ID NO: 53 or SEQ ID NO: 109, respectively, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity.

The monoclonal antibodies of the present invention may, for example, be prepared by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or may be prepared by recombinant DNA methods. Monoclonal antibodies can also be isolated from antibody phage libraries using the methods described in, for example, Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. For example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B cells obtained from mice immunized with an antigen of interest, for example, in the form of cells expressing this antigen on the surface, or a nucleic acid encoding the antigen of interest. Monoclonal antibodies can also be prepared from hydridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, rabbits, dogs, primates, and the like.

In one embodiment, the antibody of this invention is a human antibody. Human monoclonal antibodies directed against tissue factor can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse immune system. Such transgenic and transchromosomal mice are referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as transgenic mice.

The HuMAb mouse contains miniloci of the human immunoglobulin gene that encodes rearranged sequences of the variable and constant regions (μ and γ) of the heavy chain and the variable and constant chains (k) of the human immunoglobulin light chain, together with targeted mutations that inactivate the endogenous loci of the μ and k chains. (Lonberg, N. et al., Nature 368, 856-859 (1994)). Thus, these mice show reduced expression of mouse IgM or K and, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG, K monoclonal antibodies (Lonberg, N. et al. ( 1994), supra discussed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N. Ann. N. Y. Acad. Sci 764 536-546 (1995)). The production of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al. , J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397 , WO 92/22645, WO 92/03918 and WO 01/09187.

HCo7 mice have JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), CMD disruption in their endogenous heavy chain genes (as described in Example 1 WO 01/14424), the human KCo5 kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and the human HCo7 heavy chain transgene (as described in US 5,770,429).

HCo12 mice have JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), CMD disruption in their endogenous heavy chain genes (as described in Example 1 WO 01/14424), the human KCo5 kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and the human HCo7 heavy chain transgene (as described in US 5,770,429) and the heavy chain transgene human HCo7 (as described in Example 2 of WO 01/14424).

In the KM mouse line, the endogenous mouse kappa light chain gene was destroyed homozygous as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene was destroyed homozygous as described in Example 1 of WO 01/ 09187. This mouse line carries the human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse line also carries a human heavy chain transchromosome consisting of the hCF fragment of chromosome 14 (SC20) as described in WO 02/43478.

Splenocytes from these transgenic mice can be used to secrete human monoclonal antibodies according to well known methods. Human monoclonal or polyclonal antibodies of the present invention, or antibodies of the present invention derived from other species, can also be generated transgenically by generating another mammal (non-human) or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest, and making it antibodies from them in a recoverable form. In connection with transgenic production in mammals, antibodies can be produced in the milk of goats, cows or other mammals and extracted from them. See, for example, US 5827690, US 5756687, US 5750172 and US 5741957.

In addition, human antibodies of the present invention or antibodies of the present invention from other species can be generated using display-type technologies including, but not limited to, phage display, retroviral display, ribosomal display, and other methods using methods well known in the art. region, and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such methods are well known in the art (see, e.g., Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosome display), Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. Acids Research 21, 1081-1085 ( 1993), Hogenboom et al., Immunol Reviews 130, 43-68 (1992), Chiswell and McCafferty TI BTECH 10, 80-84 (1992), and US 5,733,743). If display technologies are used to generate antibodies that are not human antibodies, such antibodies can be humanized.

The antibody of this invention may be an antibody of any isotype. Isotype selection will generally be guided by desired effector functions such as ADCC induction. Examples of isotypes are IgG1, IgG2, IgG3 and IgG4. Human light chain constant regions, kappa or lambda can be used. If desired, the class of the anti-TF antibody of the present invention can be switched by known methods. For example, an antibody of the invention that is originally an IgM may be class-switched to an IgG antibody of the invention. Further, class switching techniques can be used to convert one IgG subclass to another, for example from IgG1 to IgG2. Thus, the effector function of the antibodies of the present invention can be changed by isotype switching, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibody for various therapeutic applications. In one embodiment, an antibody of this invention is an Ig1 antibody, eg IgG1,K.

In one embodiment, the antibody of this invention is a full length antibody, preferably an IgG1 antibody, in particular an IgG1,κ antibody. In another embodiment, an antibody of this invention is an antibody fragment or a single chain antibody.

Antibody fragments can, for example, be obtained by fragmentation using conventional methods, and these fragments are screened for applicability in the same manner as described here for whole antibodies. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be processed to reduce disulfide bridges to give Fab' fragments. Fab fragments can be obtained by treating an IgG antibody with papain; Fab' fragments can be obtained by pepsin digestion of an IgG antibody. The F(ab') fragment can also be obtained by linking the Fab', described below, via a thioether bond or a disulfide bond. A Fab' fragment is an antibody fragment obtained by cutting the disulfide bond of the F(ab') ₂ hinge region. The Fab' fragment can also be obtained by treating the F(ab')2 fragment with a reducing agent such as dithiothreitol. Antibody fragments can also be generated by expression of nucleic acids encoding such fragments in recombinant cells (see, for example, Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimeric gene encoding part of the F(ab')2 fragment could include DNA sequences encoding a CH1 domain and an H chain hinge region followed by a translation stop codon to produce such a truncated antibody fragment molecule.

In one embodiment, the anti-TF antibody is a monovalent antibody, preferably the monovalent antibody described in WO 2007059782 (Genmab) (incorporated here by reference) having a deletion of the hinge region. Thus, in one embodiment, the antibody is monovalent, wherein said anti-TF antibody is constructed in a manner comprising:

i) providing a nucleic acid construct encoding a light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen-specific anti-TF antibody and a nucleotide sequence

- 19 041176 a entity encoding an Ig constant CL region, wherein said nucleotide sequence encoding the VL region of the selected antigen-specific antibody and said nucleotide sequence encoding the Ig CL region are operably linked together, and where, in the case of the IgG1 subtype, the nucleotide sequence , encoding a CL region, has been modified such that the CL region does not contain amino acids capable of forming disulfide bonds or covalent bonds with other peptides containing the identical amino acid sequence of the CL region, in the presence of polyclonal human IgG or when administered to an animal or human ;

ii) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VH region of the selected antigen-specific anti-TF antibody and a nucleotide sequence encoding the Ig constant CH region, wherein said nucleotide sequence encoding this CH region has been modified so that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of this CH region, such as the CH3 region, do not contain any amino acid residues that are involved in the formation of disulfide bonds or covalent or stable non-covalent heavy chain linkages with other peptides containing the identical amino acid sequence of the human Ig CH region, in the presence of a human polyclonal IgG or when administered to an animal or human, said nucleotide sequence encoding the VH region of the choice said antigen-specific antibody and said nucleotide sequence encoding the CH region of said Ig are operably linked together;

iii) providing a cell expression system for obtaining said monovalent antibody;

iv) providing said monovalent antibody by co-expression of nucleic acid constructs (i) and (ii) in cells of this cell expression system (iii).

Similarly, in one embodiment, the anti-TF antibody is a monovalent antibody that contains:

(i) the variable region of an antibody of this invention as described herein, or an antigen-binding portion of said region, and (ii) a C _H region of an immunoglobulin or fragment thereof, comprising the C _H 2 and C _H 3 regions where the CH region or fragment thereof was modified in such a way that the region corresponding to the hinge region, and if this immunoglobulin is not an immunoglobulin of the IgG4 subtype, other regions of this CH region, such as the C _H 3 region, do not contain any amino acid residues that are capable of forming disulfide bonds with an identical CH region or other covalent or stable non-covalent intermediate heavy chains with an identical CH region in the presence of human polyclonal IgG.

In a further embodiment, the heavy chain of a monovalent anti-TF antibody has been modified such that the entire hinge region has been deleted.

In a further embodiment, said monovalent antibody is an antibody of the IgG4 subtype (see SEQ ID NO: 114, hinge-free variant of SEQ ID NO: 113), but the C _H 3 region has been modified such that one or more of the following amino acid substitutions have been made : Thr (T) at position 234 has been replaced by Ala (A); Leu (L) at position 236 was replaced by Ala (A); Leu (L) at position 236 was replaced by Val (V); Phe (F) at position 273 has been replaced by Ala (A); Phe (F) at position 273 has been replaced by Leu (L); Tyr (Y) at position 275 has been replaced by Ala (A).

In another additional embodiment, the sequence of said monovalent antibody has been modified such that it does not contain any N-linked glycosylation acceptor sites.

The anti-TF antibodies of this invention also include single chain antibodies. Single chain antibodies are peptides in which the heavy and light chain Fv regions are linked. In one embodiment, the invention provides a single chain Fv (scFv) wherein the heavy and light chains in the Fv of the anti-TF antibodies of this invention are connected by a flexible peptide linker (typically about 10, 12, 15 or more amino acid residues) into a single peptide chain. Methods for making such antibodies are described, for example, in US 4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science 242, 423-426 (1988), Huston et al., PNAS USA 85, 5879-5883 (1988) and McCafferty et al., Nature 348, 552-554 ( 1990). This single chain antibody can be monovalent if only a single VH and VL is used, bivalent if two VH and VL are used, or polyvalent if more than two VH and VL are used.

In one embodiment, the anti-TF antibody of this invention is deficient in terms of effector function of the antibody. Such antibodies are particularly useful when using this antibody in stimulating the immune system through blocking the inhibitory effects of TF. For such applications, it may be advantageous that the antibody has no effector functions.

- 20 041176 ions, such as ADCC, as they can lead to undesirable cytotoxicity.

In one embodiment, the effector-deficient anti-TF antibody is a stabilized IgG4 antibody. Examples of suitable stabilized IgG4 antibodies are antibodies in which the arginine at position 409 in the human heavy chain constant region of IgG4, which is listed in the EU index as in Kabat et al., is replaced by a lysine, threonine, methionine or leucine, preferably lysine (as described in WO 2006033386 (Kirin)), and/or in which the hinge region contains the sequence Cys-Pro-Pro-Cys.

In a further embodiment, the stabilized IgG4 anti-TF antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of Lys, Ala, Thr, Met and Leu at position 409 and/or a residue selected from the group consisting of Ala, Val, Gly, Ile and Leu at position 405, and wherein said antibody optionally contains one or more substitutions, deletions and/ or insertions, but does not contain the Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said antibody contains a Lys or Ala residue at position corresponding to 409, or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1 or human IgG3.

In another further embodiment, the stabilized IgG4 anti-TF antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of Lys, Ala, Thr, Met and Leu at position 409 and/or a residue selected from the group consisting of Ala, Val, Gly, Ile and Leu at position 405, and wherein said antibody optionally contains one or more substitutions, deletions, and /or insertions and where the specified antibody contains the sequence of Cys-Pro-Pro-Cys in the hinge region. Preferably, said antibody contains a Lys or Ala residue at position corresponding to 409, or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1, human IgG2, or human IgG3.

In a further embodiment, the effector-deficient anti-TF antibody is a He-IgG4-type antibody, such as IgG1, IgG2, or IgG3, that has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or eliminated. Such mutations have been, for example, described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001).

In a further embodiment, an antibody of this invention is conjugated to another moiety, such as a cytotoxic moiety, a radioisotope, or a drug. Such antibodies can be obtained by chemical conjugation of another particle with the N-terminal side or C-terminal side of an anti-TF antibody or fragment thereof (for example, the H chain, L chain of an anti-TF antibody or its anti-TF-specific/ selective fragment) (see, for example, Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated antibody derivatives may also be generated by conjugation of internal residues or sugars, if desired.

Generally, the anti-TF antibodies described herein may be modified by incorporating any suitable number of such modified amino acids and/or association with such conjugated substituents. Suitability in this context is generally defined by the ability to at least substantially retain the TF selectivity and/or TF specificity associated with the underivatized parental anti-TF antibody. The inclusion of one or more amino acids may be beneficial, for example, in increasing the serum half-life of the polypeptide, decreasing the antigenicity of the polypeptide, or increasing the storage stability of the polypeptide. Amino acids are modified, for example, co-translationally or post-translationally during recombinant production (eg, N-linked glycosylation in N-X-S/T motifs during expression in mammalian cells), or modified synthetically. Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenylated amino acid (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEGylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like. References sufficient to guide the skilled artisan in amino acid modification are provided in abundance in the literature. Exemplary protocols can be found in Walker (1998) Protein Protocols On Cd-Rom, Humana Press, Towata, NJ. The modified amino acid may, for example, be selected from a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatized agent.

Anti-TF antibodies can also be chemically modified by covalent conjugation with a polymer, for example, to increase their circulating half-life. Exemplary polymers and methods for attaching them to peptides are illustrated in, for example, US 4,766,106, US 4,179,337, US 4,495,285, and US 4,609,546. 1000 is about 40000, such as about 2000 - about 20000, for example about 3000-12000 g/mol).

In one embodiment, the invention provides an anti-TF antibody that is conjugated to a second molecule that is selected from a radionuclide, an enzyme, an enzyme substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide label, or a magnetic particle. In one embodiment, an anti-TF antibody may be conjugated to one or more antibody fragments, nucleic acids (oligonucleotides), nucleases, hormones, immunomodulators, chelators, boron compounds, photoactive agents, dyes, and the like. These and other suitable agents may be associated directly or indirectly with the anti-TF antibody of the present invention. One example of indirect second agent binding is spacer particle binding. These spacers can in turn be insoluble and soluble (see, for example, Diener et al., Science 231, 148 (1986)) and can be selected to facilitate drug release from the anti-TF antibody at the target site. and/or under specific conditions. Additional examples of agents that can be associated with an anti-TF antibody include lectins and fluorescent peptides.

In one embodiment, anti-TF antibodies are provided that contain one or more radiolabeled amino acids. A radiolabeled anti-TF antibody can be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature). Non-limiting examples of labels for polypeptides include, but are not limited to, ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc and ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re. Methods for making radiolabeled amino acids and related peptide derivatives are known in the art (see, for example, Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581; US 4,735,210; US 5,101,827; US 5,102,990 (US RE 35,500);

In one embodiment, the anti-TF antibody of this invention comprises a conjugated nucleic acid or a nucleic acid associated molecule. In one such aspect of the invention, the conjugated nucleic acid is a cytotoxic ribonuclease. In one embodiment, the conjugated nucleic acid is an antisense nucleic acid (e.g., an S10010-targeted antisense molecule, which may also be an independent component in the combination composition or combination route of administration of the present invention - see, e.g., Zhang et al., J Biol Chem 279(3), 2053-62 (2004)). In one embodiment, the conjugated nucleic acid is an inhibitory RNA molecule (eg, a siRNA molecule). In one embodiment, the conjugated nucleic acid is an immunostimulatory nucleic acid (eg, an immunostimulatory CpG motif-containing DNA molecule). In one embodiment, the conjugated nucleic acid is an expression cassette encoding the expression of a tumor suppressor gene, a cancer vaccine, a cancer cytokine, or an apoptotic agent. Such derivatives may also contain a conjugation of a nucleic acid encoding the expression of one or more cytotoxic proteins such as plant toxins and bacterial toxins.

In one embodiment, the anti-TF antibody is conjugated to a functional nucleic acid molecule. Functional nucleic acid molecules include antisense molecules, interfering nucleic acid molecules (eg, siRNA molecules), aptamers, ribozymes, triplex-forming molecules, and outer guide sequences. These functional nucleic acid molecules may act as effectors, inhibitors, modulators, and promoters of the specific activity possessed by the target molecule, or the functional nucleic acid molecules may have de novo activity independent of any other molecules.

In another embodiment, an anti-TF antibody of this invention is conjugated to an aptamer.

In another embodiment, the present invention provides an anti-TF antibody that is conjugated to a ribozyme.

Any method known in the art can be used to conjugate an anti-TF antibody to conjugated molecule(s) such as those described above, including those described by Hunter et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982). Numerous types of cytotoxic compounds can be attached to proteins using a reactive group on the cytotoxic compound, or using a crosslinker. A common reactive group that will form a stable covalent bond in vivo with an amine is isothiocyanate (Means et al., Chemical modifications of proteins (Holden-Day, San Francisco 1971) pp. 105-110). This group preferentially interacts with the ε- 22 041176 amine group of lysine. Maleimide is a commonly used reactive group to form a stable in vivo covalent bond with a sulfhydryl group on cysteine (Ji., Methods Enzymol 91, 580-609 (1983)). Monoclonal antibodies are usually not capable of forming covalent bonds with radioactive metal ions, but they can be attached to an antibody directly using chelating agents that are covalently linked to these antibodies. Chelating agents can be attached through amine (Meares et al., Anal. Biochem. 142, 68-78 (1984)) and sulfhydryl groups (Koyama, Chem. Abstr. 120, 217262t (1994)) amino acid residues, as well as through carbohydrate groups (Rodwell et al., PNAS USA 83, 2632-2636 (1986), Quadri et al., Nucl. Med. Biol. 20, 559-570 (1993)). Because these chelating agents contain two types of functional groups, one for binding metal ions and another for attaching a chelate to an antibody, they are commonly referred to as bifunctional chelating agents (Sundberg et al., Nature 250, 587588 (1974)).

In one embodiment, the invention provides an anti-TF antibody, such as a human anti-TF antibody, conjugated to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. Such conjugates are referred to herein as immunoconjugates. Immunoconjugates that include one or more cytotoxins are called immunotoxins.

A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells (eg, kills cells). For a description of these classes of drugs that are well known in the art and their mechanisms of action, see Goodman et al., Goodman and Gilman's The Pharmacological Basis Of Therapeutics, ^8th Ed., Macmillan Publishing Co., 1990. relating to the preparation of antibody immunotoxins are provided, for example, in Vitetta, Immunol. Today 14, 252 (1993) and US 5194594.

Suitable therapeutic agents for the formation of the immunoconjugates of this invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1- dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannite, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin), antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mitramycin in, mitomycin, mitoxandrone, plicamimin, anthramycin (AMC)), diphtheria toxin and related molecules (such as diphtheria toxin A chain and active fragments thereof, and hybrid molecules), ricin toxin (such as ricin A or deglycosylated ricin A chain toxin ), cholera toxin, Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT-toxin, C3-toxin, Shiga-toxin, pertussis toxin, tetanus toxin, Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modecin, gelanin, abrin A-chain, modeccin achain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria inhibitor officinalis, gelonin, mitgellin, restrictocin, fenomycin and enomycin toxins. Other suitable conjugated molecules include ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, American phytolacca antiviral protein, diphtheria toxin, and Pseudomonas endotoxin endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that can be administered in combination with an anti-TF antibody of this invention, described elsewhere herein, may also be candidates for therapeutic particles useful for conjugation with an anti-TF antibody of this invention.

In one embodiment, an anti-TF antibody of the present invention is attached to a linker chelator, such as tiuxetane, which allows the antibody to be conjugated to a radioactive isotope.

In a further aspect, this invention relates to a bispecific molecule comprising an anti-TF antibody of this invention as described above and a second binding specificity, such as binding specificity for a human effector cell, human Fc receptor, or T cell receptor, or binding specificity for another TF epitope.

The bispecific molecules of the present invention may further include a third binding specificity, in addition to anti-TF binding specificity and binding specificity for human effector cell, human Fc receptor or T cell receptor.

Exemplary bispecific antibody molecules of this invention comprise:

(i) two antibodies, one with specificity for TF and the other with specificity for a second target, which are conjugated together,

- 23 041176 (ii) a single antibody that has one chain specific for TF and a second chain specific for the second molecule, and (iii) a single chain antibody that has specificity for TF and the second molecule.

Typically, this second target/second molecule is a molecule other than TF. In one embodiment, this second molecule is a cancer antigen/tumor associated antigen such as carcinoembryonic antigen (CEA), prostate specific antigen (PSA), RAGE (kidney antigen), α-fetoprotein, CAMEL (CTL-recognizable antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3 and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE and SAGE), mucin antigens (e.g. , MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75, Cmyc, Marti, MelanA, MUM-I, MUM-2, MUM-3, HLA-B7 and Ep-CAM. In one embodiment, this second molecule is a cancer-associated integrin, such as the α5β3 integrin. In one embodiment, this second molecule is an angiogenic factor or other cancer-associated growth factor such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), angiogenin and its receptors, in particular receptors associated with cancer progression (eg one of the HER1-HER4 receptors, c-met or RON). Other cancer progression-associated proteins discussed herein may also be suitable second molecules.

In one embodiment, the bispecific antibody of the present invention is a diabody. Bispecific antibodies also include crosslinked or heteroconjugate antibodies. For example, one of the antibodies in a heteroconjugate can be linked to avidin and the other to biotin. Such antibodies have, for example, been proposed for targeting cells of the immune system to unwanted cells (see, for example, US 4676980). Heteroconjugate antibodies can be produced using any suitable cross-linking methods.

In a further aspect, this invention relates to an expression vector encoding an antibody of this invention.

In one embodiment, the expression vector of this invention contains a nucleotide sequence encoding one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 1-112.

In another specific embodiment, the expression vector of this invention contains a nucleotide sequence encoding one or more VH amino acid sequences selected from the group consisting of SEQ ID NO: 9, 1, 5, 13, 17, 21, 25, 29, 33, 37 , 41, 45, 49 and 53.

In one specific embodiment, the expression vector of this invention contains a nucleotide sequence encoding one or more VH CDR3 amino acid sequences selected from the group consisting of SEQ ID NOS 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 , 44, 48, 52 and 56.

In another specific embodiment, the expression vector of this invention contains a nucleotide sequence encoding one or more VL amino acid sequences selected from the group consisting of SEQ ID NOs: 65, 57, 61, 69, 73, 77, 81, 85, 89, 93 , 97, 101 and 105.

In another embodiment, the expression vector of this invention contains a nucleotide sequence encoding one or more VL CDR3 amino acid sequences selected from the group consisting of SEQ ID NO: 60, 64, 68, 72, 76, 80, 84, 88, 92, 96 , 100, 104 and 108.

In one specific embodiment, the expression vector of this invention comprises a nucleotide sequence encoding variants of one or more of the above amino acid sequences, said variants having at most 25 amino acid modifications, e.g. 20, e.g. at most 15, 14, 13, 12, or 11 amino acid modifications. modifications, e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acid modifications such as deletions or insertions, preferably substitutions such as conservative substitutions, or at least 80% identity to any of specified sequences, such as at least 85% identity or 90% identity or 95% identity, for example, 96% identity or 97% identity or 98% identity or 99% identity of any of the above amino acid sequences.

In a further embodiment, the expression vector contains a nucleotide sequence encoding a light chain, heavy chain, or both light and heavy chain constant region of an antibody, eg, a human antibody.

Such expression vectors can be used to recombinantly produce the antibodies of this invention.

An expression vector in the context of this invention may be any suitable vector, including chromosomal, non-chromosomal, and containing a synthetic nucleic acid.

- 24 041176 vectors (nucleic acid sequence containing a suitable set of expression regulatory elements). Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the anti-TF antibody is contained in a naked DNA or RNA vector, including, for example, a linear expression element (as described, for example, Sykes and Johnston, Nat Biotech 12, 355-59 (1997) ), a compact nucleic acid vector (described, for example, in US 6077835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18 or pUC 118/119, a midge nucleic acid vector of minimal size ( described, e.g., Schakowski et al., Mol Ther 3, 793-800 (2001)), or a precipitated nucleic acid vector construct such as a CaP04 precipitated construct (e.g., described in WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 2, 603 (1981)). Such nucleic acid vectors and their use are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

In one embodiment, this vector is suitable for the expression of an anti-TF antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989), pET vectors (Novagen, Madison WI), etc.) .

The expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, those containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (review: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

The nucleic acid and/or vector may also contain a nucleic acid sequence encoding a secretion/localization sequence that can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space, or to the cell culture medium. Such sequences are known in the art and include secretion leader or signal sequence peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transport sequences, chloroplast transport sequences), membrane localization/anchor sequences (e.g. , stop sequences, anchor GPI sequences), etc.

In the expression vector of this invention, the anti-TF antibody encoding nucleic acids may contain any suitable promoter, enhancer and other expression facilitating elements or be associated with any suitable promoter, enhancer and other expression facilitating elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer, as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), efficient poly(A) termination sequences, an origin of replication for a plasmid product in E. coli, an antibiotic resistance gene as a selectable marker and/or a convenient cloning site (eg a polylinker). Nucleic acids may also contain an inducible promoter as opposed to a constitutive promoter such as CMV IE (one of skill in the art will appreciate that such terms are actually descriptors for the degree of expression of a gene under certain conditions).

In one embodiment, an expression vector encoding an anti-TF antibody can be placed in a host cell or host animal and/or delivered to the host cell or host animal via a viral vector.

In another additional aspect, this invention relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody of this invention, defined here, or to a bispecific molecule of this invention, defined here. Examples of host cells include yeast cells, bacterial cells, and mammalian cells such as CHO or HEK cells. For example, in one embodiment, the invention provides a cell comprising a nucleic acid stably integrated into the cellular genome that contains a sequence encoding the expression of an anti-TF antibody of the invention. In another embodiment, this invention provides a cell containing a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which contains a sequence encoding an anti-TF antibody of this invention.

In a further aspect, this invention relates to a hybridoma that produces an antibody of this invention as defined herein. In yet another aspect, this invention relates to a transgenic (non-human) animal containing nucleic acids encoding a human heavy chain and a human light chain, wherein the animal or plant produces an antibody of the invention. The generation of such hybridomas and transgenic animals has been described above.

In the following aspect, this invention relates to a method for producing an anti-TF antibody of this invention, comprising the steps:

a) culturing the hybridoma or host cell of this invention as described above, and

b) purifying the antibodies of this invention from the culture media.

In a further main aspect, this invention relates to an anti-TF antibody as defined herein, or a bispecific molecule as defined herein, for use as a drug.

In yet another aspect, this invention relates to a pharmaceutical composition comprising an anti-TF antibody as defined herein, or a bespecifically molecule as defined herein, and a pharmaceutically acceptable carrier.

This pharmaceutical composition may be formulated with pharmaceutically acceptable carriers or diluents, as well as any known adjuvants and excipients, in accordance with conventional methods, such as those described in Remington The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.

These pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, should be appropriate for the chosen compound of this invention and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the absence of a significant adverse effect on the desired biological properties of the selected compound or pharmaceutical composition of this invention (for example, less than a significant effect (10% or less relative inhibition, 5% or less relative inhibition, and etc.)

The pharmaceutical composition of this invention may also include diluents, excipients, salts, buffers, detergents (for example, a non-ionic detergent such as Tween-20 or Tween40), stabilizers (for example, sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers and/or other materials suitable for inclusion in the pharmaceutical composition.

In cancer cells such as human colorectal cancer cells, TF expression has been reported to be under the control of 2 major neoplastic degenerative events triggering disease progression (activation of the K-ras oncogene and inactivation of the tumor suppressor p53, in a MEK/mitogen-activated protein kinase-dependent manner). (MAPK) and phosphatidylinositol 3-kinase (PI3K) (Yu et al. (2005) Blood 105: 1734.

Cancer cells overexpressing TF may be particularly good targets for the anti-TF antibody of this invention, as more antibodies per cell can be bound. Thus, in one embodiment, the cancer patient to be treated with an anti-TF antibody of this invention is a patient, for example, having pancreatic lacquer, lung cancer, or colorectal cancer, who has been diagnosed as having one or more mutations in K-Ras and/ or one or more mutations in p53 in their tumor cells.

In an alternative embodiment, the patient to be treated with the anti-TF antibodies of this invention is a patient, for example, with pancreatic lacquer, lung cancer, or colorectal cancer, which does not have a mutation in K-Ras. Without being bound by any particular theory, the present inventors consider it possible that certain tumor cells having K-Ras activation are less responsive to anti-TF antibody treatment, since the effects of anti-TF antibodies on intracellular signaling mechanisms may be less effective in cells in which K-Ras is activated.

Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may vary so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, particular composition and administration regimen, without causing toxicity to that patient. The selected dose level will depend on various pharmacokinetic factors, including the activity of the specific compositions of this invention used, or its amide, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and/or materials used. in combination with the specific compositions used, age, sex, weight, condition, general health and previous medical history of the patient to be treated, and similar factors well known in the medical field.

This pharmaceutical composition may be administered using any suitable method and regimen. Suitable methods of administering a compound of this invention in vivo and in vitro are well known in the art and can be selected by one of ordinary skill in the art.

In one embodiment, the pharmaceutical composition of this invention is administered parenterally.

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The phrases parenteral administration and parenteral administration as used herein means methods other than enteral (intestinal) and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like, which are physiologically compatible with the compound of this invention.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of this invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and their suitable mixtures, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil and sesame oil, colloidal solutions of carboxymethyl cellulose, gum tragacanth and injectable organic esters such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except where any conventional medium or agent is incompatible with this active compound, their use in the pharmaceutical compositions of the present invention is contemplated.

The required fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

The pharmaceutical compositions of this invention may also contain pharmaceutically acceptable antioxidants, for example (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediamine tretaacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The pharmaceutical compositions of this invention may also contain isotonicity agents such as sugars, polyalcohols such as mannitol, sorbitol, glycerin, or sodium chloride in these compositions.

The pharmaceutical compositions of this invention may also contain one or more adjuvants suitable for the chosen route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may increase the shelf life or efficacy of the pharmaceutical composition. The compounds of this invention may be formulated with carriers that will protect the compound against rapid release, such as controlled release forms including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or with wax, or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of this invention can be formulated to ensure correct distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except where any conventional medium or agent is incompatible with this active compound, their use in the pharmaceutical compositions of the present invention is contemplated. Additional active compounds may also be included in these compositions.

Pharmaceutical compositions for injection should generally be sterile and stable under the conditions of preparation and storage. This composition can be prepared as a solution, micro

- 27 041176 emulsion, liposomes or other ordered structure suitable for high drug concentration. This carrier may be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The required fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. In many cases it will be preferred to include isotonic agents, eg sugars, polyalcohols such as glycerol, mannitol, sorbi, or sodium chloride, in this composition. Prolonged absorption of injectable compositions can be caused by the inclusion in this composition of an agent that delays absorption, for example, monostearate salts, and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one ingredient or combination of ingredients, such as those listed above, if necessary, followed by microfiltration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the base dispersion medium and the required other ingredients, for example from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum drying (lyophilization) which yields a powder of the active ingredient plus any additional active ingredient from its previously sterile filtered solution.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with a single ingredient or combination of ingredients such as those listed above, if necessary, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the base dispersion medium and the required other ingredients, for example from the ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum drying (lyophilization) which yields a powder of the active ingredient plus any additional active ingredient from its pre-sterile filtered solution.

The pharmaceutical composition of this invention may contain one compound of this invention or a combination of compounds of this invention.

As described above, in another aspect, this invention relates to an antibody of this invention, as defined herein, or a bispecific molecule of this invention, as defined herein, for use as a drug.

The anti-TF antibodies of this invention can be used for a number of purposes. In particular, the antibodies of this invention can be used to treat various forms of cancer. In one aspect, the anti-TF monoclonal antibodies of this invention are used to treat various types of solid cancer such as tumors of the central nervous system, head and neck cancer, lung cancer (such as non-small cell lung cancer), breast cancer, esophageal cancer, gastric cancer. , liver and biliary cancer, pancreatic cancer, colorectal cancer, bladder cancer, kidney cancer, prostate cancer, endometrial cancer, ovarian cancer, malignant melanoma, sarcoma (soft tissue eg. bones and muscles), tumors of unknown primary origin (t ie unknown primary causes), leukemia, bone marrow cancer (such as multiple myeloma), acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin's lymphoma, skin cancer, glioma, cancer of the brain, uterus and rectum. In addition, autoimmune inflammation such as myopathies or multiple sclerosis may be targeted by the anti-TF monoclonal antibodies of the present invention.

Monoclonal anti-TF antibodies of the present invention may also be useful in the treatment of hemostasis.

Cancer-related hemostatic disorders may also be a target of this invention.

In addition, diseases with inflammation such as myopathies, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, spondylarthropathies, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy, reactive arthropathy, infectious or post-infectious arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, glomerulonephritis, end-stage renal disease, cystic fibrosis, chronic obstructive pulmonary disease (COPD), asthma, allergic asthma, bronchitis, acute bronchiolitis, chronic bronchiolitis, idiopathic pulmonary fibrosis, or multiple sclerosis may be targeted by monoclonal anti-TF -antibodies of the present invention.

Monoclonal anti-TF antibodies of the present invention may also be useful in the treatment of hemostasis.

Cancer-associated hemostatic disorders may also be a target for this invention.

Vascular diseases such as vascular restenosis, myocardial vascular disease, cerebral vascular disease, retinopathy, and macular degeneration, including but not limited to wet AMD, can be treated with monoclonal anti-TF antibodies.

The anti-TF monoclonal antibodies of the present invention may also be useful in the treatment of patients with cardiovascular risk such as atherosclerosis, hypertension, diabetes, dyslipidemia and acute coronary syndrome, including but not limited to acute myocardial infarction, stroke.

The anti-TF monoclonal antibodies of the present invention may also be useful for inhibiting thrombosis such as DVT (deep vein thrombosis), renal embolism, pulmonary embolism, arterial thrombosis, or for the treatment of thrombosis occurring after arterial bypass surgery, peripheral vascular bypass or coronary artery bypass grafting. artery, arteriovenous bypass, removal of an implant, such as a stent or catheter.

Monoclonal anti-TF antibodies of the present invention may also be useful for inhibiting renal ischemic reperfusion injury.

The anti-TF monoclonal antibodies of the present invention may also be useful in the treatment of hyperlipoproteinemia, hyperparathyroidism.

The anti-TF monoclonal antibodies of the present invention may also be useful in the treatment of vasculitis, ANCA positive vasculitis, Behçet's disease.

The anti-TF monoclonal antibodies of the present invention may also be useful in blocking trauma-induced respiratory failure such as acute respiratory distress syndrome, acute lung injury.

The anti-TF monoclonal antibodies of the present invention may also be useful in the treatment of various thromboembolic disorders such as those arising from angioplasty, myocardial infarction, unstable angina and coronary artery stenoses.

The anti-TF monoclonal antibodies of the present invention may also be useful prophylactically for the treatment of TF-mediated complications such as sepsis or pneumonia.

The monoclonal anti-TF antibodies of the present invention may also be useful as a prophylactic treatment for atherosclerotic patients at risk of thrombosis.

The anti-TF monoclonal antibodies of the present invention may also be useful in the treatment of graft versus host disease.

The anti-TF monoclonal antibodies of the present invention may also be useful for enhancing beta cell engraftment in islet cell transplantation to prevent cardiac vasopathy (CAV), to prevent acute graft rejection.

The anti-TF monoclonal antibodies of the present invention may also be useful in the treatment of diseases in which circulating tissue factor-revealing microparticles are present, such as, but not limited to, vascular thrombosis, type II diabetes, AMI, pulmonary arterial hypertension.

Similarly, this invention relates to a method for inhibiting the growth and/or proliferation of a tumor cell expressing TF, comprising administering to an individual in need thereof, an antibody or a bispecific molecule of this invention. In one embodiment, said tumor cell is involved in a cancer such as prostate cancer, lung cancer (such as non-small cell lung cancer), breast cancer, colorectal cancer (such as metastatic colorectal cancer), pancreatic cancer, ovarian cancer, cutaneous melanoma, leukemic bone marrow cancer, chronic lymphoblastic leukemia and non-Hodgkin's lymphoma, skin cancer, prostate cancer, glioma, cancer of the brain, kidney, uterus, bladder and rectum.

This invention also relates to the use of a monoclonal antibody that binds to human TF for the preparation of a medicament for the treatment of cancer, such as one of the aforementioned specific types of cancer.

In one embodiment, the selection of patients to be treated with an anti-TF antibody is based on the level of tissue factor (TF) in their urine and/or blood. In one specific embodiment, the patient to be treated has a relatively high level of TF in the urine and/or blood. For example, a particular patient to be treated may have a urinary TF level greater than 20 ng/ml, such as greater than 40 ng/ml, such as greater than 100 ng/ml, such as greater than 200 ng/ml. ml. Alternatively or additionally, the serum TF level of these patients may be greater than 100 pg/ml, such as greater than 200 pg/ml. It can be determined using ELISA.

In a further embodiment of the methods of treatment of the present invention, the effectiveness of the treatment is monitored during this therapy, for example, at certain time points. In one embodiment, this efficacy can be monitored by measuring the level of TF in urine or blood, for example, using ELISA. In another embodiment, this efficacy may be determined by visualization of the disease area, eg by performing one or more PET-CT scans, eg using a labeled anti-TF antibody, such as a labeled anti-TF antibody of the present invention. In addition, labeled anti-TF antibodies, such as the labeled anti-TF antibodies of this invention, could be used to detect TF-producing tumors, for example, using a PET-CT scan.

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Dosing regimens in the above treatments are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, several divided doses over time, or the dose may be proportionally decreased or increased as needed by the therapeutic situation. Parenteral compositions may be formulated in unit dosage form for ease of administration and dose uniformity. Dosage unit form refers in this context to physically discrete units suitable as unitary (unit) doses for subjects to be treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The guidance regarding dosage unit forms of this invention is dictated by (and directly dependent on) (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the disadvantages present in the field of compounding such an active compound for treatment in sensitivity in individuals.

Effective doses and dosing schedules for anti-TF antibodies depend on the disease or condition being treated and can be determined by those skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of this invention is the range of about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, such as about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, such as about 0.5, about 0.3, about 1 or about 3 mg/kg.

A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian might start with doses of the anti-TF antibody used in a pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, a suitable daily dose of a composition of this invention will be the amount of this compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Administration may be intravenous, intramuscular, intraperitoneal, or subcutaneous, and administered, for example, proximal to the target site. If desired, this effective daily dose may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. Although the compound of the present invention may be administered alone, it is preferable to administer the compound as a pharmaceutical composition as described above.

In one embodiment, anti-TF antibodies may be administered by infusion at a weekly dose of 10 to 500 mg/m ² , eg 200 to 400 mg/m ² . Such administration may be repeated eg 1-8 times, eg 3-5 times. This administration may be by continuous infusion over a period of 2-24 hours, such as 2-12 hours.

In one embodiment, the anti-TF antibodies may be administered by slow continuous infusion over an extended period, such as over 24 hours, to reduce toxic side effects.

In one embodiment, anti-TF antibodies may be administered at a weekly dose of 250 to 2000 mg, such as, for example, 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg, or 2000 mg, up to 8 times, for example, 4 to 6 times. This administration may be by continuous infusion over a period of 2 to 24 hours, eg 2 to 12 hours. This schedule may be repeated one or more times as needed, eg after 6 months or 12 months. This dose can be determined or adjusted by measuring the amount of a compound of this invention in the blood after administration, for example by taking a biological sample and using anti-idiotypic antibodies that target the antigen-binding region of anti-TF antibodies of this invention.

In one embodiment, anti-TF antibodies may be administered via maintenance therapy, eg once a week for a period of 6 months or more.

In one embodiment, anti-TF antibodies may be administered via a regimen comprising one infusion of an anti-TF antibody of the present invention followed by an infusion of a radioisotope-conjugated anti-TF antibody of the present invention. This administration schedule can be repeated, for example, after 7-9 days.

As non-limiting examples, treatment in accordance with this invention can be provided as a daily dose of a compound of this invention in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1 , 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg/kg daily on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 after the start of treatment, or any combination thereof, using a single dose or divided doses every 24, 12, 8, 6 , 4 or 2 hours, or any combination thereof.

An effective amount for tumor therapy can also be measured by its ability to stabilize the progression of the disease. The ability of a compound to inhibit cancer can be assessed in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be assessed by testing the ability of the compound to inhibit cell growth or induce apoptosis by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may reduce the size of a tumor or otherwise relieve symptoms in a subject. One of ordinary skill in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition chosen or the particular route of administration chosen.

The anti-TF antibody may also be administered prophylactically to reduce the risk of cancer, delay the onset of an event in cancer progression, and/or reduce the risk of recurrence when the cancer is in remission. This may be particularly applicable in patients in whom it is difficult to locate a tumor known to be present due to other biological factors.

Anti-TF antibodies can also be administered in combination therapy, ie. in combination with other therapeutic agents relevant to the disease or condition being treated. Thus, in one embodiment, the antibody drug is for combination with one or more additional agents, such as a cytotoxic, chemotherapeutic, or anti-angiogenic agent.

Such combined administration may be simultaneous, separate or sequential. For simultaneous administration, these agents may be administered as a single composition or as separate compositions, respectively. Thus, the invention also provides methods for treating a disorder involving cells expressing TF as described above, comprising administering an anti-TF antibody of the invention combined with one or more additional therapeutic agents described below.

In one embodiment, the invention provides a method of treating a disorder involving cells expressing TF in a subject, comprising administering a therapeutically effective amount of an anti-TF antibody of the invention and at least one chemotherapeutic agent to the subject in need thereof.

In one embodiment, the invention provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of an anti-TF antibody of the invention and at least one chemotherapeutic agent to a subject in need thereof.

In one embodiment, the present invention provides for the use of an anti-TF antibody of the present invention for the preparation of a pharmaceutical composition for administration with at least one chemotherapeutic agent for the treatment of cancer.

In one embodiment, such a chemotherapeutic agent may be selected from an antimetabolite such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine, and similar agents.

In one embodiment, such a chemotherapeutic agent may be selected from an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin and similar agents.

In one embodiment, such a chemotherapeutic agent may be selected from an antimitotic agent such as taxanes, such as docetaxel and paclitaxel, and vinca alkaloids (Vinca), such as vindesine, vincristine, vinblastine, and vinorelbine.

In one embodiment, such a chemotherapeutic agent may be selected from a topoisomerase inhibitor such as topotecan or irinotecan.

In one embodiment, such a chemotherapeutic agent may be selected from a cytotoxic drug such as etoposide and teniposide.

In one embodiment, such a chemotherapeutic agent can be selected from a growth factor inhibitor such as an ErbB 1 (EGFR) inhibitor (such as Iressa, Erbitux (cetuximab), Tarceva and related agents), an ErbB2 (Her2/neu) inhibitor (such as herceptin and related agents), and related agents.

In one embodiment, such a chemotherapeutic agent may be selected from a tyrosine kinase inhibitor such as imatinib (Glivec, Gleevec STI571), lapatinib, PTK787/ZK222584 and similar agents.

In one embodiment, the present invention provides a method for treating a disorder,

- 31 041176 comprising cells expressing TF in a subject, comprising administering a therapeutically effective amount of an anti-TF antibody of the present invention and at least one inhibitor of angiogenesis, neovascularization and/or other vascularization, to a subject in need thereof.

Examples of such angiogenesis inhibitors are urokinase inhibitors, matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY 12-9566, AG 3340, BMS-275291 and similar agents), endothelial cell migration and proliferation inhibitors (such as TNP-470, squalamine, 2 -methoxyestradiol, combretastatins, endostatin, angiostatin, penicillamine, SCH66336 (ScheringPlough Corp, Madison, NJ), R115777 (Janssen Pharmaceutica, Inc, Titusville, NJ) and related agents), angiogenic growth factor antagonists (such as ZD6474, SU6668, antibodies against angiogenic agents and/or their receptors (such as VEGF, bFGF and angiopoietin-1), thalidomide, thalidomide analogs (such as CC-5013), Sugen 5416, SU5402, antiangiogenic ribozyme (such as angiozyme), interferon α (such as interferon a2a), suramin and related agents), VEGF-R kinase inhibitors and other anti-angiogenic tyrosine kinase inhibitors (such as SUO11248), endothelium-specific integrin/survival signaling inhibitors (such as acvitaxin and related agents), copper antagonists/chelators (such as tetrathiomolybdate, captopril and related agents), carboxyamidotriazole (CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E, and nucleotide molecules that inhibit angiogenesis (such as antisense-VEGF cDNA, cDNA encoding angiostatin, cDNA encoding p53, and cDNA encoding defective VEGF receptor-2) and similar agents.

Other examples of such inhibitors of angiogenesis, neovascularization and/or other vascularization are antiangiogenic heparin derivatives and related molecules (e.g. heparinase III), temozolomide, NK4, macrophage migration inhibitory factor (MIF), cyclooxygenase-2 inhibitors, hypoxia inducible factor 1 inhibitors, antiangiogenic soy isoflavones, oltipraz, fumagilin and their analogs, somatostatin analogs, pentosan polysulfate, sodiumtecogalan, dalteparin, tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin, antibodies against other relevant targets (such as anti-alpha-y/beta-3 -integrin and anti-kininostatin-mAb) and similar agents.

In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody to treat the disorders described above may be an anti-cancer immunogen such as a cancer antigen/tumor-associated antigen (e.g., epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1 ), carcinoembryonic antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (e.g., human papillomavirus vaccines), tumor-derived proteins heat shock and similar agents A number of other suitable cancer antigens/tumor associated antigens described elsewhere herein and similar molecules known in the art may also or alternatively be used in such an embodiment. such as BEC2 anti-idiotypic antibodies, Mitumomab, CeaVac and related anti-idiotypic anti-idiotypic antibodies, anti-MG7 anti-idiotypic antibodies, and other anti-cancer anti-idiotypic antibodies (see, e.g., Birebent et al., Vaccine. 21(15), 1601-12 (2003), Li et al., Chin Med J (Engl). 114(9), 962-6 (2001), Schmitt et al., Hybridoma. 13(5), 389-96 (1994), Maloney et al., Hybridoma. 4(3), 191209 (1985), Raychardhuri et al., J Immunol. 137(5), 1743-9 (1986), Pohl et al., Int J Cancer. 50(6), 958-67 (1992), Bohlen et al., Cytokines Mol Ther 2(4), 231-8 (1996) and Maruyama, J Immunol Methods. 264(1-2), 12133 (2002)). Such anti-idiotype Abs may optionally be conjugated to a carrier, which may be a carrier with a synthetic (usually inert) molecule, a protein (e.g., shellfish hemocyanin (KLH) (see, e.g., Ochi et al., Eur J Immunol. IZ(H) , 1645-8 (1987)) or a cell (eg, an erythrocyte - see, for example, Wi et al., J Immunol Methods. 122(2), 227-34 (1989)).

In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody to treat the disorders described above may be an anti-cancer cytokine, a chemokine, or a combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23 , IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (eg, INFa2b), IFNe, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines may include Glu-Leu-Arg (ELR) negative chemokines such as IP-10, MCP3, MIG, and SDF-1a from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins. These and other methods or uses involving the naturally occurring peptide-encoding nucleic acids described herein may alternatively or additionally be carried out by methods of down-regulating gene activation and homologous gene recombination, such as those described in US 5,968,502, US 6,063,630 and US 6187305 and EP 0505500.

In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody to treat the disorders described above may be a cell cycle control/apoptosis regulator (or regulatory agent). Cell cycle/apoptosis control regulator may

- 32 041176 include molecules that target and modulate cell cycle control/apoptosis regulators such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol ( L868275, HMR1275), 7hydroxystaurosporine (UCN-01, KW-2401) and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (such as BIBR1532, SOT-095, GRN163 and the compositions described, for example, in US 6440735 and US 6713055). Non-limiting examples of molecules that interfere with apoptosis pathways include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFN, and antisense Bcl-2.

In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody to treat the disorders described above may be a hormone regulating agent, such as agents useful in anti-androgen and anti-estrogen therapy. Examples of such hormone regulating agents are tamoxifen, idoxifen, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl-estradiol/estinil, antiandrogen (such as flutamind/eulexin), progestin (such as hydroxyprogesterone caproate, medroxyprogesterone/prover, megestrol acetate/megase ), adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone releasing factor (and its analogs and other LHRH agonists such as buserelin and goserelin), aromatase inhibitor (such as anastrozole/arimidex, aminoglutethimide/citraden, exemestane), inhibitor hormone (such as ostreotide/sandostatin); and similar agents.

In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody to treat the disorders described above may be an anti-anergic agent (e.g., small molecule compounds, proteins, glycoproteins, or antibodies that destroy resistance to tumor and cancer antigens) . Examples of such compounds are molecules that block CTLA-4 activity such as MDX-010 (ipilimumab) (Phan et al., PNAS USA 100, 8372 (2003)).

In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody to treat the disorders described above can be a tumor suppressor gene-containing nucleic acid or vector, such as a replication-defective adenovirus encoding wild-type human recombinant p53/SCH58500, and so on. .d.; antisense nucleic acids targeting oncogenes, mutated or dysregulated genes; or siRNAs targeting mutated or deregulated genes. Examples of tumor suppressor targets include, for example, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1 and DCC.

In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody to treat the disorders described above may be an anti-cancer nucleic acid such as antisense nucleic acid (augmerosen/03139), LY900003 (ISIS 3521), ISIS 2503, OGX-011 (ISIS 112989), LE-AON/LEraf-AON (liposome-encapsulated c-raf/ISIS-5132 antisense oligonucleotide), MG98, and other antisense nucleic acids that target PKCa, clusterin, IBP, protein kinase GF A, cyclin D1, or Bcl-2h.

In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody to treat the disorders described above may be an anti-cancer inhibitory RNA molecule (see, e.g., Lin et al., Curr Cancer Drug Targets. 1(3), 241 -7 (2001), Erratum in: Curr Cancer Drug Targets 3(3), 237 (2003), Lima et al., Cancer Gene Ther 11(5), 309-16 (2004), Grzmil et al. Int J Oncol 4(1), 97-105 (2004), Collis et al, Int J Radiat Oncol Biol Phys 57(2 Suppl), S144 (2003), Yang et al, Oncogene 22(36) , 5694-701 (2003) and Zhang et al., Biochem Biophys Res Commun 303(4), 1169-78 (2003)).

The combination administration compositions and methods of this invention also include the administration of nucleic acid vaccines, such as naked DNA vaccines encoding such cancer antigens/tumor-associated antigens (see, for example, US 5589466, US 5593972, US 5703057, US 5879687, US 6235523 and US 6387888). In one embodiment, this combined route of administration and/or combined composition comprises an autologous vaccine composition. In one embodiment, this combined composition and/or method of combined administration includes a vaccine of whole cells or cytokine-expressing cells (e.g., recombinant IL-2-expressing fibroblasts, recombinant cytokine-expressing dendritic cells, etc.) (see, for example, , Kowalczyk et al., Acta Biochim Pol. 50(3), 613-24 (2003), Reilly et al., Methods Mol Med. 69, 233-57 (2002) and Tirapu et al., Curr Gene Ther. 2 (1), 79-89 (2002) Another example of such an autologous cell approach that can be used in the combination methods of the present invention is the MyVax® Personalized Immunotherapy (formerly called GTOP-99) method (Genitope Corporation - Redwood City, CA , USA).

In one embodiment, the invention provides combination compositions and combination routes of administration in which an anti-TF antibody is combined or co-administered with a virus, viral proteins, and the like. Replication-deficient viruses that are typically capable of one or only a few rounds of replication in vivo and that target tumor cells may be useful components of such compositions and methods, for example. Such viral agents may contain or be associated with nucleic acids that contain immunostimulants such as GM-CSF and/or IL-2. Both naturally occurring oncolytic and such recombinant oncolytic viruses (eg, HSV-1 viruses, reoviruses, replication deficient and replication sensitive adenovirus, etc.) can be useful components of such methods and compositions. Thus, in one embodiment, the invention provides combination compositions and combination routes of administration, wherein an anti-TF antibody is combined or co-administered with an oncolytic virus. Examples of such viruses include adenoviruses and herpesviruses, which may or may not be modified viruses (see, for example, Shah et al., J Neurooncol. 65(3), 203-26 (2003), Stiles et al. , Surgery 134(2), 357-64 (2003), Sunarmura et al., Pancreas 28(3), 326-9 (2004), Teshigahara et al., J Surg Oncol 85(1), 42- 7 (2004), Varghese et al., Cancer Gene Ther. 9(12), 967-78 (2002), Wildner et al., Cancer Res. 59(2), 410-3 (1999), Yamanaka, Int J Oncol 24(4), 919-23 (2004) and Zwiebel et al., Semin Oncol 28(4), 336-43 (2001).

Combination compositions and combination routes of administration of the present invention may also include whole cell methods and adoptive immunotherapy methods. For example, such methods may involve infusion or re-infusion of immune system cells (e.g., tumor-infiltrating lymphocytes (TILs), such as CD4+ and/or CD8+ T cells (e.g., T cells increased with tumor-specific antigens and/or other genetic enhancements) , antibody-expressing B cells, or other antibody-producing/antigen-presenting, dendritic cells (e.g., anti-cytokine-expressing recombinant dendritic cells, dendritic cells cultured with a DC propagating agent such as GM-CSF and/or Flt3-L , and/or tumor-associated antigen-loaded dendritic cells), anti-tumor NK cells, so-called hybrid cells, or combinations thereof. Cell lysates may also be used in such methods and compositions. Cell-based vaccines in clinical trials that may be applicable in such aspects include lysates of Canvaxin™, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cells. derived from cancer cells and mixtures thereof (see, for example, Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionally mixed with adjuvants such as alum, may also be components in such methods and combined compositions.

In one embodiment, an anti-TF antibody may be delivered to a patient in combination with an internal vaccination method. Internal vaccination refers to the induced death of tumor or cancer cells, such as drug-induced or radiation-induced death of tumor cells in a patient, which usually results in the induction of an immune response directed towards (i) the tumor cells as a whole, or (ii) parts of the tumor cells. including (a) secreted proteins, glycoproteins or other products, (b) membrane-associated proteins or glycoproteins or other components associated with or incorporated into membranes, and/or c) intracellular proteins or other intracellular components. The immune response induced by internal vaccination may be humoral (ie, antibody-complement-mediated) or cell-mediated (eg, the development and/or increase of endogenous T-lymphocytes that recognize internally killed tumor cells or parts thereof). In addition to radiotherapy, non-limiting examples of drugs and agents that can be used to induce said tumor cell death and internal vaccination are conventional chemotherapeutic agents, cell cycle inhibitors, anti-angiogenesis drugs, monoclonal antibodies, apoptosis-inducing agents, and signal transduction inhibitors.

Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with an anti-TF antibody to treat the disorders described above are differentiation-inducing agents, retinoic acid analogs (such as all-trans retinoic acid, 13- cis-retinoic acid and related agents), vitamin D analogs (such as seocalcitol and related agents), ErbB3, ErbB4, IGF-IR inhibitors, insulin receptor, PDGFRa, PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA , TRKC, c-met, Ron, Sea, Tie, Tie2, Eph, Ret, Ros, AIk, LTK, PTK7 and similar agents.

Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with an anti-TF antibody to treat the disorders described above are cathepsin B, cathepsin D dehydrogenase activity modulators, glutathione S transferase (such as glutaacylcysteine synthetase and lactate dehydrogenase) , and similar agents.

Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with an anti-TF antibody to treat the disorders described above are estramustine and epirubicin.

Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with an anti-TF antibody to treat the disorders described above are an HSP90 inhibitor such as 17-allylamino-geld-anamycin, an antibody directed 041176 against tumor antigen such as PSA, CA125, KSA, etc., integrins such as β 1 integrin, VCAM inhibitors and similar agents.

Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with an anti-TF antibody to treat the disorders described above are calcineurin inhibitors (such as valspodar, PSC 833 and other MDR-1 or p-glycoprotein inhibitors). ), TOR inhibitors (such as sirolimus, everolimus and rapomycin) and inhibitors of lymphocyte homing mechanisms (such as FTY720), and agents with actions on cell signaling such as adhesion molecule inhibitors (such as anti-LFA, etc. .).

In one embodiment, an anti-TF antibody of this invention is used in combination with one or more other therapeutic antibodies such as bevacizumab (Avastin®), zalutumumab, cetuximab (Erbitux®), panitumumab (Vectibix™), ofatumumab, zanolimumab, daratumumab , ranibizumab (Lucentis®), Zenapax, Simulest, Remicade, Humira, Tysabri, Xolair, raptiva, nimotuzumab, rituximab and/or trastuzumab (Herceptin®). Other therapeutic antibodies that can be used in combination with an antibody of this invention are those described in WO 98/40408 (antibodies that can bind native human TF), WO 04/094475 (antibodies that can bind to human tissue factor that do not inhibit factor-mediated blood coagulation compared to normal plasma controls), WO 03/093422 (antibodies that bind with higher affinity to the TF:VIIa complex than to TF alone) or WO 03/037361 (TF agonist or antagonist for treatment associated with apoptosis).

In another embodiment, two or more of the different antibodies of this invention described above are used in combination to treat a disease. Particularly interesting combinations include two or more non-competing antibodies. Thus, in one embodiment, the patient is treated with a combination of a cross-block group I antibody as defined herein with a group II or III antibody as defined herein. In another embodiment, the patient is treated with a combination of a group II antibody, as defined herein below, with a group III antibody. Such combination therapy may result in the binding of an increased number of antibody molecules per cell, which may lead to increased efficacy, for example, through activation of complement-mediated lysis.

In one embodiment, the anti-TF antibody may be administered in connection with the delivery of one or more agents that stimulate entry of the anti-TF antibody or combination composition into the interior of the tumor. Such methods can, for example, be performed in association with the delivery of relaxin, which is able to relax the tumor (see, for example, US 6719977). In one embodiment, an anti-TF antibody of the present invention may be linked to a cell penetrating peptide (CPP). Cell-penetrating peptides and related peptides (such as engineered cell-penetrating antibodies) are described, for example, in Zhao et al., J Immunol Methods. 254(1-2), 137-45 (2001), Hong et al., Cancer Res. 60(23), 6551-6 (2000). Lindgren et al., Biochem J. 377(Pt 1), 69-76 (2004), Buerger et al., J Cancer Res Clin Oncol. 129(12), 669-75 (2003), Pooga et al., FASEB J. 12(1), 67-77 (1998) and Tseng et al., Mol Pharmacol. 62(4), 864-72 (2002).

In one embodiment, the invention provides a method of treating a disorder involving cells expressing TF in a subject, comprising administering a therapeutically effective amount of an anti-TF antibody and at least one anti-inflammatory agent to the subject in need thereof.

In one embodiment, such an anti-inflammatory agent may be selected from aspirin and other salicylates, Cox-2 inhibitors (such as rofecoxib and celecoxib), NSAIDs (such as ibuprofen, fenoprofen, naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone , etodolac, oxaprozin and indomethacin), anti-IL6R antibodies, anti-IL8 antibodies (e.g. antibodies described in WO 2004058797, e.g. 10F8), anti-IL15 antibodies (e.g. antibodies described in WO 03017935 and WO 2004076620), anti-IL15R antibodies, anti-CD4 antibodies (eg, zanolimumab), anti-CD11a antibodies (eg, efalizumab), anti-alpha-4/beta-1 integrin (VLA4) antibodies (eg, natalizumab), CTLA4-Ig for the treatment of inflammatory diseases, prednisone, prednisone, disease-modifying antirheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine, sulfasalazine, pyrimidine synthesis inhibitors (such as leflunomide), IL-1 receptor blocking agents (such as anaki nra), TNF-α blocking agents (such as etanercept, infliximab and adalimumab) and similar agents.

In one embodiment, such an immunosuppressive and/or immunomodulatory agent may be selected from cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids such as prednisone, methotrexate, gold salts, sulfasalazine, antimalarial agents, brequinar, leflunomide, mizoribine, 15 -deoxypergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentine, thymosin-α and similar agents.

In one embodiment, such an immunosuppressive and/or immunomodulatory agent may be selected from immunosuppressive antibodies such as antibodies that bind to the p75 IL-2-35 041176 receptor, anti-CD25 antibodies (for example, the antibodies described in WO 2004045512 such as AB1, AB7,

AB11 and AB12) or antibodies that bind, for example, MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40,

CD45, IFNy, TNF-α, IL-4, IL-5, IL-6R, IL-7, IL-8, IL-10, CD11a or CD58, or antibodies binding to their ligands.

In one embodiment, such an immunosuppressive and/or immunomodulatory agent may be selected from soluble molecules IL-15R, IL-10 B7 (B7-1, B7-2, variants and fragments thereof), ICOS and OX40, an inhibitor of the negative regulator of T- cells (such as an anti-CTLA4 antibody) and similar agents.

In one embodiment, the invention provides a method of treating a disorder involving cells expressing TF in a subject, comprising administering a therapeutically effective amount of an anti-TF antibody and an anti-C3b(i) antibody to the subject in need thereof.

In one embodiment, the therapeutic agent for use in combination with anti-TF antibodies to treat the disorders described above may be selected from histone deacetylase inhibitors (e.g., phenylbutyrate) and/or DNA repair agents (e.g., DNA repair enzymes and related compositions such as dimericin).

Methods of the present invention for treating the disorder described above, comprising administering a therapeutically effective amount of an anti-TF antibody, may also include anti-cancer photodynamic therapy (e.g., anti-cancer laser therapy which may optionally be practiced using a photosensitizing agent, see e.g., Zhang et al., J Control Release 93(2), 141-50 (2003)), anti-cancer sonic (acoustic) wave and shock wave therapies (see e.g. Kambe et al., Hum Cell 10(1) , 87-94 (1997)), and/or anti-cancer nutraceutical therapy (see e.g. Roudebush et al., Vet Clin North Am Small Anim Pract. 34(1), 249-69, viii (2004) and Rafi, Nutrition 20(1), 78-82 (2004) Similarly, an anti-TF antibody can be used to formulate a pharmaceutical composition for the treatment of the disorder described above, for administration with anti-cancer photodynamic therapy (e.g., anti-cancer drug). laser therapy - which may optionally be practiced using a photosensitizing agent, anti-cancer using sound (acoustic) wave and shock wave therapies and/or anti-cancer nutraceutical therapy.

In one embodiment, the invention provides a method of treating a disorder involving cells expressing TF in a subject, comprising administering a therapeutically effective amount of an anti-TF antibody, such as an anti-TF antibody of the present invention, and radiotherapy to the subject in need thereof.

In one embodiment, this invention provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of an anti-TF antibody, such as an anti-TF antibody of this invention, and radiotherapy to a subject in need thereof.

In one embodiment, this invention provides for the use of an anti-TF antibody, such as an anti-TF antibody of the present invention, for the preparation of a pharmaceutical composition for the treatment of cancer to be administered in combination with radiotherapy.

Radiotherapy may include radiation or the associated administration of radiopharmaceuticals to a patient. The radiation source may be either external or internal to the patient being treated (irradiation may be, for example, in the form of external beam radiation therapy (EBRT) or close focus radiation therapy (BT)). Radioactive elements that can be used in the practice of such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131 and indium-111.

In a further embodiment, this invention provides a method for treating or preventing cancer comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TF antibody, such as an anti-TF antibody of the present invention, in combination with surgery.

As described above, the pharmaceutical composition may be administered in combination therapy, i. in combination with one or more agents relevant to the disease or condition being treated, either as separate pharmaceutical compositions or with a compound of this invention co-formulated with one or more additional therapeutic agents described above. Such combination therapies may require lower doses of the compound of the present invention and/or co-administered agents to avoid potential toxicities or complications associated with various monotherapies.

In addition to the above, other interesting combination therapies include the following:

For the treatment of pancreatic cancer, an anti-TF antibody in combination with an antimetabolite such as 5-fluorouracil and/or gemcitabine, possibly in combination with one or more compounds selected from 90Y-hPAM4, ARC-100, ARQ-197, AZD-6244 , bardoxolone-methyl, cixutumumab, (IMCA12), folithixorin-calcium, GVAX, ipilimumab, KRX-0601, merbaron, MGCD-0103, MORAb-009,

- 36 041176

PX-12, Rh-Apo2L, TLN-4601, Trabedersen, Volociximab (M200), WX-671, Pemetrexed, Rubitecan, Ixabepilone, OCX-0191Vion, 216586-46-8, Lapatinib, Matutsumab, Imatinib, Sorafinib, Trastuzumab, Exabepilone , erlotinib, avastin, and cetuximab.

For the treatment of colorectal cancer, an anti-TF antibody in combination with one or more compounds selected from gemcitabine, bevacizumab, FOLFOX, FOLFIRI, XELOX, IFL, oxaliplatin, irinotecan, 5-FU/LV, capecitabine, UFT, EGFR targeting agents, such as cetuximab, panitumumab, zalutumumab, nimotuzumab; VEGF inhibitors or tyrosine kinase inhibitors such as sunitinib.

For the treatment of breast cancer, an anti-TF antibody in combination with one or more compounds selected from antimetabolites, anthracyclines, taxanes, alkylating agents, antihormonal epothilones (femara, tamoxifen, etc.), ErbB2 (Her2/neu) inhibitors ( such as herceptin and related agents), CAF/FAC (cyclophosphamide, doxorubicin, 5FU) AC (cyclo, doxo), CMF (cyclo, methotrexate, 5FU), Docetaxel+capecitabine, GT (paclitaxel, gemcitabine) FEC (cyclo, epi, 5FU) in combination with Herceptin: Paclitaxel ± carboplatin, Vinorelbine, Docetaxel, CT in combination with lapatinib; Capecitabine.

For bladder treatment, an anti-TF antibody in combination with one or more compounds selected from antimetabolites (gemcitabine, alimta, methotrexmate), platinum analogs (cisplatin, carboplatin), EGFr inhibitors (such as cetuximab or zalutumumab), VEGF inhibitors (such such as Avastin), doxorubicin, tyrosine kinase inhibitors such as gefitinib, trastuzumab, an antimitotic agent such as taxanes such as paclitaxel, and Vinca alkaloids such as vinblastine.

For the treatment of prostate cancer, an anti-TF antibody in combination with one or more compounds selected from: hormonal/antihormonal therapies; such as antiandrogens, luteinizing hormone releasing hormone (LHRH) agonists, and chemotherapeutic agents such as taxanes, mitoxatron, estramustine, 5FU, vinblastine, ixabepilone.

For the treatment of ovarian cancer, an anti-TF antibody in combination with one or more compounds selected from an antimitotic agent such as taxanes and Vinca alkaloids, caelix, topotecan.

Diagnostic applications

The anti-TF antibodies of this invention can also be used for diagnostic purposes. Thus, in an additional aspect, this invention relates to a diagnostic composition containing an anti-TF antibody defined here.

In one embodiment, an anti-TF antibody of the present invention can be used in vivo or in vitro to diagnose diseases in which activated cells expressing TF play an active role in pathogenesis by detecting levels of TF or levels of cells that contain TF on the surface. their membranes. This can be achieved, for example, by contacting the test sample, optionally together with a control sample, with an anti-TF antibody under conditions that allow the formation of a complex between this antibody and TF. The formation of the complex is then detected (eg using ELISA). When using a control sample along with a test sample, the complex is detected in both samples, and any statistically significant difference in the formation of complexes between these samples is indicative of the presence of TF in the test sample.

Thus, in a further aspect, this invention relates to a method for detecting the presence of a TF antigen or a TF expressing cell in a sample, comprising contacting the sample with an anti-TF antibody of this invention or a bispecific molecule of this invention under conditions that allow the formation of a complex between these antibody and TF; and analyzing whether a complex has been formed.

In one embodiment, this method is performed in vitro.

More specifically, the invention provides methods for identifying and diagnosing invasive cells and tissues and other target cells of the anti-TF antibodies of the invention, and for monitoring the progress of therapeutic treatment, post-treatment status, cancer risk, cancer progression, and the like.

In one example of such a diagnostic assay, the present invention provides a method for diagnosing the level of invasive cells in a tissue, comprising the formation of an immunocomplex between an anti-TF antibody and potential TF-containing tissues, and detection of the formation of this immunocomplex, where the formation of the immunocomplex is correlated with the presence of invasive cells in that tissue. . This contacting can be done in vivo, using labeled isolated antibodies and standard imaging techniques, or can be done in vitro on tissue samples.

Anti-TF antibodies can be used to detect TF-containing peptides and peptide fragments in any suitable biological sample by any suitable method. Examples of conventional immunoassays provided by the present invention include, without limitation, ELISA, RIA, FACS assays, plasmon resonance assays, chromatographic assays, tissue immunohistochemistry, Western blotting, and/or immunoprecipitation using aHTu-TF antibody. . The anti-TF antibodies of the present invention can be used to detect TF and TF fragments from humans. Suitable labels for anti-TF antibody and/or secondary antibodies used in such methods include, without limitation, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include ¹²⁵ I, ¹³¹ I, ³⁵ S, and ³ H.

Anti-TF antibodies can also be assayed in a biological sample by a competitive immunoassay using TF peptide standards labeled with a detectable substance and an unlabeled anti-TF antibody. In such an assay, the biological sample, labeled TF peptide standard(s) and anti-TF antibody are combined and the amount of labeled TF standard bound to the unlabeled anti-TF antibody is determined. The amount of TF peptide in a biological sample is inversely proportional to the amount of labeled TF standard bound to the anti-TF antibody.

Anti-TF antibodies are particularly useful in in vivo imaging of tumors. In vivo imaging of TF-associated tumors can be performed by any suitable method. For example, ⁹⁹ T labeling or labeling with another gamma ray emitting isotope can be used to label anti-TF antibodies in tumors or secondarily labeled (e.g. FITC-tagged) anti-TF antibody:TF complexes from tumors and imaged with a gamma scintillation camera (e.g. , Elscint Apex 409ECT), usually using a high resolution low energy collimator or a low energy collimator for all purposes. The stained tissues are then scored for radioactivity as an indicator of the amount of TF-associated peptides in that tumor. Images obtained using such methods can be used to assess the biodistribution of TF in a patient, mammal or tissue, for example in the context of using TF or TF fragments as a biomarker for the presence of invasive cancer cells. Variations of this method may include the use of magnetic resonance imaging (MRI) to improve imaging compared to gamma camera methods. Similar methods and principles of immunoscintigraphy are described, for example, in Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging And Therapy (Plenum Press 1988), Chase, Medical Applications of Radioisotopes, in Remington's Pharmaceutical Sciences, 18th Edition, Gennaro et al., ( eds.), pp. 624-652 (Mack Publishing Co., 1990), and Brown, Clinical Use of Monoclonal Antibodies, in Biotechnology And Pharmacy 227-49, Pezzuto et al., (eds.) (Chapman & Hall 1993). Such images can also be used to target delivery of other anticancer agents, examples of which are described here (eg, apoptotic agents, toxins, or formulations of the CHOP chemotherapy program). In addition, such images may also or alternatively serve as the basis for surgical methods to remove tumors. Moreover, such in vivo imaging techniques may allow identification and localization of a tumor in a situation where a patient is identified as having a tumor (due to the presence of other biomarkers, metastases, etc.) but the tumor cannot be identified by conventional analytical methods. All of these methods are features of the present invention.

The in vivo imaging and other diagnostic methods provided by the present invention are particularly useful in detecting micrometastases in a human patient (eg, in a patient not previously diagnosed as having cancer, or in a patient in cancer recovery/remission). For example, it has been demonstrated that carcinoma cancer cells, which comprise up to 90% of all cancer cells, stain very well with anti-TF antibody conjugate compositions. Anti-TF monoclonal antibody detection described herein can be indicative of the presence of carcinomas that are aggressive/invasive, and also or alternatively provide an indication of the possibility of using a related anti-TF monoclonal antibody against such micrometastases.

In one embodiment, the invention provides an in vivo imaging method wherein an anti-TF antibody of the invention is conjugated with a detection stimulating x-ray opaque agent, the conjugated antibody is administered to a host, e.g., by injection into the blood stream, and the presence and location of the labeled antibody is assayed. in this host. Through this method and any other diagnostic method provided herein, the present invention provides a method for screening for the presence of disease-associated cells in a human patient or a biological sample from a human patient.

For diagnostic imaging, radioactive isotopes can be linked to an anti-TF antibody either directly or indirectly using an intermediate functional group. Useful functional intermediates include chelators such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, for example, US

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5057313).

In addition to radioisotopes and X-ray opaque agents, diagnostic methods can be performed using anti-TF antibodies that are conjugated to dyes (eg, biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, and enhancers (eg, paramagnetic ions). ) for magnetic resonance imaging (MRI) (see, for example, US Patent No. 6,331,175, which describes MRI methods and the preparation of antibodies conjugated to an MRI enhancing agent). Such diagnostic/detecting agents may be selected from agents for use in magnetic resonance imaging and fluorescent compounds. To load an anti-TF antibody with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which a plurality of chelating groups are attached to bind these ions. Such a tail may be a polymer such as polylysine, a polysaccharide or other derivatized or derivatized chain having side groups to which chelating groups can be attached, such as, for example, porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and the like. groups known to be applicable for this purpose. Chelates can be coupled to anti-TF antibodies using standard chemistry.

Thus, the present invention provides diagnostic anti-TF antibody conjugates in which the anti-TF antibody is conjugated to a contrast agent (such as an agent for magnetic resonance imaging, computed tomography or ultrasound contrast enhancing agent) or a radionuclide, which may be, for example, gamma, beta, alpha, Auger electron or positron emitting isotope.

In a further aspect, this invention relates to a kit for detecting the presence of a TF antigen or a TF expressing cell in a sample, comprising an anti-TF antibody of the invention or a bispecific molecule of the invention; and instructions for using this kit.

In one embodiment, this invention provides a cancer diagnostic kit comprising a container containing an anti-TF antibody and one or more reagents for detecting the binding of the anti-TF antibody to a TF peptide. Reagents may include, for example, fluorescent labels, enzymatic labels, or other detectable labels. These reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions, where these enzymatic reactions produce a product that can be visualized. In one embodiment, the invention provides a diagnostic kit comprising one or more anti-TF antibodies of the invention in labeled or unlabeled form in a suitable container(s), incubation reagents for indirect assay, and substrates or derivatizing agents for detection in such assay. , depending on the nature of this label. Control reagent(s) and instructions for use may also be included.

Diagnostic kits can also be supplied for use with an anti-TF antibody, such as a conjugated/labeled anti-TF antibody, to detect cellular activity or to detect the presence of TF peptides in a tissue sample or host. In such diagnostic kits, as well as kits for therapeutic applications described elsewhere herein, the anti-TF antibody can typically be provided in lyophilized form in a container, either alone or together with additional antibodies specific for the target cell or peptide- targets. Typically also included are a pharmaceutically acceptable carrier (eg, an inert diluent) and/or components thereof, such as Tris, a phosphate or carbonate buffer, stabilizers, preservatives, biocides, inert proteins, eg, serum albumin, or the like. (usually in a separate mixing container) and additional reagents (also usually in a separate container(s)). Some kits also include a secondary antibody capable of binding to the anti-TF antibody, which is usually present in a separate container. This second antibody is typically tagged and prepared in a manner similar to that of the anti-TF antibody of the present invention. Using the methods described above and elsewhere herein, anti-TF antibodies can be used to identify subpopulations of cancer/tumor cells and characterize such cells and related tissues/growth.

In situ detection can be performed by taking a histological sample from a patient and providing a combination of labeled anti-TF antibodies of the present invention to that sample. This anti-TF antibody of the present invention can be provided by applying or layering this labeled anti-TF antibody of the present invention onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of TF or TF fragments, but also the distribution of such peptides in the test tissue (for example, in the context of evaluating the spread of cancer cells). With the use of this invention, those skilled in the art will readily appreciate that any of a wide variety of histological methods (such as staining procedures) can be modified to achieve such in situ detection.

In a further aspect, this invention relates to an anti-idiotypic antibody that binds to an anti-TF antibody of this invention described herein.

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An anti-idiotypic (Id) antibody is an antibody that recognizes unique determinants normally associated with an antibody's antigen-binding site. An Id antibody can be obtained by immunizing an animal of the same species and genetic type as the source of the anti-TF mAb with the mAb to which the anti-Id was obtained. The immunized animal can usually recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody against those idiotypic determinants (anti-Id antibodies). Such antibodies are described, for example, in US 4,699,880. Such antibodies are additional features of the present invention.

An anti-I antibody can also be used as an immunogen to induce an immune response in another animal to produce a so-called anti-anti-I antibody. The anti-anti-Id may be epitopically identical to the parent mAb that induced the anti-Id. Thus, using antibodies to the idiotypic determinants of the mAb, it is possible to identify other clones expressing antibodies of identical specificity. The anti-Id antibodies may be varied (producing thereby anti-Id antibody variants) and/or derivatized in any suitable manner, such as the methods described elsewhere herein in relation to the anti-TF antibodies of the present invention. For example, anti-Id-mAbs can be coupled to a carrier such as shellfish hemocyanin (KLH) and used to immunize BALB/c mice. Sera from these mice will typically contain anti-anti-Id antibodies that have binding properties similar, if not identical, to those of the original/original TF antibody.

The present invention is further illustrated by the following examples, which should not be construed as further limiting.

Examples

Example 1 Tissue Factor (TF) Expression Constructs

Fully codon-optimized constructs were generated to express TF or its extracellular domains in HEK, NSO or CHO cells. The proteins encoded by these constructs are identical to Genbank accession NP_001984 for TF. These constructs contained suitable restriction sites for cloning and an optimal Kozak sequence (Kozak, 1987). These constructs were cloned into the mammalian expression vector pEE13.4 (Lonza Biologies) (Bebbington, Renner et al., 1992) to give pEE13.4TF. PCR was used to amplify the extracellular domain (ECD) (amino acids 1-251) coding portion of TF from this synthetic construct, with the addition of a C-terminal His tag containing 6 His residues (TFECDHis). This construct was cloned into pEE13.4 and fully sequenced to validate this construct.

Example 2 Transient Expression in HEK-293F Cells

Freestyle™ 293-F cells (HEK-293 subclone adapted to suspension growth and chemically defined Freestyle medium (HEK-293F)) were obtained from Invitrogen and transfected with the appropriate DNA plasmid using 293fectin (Invitrogen) according to the manufacturer's instructions. In the case of antibody expression, appropriate heavy chain and light chain vectors were co-expressed as described in Example 10.

Example 3 Semi-stable expression in NSO cells pEE13.4TF was stably transfected in NSO cells and stable cells were selected for growth in the absence of glutamine and in the presence of 7.5 μM methylsulfoximine (MSX). A pool of clones was grown in suspension culture with selection pressure maintained. Pools were tested for TF expression by FACS analysis and stored for later use.

Example 4 Stable Expression in CHO Cells pEE13.4TF was stably transfected in CHO-K1SV cells (Lonza Biologies) and stable clones were selected for growth in the absence of glutamine and in the presence of 50 μM MSX. Individual clones were scraped and expanded and tested for TF expression by FACS analysis as described below. High-expressing clones were selected and stored for later use.

Example 5 Purification of His-tagged TF

TFECDhis was expressed in HEK-293F cells. His-tag in TFECDHis allows purification by affinity chromatography with immobilized metal. In this process, a chelator fixed on a chromatographic column was loaded with Co ²⁺ cations. The TFECDHis-containing supernatant is incubated with this resin in batch mode (ie, in solution). The His-tagged protein binds strongly to the resin beads, while other proteins present in the supernatant do not bind strongly. After incubation, these beads are removed from the supernatant and packed into a column. This column is washed to remove loosely bound proteins. The highly bound TFECDHis proteins are then eluted with a buffer containing imidazole which competes with His binding to Co ²⁺ . The eluent is removed from this protein by changing the buffer on a desalting column.

Example 6 Transgenic Mice Immunization Procedure

HuMab mice were immunized every two weeks with alternating immunizations with 5x106 semi-stable transfected NSO-TF cells or 20 μg TFECDHis protein. A total of eight immunizations were performed, four intraperitoneal (IP) and four subcutaneous (SC) immunizations in the base.

- 40 041176 tail. The first immunization with cells was performed in complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA). For all other immunizations, cells were injected with IP in PBS and TFECDHis were injected with SC using incomplete Freund's adjuvant (IFA; Difco Laboratories, Detroit, MI, USA). When serum titers were found to be sufficient (1/50 serum dilution was found to be positive in the antigen-specific screen assay as described in Example 7 for at least 2 consecutive biweekly screening events), mice were further immunized twice intravenously (IV) 10 μg of TFECDHis protein in 100 μl of PBS, 4 and 3 days before the fusion. The first immunization with cells was performed in CFA, for all other (7) immunizations cells were injected with IP in PBS. When titers were found to be sufficient, mice were immunized additionally twice IV with 1×10 ⁶ transiently semi-stably transfected NSO-TF cells in 100 μl of PBS, 4 and 3 days before confluence.

When serum titers were found to be sufficient (as determined above), mice were additionally immunized twice intravenously (IV) with 10 μg of TFECDHis protein in 100 μl of PBS, 4 and 3 days before confluence.

Example 7 Homogeneous Antigen Specific Screening Assay

The presence of anti-TF antibodies in the sera of immunized mice or HuMab (human monoclonal antibody) in hybridoma or transfectoma culture supernatant was determined by homogeneous antigen-specific screening assays (four quadrants) using Fluorometric Microvolume Analysis (FMAT) Technology; Applied Biosystems, Foster City, CA, USA ).

For this, a combination of 3 cell-based assays and one bead-based assay was used. In cell-based assays, binding to TH1015-TF (HEK-293F cells transiently expressing TF; prepared as described above) and A431 (which express TF on the cell surface) as well as wild-type HEK293 cells (which do not express TF , negative control). In a bead-based assay, binding to biotinylated TF bound to a streptavidin bead (SB1015-TF) was determined.

Samples were added to cells/beads for binding to TF. HuMab binding was then detected using a fluorescent conjugate (Goat anti-anti-IgG-Cy5-human; Jackson ImmunoResearch). Mouse anti-human TF antibody (ERL; linked to Alexa647 in Genmab) was used as positive control, pooled mouse HuMAb serum and mouse chrompure-Alexa647 antibody were used as negative controls. These samples were scanned using the Applied Biosystems 8200 Cellular Detection System (8200 CDS) and count x fluorescence was used for reading.

Example 8 Generation of HuMab Hybridoma

HuMab mice with sufficient development of an antigen-specific titer (determined as described above) were euthanized and the spleen and lymph nodes flanking the abdominal aorta and vena cava were harvested. Fusion of splenocytes and lymph node cells with a mouse myeloma cell line was performed by electrofusion using the CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie, MD, USA), essentially according to the manufacturer's instructions. Hybridoma-derived HuMab selection and culture were performed based on standard protocols (e.g., as described in Coligan J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M. and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons, Inc., 2006).

Example 9 Mass Spectrometry of Purified Antibodies

Small aliquots of 0.8 ml antibody-containing supernatant from the 6-well or Hyperflask phase were purified using PhyTip columns containing Protein G-resin (PhyNexus Inc., San Jose, USA) on a Sciclone ALH 3000 workstation (Caliper Lifesciences, Hopkinton, USA). ). PhyTtip columns were used according to the manufacturer's instructions, but the buffers were replaced with: PBS binding buffer (B. Braun, Medical B. V., Oss, Netherlands) and 0.1 M Glycine-HCl pH 2.7 elution buffer (Fluka Riedel-de Haen, Buchs, Germany). After purification, the samples were neutralized with 2 M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands). Alternatively, in some cases, large volumes of culture supernatants were purified using protein A affinity column chromatography.

After purification, the samples were placed in a 384-well plate (Waters, 100 μl square well plate, part# 186002631). Samples were deglycosylated overnight at 37°C with N-glycosidase F (Roche cat no 11365177001. DTT (15 mg/ml) was added (1 µl per well and incubated for 1 hour at 37°C. Samples (5 or 6 µl) desalted on Acquity UPLC™ (Waters, Milford, USA) with BEN300 C18, 1.7 µm, 2.1x50 mm column at 60° C. MQ-water and LC-MS grade acetonitrile (Biosolve, cat no 01204101, Valkenswaard, The Netherlands) with 0.1% formic acid in both cases (Fluka, cat no 56302, Buchs, Germany) were used as eluents A and B, respectively. (Bruker, Bremen, Germany) operating in positive ion mode Before analysis, the 9003000 m/z scale was calibrated with an ES tuning mixture (Agilent Technologies, Santa Clara, USA) The mass spectra were deconvolved using DataAnalysis™ v.

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3.4 (Bruker) using the Maximum Entropy search algorithm for molecular weights between and 80 kDa.

After deconvolution, the resulting masses of heavy and light chains for all samples were compared to find duplicate antibodies. In the comparison of heavy chains, the possible presence of variants with C-terminal lysines was taken into account. This led to a list of unique antibodies, where uniqueness is defined as a unique combination of heavy and light chains. In the event that duplicate antibodies were found, the results from other tests were used to decide which was the best material to continue experimenting with.

MS analysis of the molecular weights of the heavy and light chains of 118 TF-specific hybridomas yielded 70 unique antibodies (unique heavy chain/light chain combinations). They were characterized in a series of functional assays identifying 14 major candidate TF-specific antibodies.

Example 10 Anti-TF-HuMab variable domain sequence analysis and cloning in expression vectors

Total anti-TF-HuMab RNA was prepared from 5x10 ⁶ hybridoma cells and 5'-RACE Complementary DNA (cDNA) was prepared from 100 ng of total RNA using the SMART RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. The VH (heavy chain variable region) and VL (light chain variable region) coding regions were amplified by PCR and cloned into the pCR-Blunt II-TOPO vector (Invitrogen) using the Zero Blunt PCR cloning kit (Invitrogen). For each HuMab, 16 VL clones and 8 VH clones were sequenced. These sequences are shown in the Sequence Listing and in FIG. 1 here.

Tab. 1A and table. 1B (below) provide an overview of antibody sequence information and most homologous germline sequences.

Table 1A. Heavy chain homology

AL V-ΓΕΗ and allele identity of the V region, % J-ΓΕΗ and allele D-GENE and allele CDR-IMGT length 003 IGHVl-69*02, or IGHV1-69*O4 97.57% (281/288 nt) YUSh4*02 IGHD6-13*01 [8,8,11] 098 IGHVl-69*04 95.49% (275/288 nt) SWZ*02 IGHD2-21*02 [8,8,11] 011 IGHV3-23*01 96.53% (278/288 nt) YUSh4*02 IGHD 1-26*01 [8,8,11] 017 IGHV3-23*01 98.26% (283/288 nt) IGHJ2*01 IGHD2-15*01 [8,8,13] 092 IGHV3-23*01 97.92% (282/288 nt) IGHJ4*02 IGHD7-27*01 [8,8,11] 101 IGHV3-23*01 95.83% (276/288 nt) IGHJ4*02 IGHD7-27*01 [8,8,11] 025 IGHV3-30-3*01 97.57% (281/288 nt) IGHJ4*02 IGHD7-27*01 [8,8,13] 109 IGHV3-30-3*01 96.18% (277/288 nt) IGHJ4*02 IGHD7-27*01 [8,8,13] 111 IGHV3-30-3*01 97.57% (281/288 nt) IGHJ4*02 IGHD3-10*01 [8,8,13] 114 IGHV3-33*O1, or IGHV3-33*03 94.44% (272/288 nt) IGHJ6*02 IGHD3-10*01 [8,8,12] 013 IGHV5-51*01 99.65% (287/288 nt) IGHJ3*02 IGHD6-13*01 [8,8,19] Table 1B. Light chain homology AL V-GENE and allele V-region identity % (nt) J-GENE and CDR-IMGT length allele 003 IGKV1-13*O2 99.28% (277/279 nt) IGKJ4*01 [6.3.9] 011 IGKV1D-16*O1 98.57% (275/279 nt) IGKJ2*01 [6.3.9] 013 IGKV1D-16*O1 98.57% (275/279 nt) IGKJ5*01 [6.3.9] 092 IGKV1D-16*O1 99.28% (277/279 nt) IGKJ2*01 [6.3.10] 098 IGKV1D-16*O1 100.00% (279/279 nt) IGKJ2*01 [6.3.9] 101 IGKV1D-16*O1 100.00% (279/279 nt) IGKJ2*01 [6.3.10] 025 IGKV3-ll*01 100.00% (279/279 nt) IGKJ4*01 [6.3.9] 109 IGKV3-ll*01 99.64% (278/279 nt) IGKJ4*01 [6.3.9] 017 IGKV3-20*01 99.29% (280/282 nt) IGKJl*01 [7.3.9] 114 IGKV3-20*01 99.65% (281/282 nt) IGKJ4*01 [7.3.8]

Links to the sequence list:

VH-district SEQ ID NO: 1 VH013 SEQ ID No: 2 VH013, CDR1 SEQ ID No: 3 VH013, CDR2 SEQ ID NO: 4 VH013, CDR3 SEQ ID NO: 5 VH 114 SEQ ID No: 6 VH114,CDR1

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SEQ W No: 7 VH114,CDR2 SEQ ID NO: 8 VH114,CDR3 SEQ ID NO: 9 VH011 SEQ ID NO: 10 VH011, CDR1 SEQ ID NO: 11 VH011, CDR2 SEQ ID NO: 12 VH011, CDR3 SEQID No: 13 VH017-D12 SEQ ID NO: 14 VH 017-D12 , CDR1 SEQIDNo: 15 VH 017-D12, CDR2 SEQID No: 16 VH 017-D12, CDR3 SEQID No: 17 VH 042 SEQID No: 18 VH042, CDR1 SEQIDNo: 19 VH042, CDR2 SEQ ID No: 20 VH042, CDR3 SEQID No: 21 VH 092-A09 SEQ ID NO: 22 VH 092-A09, CDR1 SEQ ID NO: 23 VH 092-A09, CDR2 SEQ ID NO: 24 VH 092-A09, CDR3 SEQ ID NO: 25 VH 101 SEQ ID NO: 26 VH 101, CDR1 SEQ ID NO: 27 VH 101, CDR2 SEQ ID NO: 28 VH 101, CDR3 SEQ ID NO: 29 VH003 SEQ ID No: 30 VH003, CDR1 SEQID No: 31 VH003, CDR2 SEQ ID NO: 32 VH003, CDR3 SEQ ID NO: 33 VH025 SEQ ID NO: 34 VH025, CDR1 SEQIDNo: 35 VH025, CDR2 SEQ ID NO: 36 VH025, CDR3 SEQ ID NO: 37 VH 109 SEQID No: 38 VH 109, CDR1 SEQ ID NO: 39 VH 109, CDR2 SEQ ID NO: 40 VH 109, CDR3 SEQID No: 41 VH044 SEQ ID NO: 42 VH044, CDR1 SEQ ID NO: 43 VH044, CDR2 SEQ ID NO: 44 VH044, CDR3 SEQ ID NO: 45 VH 087-Lg6 SEQ ID NO: 46 VH 087-Lg6, CDR1

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SEQ W No: 47 VH 087-Lg6, CDR2 SEQ ID NO: 48 VH 087-Lg6, CDR3 SEQ ID NO: 49 VH098 SEQ ID NO: 50 VH098, CDR1 SEQ W No: 51 VH098, CDR2 SEQ ID NO: 52 VH098, CDR3 SEQ ID NO: 53 VH 111 SEQ ID NO: 54 VH 111, CDR1 SEQ ID NO: 55 VH 111, CDR2 SEQ ID NO: 56 VH 111, CDR3

VL-district SEQ ID NO: 57 VL013 SEQ ID NO: 58 VL013, CDR1 SEQ ID NO: 59 VL013, CDR2 SEQID No: 60 VL013, CDR3 SEQ ID NO 61 VL 114 SEQ ID NO 62 VL 114, CDR1 SEQ ID NO 63 VL 114, CDR2 SEQ ID NO 64 VL 114, CDR3 SEQ ID NO: 65 VL011 SEQ ID NO: 66 VL011, CDR1 SEQ ID NO: 67 VL011, CDR2 SEQ ID NO: 68 VL011, CDR3 SEQ ID NO: 69 VL 017-D12 SEQ ID NO: 70 VL 017-D12 , CDR1 SEQ W No: 71 VL 017-D12 , CDR2 SEQ ID NO: 72 VL 017-D12 , CDR3 SEQ ID NO 73 VL 042 SEQ ID NO 74 VL042, CDR1 SEQ ID NO 75 VL042, CDR2 SEQ ID NO 76 VL042, CDR3 SEQ ID NO: 77 VL 092-A09 SEQ ID NO: 78 VL 092-A09, CDR1 SEQ ID NO: 79 VL 092-A09, CDR2 SEQ ID NO: 80 VL 092-A09, CDR3 SEQ W No: 81 VL 101 SEQ ID NO: 82 VL 101, CDR1 SEQ ID NO: 83 VL 101, CDR2 SEQ ID NO: 84 VL 101, CDR3 SEQ ID NO: 85 VL003

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SEQ W No: 86 VL003, CDR1 SEQ W No: 87 VL003, CDR2 SEQ W No: 88 VL003, CDR3 SEQ W No: 89 VL025 SEQ W No: 90 VL025, CDR1 SEQID No: 91 VL025, CDR2 SEQ W No: 92 VL025, CDR3 SEQ W No: 93 VL 109 SEQ W No: 94 VL 109, CDR1 SEQ ID NO: 95 VL 109, CDR2 SEQ ID NO: 96 VL 109, CDR3 SEQ ID NO: 97 VL 044 SEQ ID NO: 98 VL044, CDR1 SEQ ID NO: 99 VL044, CDR2 SEQIDNo: 100 VL044, CDR3 SEQID No: 101 VL 087 SEQID No: 102 VL087, CDR1 SEQID No: 103 VL087, CDR2 SEQID No: 104 VL087, CDR3 SEQ ID NO: 105 VL 098 SEQID No: 106 VL098, CDR1 SEQID No: 107 VL098, CDR2 SEQID No: 108 VL098, CDR3 SEQID No: 109 VL 111 SEQID No: 110 VL 111, CDR1 SEQID No: 111 VL 111, CDR2 SEQID No: 112 VL 111, CDR3

Example 11 Antibody Purification

The culture supernatant was filtered through 0.2 µm filters with no exits and loaded onto a 5 ml Protein A column (rProtein A FF, Amersham Bioscience) and eluted with 0.1 M citric acid NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl , pH 9 and dialyzed overnight against 12.6 mM NaH2PO4 140 mM NaCl, pH 7.4 (B. Braun). After dialysis, the samples were sterile filtered through 0.2 µm filters without outlets. Purity was determined by SDS-PAGE and concentration was measured by nephelometry and absorbance was measured at 280 nm. Aliquots of purified antibodies were stored at -80°C. After thawing, aliquots of purified antibodies were stored at 4°C. Mass spectrometry was performed to identify the molecular weight of the heavy and light chains expressed by the hybridomas as described in Example 9.

Example 12 Antibody Cross-Competition Studies Using Sandwich ELISA

The wells of the ELISA plate were coated overnight at +4°C with each of the anti-TF-HuMab (0.5 or 2 μg/ml 100 μl per well) diluted in PBS. These ELISA wells were washed with PBS, blocked for 1 h at room temperature with 2% (v/v) chicken serum (Gibco, Paisley, Scotland) in PBS, and washed again with PBS. Then 50 µl of anti-TF-HuMab (10 µg/ml) was added followed by 50 µl of TFECDHis (0.5 or 1 µg/ml) (generated in Genmab; example 5) and incubated for 1 h at room temperature (at shaking). The plates were washed 3 times with PBS (PBS+0.05% Tween) and incubated with 1:2000 diluted BAM050 anti-his-biotin for one hour at room temperature (with shaking). The plates were washed and incubated with Streptavidin-poly-HRP (Sanquin, Amsterdam, The Netherlands) for 20 min at room temperature and washed again. This reaction was further developed with ABTS (Roche Diagnostics) at room temperature in the dark, stopped after 15 min by the addition of 2% (w/v) oxalic acid, and the absorbance was measured at 405 nm.

Tab. 2 shows that 3 cross-block groups (groups of antibodies competing with each other for TFECDHis binding) could be identified, with antibodies 013, 044 and 087-Lg6 belonging to the same cross-block group (group I), antibodies 011 , 017-D12, 42, 092-A09 and 101 belong to another cross-block group (group II) and antibodies 003, 025, 109 and 111 belong to the third cross-block group (group III). Antibody 114 was found to compete for TFECDHis binding with both cross-block group II and III antibodies. The binding of antibody 098 to TFECDHis could be overcome by both cross-block group II and III antibodies.

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Table 2 Competition of anti-TP antibodies for binding to TAECDHis

1 II 0.5 µg of coating 2 µg coverage 2 µg coverage 0.5 µg coverage 0.5 µg of coating 0.5 µg of coating 42 0.5 µg of coating 0.5 µg of coating Competing antibody (10 µg/ml) 13 44 087-Lg6 eleven 017-D12 092-A09 101 13 19 5 28 101 100 98 BY 98 44 93 40 29 109 96 96 103 109 087-Lg6 91 54 41 103 93 95 109 93 eleven 96 143 929 20 34 35 21 23 017-D12 97 143 995 14 25 20 8 12 42 99 143 931 18 28 27 10 17 092-A09 95 143 995 22 37 37 32 24 101 96 100 714 10 15 15 10 12 114 101 143 995 21 34 34 19 22 98 95 143 995 90 93 97 91 86 3 84 118 770 100 95 91 96 88 25 102 143 995 117 96 108 111 100 109 96 143 995 101 100 101 99 102 111 89 143 995 BY 93 102 95 108 p/w HI 0.5 µg of coating 2 µg coverage 0.5 µg coverage 0.5 µg coverage 0.5 µg coverage 2 µg coverage Competing antibody (10 µg/ml) 114 98 3 25 109 111 13 105 320 85 89 BUT 175 44 105 330 80 108 94 175 087-Lg6 107 210 88 105 103 115 eleven 19 9 103 104 109 175 017-D12 9 9 100 108 97 175 42 24 8 98 93 111 155 092-A09 22 10 103 108 101 175 101 eleven 7 96 108 106 118 114 13 9 100 47 26 5 98 94 24 103 94 86 35 3 102 10 33 22 10 6 25 28 10 48 34 eleven 6 109 44 9 62 51 17 6 111 99 37 89 104 93 43

White boxes indicate no competition for binding, light gray boxes indicate partial competition for binding, and dark gray boxes indicate competition for binding to TFECDHis.

Example 13 Anti-TF-HuMab Binding to TF Extracellular Domain in ELISA

The specificity of the obtained anti-TF-HuMab was evaluated using ELISA. ELISA plates (MicroIon; Greiner Bio-One) were coated overnight at +4°C with 0.5 μg/ml TFECDHis in PBS, pH 7.4. Coated ELISA plates were emptied and blocked for one hour at room temperature with 2% (v/v) chicken serum (Gibco, Paisley, Scotland) in PBS and washed in PBS containing 0.05% Tween 20 (PBS). Then, HuMab serially diluted in PBS (PBS supplemented with 2% (v/v) chicken serum and 0.05% (v/v) Tween-20) were incubated for 1 h at room temperature under shaking conditions (300 v/v). /min). Bound HuMabs were detected using HRP-conjugated goat anti-human IgG (Jackson ImmunoResearch) diluted 1:5000 in PBS, which were incubated for 1 hour at room temperature under shaking conditions (300 rpm). This reaction was further developed with ABTS (Roche Diagnostics) at room temperature in the dark, stopped after 15-30 min by addition of 2% (w/v) oxalic acid, and then the absorbance was measured at 405 nm. HuMab-KLH (human monoclonal antibody against KLH (shellfish hemocyanin)) was used as a negative control. Mouse anti-human TF antibody (ERL) was used as a positive control. (HRP-labeled anti-mouse IgG conjugate). Binding curves were analyzed using non-linear regression (sigmoidal dose-response curve with variable slope) using GraphPad Prism V4.03 software.

As seen in FIG. 3, all of these anti-TP antibodies bound TFECDHis. The EC ₅₀ values for these HuMabs are the average of 3 experiments and range between 0.09 and 0.46 nM (Table 3 below).

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Group HuMab TF EC50 nM I 13 0.24 I 44 0.14 I 87-Lg6 0.09 II eleven 0.16 II 017-D12 0.25 II 42 0.23 II 092-A09 0.18 II 101 0.28 p/w 98 0.13 p/w 114 0.17 III 3 0.46 III 25 0.34 III 109 0.27 III 111 0.11

Example 14 Anti-TF-HuMab Binding to Membrane Bound TF

Binding of anti-TF-HuMab to membrane-bound TF was determined by FACS analysis using TF-transfected CHO cells, or TF-expressing tumor cell lines MDA-MB231, (luciferase-transfected) A431 and Bx-PC3.

Cells were resuspended in PBS (2x10 ⁶ cells/ml), placed in 96-well V-bottom plates (50 μl per well). 50 µl of serially diluted HuMab in FACS buffer (PBS supplemented with 0.1% BSA and 0.02% Na-azide) was added to these cells and incubated for 30 min on ice. After washing three times with FACS buffer, 50 μl of phycoerythrin (PE)-conjugated goat anti-human IgGFc (Jackson ImmunoResearch) diluted 1:100 in FACS buffer was added. After 30 min on ice (in the dark), cells were washed three times and specific HuMab binding was detected by flow cytometry on a FACSCalibur (BD Biosciences). HuMab-KLH was used as a negative control. A mouse anti-TE antibody followed by a PE-conjugated anti-mouse IgGFc antibody was used as a positive control. Binding curves were analyzed using non-linear regression (sigmoidal dose-response curve with variable slope) using GraphPad San Diego, CA, USA software.

Fig. 4 shows an example of TF-specific HuMab binding curves to MDA-MB-231 cells. Tab. 4 gives an overview of the EC ₅ o values of TF-specific HuMab binding to TF-transfected CHO (S1015-TF), MDA-MB-231, A431 and Bx-PC3 cells.

Table 4 Overview of EC ₅₀ and maximum mean fluorescence index (max MFI) values determined by FACS binding analysis of TF-specific HuMabs to various cell types

MDA-MB-231 Vx-RSZ A431 S1015-TF-012 group HuMab TF EC50 Max MFI EC50 Max MFI EC50 Max MFI EC50 Max MFI I 13 1.58 2451 1.86 1305 8.04 3622 1.07 5207 I 44 0.87 1881 1.88 1136 1.45 2646 2.13 5021 I 87-Lg6 8.28 1107 7.19 1030 HT HT HT HT II eleven 0.47 2143 1.01 1280 0.20 2606 1.32 5654 II 017-D12 1.33 2401 1.61 1422 1.24 3296 1.21 5792 II 42 0.25 1518 2.45 1701 HT HT HT HT II 092-A09 0.53 2290 0.84 1262 0.83 31.37 1.32 5409 II 101 0.85 2071 2.25 1220 3.16 2934 1.77 5859 II/III 98 0.99 1956 1.38 1151 1.40 2755 0.96 5229 II/III 114 0.47 2438 0.80 1407 0.90 3433 1.72 6095 III 3 3.20 1798 4.98 1106 6.94 2530 2.06 4247 III 25 0.69 2254 0.88 1320 5D9 3170 0.73 5808 III 109 2.16 2052 4.04 1324 1.74 3124 0.92 5629 III 111 1.03 1774 1.83 1128 2.88 3043 0.55 5353

EC ₅₀ values are given in nM. Max MFI for MDA-MB-231, BxPCS and A431 cells at 30 µg/ml antibody, for S1015-TF at 7.5 µg/ml antibody.

Example 15 Inhibition of FVIIa Binding to TF

Inhibition of FVIIa binding to TFECDHis by TF-HuMab was measured by ELISA. ELISA plates were coated overnight with TFECDHis (0.5 μg/ml, 10 μl per well). The plates were emptied, blocked with PBS containing 2% (v/v) chicken serum (1h at room temperature), and emptied again. 4-fold serial dilutions of TF-HuMab or HuMab-KLH (negative control) were added to these wells followed by the addition of FVIIa at EC ₅ o concentration (100 nM) and the plates were incubated for 1 h at room temperature (with shaking, 300 rpm). The plates were washed and incubated with rabbit aHTH-FVIIa antibody (2.5 μg; Abeam) as described above. The plates were washed and incubated with porcine anti-rabbit IgG-HRP (1:2,500; DAKO). After washing, immune complexes were visualized using ABTS as a substrate. The reaction was stopped by adding 2% v/v oxalic acid followed by measurement of absorbance at 405 nm using an ELISA reader. The antibody concentration required to obtain 50% inhibition (1C ₅₀ ) was calculated using the program

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GraphPad prism (Nonlinear Regression Analysis).

Fig. 5 shows that antibodies from cross-block groups II and III effectively inhibited binding

FVIIa with TF, while antibodies from cross-block group I did not (or inhibited to a much lesser extent) FVIIa binding.

Table 5. IC ₅ o values and maximum inhibition values (percentage) of inhibition of FVII binding to TF TF-specific HuMab

Group HuMab TF IC50 nM maximum inhibition I 13 19.3 27 I 44 0.8 54 I 87-Lg6 pa 35 II eleven 1.1 91 II 017-D12 1.9 90 II 42 2.7 88 II 092-A09 1.5 90 II 101 0.6 84 F/W 98 0.8 85 F/W 114 1.3 90 III 3 1.9 89 III 25 2.1 90 III 109 1.7 90 III 111 1.7 79

Tab. 5 shows IC50 values and maximum inhibition values (percentage) of inhibition of FVIIa binding to TF-specific HuMab.

Example 16 Inhibition of Phosphorylation of FVIIa-Induced ERK

After binding of coagulation factor Vila (FVIIa) to TF, mitogen-activated kinase (p42 and p44 MAPK or ERK1 and ERK2) phosphorylation is initiated. The A431 epidermoid carcinoma cell line expresses high levels of TF, and after stimulation with FVIIa, optimal (3-5 fold) ERK phosphorylation (ERK-P) as measured using the AlphaScreen Surefire ERK assay (Perkin Elmer) is induced within 10 minutes.

A431 cells (30,000 cells per well) were plated in 96-well TC plates and cultured overnight (37°C, 5% CO ₂ , 85% humidity) in serum-free medium (RPMI containing 20% HSA and penicillin/streptomycin) . The medium was then replaced with DMEM (no additives) and the cells were incubated for 1.5 h. 3-fold serial dilutions of TF-HuMab or HuMab-KLH were added and the cells were incubated for 0.5 h. The cells were then stimulated with FVIIa at a concentration of EC ₅₀ ( 50 nM, 10 min, 37°C, 5% CO ₂ , humidity 85%). Cells were washed once with PBS and lysed using 25 μl of lysis buffer (Perkin Elmer, Surefire kit). Lysates were centrifuged (3 min, 330 xg, RT). Four μl of the supernatant was transferred to 384-well Proxyplates (Perkin Elmer). 7 μl of reaction buffer/activation buffer containing AlphaScreen beads (Perkin Elmer, Surefire kit) was added and the plates were incubated in the dark for 2 hours at room temperature. Plates were read using the Surefire Plus protocol from EnVision technology.

Fig. 6 shows that, as measured using the AlphaScreen Surefire ERK assay, antibody 013 does not inhibit FVIIa-induced phosphorylation, 044 and 111 modestly inhibit ERK phosphorylation, and all other antibodies effectively block ERK phosphorylation.

Tab. 6 shows IC ₅ o values and maximum inhibition values (percentage) of inhibition of FVIIa-induced ERK phosphorylation by TF-specific HuMab measured using the AlphaScreen Surefire ERK assay.

Table 6. IC ₅₀ values and maximum inhibition values (percentage) of inhibition of FVIIa-induced ERK phosphorylation (measured using the AlphaScreen assay

Surefire ERK) TF-specific HuMab

Group HuMab TF IC50 nM % maximum inhibition I 13 9.11 26 I 44 >66.6 45 I 87-Lg6 NT NT II eleven 0.79 69 II 017-D12 2.01 65 II 42 NT NT II 092-A09 1.27 68 II 101 1.05 57 F/W 98 1.89 64 F/W 114 1.08 68 III 3 7.99 63 III 25 2.16 66 III 109 2.42 72 III 111 >66.6 52

AlphaScreen Surefire ERK assay results were confirmed by Western blot analysis

-48041176 using cell lines HaCaT and BxPCS. 30,000 cells/well were seeded in DMEM containing minimal serum concentrations (fasting medium) and cultured overnight. Cells were additionally cultured for 2 h in DMEM without serum, anti-TF antibodies were added during the last 30 min of culture. Cells were stimulated with 0, 10, or 50 nM FVIIa for 10 min (37° C.) and then lysed in cell lysis buffer (50 μl lysis buffer per well, 30-60 min lysis under shaking conditions, at room temperature). 25 μl of SDS-containing sample buffer was added to each sample. Samples were applied to SDS-PAGE electrophoresis gels, electrophoresed and blotted using standard Western blot procedures. Blots were blocked with TBSTIx-containing 5% non-assay protein (ELK) for 1 h at room temperature. Blots were incubated with rabbit anti-ERK-P antibody (overnight, 4°C). Blots were washed with TBSTIx and incubated with anti-rabbit IgG-HRP antibody (1 h at room temperature), washed, developed with HRP substrate, and imaged using the Optigo Ultima Imaging system (Isogen Life Sciences).

Fig. 6a shows results in BxPC3 cells for a sub-panel of antibodies. ERK fosorylation induced by 10 nM FVIIa was not inhibited by antibody 013, while it was effectively inhibited by antibodies 111, 044 and 025 (the latter as an example for all other TF-specific HuMabs described here). More strongly induced ERK phosphorylation (50 nM FVIIa) was not inhibited by antibodies 013, 111 and 044, but was inhibited by antibody 025.

Example 17 Inhibition of FVIIa-Induced IL-8 Release

The ability of TF-specific HuMabs to inhibit FVIIa-induced IL-8 release was tested using MDA-MB-231 cells. Cells were seeded in 96-well plates (60,000 cells per well) and cultured (overnight, 37°C, 5% CO ₂ ) in DMEM containing CS, sodium pyruvate, L-glutamine, MEM NEAA and penicillin/streptomycin. Tissue culture medium was removed, cells were washed twice with serum-free calcium-rich medium (DMEM containing penicillin/streptomycin) and cultured in this medium for an additional 105 min. Serial dilutions of antibodies were added and cells were cultured for 15 min. FVIIa (Novo Nordisk; final concentration 10 nM) was added and cells were cultured for 5 hours. The supernatant was removed and centrifuged (300xg, RT). IL-8 concentrations in the supernatant were measured using an IL-8 ELISA kit according to the manufacturer's protocol (Sanquin).

Fig. 7 shows that antibodies from cross-block groups II and III effectively inhibited FVIIa-induced release of IL-8 by MDA-MB-231 cells, with the exception of antibody 111 from cross-block group III. Antibodies from cross-block group I (013, 044 and 87-Lg6) all did not inhibit FVIIa-induced release of IL-8.

Tab. 7 shows IC ₅₀ values and maximum inhibition values (percentage) of inhibition of FVIIa-induced IL-8 release by TF-specific HuMab.

Table 7. IC ₅₀ values and maximum inhibition values (percentage) of inhibition of FVIIa-induced IL-8 release by TF-specific HuMab

Group HuMab TF IC50 nM maximum inhibition I 13 pa -0.3 I 44 74.6 17.2 I 87-Lg6 pa 4.3 II eleven 9.4 61.7 II 017-D12 9.0 65.8 II 42 14.9 53.7 II 092-A09 28.2 66.6 II 101 22.7 74.9 F/W 98 9.3 59.0 F/W 114 9.2 71.5 III 3 23.7 76.2 III 25 23.1 75.6 III 109 13.6 70.4 III 111 >200 40.1

Example 18 Inhibition of FXa Generation

The ability of TF-specific HuMabs to inhibit Fxa generation was tested in an assay in which the conversion of FX to Fxa by the TF/FVIIa complex is measured using a colorimetric Fxa-specific substrate. TF (Innovin) was added to flat-bottomed 96-well plates along with serial dilutions of TF-specific HuMab, positive control (mouse anti-TF), or negative control (HuMab-KLH) (all diluted in Hepes buffer containing 3 mM CaCl ₂ . The plates were incubated for 30 min at room temperature and FVIIa (final concentration 1 nM) and FX (ERL; final concentration 200 nM) were added.The plates were incubated for 30 min at 37° C. 50 μl from each well was transferred into a 96-well plate, containing (preheated, 37°C) stop buffer (5 mM EDTA in 100 ml Hepes buffer) Fxa-specific substrate Chromogenix-49 041176 was added

2765 (Instrumation Laboratory Company), plates were incubated for 60 min at 37°C and measured

OD ₄₀₅ im at 37°C.

Fig. 8 shows that 017-D12 antibody strongly inhibited FXa generation, 013 showed intermediate inhibition and other antibodies showed no inhibition of FXa generation.

FXa.

Tab. 8 shows IC ₅₀ values and maximum inhibition values (percentage) of inhibition of FXa generation by TF-specific HuMab.

Table 8. IC ₅₀ values and maximum inhibition values (percentage) of inhibition of ___________ FXa generation by TF-specific HuMab______

group HuMab TF IC50 nM maximum inhibition % I 13 0.05 31 I 44 ΝΑ 3 I 87-Lg6 NT NT II eleven 0.05 26 II 017-D12 0.28 84 II 42 NT NT II 092-A09 0.30 21 II 101 nt NT II/III 98 0.43 14 II/III 114 0.24 21 III 3 0.07 21 III 25 0.30 19 III 109 0.09 18 III 111 0.07 7

Example 19 Inhibition of blood coagulation

Inhibition of blood coagulation by TF-HuMab was measured in the TF-induced clotting time assay. Mixtures of 17 µl 100 mM CaCl ₂ (final concentration 17 mM), 10 µl 1:100 innovine (final concentration 1:1000), 23 µl 1x FIEPES buffer and 50 µl serially diluted antibody were prepared in 96-well plates. 50 μl of human plasma was added to the wells of Immulon 2B plates (Thermo Electron). 50 μl of the prepared antibody mixtures were added to Immulon 2b plates and the development of coagulation at 405 nm was measured every 15 s for 25 min using a kinetic plate reader. The increase in optical density was plotted against time and the clotting time (t1/2) was calculated. The clotting time was built depending on the concentration of antibodies. The _IC50 of antibody-induced coagulation inhibition was calculated from these data by non-linear regression analysis using GraphPad Prism.

Fig. 9 shows that antibodies 044, 087 and 111 did not inhibit TF-induced blood coagulation, while all other antibodies did.

Tab. 9 shows the IC ₅₀ values of inhibition of blood coagulation by TF-specific HuMab.

Table 9. IC ₅₀ values for inhibition of blood coagulation by TF-specific HuMab

group HuMab TF IC50hM I 13 0.6 I 44 NA I 87-Lg6 NA II eleven 1.6 II 017-D12 2.6 II 42 1.5 II 092-A09 0.2 II 101 0.7 II/III 98 1.1 II/III 114 0.4 III 3 7.3 III 25 2.3 III 109 7.6 III 111 NA

Example 20 Antibody-Dependent Cell-Mediated Cytotoxicity

Preparation of target cells:

TF-expressing target cells (5 x 10 ⁶ Bx-PC3 cells, MDA-MB-231 cells, or A431 cells) were harvested, washed (twice in PBS, 1500 rpm, 5 min) and harvested in 1 ml RPMI 1640 culture medium, supplemented with Cosmic Calf serum, sodium pyruvate, L-glutamine, MEM NEAA, and penicillin/streptomycin, to which 100 μCi of ⁵¹ Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands) was added. This mixture was incubated in a shaking water bath for 1 hour at 37°C. After washing the cells (twice in PBS, 1500 rpm, 5 min), the cells were resuspended in culture medium and viable cells were counted as a trypan blue dye exclusion method. Viable cells were adjusted to a concentration of 1 x 10 ⁵ cells/ml.

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Preparation of effector cells:

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats (Sanquin, Amsterdam, The Netherlands) using standard Ficoll gradient centrifugation according to the manufacturer's instructions (lymphocyte separation medium; Lonza, Verviers, France). After resuspending the cells in the culture medium, the cells were counted by the trypan blue displacement method and adjusted to a concentration of 1x107 cells/ml.

Establish ADCC:

µl of ⁵¹ Cr-labeled target cells were transferred to microtiter wells and 50 µl of serially diluted antibody diluted in culture medium was added. Cells were incubated (RT, 15 min) and 50 μl of effector cells were added to give an effector:target ratio of 100:1. To determine the maximum level of lysis, 100 µl of 5% Triton-X100 was added instead of effector cells; to determine the spontaneous level of lysis, 100 μl of the culture medium was added; to determine the level of antibody-independent lysis, 50 μl of effector cells and 50 μl of culture medium were added). The cells were then incubated overnight at 37°C, 5% CO2. After centrifugation of these cells (1200 rpm, 3 min), 75 μl of the soup was transferred into micron tubes. The released ⁵¹ Cr was counted in a gamma counter and the percentage of antibody-mediated lysis was calculated as follows: ((imp/min of sample-imp/min of antibody-independent lysis)/(imp/min of maximum lysis-imp/min of spontaneous lysis))x100%, where cpm is the number of pulses per minute.

Fig. 10 shows that all TF-HuMabs tested induced lysis of Bx-PC3 cells by ADCC, albeit with varying efficiencies (EC ₅₀ ).

Tab. 10 shows EC ₅₀ values (nM) of ADCC of various cell lines with TF-specific HuMab.

Table 10. EC ₅₀ values (nM) of ADCC of various cell lines with TF-specific HuMab MDA-MB-231 Bx-RS3 A431

group HuMab TF EU50 EC50 EC50 I 13 0.06 0.07 0.11 I 44 0.08 0.12 0.19 I 87-Lg6 nt nt nt II eleven 0.07 0.22 0.06 II 017-D12 0.14 0.13 0.18 II 42 nt nt nt II 092-A09 0.11 0.13 0.22 II 101 0.10 0.09 0.01 Will 98 0.15 0.02 0.07 Will 114 0.07 0.07 0.08 III 3 0.29 0.17 0.58 III 25 0.24 0.15 0.16 III 109 0.12 0.06 0.13 III 111 0.84 0.22 1.56

Example 21 Complement Deposition

The deposition of complement fragments C3c and C4c to TF-HuMab incubated target cells was measured by FACS analysis. TF-expressing target cells (Bx-PC3 or MDAMB-231 cells) were seeded in 96-well round bottom plates (1 x 10 ⁵ cells per well) in RPMI containing 1% BSA. Antibody (30 μg/ml) was added and cells were incubated at room temperature for 15 min. 25 μl of pooled human serum was added as a source of complement, heat inactivated human serum was used to determine spontaneous complement fixation. Cells were incubated at 37°C for 45 min. Cells were washed once and incubated with anti-human C3c-FITC or anti-human C4c-FITC (DAKO) in FACS buffer and incubated for 30 min on ice. Samples were analyzed using FACS Canto.

Fig. 11 shows that antibodies from cross-block group I did not induce C3c or C4c deposits on either BxPC3 cells or MDA-MB-231 cells. All tested antibodies from cross-block group II did not induce deposits of C3c and C4c, as well as antibodies from cross-block group III, with the exception of antibody 003.

Example 22 Avidity/Affinity Studies

Definition of affinity:

Antibody binding to TF was analyzed by surface plasmon resonance in a BIAcore 3000 (GE Healthcare). TFECDHis was used for this analysis. HuMab antibodies (500 resonance units) were immobilized on the CM-5 sensor chip according to the manufacturer's recommended procedure. Briefly, after activation using EDC and NHS, HuMab antibody was injected onto this activated surface of CM-5 in 10 mM sodium acetate, pH ranging from 4.0 to 5.5 at 5 µl/min, followed by the addition of 1 M ethanolamine for deactivation. . Concentration series of TFECDHis in HBS-EP buffer were injected onto immobilized antibodies at a flow rate of 30 μl/min for 180 s. HuMab surface regeneration was performed by injection of 10 mM Glycine-HCl or 10 mM sodium acetate pH

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3.0. Kinetic analysis was performed using double reference subtraction and 1:1 model (langmuir) binding analysis.

Tab. 11 shows for most HuMabs a certain affinity in the (sub) nanomolar range. Kinetic parameters could not be determined for all antibodies. 044 did give a high variation in dissociation rates (kd) and had high residues, which means that the fit of these curves was not good. 098, 111 and 087-Lg6 had dissociation rates that were too high to be measured with Biacore 3000. na not assessed >10 ^-3 s ^-1

Table. 11. Kinetic constants of TF-HuMab for reactivity with ______________TFECDHis - affinity measurements_________

group HuMabTF affinity nM ka(l/Mc) kd (1/s) I 13 2.78 5.67E+05 1.57E-03 I 44 η. A. 8.77E+04 variables I 87-Lg6 η. A. 5.91 E+05 n. A. II eleven 3.15 2.86E+05 9.02Е-04 II 017-D12 2.55 1.02E+05 2.59Е-04 II 42 4.22 1.64E+05 6.90E-04 II 092-A09 14.1 1.42E+05 2.00Е-03 II 101 3.4 3.18E+05 1.07Е-03 F/W 98 η. A. 2.90E+05 n. A. F/W 114 eleven 1.77E+05 1.95E-03 III 3 4.51 2.33E+05 1.26E-03 III 25 1.97 3.29E+05 6.50Е-04 III 109 4.75 1.65E+05 7.77E-04 III 111 η. A. 2.13E+05 n. A.

Definition of avidity:

Binding of TF (TFECDHis) to TF-specific HuMabs was determined essentially as described above, with TFECDHis being immobilized on a CM-5 sensor chip (300 resonant units) and a range of Humab antibody concentrations used for kinetic analysis. Kinetic analysis was performed using double reference subtraction and 1:1 model (langmuir) binding analysis.

Tab. 12 shows avidity measurements for antibodies 11, 98, 109, and 111. While affinity measurements for 98 and 111 showed high dissociation rates (above Biacore detection limits (i.e., >10 ^-3 )), avidity determination revealed interactions in the nanomolar range.

Table 12. Kinetic constants TFECDHis for reactivity with TF-HuMab - avidity measurements

group HuMab TF avidity nM II eleven 0.47 F/W 98 4.85 III 109 0.01 III 111 0.11

Example 23 Immunohistochemical Binding Assay with Normal Human Tissues and Pancreatic Tumors

Binding of TF-HuMab to various human tissues known to express TF (colon, heart, kidney, skin, lung and brain) was determined by immunohistochemistry (IHC).

IHC on frozen tissue

Frozen tissue sections were cut (4-6 µm thick) and fixed in acetone. Endogenous tissue peroxidase (PO) was blocked and tissue slides were preincubated with normal human serum to prevent aseptic binding of later applied antibodies to endogenous Fc receptors. Mouse Abs directed against human TF (and mouse Abs as a negative control) were applied to these tissues at optimal dilution and then detected using Powervision-PO (goat anti-mouse/rabbit IgG-PO). TF-specific HuMab was bound to Fab' goat anti-human IgG (Fc)-FITC and then applied to frozen tissue slides at 3 dilutions, including a predetermined optimal dilution. The HuMab-Fab-FITC complex was then detected with rabbit anti-FITC and Powervision-PO. PO activity was visualized using AEC as a substrate and the nucleus was visualized with hematoxylin. Staining was analyzed under a bright field microscope.

IHC with mouse Ab on formalin-fixed and paraffin-embedded (FFPE) tissue

Biopsies of FFPE tissue were cut at 4 μm, deparaffinized, blocked for endogenous tissue peroxidase, and subjected to antigen reconstitution ((pH6, citrate buffer). Prior to incubation with mouse Ab, tissue slides were preincubated with normal human serum to prevent aseptic binding to endogenous Fc- receptors.

- 52 041176 directed against human TF (and mouse AB as a negative control) were applied to these tissue slides at optimal dilution and then detected using Powervision-PO (goat anti-mouse/rabbit IgG-PO). PO activity was visualized using AEC as a substrate and the nucleus was visualized with hematoxylin. Staining was analyzed under a bright field microscope.

Fig. 12 shows an example of antibody 013 (positive stain), OH (positive stain), 114 (positive stain) and 111 (intermediate stain) binding to the glomeruli of the kidney. Antibody 098 and 044 do not bind glomeruli.

Tab. 13 gives an overview of the staining results for all TF-HuMabs in all human kidney tissues tested.

Table 13.1 HC staining of human glomeruli

group HuMab TF IHC human glomeruli I 13 + I 44 - I 87-Lg6 nt II eleven + II 017-D12 + II 42 nt II 092-A09 nt II 101 + p/w 98 - plp 114 + III 3 + III 25 nt III 109 + III 111 +/-

Tab. 14 provides an overview of staining results for selected TF-specific HuMab in human kidney, colon, heart, brain, and skin, and human pancreatic tumors.

Table 14. IHC staining of normal tissue and human pancreatic tumors

Ab human kidney human colon human heart human brain Human skin Pankr. tumor 13 renal corpus + basement membrane++ - + epidermis + +++ 114 renal corpus ++ basement membrane++ - ++ epidermis ++++ eleven renal corpus + basement membrane++ - ++ p.a. (+) +++ 44 - basement membrane+ - +/- p.a. ++ 98 - basement membrane+ - +/- p.a. (+) +++ 111 renal corpus +/- basement membrane+ - + p.a. +++

HS binding analysis of TF-HuMab to human pancreatic tumors revealed positive staining for all TF-HuMab (illustrated in FIG. 13).

Example 24 Treatment of established MDA-MB-231 tumor xenograft in SCID mouse mammary fat pads

The in vivo efficacy of TF-HuMab was determined in established orthotopic tumors of MDA-MB-231 xenografts in SCID mice. 2x10 ⁶ tumor cells in PBS were injected sc into the 2nd mammary fat pad of female SCID mice followed by treatment with TFHuMab or control mAb (HuMab-KLH) starting when tumors became measurable. Antibodies were injected on day 21 (260 μg/mouse), day 28 (130 μg/mouse and day 42 (130 μg/mouse). Tumor volume was determined at least 2 times per week. Volumes (mm ³ ) were calculated from caliper measurements ( PLEXX) as 0.52x(length)x(width) ² .

Fig. 14 shows that antibodies 114, 111, 013, 098, 011 and 044 were all effective in inhibiting the growth of established MDA-MB-231 tumors.

Example 25 Pilot Repetitive Dosing of TF-Specific HuMab in Canine Monkeys

To obtain initial information on the toxicology of TF-specific HuMab, including evaluating the ability of antibodies to interfere with the coagulation cascade and therefore increase the risk of bleeding in exposed animals, a pilot repeated dosing study was performed in dog-headed monkeys.

Two male and two female cynomolgus monkeys (Macaca fascicularis), approximately 2 years old, received intravenous injections of antibody 011:

-53 041176 study day 1: 0 mg/kg (vehicle only) day 8: 1 mg/kg; 1 ml/min day 15: 10 mg/kg; 1 ml/min day 22: 100 mg/kg; 1 ml/min

These animals were observed until day 27, at which time the animals were euthanized for necropsy and histological evaluation of organs.

The main endpoints of this study were clinical observations: measured daily, signs of bleeding from the gums, eyes;

functional bleeding time and blood loss: determined at days 1, 8, 15 and 22 (1, 24 and 120 hours post-dose) and two pre-test time points;

blood/blood traces/clots: non-staining of all tissues (determined in tissues obtained at the final sacrifice);

blood in urine, faeces, vomit: determined daily/weekly.

No apparent toxicity was observed with repeated, escalating doses of antibody 011. Animals showed no clinical symptoms and there was no indication of cytokine release. In addition, there were no visible clinical symptoms of an impaired coagulation system or systemic bleeding. At the 1 hour post-dose time point, mean bleeding time on day 22 was significantly higher than that observed on day 1 (p=0.012). There were no other statistically significant differences between days 8, 15 and 22 compared to day 1. In addition, it was found that there was no apparent major organ toxicity and no deleterious hematological effects. The preliminary conclusion from the histological evaluation of tissues from this study is that there were no histological findings in the four treated animals that could be attributed to test antibody treatment.

Fig. 15 shows individual data points for each animal (duplicate samples) versus time. Bleeding time for 4 animals was determined on days 1, 8, 15 and 22 (1, 24 and 120 hours) and at two time points before the study.

Example 26 Preventive and Therapeutic Treatment of BxPC3 Tumor Xenografts in SCID Mice

The in vivo efficacy of TF-HuMab in the preventive or therapeutic treatment of BxPC3 cell xenografts in SCID mice was determined. 10x106 BxPC3 tumor cells in PBS were injected sc into female SCID mice, followed by treatment with TF-HuMab or control mAb (HuMab-KLH). For prophylactic treatment, antibodies (400 μg/mouse) were injected ip 1 hour after tumor induction. For therapeutic treatment, antibody injection (300 μg/mouse) was started on day 8 after tumor induction, followed by weekly antibody injections (150 μg/mouse). Tumor volume was determined at least 2 times a week. Volumes (mm ³ ) were calculated from caliper measurements (PLEXX) as 0.52x (length) x (width) ² .

Fig. 16 shows that TF-specific HuMabs are able to prevent as well as treat BxPC3 xenograft tumors.

Example 27 DNA shuffling between mouse and human TF to determine domains important for anti-TF-HuMab binding

DNA shuffling between human and mouse TF was performed to determine domains important for anti-TF-HuMab binding to human TF. Shuffle constructs were generated from DNA encoding human TF by replacing human domains with mouse domains, and from DNA encoding mouse TF by replacing mouse domains with human domains. If a domain in human TF is important for anti-TF-HuMab binding, binding will be lost when that domain is replaced with a mouse domain. Human and mouse TFs are 57% homologous at the protein level. Fig. 17A and 17B show constructs for human TF containing mouse domains (TFh containing TFmm domains) and for mouse TF containing human TF domains. HEK293F cells were transiently transfected with these constructs or with a single vector (pcDNA3.3SP; pseudo). FACS analysis was performed essentially as described by supra with 30 μg/ml of purified starting material. HuMab-KLH was used as a control Ab.

Fig. 17 shows that all but one anti-TF-HuMab only binds to human TF and not to mouse TF. HuMab-TF-003 shows some binding to mouse TF.

Fig. 18A-O shows the results of binding of various anti-TF-HuMabs to constructs expressed on HEK293F cells. These results are summarized in Table. 15. In this table, anti-TFHuMabs are classified into groups based on domains on human TF that are important for the binding of these HuMabs.

- 54 041176

Shuffled TFh constructions HuMab who discover reduced binding 1-41mm 42-84 mm Nothing 11, 17, 42, 92, 98, 101, 111 85-122 mm 123-137 mm 25, 42, 98, 109, 111 44.114 185-225 mm 13, 27, 44, 87 226-250 mm 44 Groups based on linking shuffled constructs HuMab is in this group 1.42-84 2.42-84 + 85-122 11, 17, 92, 101 42, 98, 111 3.85-122 25.109 4. 123-137 5.185-225 114 13, 27, 87 6. 123-137 + 185-225 + 226-250 44

Table 15

Example 28 Anti-TF-HuMab Fab Fab Binding to TF Extracellular Domain by ELISA and to Cellular TF on BxPC3 Cells by FACS

The binding of anti-TF-HuMab Fab fragments to TF was measured by ELISA (coated extracellular domain of TF) and by FACS (TF on BxPC3 cells). ELISA was performed essentially as described by supra. Bound Fab fragments were detected using an FtRP-conjugated donkey anti-human H+L antibody. FACS analysis was performed essentially as described by supra. FITC-conjugated goat anti-human IgG (H+L) (Jackson) was used to detect associated prime candidates. Fluorescence was measured on FACSCantoII. Binding curves were analyzed as described by supra using GraphPad Prism 5 software.

Fig. 19 shows less binding of HuMab-TF-098 and -111 Fab fragments to the extracellular domain of TF compared to -011 Fab fragments measured by ELISA.

Fig. 20 shows less binding of HuMab-TF-098 and -111 Fab fragments to the TF cellular domain compared to -011 Fab fragments measured by FACS on BxPC3 cells.

Tab. 16 shows EC ₅₀ values of HuMab-TF Fab fragments for binding to the extracellular domain of TF according to ELISA and to cellular TF according to FACS on BxPC3 cells.

Table 16 Overview of EC50 values for binding of HuMab-TF Fab fragments to extracellular TF by ELISA and to cellular TF by FACS on BxPC3 cells

HuMab-TF EC50 (ELISA) EU50 (FACS) HE 0.04 0.132 013 0.03 0.301 044 0.59 8.040 098 1.98 n.a. 109 0.02 0.143 111 3.14 pa

EC ₅₀ values are given in µg/ml.

na - could not be calculated.

Example 29 Anti-TF-HuMab Binding to Cell Lines Expressing Different Levels of TF

The binding of anti-TF-HuMab to membrane-bound TF on cell lines expressing different levels of TF was determined by FACS analysis essentially as described by supra. A mouse anti-TF antibody followed by a PE-conjugated anti-mouse IgGFc antibody was used as a positive control. Fluorescence was measured on FACSCantoII. Binding curves were analyzed essentially as described by supra using GraphPad Prism 5 software. The number of TF molecules on cell lines was determined using a Qifi kit (Dako, Glostrup, Denmark) according to the manufacturer's instructions. It was determined that SW480 cells express ~20,000 TF molecules per cell, SK-OV-3 cells express ~60,000 molecules per cell, AsPC1 cells express ~175,000 molecules per cell, and MDA-MB-231 cells express ~900,000 molecules per cell.

Fig. 21 - HuMab-TF-98 and -111 show similar binding characteristics to HuMab-TF-11, -13 and -109 of the MDA-MD-231 cell line expressing high amounts of TF. In cell lines with fewer TF molecules per cell, such as SK-OV-3 and SW480 cell lines, HuMab-TF-98 and 111 show different binding characteristics compared to other HuMab-TF antibodies.

- 55 041176

Sequence listing <110> Genmab < 120> Tissue factor <130> P57 <160> 112 < 170> Patentin version 3.2 <210> 1 <211> 126 < 212> protein < 213> Homo sapiens < 400> 1

Glu Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Glu 15 1015

Ser Leu Lys He Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 2530

Trp He Gly Trp Vai Arg Gin Met Pro Gly Lys Gly Leu Glu Trp Met 35 4045

Gly lie lie Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 5560

Gin Gly Gin Vai Thr lie Ser Ala Asp Lys Ser lie Ser Thr Ala Tyr 65 70 7580

Leu Gin Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 9095

Ala Arg lie His Arg Gly Ala Gly Tyr Ser Ser Ser Ser Trp Pro Gly Ala

100 105110

Phe Asp lie Trp Gly Gin Gly Thr Met Vai Thr Vai Ser Ser

115 120125 < 210> 2 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 2

Gly Tyr Ser Phe Thr Ser Tyr Trp 1 5

- 56 041176 <210> 3 <211> 8 <212> protein <213> Homo sapiens <400> 3

He Tyr Pro Gly Asp Ser Asp Thr 1 5 < 210> 4 < 211> 19 < 212> protein < 213> Homo sapiens < 400> 4

Ala Arg He His Arg Gly Ala Gly Tyr Ser Ser Ser Ser Trp Pro Gly Ala 15 10 15

Phe Asp lie < 210> 5 <211> 119 < 212> protein < 213> Homo sapiens < 400> 5

Gin Vai Gin Leu Vai Glu Ser Gly Gly Gly Vai Vai Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Vai Ala Ser Gly Phe Thr Vai Ser Asn Asp 20 2530

Gly Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

4045

Ala Leu lie Trp Tyr Asp Gly Vai Asn Lys Asn Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr lie Ser Arg Asp Lys Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Arg Pro Gly Thr Phe Tyr Gly Leu Asp Vai Trp Gly Gin Gly

100 105110

- 57 041176

Thr Thr Vai Thr Vai Ser Ser

115 <210> 6 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 6

Gly Phe Thr Vai Ser Asn Asp Gly 1 5 < 210> 7 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 7

He Trp Tyr Asp Gly Vai Asn Lys 1 5 < 210> 8 < 211> 12 < 212> protein < 213> Homo sapiens < 400> 8

Ala Arg Arg Pro Gly Thr Phe Tyr Gly Leu Asp Vai

5 10 <210> 9 <211> 118 <212> protein <213> Homo sapiens <400> 9

Glu Vai Gin Leu Leu Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly Gly 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 2530

Ala Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

4045

Ser Ser He Ser Gly Ser Gly Asp Tyr Thr Tyr Tyr Thr Asp Ser Vai 50 5560

- 58 041176

Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Ser Pro Trp Gly Tyr Tyr Leu Asp Ser Trp Gly Gin Gly Thr

100 105110

Leu Vai Thr Vai Ser Ser

115 <210> 10 <211> 8 <212> protein <213> Homo sapiens <400> 10

Gly Phe Thr Phe Ser Asn Tyr Ala 1 5 < 210> 11 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 11

He Ser Gly Ser Gly Asp Tyr Thr 1 5 < 210> 12 < 211> 11 < 212> protein < 213> Homo sapiens < 400> 12

Ala Arg Ser Pro Trp Gly Tyr Tyr Leu Asp Ser

5 10 <210> 13 <211> 120 <212> protein <213> Homo sapiens <400> 13

Glu Vai Gin Leu Leu Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly Gly 15 10 15

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

- 59 041176

2530

Ala Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

4045

Ser Ala He Ser Gly Ser Gly Asp Ser Thr Asn Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Lys Asp Gly Tyr Phe Leu Leu Trp Tyr Phe Asp Leu Trp Gly Arg

100 105110

Gly Thr Leu Vai Thr Vai Ser Ser

115120 <210> 14 <211> 8 <212> protein <213> Homo sapiens <400> 14

Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 < 210> 15 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 15 lie Ser Gly Ser Gly Asp Ser Thr

5 <210> 16 <211> 13 <212> protein <213> Homo sapiens <400> 16

Ala Lys Asp Gly Tyr Phe Leu Leu Trp Tyr Phe Asp Leu

5 10 <210> 17

- 60 041176 <211> 118 <212> protein <213> Homo sapiens <400> 17

Glu Vai Gin Leu Leu Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly Gly 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 2530

Gly Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

4045

Ser Vai He Ser Gly Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Lys Ala Pro Trp Thr Tyr Tyr Phe Asp Tyr Trp Gly Gin Gly Thr

100 105110

Leu Vai Thr Vai Ser Ser

115 < 210> 18 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 18

Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 19 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 19 lie Ser Gly Ser Gly Gly Thr Thr

5 <210> 20

- 61 041176 <211> 11 <212> protein <213> Homo sapiens <400> 20

Ala Lys Ala Pro Trp Thr Tyr Tyr Phe Asp Tyr

5 10 <210> 21 <211> 118 <212> protein <213> Homo sapiens <400> 21

Glu Vai Gin Leu Leu Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly Gly 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 2530

Ala Met Ser Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

4045

Ser Ser He Ser Gly Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Lys Thr Pro Trp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gin Gly Thr

100 105110

Leu Vai Thr Vai Ser Ser

115 <210> 22 <211> 8 <212> protein <213> Homo sapiens <400> 22

Gly Phe Thr Phe Asn Asn Tyr Ala 1 5 <210> 23

- 62 041176 <211> 8 <212> protein <213> Homo sapiens <400> 23

He Ser Gly Ser Gly Gly Arg Thr 1 5 < 210> 24 < 211> 11 < 212> protein < 213> Homo sapiens < 400> 24

Ala Lys Thr Pro Trp Gly Tyr Tyr Phe Asp Tyr

5 10 <210> 25 <211> 118 <212> protein <213> Homo sapiens <400> 25

Glu Vai Gin Leu Leu Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly Gly 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 2530

Ala Met Ser Trp Vai Arg Gin Ala Pro Ala Lys Gly Leu Asp Trp Vai

4045

Ser Gly lie Ser Gly Ser Gly Vai Thr Thr Tyr Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Phe Cys 85 9095

Ala Lys Thr Pro Trp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gin Gly lie

100 105110

Leu Vai Ala Vai Ser Ser

115 <210> 26

- 63 041176 <211> 8 <212> protein <213> Homo sapiens <400> 26

Gly Phe Thr Phe Ser Asn Tyr Ala 1 5 <210> 27 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 27

He Ser Gly Ser Gly Vai Thr Thr 1 5 < 210> 28 < 211> 11 < 212> protein < 213> Homo sapiens < 400> 28

Ala Lys Thr Pro Trp Gly Tyr Tyr Phe Asp Tyr

5 10 <210> 29 <211> 118 <212> protein <213> Homo sapiens <400> 29

Gin Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Ser 15 1015

Ser Vai Lys Vai Ser Cys Lys Ala Pro Arg Gly Thr Phe Ser Tyr Tyr 20 2530

Thr lie Ser Trp Vai Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met

4045

Gly Arg lie lie Pro lie Leu Gly Vai Ala Asn Tyr Ala Gin Lys Phe 50 5560

Gin Gly Arg Vai Thr lie Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 7580

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

- 64 041176

Ala Arg Glu Gly Asp Arg Arg Tyr Phe Asp Tyr Trp Gly Gin Gly Thr

100 105 110

Leu Vai Thr Vai Ser Ser

115 <210> 30 <211> 8 <212> protein <213> Homo sapiens <400> 30

Arg Gly Thr Phe Ser Tyr Tyr Thr 1 5 < 210> 31 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 31

He lie Pro lie Leu Gly Vai Ala 1 5 < 210> 32 < 211> 11 < 212> protein < 213> Homo sapiens < 400> 32

Ala Arg Glu Gly Asp Arg Arg Tyr Phe Asp Tyr

5 10 <210> 33 <211> 120 <212> protein <213> Homo sapiens <400> 33

Gin Vai Gin Leu Vai Glu Ser Gly Gly Gly Vai Vai Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 2530

Ala Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

4045

- 65 041176

Ala Vai He Ser Asn Asp Gly Tyr Asn Asp Tyr Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr Vai Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Asp Gly Gin Leu Gly Arg Gly Tyr Phe Asp Tyr Trp Gly Gin

100 105110

Gly Thr Leu Vai Thr Vai Ser Ser

115120 <210> 34 <211> 8 <212> protein <213> Homo sapiens <400> 34

Gly Phe Thr Phe Ser Arg Tyr Ala 1 5 < 210> 35 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 35

He Ser Asn Asp Gly Tyr Asn Asp 1 5 < 210> 36 < 211> 13 < 212> protein < 213> Homo sapiens < 400> 36

Ala Arg Asp Gly Gin Leu Gly Arg Gly Tyr Phe Asp Tyr

5 10 <210> 37 <211> 120 <212> protein <213> Homo sapiens <400> 37

- 66 041176

Gin Vai Gin Leu Vai Glu Ser Gly Gly Gly Vai Vai Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Pro Ala Ser Gly Phe Thr Phe Ser He Tyr 20 2530

Ala Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

4045

Ala Vai Vai Ser Asn Asp Gly Tyr Asn Lys Tyr Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Asp Gly Gin Leu Gly Arg Gly Tyr Phe Asp Tyr Trp Gly Gin

100 105110

Gly Thr Leu Vai Thr Vai Ser Ser

115120 <210> 38 <211> 8 <212> protein <213> Homo sapiens <400> 38

Gly Phe Thr Phe Ser lie Tyr Ala 1 5 < 210> 39 < 211> 8 < 212> protein <213> Homo sapiens <400> 39

Vai Ser Asn Asp Gly Tyr Asn Lys 1 5 <210> 40 <211> 13 <212> protein <213> Homo sapiens <400> 40

- 67 041176

Ala Arg Asp Gly Gin Leu Gly Arg Gly Tyr Phe Asp Tyr 1 5 10 <210> 41 <211> 118 <212> protein <213> Homo sapiens <400> 41

Gin Vai Gin Leu Vai Glu Ser Gly Gly Gly Vai Vai Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 2530

Ala Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai

4045

Ala Vai He Pro Tyr Asp Gly Asp Asn Lys Tyr Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Glu Asp Trp Gly Leu Glu Vai Asp Tyr Trp Gly Gin Gly Ala

100 105110

Leu Vai Thr Vai Ser Ser

115 <210> 42 <211> 8 <212> protein <213> Homo sapiens <400> 42

Gly Phe Thr Phe Ser Asp Tyr Ala 1 5 <210> 43 <211> 8 <212> protein <213> Homo sapiens <400> 43

- 68 041176

He Pro Tyr Asp Gly Asp Asn Lys 1 5 <210> 44 <211> 11 <212> protein <213> Homo sapiens <400> 44

Ala Arg Glu Asp Trp Gly Leu Glu Vai Asp Tyr

5 10 <210> 45 <211> 121 <212> protein <213> Homo sapiens <400> 45

Glu Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Glu 15 1015

Ser Leu Lys He Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Cys

2530

Trp lie Gly Trp Vai Arg Gin Met Pro Gly Lys Gly Leu Glu Trp Met

4045

Gly lie lie Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 5560

Gin Gly Gin Vai Thr lie Ser Ala Asp Lys Ser lie Ser Thr Ala Tyr 65 70 7580

Leu Gin Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 9095

Ala Arg His Lys Leu Gly Met Asp His Asp Ala Phe Asp lie Trp Gly

100 105110

Gin Gly Thr Met Vai Thr Vai Ser Ser

115120 <210> 46 <211> 8 <212> protein <213> Homo sapiens <400> 46

- 69 041176

Gly Tyr Ser Phe Thr Ser Cys Trp 1 5 <210> 47 <211> 8 <212> protein <213> Homo sapiens <400> 47

He Tyr Pro Gly Asp Ser Asp Thr 1 5 <210> 48 <211> 14 <212> protein <213> Homo sapiens <400> 48

Ala Arg His Lys Leu Gly Met Asp His Asp Ala Phe Asp He

5 10 <210> 49 <211> 118 <212> protein <213> Homo sapiens <400> 49

Gin Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Arg Lys Pro Gly Ser 15 1015

Ser Vai Lys Vai Ser Cys Lys Ala Ser Gly Gly Ser Phe Asn Asn Tyr

2530

Pro lie Phe Trp Vai Arg Gin Ala Pro Gly Gin Gly Phe Glu Trp Met

4045

Gly Arg lie lie Pro lie Leu Gly lie Thr Ala Tyr Ala Gin Lys Phe 50 5560

Gin Gly Arg Vai Thr lie Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 7580

Met Glu Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Gly Gly Asp Asp Leu Asp Ala Phe Asp lie Trp Gly Gin Gly Thr

100 105110

- 70 041176

Met Vai Ser Vai Ser Ser

115 <210> 50 <211> 8 <212> protein <213> Homo sapiens <400> 50

Gly Gly Ser Phe Asn Asn Tyr Pro 1 5 <210> 51 <211> 8 <212> protein <213> Homo sapiens <400> 51

He lie Pro lie Leu Gly lie Thr 1 5 <210> 52 <211> 11 <212> protein <213> Homo sapiens <400> 52

Ala Gly Gly Asp Asp Leu Asp Ala Phe Asp lie

5 10 <210> 53 <211> 120 <212> protein <213> Homo sapiens <400> 53

Gin Vai Gin Leu Vai Glu Ser Gly Gly Gly Vai Vai Gin Pro Gly Arg 15 1015

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

2530

Ala Met Tyr Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Asp Trp Vai

4045

Ala Vai He Ser Asn Asp Gly lie Asn Lys Tyr Tyr Ala Asp Ser Vai 50 5560

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

- 71 041176

70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Asp His Thr Met Vai Arg Gly Ala Phe Asp Tyr Trp Gly Gin

100 105110

Gly Thr Leu Vai Thr Vai Ser Ser

115120 <210> 54 <211> 8 <212> protein <213> Homo sapiens <400> 54

Gly Phe Thr Phe Asn Arg Tyr Ala 1 5 <210> 55 <211> 8 <212> protein <213> Homo sapiens <400> 55

He Ser Asn Asp Gly He Asn Lys 1 5 < 210> 56 < 211> 13 < 212> protein < 213> Homo sapiens < 400> 56

Ala Arg Asp His Thr Met Vai Arg Gly Ala Phe Asp Tyr

5 10 <210> 57 <211> 107 <212> protein <213> Homo sapiens <400> 57

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 15 10 15

Asp Arg Vai Thr lie Thr Cys Arg Ala Ser Gin Gly lie Ser Arg Trp 20 25 30

- 72 041176

Leu Ala Trp Tyr Gin Gin Lys Pro Glu Lys Ala Pro Lys Ser Leu He

4045

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asp Ser Asp Pro He

9095

Thr Phe Gly Gin Gly Thr Arg Leu Glu lie Lys

100105 <210> 58 <211> 6 <212> protein <213> Homo sapiens <400> 58

Gin Gly lie Ser Arg Trp 1 5 < 210> 59 < 211> 3 < 212> protein < 213> Homo sapiens < 400> 59

Ala Ala Ser < 210> 60 < 211> 9 < 212> protein < 213> Homo sapiens < 400> 60

Gin Gin Tyr Asp Ser Asp Pro lie Thr

5 <210> 61 <211> 107 <212> protein <213> Homo sapiens <400> 61

- 73 041176

Glu He Vai Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 1015

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Ser Ser Ser

2530

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu

4045 lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 5560

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu 65 70 7580

Pro Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Leu 85 9095

Thr Phe Gly Gly Gly Thr Lys Vai Glu lie Lys

100105 <210> 62 <211> 7 <212> protein <213> Homo sapiens <400> 62

Gin Ser Vai Ser Ser Ser Tyr 1 5 <210> 63 <211> 3 <212> protein <213> Homo sapiens <400> 63

Gly Ala Ser < 210> 64 < 211> 8 < 212> protein < 213> Homo sapiens < 400> 64

Gin Gin Tyr Gly Ser Ser Leu Thr 1 5

- 74 041176 <210> 65 <211> 107 <212> protein <213> Homo sapiens <400> 65

Asp He Gin Met Thr Gin Ser Pro Ser Leu Ser Ala Ser Ala Gly 15 1015

Asp Arg Vai Thr lie Thr Cys Arg Ala Ser Gin Gly lie Ser Ser Arg

2530

Leu Ala Trp Tyr Gin Gin Lys Pro Glu Lys Ala Pro Lys Ser Leu lie 35 4045

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Tyr Pro Tyr 85 9095

Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys

100105 <210> 66 <211> 6 <212> protein <213> Homo sapiens <400> 66

Gin Gly lie Ser Ser Arg 1 5 < 210> 67 < 211> 3 < 212> protein < 213> Homo sapiens < 400> 67

Ala Ala Ser 1 <210> 68 <211> 9 <212> protein

- 75 041176 <213> Homo sapiens <400> 68

Gin Gin Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 69 <211> 108 <212> protein < 213> Homo sapiens < 400> 69

Glu He Vai Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 1015

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Arg Ser Ser 20 2530

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu

4045 lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 5560

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu 65 70 7580

Pro Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 9095

Arg Thr Phe Gly Gin Gly Thr Lys Vai Glu lie Lys

100105 < 210> 70 < 211> 7 < 212> protein < 213> Homo sapiens < 400> 70

Gin Ser Vai Arg Ser Ser Tyr 1 5 < 210> 71 < 211> 3 < 212> protein < 213> Homo sapiens < 400> 71

- 76 041176

Gly Ala Ser 1 <210> 72 <211> 9 <212> protein <213> Homo sapiens <400> 72

Gin Gin Tyr Gly Ser Ser Pro Arg Thr 1 5 <210> 73 <211> 108 <212> protein < 213> Homo sapiens < 400> 73

Glu He Vai Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 1015

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Gly Ser Ser 20 2530

Ser Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu

4045

He Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser

5560

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu 65 70 7580

Pro Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 9095

Arg Thr Phe Gly Gin Gly Thr Lys Vai Glu lie Lys

100105 < 210> 74 < 211> 7 < 212> protein < 213> Homo sapiens < 400> 74

Gin Ser Vai Gly Ser Ser Ser 1 5

- 77 041176 <210> 75 <211> 3 <212> protein <213> Homo sapiens <400> 75

Gly Ala Ser < 210> 76 < 211> 9 < 212> protein <213> Homo sapiens <400> 76

Gin Gin Tyr Gly Ser Ser Pro Arg Thr 1 5 <210> 77 <211> 107 <212> protein <213> Homo sapiens <400> 77

Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 15 1015

Asp Arg Vai Thr He Thr Cys Arg Ala Ser Gin Gly lie Ser Ser Arg

2530

Leu Ala Trp Tyr Gin Gin Lys Pro Glu Lys Ala Pro Lys Ser Leu lie 35 4045

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Tyr Pro Tyr 85 9095

Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys

100105 <210> 78 <211> 6 <212> protein <213> Homo sapiens

- 78 041176 <400> 78

Gin Gly He Ser Ser Arg 1 5 <210> 79 <211> 3 <212> protein <213> Homo sapiens <400> 79

Ala Ala Ser 1 < 210> 80 < 211> 9 < 212> protein <213> Homo sapiens <400> 80

Gin Gin Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 81 <211> 108 <212> protein <213> Homo sapiens <400> 81

Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 15 1015

Asp Arg Vai Thr lie Thr Cys Arg Ala Ser Gin Gly lie Ser Ser Trp

2530

Leu Ala Trp Tyr Gin Gin Lys Pro Glu Lys Ala Pro Lys Ser Leu He

4045

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Tyr Pro Leu 85 9095

Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys

- 79 041176

100 105 <210> 82 <211> 6 <212> protein <213> Homo sapiens <400> 82

Gin Gly He Ser Ser Trp 1 5 < 210> 83 < 211> 3 < 212> protein < 213> Homo sapiens < 400> 83

Ala Ala Ser 1 < 210> 84 < 211> 10 < 212> protein < 213> Homo sapiens < 400> 84

Gin Gin Tyr Asn Ser Tyr Pro Leu Tyr Thr

5 10 <210> 85 <211> 107 <212> protein <213> Homo sapiens <400> 85

Ala He Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 15 1015

Asp Arg Vai Thr lie Thr Cys Arg Ala Ser Gin Asp He Ser Ser Ala 20 2530

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie

4045

Tyr Asp Ala Ser lie Leu Glu Ser Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

- 80 041176

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Phe Asn Ser Tyr Pro Leu 85 90 95

Thr Phe Gly Gly Gly Thr Lys Vai Glu He Lys

100 105 <210> 86 <211> 6 <212> protein <213> Homo sapiens <400> 86

Gin Asp He Ser Ser Ala 1 5 < 210> 87 < 211> 3 < 212> protein < 213> Homo sapiens < 400> 87

Asp Ala Ser < 210> 88 < 211> 9 < 212> protein <213> Homo sapiens <400> 88

Gin Gin Phe Asn Ser Tyr Pro Leu Thr 1 5 <210> 89 <211> 107 <212> protein <213> Homo sapiens <400> 89

Glu lie Vai Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 15 1015

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Ser Ser Tyr 20 2530

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie 35 4045

- 81 041176

Tyr Asp Ala Ser Asn Arg Ala Thr Gly He Pro Ala Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Glu Pro 65 70 7580

Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Leu 85 9095

Thr Phe Gly Gly Gly Thr Lys Vai Glu lie Lys

100105 <210> 90 <211> 6 <212> protein <213> Homo sapiens <400> 90

Gin Ser Vai Ser Ser Tyr 1 5 <210> 91 <211> 3 <212> protein <213> Homo sapiens <400> 91

Asp Ala Ser 1 < 210> 92 < 211> 9 < 212> protein <213> Homo sapiens <400> 92

Gin Gin Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 93 <211> 107 <212> protein <213> Homo sapiens <400> 93

Glu lie Vai Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 15 1015

Glu Arg Ala He Leu Ser Cys Arg Ala Ser Gin Ser Vai Ser Ser Tyr 20 2530

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu He

4045

Tyr Asp Ala Ser Asn Arg Ala Thr Gly He Pro Ala Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Glu Pro 65 70 7580

Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Leu 85 9095

Thr Phe Gly Gly Gly Thr Lys Vai Glu He Lys

100105 <210> 94 <211> 6 <212> protein <213> Homo sapiens <400> 94

Gin Ser Vai Ser Ser Tyr 1 5 <210> 95 <211> 3 <212> protein <213> Homo sapiens <400> 95

Asp Ala Ser < 210> 96 < 211> 9 < 212> protein <213> Homo sapiens <400> 96

Gin Gin Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 97 <211> 107 <212> protein <213> Homo sapiens

- 82 041176 <400> 97

Ala He Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 15 1015

Asp Arg Vai Thr He Thr Cys Arg Ala Ser Gin Gly lie Asn Ser Ala

2530

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 4045

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Phe Asn Ser Tyr Pro Leu 85 9095

Thr Phe Gly Gly Gly Thr Lys Vai Glu lie Lys

100105 <210> 98 <211> 6 <212> protein <213> Homo sapiens <400> 98

Gin Gly lie Asn Ser Ala 1 5 < 210> 99 < 211> 3 < 212> protein <213> Homo sapiens <400> 99

Asp Ala Ser <210> 100 <211> 9 <212> protein <213> Homo sapiens <400> 100

Gin Gin Phe Asn Ser Tyr Pro Leu Thr

- 83 041176 <210> 101 <211> 107 <212> protein <213> Homo sapiens <400> 101

Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 15 1015

Asp Arg Vai Thr lie Thr Cys Arg Ala Ser Gin Gly lie Ser Ser Trp

2530

Leu Ala Trp Tyr Gin Gin Lys Pro Glu Lys Ala Pro Lys Ser Leu lie 35 4045

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Tyr Pro Pro

9095

Thr Phe Gly Gin Gly Thr Thr Vai Glu Vai Lys

100105 <210> 102 <211> 6 <212> protein <213> Homo sapiens <400> 102

Gin Gly lie Ser Ser Trp 1 5 <210> 103 < 211> 3 < 212> protein < 213> Homo sapiens < 400> 103

Ala Ala Ser <210> 104

- 84 041176 <211> 9 <212> protein <213> Homo sapiens <400> 104

Gin Gin Tyr Asn Ser Tyr Pro Pro Thr 1 5 <210> 105 <211> 107 <212> protein < 213> Homo sapiens < 400> 105

Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 15 1015

Asp Arg Vai Thr lie Thr Cys Arg Ala Ser Gin Gly lie Ser Ser Trp

2530

Leu Ala Trp Tyr Gin Gin Lys Pro Glu Lys Ala Pro Lys Ser Leu lie 35 4045

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Tyr Pro Tyr 85 9095

Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys

100105 <210> 106 <211> 6 <212> protein <213> Homo sapiens <400> 106

Gin Gly lie Ser Ser Trp 1 5 <210> 107 <211> 3 <212> protein <213> Homo sapiens

-

Claims (19)

<400> 107 Ala Ala Ser 1 <210> 108 <211> 9 <212> protein <213> Homo sapiens <400> 108 Gin Gin Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 109 <211> 107 <212> protein < 213> Homo sapiens < 400> 109 Glu He Vai Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 15 1015 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Ser Ser Tyr 20 2530 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie 35 4045 Tyr Asp Ala Ser Asn Arg Ala Thr Gly lie Pro Ala Arg Phe Ser Gly 50 5560 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Glu Pro 65 70 7580 Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Leu 85 9095 Thr Phe Gly Gly Gly Thr Lys Vai Glu lie Lys 100105 < 210> 110 < 211> 6 < 212> protein < 213> Homo sapiens < 400> 110 Gin Ser Vai Ser Ser Tyr 1 5 <210> 111 <211> 3 <212> protein < 213> Homo sapiens <400> 111 Asp Ala Ser 1 < 210> 112 < 211> 9 < 212> protein < 213> Homo sapiens < 400> 112 Gin Gin Arg Ser Asn Trp Pro Leu Thr 1 5 CLAIM 1. Human IgG1 antibody that binds human tissue factor, comprising a V _H region containing CDRs 1, 2 and 3 with the sequences of SEQ ID NOs: 10, 11 and 12, and a VL region containing CDRs 1, 2 and 3 with the sequences of SEQ ID NOs: 66, 67 and 68, or a variant of said antibody, wherein said variant has no more than 3 conservative amino acid substitutions. 2. An antibody according to claim 1, which has one or more of the following properties: a) binds to the extracellular domain of tissue factor with an apparent affinity (EC ₅₀ ) of 3 nM or less, b) binds to mammalian cells expressing tissue factor with an apparent affinity (EC ₅₀ ) of 10 nM or less, c) capable of inducing antibody-dependent cellular cytotoxicity in A431 cells with an EC ₅₀ value of 1 nM or less, - 86 041176 d) is effective in inhibiting the growth of established MDA-MB-231 tumors and/or in inhibiting the growth of established BxPC3 tumors, e) inhibits tissue factor-induced blood coagulation with a median inhibition concentration of less than 10 nM, f) inhibits the conversion of FX to FXa by less than 50% tissue factor/FVIIa complex, g) inhibits FVIIa-induced release of IL-8 by MDA-MB-231 cells with a maximum inhibition value greater than 50%, or h) inhibits FVIIa binding to tissue factor with a maximum inhibition value greater than 80%, or i) inhibits FVIIa-induced ERK phosphorylation with a median inhibition concentration of less than 10 nM, or j) is capable of inducing C3c and C4C deposition. 3. The antibody of claim 2, wherein the mammalian cells expressing the tissue factor as defined in b) are A431 cells transfected with a construct encoding the tissue factor. 4. An antibody according to any one of claims 1-3, including: I) V _H , which a) at least 80% identical to the sequence of the V _H region of SEQ ID NO: 9, b) at least 90% identical to the sequence of the V _H region of SEQ ID NO: 9, c) at least 95% identical to the sequence of the V _H region of SEQ ID NO: 9, or d) 100% identical to the sequence of the VH region of SEQ ID NO: 9, and II) V _L , which a) at least 80% identical to the sequence of the V _L region of SEQ ID NO: 65, b) at least 90% identical to the sequence of the V _L region of SEQ ID NO: 65, c) at least 95% identical to the sequence of the V _L region of SEQ ID NO: 65, or d) 100% identical to the sequence of the V _L region of SEQ ID NO: 65. 5. Antibody according to any one of claims 1-4, containing: (a) a VH region containing the sequence of SEQ ID NO: 9 and a VL region containing the sequence of SEQ ID NO: 65, or (b) a variant of said antibody that has no more than 3 conservative amino acid substitutions. 6. An antibody according to any one of claims 1 to 5, which further has one or more of the following properties: inhibition of proliferation, inhibition of tumor angiogenesis, induction of apoptosis of tumor cells, binding to alternatively spliced tissue factor. 7. An antibody according to any one of claims 1 to 6, which is conjugated to another particle selected from a cytotoxic particle, a radioisotope, or a drug. 8. A bispecific molecule for diagnosing or treating cancer, comprising an antibody according to any one of claims 1 to 7 and a second binding specificity selected from the group consisting of a binding specificity for a human effector cell, a human Fc receptor, or a T cell receptor. 9. An expression vector for producing an antibody according to claim 1, containing a nucleotide sequence encoding the amino acid sequences V _H CDR 1, 2 and 3 SEQ ID NO: 10-12 and V _L CDR 1, 2 and 3 SEQ ID NO: 66-68 . 10. An expression vector for producing an antibody according to claim 1, containing a nucleotide sequence encoding the amino acid sequences of the VH region of SEQ ID NO: 9 and the VL region of SEQ ID NO: 65. 11. A recombinant eukaryotic or prokaryotic host cell containing the vector according to any one of claims 9 or 10, which produces an antibody according to any one of claims 1-6. 12. A pharmaceutical composition for the treatment of cancer, comprising an antibody according to any one of claims 1 to 7 or a bispecific molecule according to claim 8 and a pharmaceutically acceptable carrier. 13. The use of an antibody according to any one of claims 1 to 7 as a drug for the treatment of cancer. 14. The use of a bispecific molecule according to claim 8 as a drug for the treatment of cancer. 15. The use of an antibody according to any one of claims 1 to 7 as a drug for the treatment of cancer in combination with one or more additional therapeutic agents. 16. The use of the bispecific molecule of claim 8 as a cancer drug in combination with one or more additional therapeutic agents. 17. Use according to any one of claims 15 or 16, wherein one or more therapeutic agents is a chemotherapeutic agent. 18. The use of an antibody according to any one of claims 1 to 7 for the preparation of a medicament for the treatment of cancer. 19. The use of a bispecific molecule according to claim 8 for the preparation of a medicament for the treatment of cancer. -