NZ734153A - Human antibodies against tissue factor - Google Patents

Human antibodies against tissue factor

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Publication number
NZ734153A
NZ734153A NZ734153A NZ73415309A NZ734153A NZ 734153 A NZ734153 A NZ 734153A NZ 734153 A NZ734153 A NZ 734153A NZ 73415309 A NZ73415309 A NZ 73415309A NZ 734153 A NZ734153 A NZ 734153A
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antibody
region
seq
cancer
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NZ734153A
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Rene Hoet
Mischa Houtkamp
De Winkel Jan Van
Tom Vink
Maroeska Oudshoorn
Vibeke Breinholt
Ole Baadsgaard
Eva Ehrnrooth
Jong Rob De
Sandra Verploegen
Willem Bleeker
David Satijn
Paul Parren
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Genmab As
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Priority to NZ786274A priority Critical patent/NZ786274A/en
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Abstract

Disclsoes human antibodies against tissue factor and cells expressing tissue gactor. Further discloses use for treating MDA-MB-231 tumors, inhibition of tissue factor induced blood coagulation and FVIIa binding. In preferred embodiments, the antibodies of the invention have a high affinity towards human tissue factor, mediate antibody-dependent cellular cytotoxicity (ADCC), inhibit FVIIa binding to TF, inhibit FVIIa-induced ERK phosphorylation and IL8 release.

Description

HUMAN ANTIBODIES T TISSUE FACTOR FIELD OF THE INVENTION The present invention relates to antibodies directed to tissue factor in particular to human tissue factor, and uses of such antibodies, in particular their use in the treatment of cancer, inflammation and vascular es.
BACKGROUND OF THE ION Tissue factor (TF), also called thromboplastin, factor III or CD142 is a protein present in subendothelial , platelets, and leukocytes necessary for the initiation of thrombin formation from the zymogen prothrombin. Thrombin formation ultimately leads to the coagulation of blood. Tissue factor s cells to initiate the blood coagulation cascades, and it functions as the high-affinity receptor for the ation factor VII. The resulting complex es a catalytic event that is responsible for initiation of the coagulation protease cascades by specific limited proteolysis. Unlike the other cofactors of these protease cascades, which circulate as nonfunctional precursors, this factor is a potent initiator that is fully functional when expressed on cell surfaces.
Tissue factor is the cell surface receptor for the serine protease factor VIIa (FVIIa).
Binding of FVIIa to tissue factor has been found to start signaling processes inside the cell said signaling function playing a role in angiogenesis. Whereas 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 gain the ability to produce proteins that participate in angiogenesis, so called angiogenic growth factors, these ns are released by the tumor into nearby tissues, and stimulate new blood s to sprout from existing healthy blood vessels toward and into the tumor. Once new blood vessels enter the tumor it can y expand its size and invade local tissue and organs. Through the new blood vessels cancer cells may further escape into the circulation and lodge in other organs to form new tumors (metastases).
Further TF plays a role in inflammation. The role of TF is assumed to be mediated by blood coagulation (A. J. Chu: "Tissue factor es inflammation" in Archives of biochemistry and biophysics, 2005, vol. 440, No. 2, pp. 123-132). ingly, the inhibition of TF e.g. by monoclonal anti-TF antibodies is of significance in interrupting the coagulation-inflammation cycle in contribution to not only nflammation but also to ar diseases.
TF expression is observed in many types of cancer and is associated with more aggressive disease. Furthermore, human TF also exist in a soluble alternatively-spliced form, asHTF. It has recently been found that asHTF es tumor growth (Hobbs et 7 Thrombosis Res. 120(2) 813-821).
Antibodies binding to TF have been disclosed in the prior art: WO98/40408 discloses dies that can bind native human TF, either alone or present in a TF:VIIa complex, effectively preventing factor X binding to TF or that complex, and thereby reducing blood coagulation. It is disclosed that the antibodies may be used to alleviate thromboses following an invasive medical procedure such as arterial or cardiac surgery or to eliminate blood ation arising from us of medical entation. Further antibodies are disclosed to be employed in in vivo diagnostic methods including in vivo diagnostic imaging of native human TF. 94475 provides antibodies capable of binding to human tissue factor, which do not inhibit factor mediated blood coagulation compared to a normal plasma control.
Human antibodies are not described. It is d that the antibody may be used for treatment of cancer.
WOO3/O93422 relates to antibodies that bind with greater affinity to the TF:VIIa complex than to TF alone. Use of the antibodies as anticoagulant in the treatment of certain diseases, such as sepsis, disseminated intravascular coagulation, ischemic stroke, thrombosis, acute coronary syndromes and coagulopathy in advanced cancer is proposed.
WOOl/27079 discloses compositions and methods for inhibiting abnormal cell proliferation, ularly endothelial cell proliferation, such as cancer, abnormal development of embryos, malfunctioning of immune ses, as well as angiogenesis d to neovasularization and tumor growth. Many active substances, including antibodies, are proposed, but no specific antibodies are disclosed.
WOO3/O37361 s to us of TF agonist or antagonist for treatment d to apoptosis. 29295 relates to isolated human antibodies that immunoreact with human TF to inhibit the g of coagulation factor VIIa. However, the application does not disclose a single example of an dy having these properties.
A number of monoclonal antibody therapies are approved to treat different tumor types, including e.g. bevacizumab (Avastin®), cetuximab (Erbitux®), panitumumab (VectibixTM) and trastuzumab (Herceptin®).
SUMMARY OF THE INVENTION Although much progress has been made, there remains a need for improved methods of treating serious diseases, e.g. improved treatment of cancer, based on therapeutic antibodies.
Advantageously, the present invention may provide novel highly specific and effective human anti-TF antibodies for medical use. The dies of the invention exhibit TF binding characteristics that differ from the antibodies described in the art.
In red embodiments, the antibodies of the invention have a high affinity towards human tissue factor, e antibody-dependent cellular cytotoxicity (ADCC), inhibit FVIIa g to TF, inhibit FVIIa-induced ERK phosphorylation and IL8 release, do not or poorly inhibit coagulation.
In one embodiment, there is provided a human antibody which binds human Tissue Factor, wherein the antibody ses: a) a variable heavy (VH) region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:10, 11 and 12 and a variable light (VL) region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:66, 67 and 68.
It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.
BRIEF DESCRIPTION OF THE GS Figure 1: Alignment of sequences of the antibodies of the present invention.
CDR1, CDR2 and CDR3 according to IMGT are highlighted: sequences in italics represent the CDR1 region, underlined sequences represent the CDR2 region, bold sequences represent the CDR3 region.
Figure 2: IgG4 sequences (SEQ ID NO: 113-114) SEQ ID NO: 113: The amino acid sequence of the wild-type CH region of human IgG4.
The Sequence in italics represents the CH1 , highlighted sequence represents the hinge region, regular sequence represents the CH2 region and underlined ce ents the CH3 region.
SEQ ID NO: 114: The amino acid sequence of the hingeless CH region of a human IgG4 Figure 3: Binding of anti-TF HuMabs to the extracellular domain of TF.
Figure 4: Binding of anti-TF HuMabs to membrane bound TF.
Figure 5: tion of FVIIa binding to TF.
Figure 6: tion of FVIIa induced ERK phosphorylation Figure 6a: Inhibition of FVIIa induced ERK phosphylation Figure 7: Inhibition of FVIIa induced IL-8 release.
Figure 8: Inhibition of FXa generation.
Figure 9: tion of blood coagulation.
Figure 10: TF-HuMabs induces lysis of Bx-PC3 cells by ADCC Figure 11: Deposition of ment components C3c and C4c on target cells.
Figure 12: Immunohistochemical analysis of binding of TF-HuMabs to glomeruli. 18563328_1 (GHMatters) P34563NZ04 Figure 13: Immunohistochemical analysis of binding of TF -HuMabs to pancreatic Figure 14: In vivo efficacy of TF-HuMabs in established MDA-MB-231 tumor xenograft.
Figure 15: Bleeding time determined in cynomolgus monkeys upon intravenous ions of TF-specific HuMab 011. The dy was administered on day 1 (0 mg/kg), 8 (1 mg/kg), (10 mg/kg) and 22 (100 mg/kg).
Figure 16: In vivo efficacy of TF -HuMabs in a prophylactic and established BX-PC3 tumor xenograft.
[PAGE 4 TO FOLLOW] 18563328_1 (GHMatters) P34563NZ04 Figure 17: Shuffle constuct and TF domains Figure 18: binding of anti-TF antibodies to TF shuffle constructs Figure 19: Binding of HuMab-TF Fab fragments to extracellular domain of TF, ELISA Figure 20: Binding of HuMab-TF Fab fragments to extracellular domain of TF, FACS Figure 21: Binding profile of F HuMabs dependent on the number of TF molecules expressed.
DETAILED DESCRIPTION OF THE INVENTION Definitions The terms "tissue factor", "TF", "CD142", e factor antigen", "TF antigen" and "CD142 antigen are used interchangeably herein, and, unless specified otherwise, include any variants, isoforms and species homologs of human tissue factor which are naturally expressed by cells or are sed on cells transfected with the tissue factor gene.
The term oglobulin" 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 may all four be inter-connected by disulfide bonds. The structure of globulins has been well terized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. ). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain nt region. The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of ariability (or hypervariable s which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from terminus to carboxy-terminus in the following order: FR1, CDRl, FR2, CDRZ, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol.
Biol. fl, 901-917 (1987)). Typically, the numbering of amino acid residues in this region is according to IMGT., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, da, MD. (1991) (phrases such as variable domain residue numbering as in Kabat or ing to Kabat herein refer to this numbering system for heavy chain variable domains or light chain variable s). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of VH CDR2 and inserted es (for instance residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given dy by alignment at regions of gy of the sequence of the dy with a "standard" Kabat numbered sequence.
The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, 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 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a logical response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The nt s of the antibodies (Abs) may e the binding of the immunoglobulin 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 pathway of complement activation. An anti-TF antibody may also be a bispecific antibody, diabody, or similar le (see for instance PNAS USA fl(14), 6444-8 (1993) for a description of diabodies). , bispecific antibodies, diabodies, and the like, provided by the present invention may bind any suitable target in addition to a portion of tissue factor or tissue factor FVIIa complex. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by t, includes fragments of an antibody that retain the ability to specifically bind to the n. It has been shown that the antigen-binding function of an dy may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term ody" include (i) a Fab’ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as bed in W02007059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of a VL and VH domains, (v) a dAb fragment (Ward et al., Nature fl, 6 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;fl(11):484-90); (vi) camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan;§(1):111-24) and (vii) an isolated complementarity determining region (CDR).
Furthermore, although the two domains of the Fv nt, VL and VH, are coded for by separate genes, they may be joined, using recombinant s, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science fl, 423-426 (1988) and Huston et al., PNAS USA g, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they tively and each independently are unique features of the present invention, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present ion are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal dies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric dies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.
An TF antibody" is an antibody as described above, which binds specifically to the antigen tissue .
The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions d from human germline immunoglobulin sequences.
The human antibodies of the invention may include amino acid es not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or during gene rearrangement or by somatic mutation in vivo).
However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another ian s, such as a mouse, have been grafted onto human framework sequences.
In a red embodiment, the antibody of the invention is ed. An "isolated antibody," as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (for instance an isolated antibody that specifically binds to tissue factor is substantially free of antibodies that specifically bind ns other than tissue factor). An isolated antibody that specifically binds to an epitope, m or variant of human tissue factor may, r, have cross-reactivity to other related antigens, for instance from other species (such as tissue factor species homologs).
Moreover, an isolated antibody may be substantially free of 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 ed in a well-defined ition.
When used herein 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 g to TF, e.g. compete for TF binding in the assay described in Example 6 herein. For some pairs of antibodies, ition in the assay of Example 6 is only observed when one dy is coated on the plate and the other is used to compete, and not vice versa. The term "competes with" when used herein is also intended to cover such combinations antibodies.
The terms lonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody ition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be generated by a oma which includes a B cell obtained from a transgenic or transchromosomal nonhuman animal, such as a transgenic mouse, having a genome comprising 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 the binding of an antibody to a predetermined antigen typically is a binding with an affinity corresponding to a KD of about '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 when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ld lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for ce at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the ermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a ecific antigen may be at least 10,000 fold.
The term "kd" ), as used herein, refers to the dissociation rate constant of a ular antibody-antigen interaction. Said value is also referred to as the koff value.
The term "ka" (M'1 x sec'l), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.
The term " KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen ction.
The term "KA" (M'l), as used herein, refers to the association brium constant of a particular antibody-antigen interaction and is obtained by dividing the ka by the kd.
The present invention also es antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of an anti-TF antibody still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity/avidity and/or the specificity/selectivity of the parent dy and in some cases such an F antibody may be associated with greater affinity, selectivity and/or specificity than the parent antibody.
Such functional variants typically retain icant sequence identity to the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of ons X 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and ination of percent identity n two sequences may be lished using a mathematical thm, as described in the non-limiting examples below.
The percent identity between two nucleotide sequences may be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences may also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight e table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 4_8, 444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG re 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 a length weight of 1, 2, 3, 4, 5, or 6.
The sequence of CDR variants may differ from the sequence of the CDR of the parent dy sequences through mostly conservative substitutions; for instance at least about %, 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 (e.g., about 65-99%, such as about 96%, 97% or 98%) of the substitutions in the variant are conservative amino acid residue replacements.
The sequence of CDR variants may differ from the sequence of the CDR of the parent dy sequences through mostly vative substitutions; for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.
In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three : Amino acid residue classes for conservative substitutions Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), and Gln (Q) Aliphatic ged Residues Gly (G), Ala (A), Val (V), Leu (L), and lie (I) Non-polar ged Residues Cys (C), Met (M), and Pro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W) Alternative conservative amino acid residue substitution classes Alternative Physical and Functional Classifications of Amino Acid Residues Alcohol group-containing residues S and T Aliphatic residues I, L, V, and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Negatively charged residues D and E Polar es C, D, E, H, K, N, Q, R, S, and T Positively d residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Very small es A, G, and S Residues ed in turn A, C, D, E, G, H, K, N, Q, R, S, P, and formation T Flexible residues Q, T, K, S, G, P, D, E, and R More conservative substitutions groupings include: va|ine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, ancl gine-glutamine.
Additional groups of amino acids may also be formulated using the principles described in, e.g., Creighton (1984) Proteins: Structure and Molecular Properties (2d Ed. 1993), W.H. Freeman and Company.
In one ment of the present invention, conservation in terms of hydropathic/hydrophilic ties and residue weight/size also is substantially retained in a variant CDR as compared to a CDR of an antibody of the examples (e.g., the weight class, hydropathic score, or both of the 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 (e.g., about 65-99%) retained). For example, conservative residue substitutions may also or alternatively be based on the ement of strong or weak based weight based conservation groups, which are known in the art.
The retention of similar residues may also or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62, Open Gap=11 and Extended Gap=1).
Suitable variants typically exhibit at least about 45%, such as at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, or more (e.g., about 70-99%) rity to the parent peptide.
As used herein, "isotype" refers to the immunoglobulin class (for ce IgGl, IgGZ, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes.
The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes y consist of surface groupings of les such as amino acids or sugar side chains and usually have specific three dimensional ural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the g (also called immunodominant component of the epitope) and other amino acid es, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the ically antigen binding peptide (in other words, the amino acid e is within the footprint of the specifically n binding peptide).
As used herein, a human antibody is "derived from" a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, for instance by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library, and wherein the selected human antibody V domain sequence is at least 90%, such as at least 95%, for instance at least 96%, such as at least 97%, for instance at least 98%, or such as at least 99% identical in amino acid V domain sequence sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, outside the heavy chain CDR3, a human antibody derived from a particular human germline sequence will display no more than 20 amino acid differences, e.g. no more than 10 amino acid differences, such as no more than 9, 8, 7,6 or , for instance no more than 4, 3, 2, or 1 amino acid ence from the amino acid sequence encoded by the germline immunoglobulin gene.
As used herein, the term "inhibits growth" (e.g. referring to cells, such as tumor cells) is intended to include any able decrease in the cell growth when contacted with an anti-TF antibody as compared to the growth of the same cells not in contact with an anti-TF antibody, e.g., the inhibition of growth of a cell culture by at least about 10%, 20%, %, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Such a decrease in cell growth can occur by a variety of mechanisms, e.g. effector cell phagocytosis, ADCC, CDC, and/or apoptosis.
The term "bispecific molecule" is intended to include any agent, such as a n, peptide, or protein or peptide complex, which has two ent binding specificities. For example, the molecule may bind to, or interact with, (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "bispecific antibody" is intended to include any anti-TF antibody, which is a bispecific molecule. The term "bispecific antibodies" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two n binding sites (see for instance Holliger, P. et al., PNAS USA fl, 6444-6448 (1993), Poljak, R.J. et al., ure ;, 1121-1123 (1994)).
An "antibody deficient in effector function" or an "effector-function-deficient antibody" refers to an antibody which has a significantly reduced or no ability to activate one or more or isms, such as complement activation or Fc receptor g.
Thus, effector-function deficient antibodies have significantly reduced or no ability to mediate antibody-dependent ediated xicity (ADCC) and/or complement- ent cytotoxicity (CDC). An example of such an antibody is IgG4.
The term "monovalent antibody" means in the context of the present invention that an antibody molecule is capable of binding a single molecule of the n, and thus is not able of antigen crosslinking.
The term "stabilized IgG4 antibody" refers to an IgG4 antibody which has been modified to reduce half-molecule exchange (see van der Neut Kolfschoten M et al . (2007) Science 14;317(5844) and references n, and also Labrijn et al. (2009) Nature hnology, 27, 767-771.
As used herein, the term "effector cell" refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including tic T cells (CTLs)), 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, an effector cell is capable of inducing dy-dependent cellular cytotoxicity (ADCC), such as a natural killer cell, capable of inducing ADCC. For example, monocytes, macrophages, which express FcR are involved in specific killing of target cells and presenting ns to other components of the immune system, or binding to cells that present antigens. In some embodiments, an effector cell may phagocytose a target antigen or target cell. The expression of a ular FcR on an effector cell may be regulated by humoral factors such as cytokines. For example, expression of FcyRI has been found to be up-regulated by interferon y (IFN-y) and/or G-CSF. This enhanced sion increases the cytotoxic activity of FcyRI-bearing cells against targets. An effector cell can ytose or lyse a target antigen or a target cell.
The term "vector," as used herein, is intended to refer 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 refers to a circular double stranded DNA loop into which onal DNA ts may be ligated. Another type of vector is a viral vector, n onal DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are uced (for instance bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the sion of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression s" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the t specification, "plasmid" and r" may be used interchangeably as the d is the most commonly used form of vector. r, the present invention is intended to include such other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated s), which serve equivalent functions.
The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which an sion vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain cations may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK293 cells, NS/O cells, and lymphocytic cells.
The term "transfectoma", as used herein, includes inant eukaryotic host cells expressing the antibody, such as CHO cells, NS/O cells, HEK293 cells, plant cells, or fungi, including yeast cells.
The term "transgenic non-human animal" refers to a non-human animal having a genome comprising one or more human heavy and/or light chain transgenes or transchromosomes (either integrated or non-integrated into the animal’s natural genomic DNA) and which is capable of expressing fully human antibodies. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces human anti-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, as is the case for transgenic mice, for instance HuMAb mice, such as HCo7 or HC012 mice, or the human heavy chain transgene may be maintained extrachromosomally, as is the case for transchromosomal KM mice as described in W002/43478. Such transgenic and transchromosomal mice (collectively ed to herein as "transgenic mice") are capable of producing multiple isotypes of human monoclonal antibodies to a given n (such as IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-J recombination and isotype switching.
Transgenic, nonhuman animal can also be used for production of antibodies against a specific antigen by introducing genes encoding such specific antibody, for example by operatively linking the genes to a gene which is expressed in the milk of the animal.
"Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of , rating, arresting or eradicating g) symptoms or e .
An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of an 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 elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
An "anti-idiotypic" (Id) antibody is an antibody which recognizes unique determinants generally ated with the antigen-binding site of an antibody.
Further aspects and embodiments of the ion As described above, in a first aspect, the invention relates to a human antibody which binds human Tissue Factor.
In one ment, the antibody binds to the extracellular domain of Tissue Factor with an apparent ty (ECso) of 3 nM or less, such as 0.50 nM or less, e.g. 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 e 13.
In another embodiment, the antibody binds to mammalian cells expressing Tissue , such as A431 cells transfected with a construct encoding Tissue Factor, preferably with an apparent affinity (EC50) of 10 nM or less, e.g. 8 nM or less, such as 5 nM or less, e.g. 2 nM or less, such as 1 nM or less, e.g. 0.5 nM or less, such as 0.3 nM or less, when determined as bed 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 EC50 value of 2 nM or less, e.g. 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 e 20.
In another embodiment, the antibody is effective in inhibiting growth of established MDA-MB-231 tumors, when determined by the method described in Example 24 and/or in inhibiting growth of established BxPC3 tumors, when determined 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, e.g. less than 2 nM, such as less than 1 nM when ined as described in the assay in Example 19.
In another embodiment, the antibody does not inhibit coagulation. In an embodiment the coagulation is inhibited with a maximum of 30%, such as 25%, such as %, such as 15%, such as 10% or such as 5% compared to native level.
In a further embodiment, the antibody inhibits FVIIa binding to Tissue , preferably with a maximum inhibition value of inhibition of more than 80%, such as more than 90% when determined as described in the assay in Example 15.
In a r embodiment, the dy inhibits FVIIa-induced IL-8 release by MDA- MB-231 cells, preferably with a maximum inhibition value of inhibition of more than 40%, such as more than 50%, e.g. more than 60%, when ined in as described in the assay in Example 17.
In a further embodiment, the antibody inhibits conversion of FX into FXa by the TF/FVIIa complex, preferably by less than 50%, e.g. 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 comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:65.
In a further ment, the binding of the antibody of the invention to Tissue Factor does not involve all three of the ing residues: W in on 45, K in position 46 or Y in position 94 of Tissue Factor. In an even r embodiment, the binding does not involve any of the following residues: W in position 45, K in position 46 or Y in position 94 (these number refer to mature TF, the equivalent positions in Genbank entry NP_001984 are 77, 78 and 126).
In another embodiment of the antibody of the ion, the antibody competes for Tissue Factor binding with an dy comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93.
In a further embodiment, the antibody inhibits FVIIa induced ERK phosphorylation, preferably with 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 with 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 and do not inhibit FVII induced IL-8 e as described in the assay in Example 17 by more than maximum 10% In a further embodiment, the antibody is capable of inducing C3c and C4c deposition, preferably wherein the antibody is capable of inducing C3c and C4c deposition as determined in Example 21.
In a further embodiment, the dy Fab fragments binds to the extracellular domain of tissue factor as described in example 28 with an EC50 value of below 0.1 ug/mL., such as below 0.05 ug/mL. below 0.04 ug/mL. as ed by ELISA. , e.g.
In a further embodiment, the antibody Fab fragments binds to the extracellular domain of tissue factor as described in example 28 with an EC50 value of above 1.0 ug/mL. as measured by ELISA.
In a r embodiment, the antibody Fab fragments binds to the extracellular domain of tissue factor as described in example 28 with an EC50 value of below 10ug/mL, such as below 1 ug/mL, e.g. below 0.5 ug/mL, or below 0.2 ug/mL.
In a further embodiment, the antibody binds to human tissue factor and not murine tissue factor and shows reduced binding as compared to binding to human TF to the shuffle construct 42-84 mm, containing the human sequence for TF except for amino acid 42-84, which has been replaced with mouse sequence, as described in example 27.
In a further embodiment, the antibody binds to human tissue factor and not murine tissue factor and and shows reduced binding as compared to binding to human TF to the e construct 85-122, containing the human sequence for TF except for amino acid 85-122, which has been replaced with mouse sequence, as described in example 27.
In a further ment, the antibody binds to human tissue factor and not murine tissue factor and shows reduced binding as compared to g to human TF to the e construct 7 mm containing the human ce for TF except for amino acid 123- 137, which has been replaced with mouse sequence, as described in example 27.
In a further embodiment, the antibody binds to human tissue factor and not murine tissue factor and shows reduced binding as compared to binding to human TF to the shuffle construct 185-225 mm containing the human sequence for TF except for amino acid 185- 225, which has been replaced with mouse sequence, as described in e 27.
In a further embodiment, the antibody binds to human tissue factor and not murine tissue factor and shows reduced binding as compared to binding to human TF to both the shuffle construct 226-250 mm containing the human sequence for TF except for amino acid 226- 250, which has been replaced with mouse sequence, as described in example 27.
In a further embodiment, the antibody shows reduced binding as compared to binding to human TF to more than one shuffle uct. In an embodiment an antibody shows reduced binding to uct 42-84 mm as well as to 85-122 mm. In an embodiment an antibody shows d binding to 123-137mm as well as to construct 185-225mm. In an embodiment an antibody shows reduced binding to construct 123-137mm as well as to construct 185-225mm and further to construct 226-250mm.
In a further embodiment, the antibody is e of inducing C3c and C4c tion, preferably wherein the antibody is capable of inducing C3c and C4c deposition as determined in Example 21.
In one embodiment of the antibody of the invention, said antibody - competes for Tissue Factor g with an antibody comprising a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:65, - does not compete for Tissue Factor binding with an dy comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region sing the sequence of SEQ ID NO:93.
In a further embodiment, the antibody ses a VH CDR3 region having a) the sequence as set forth in - SEQ ID No: 12, - SEQ ID No: 16, - SEQ ID No: 20, - SEQ ID No: 24, - SEQ ID No: 28, b) a variant of any of said sequences, such as a variant having at most 1, 2, 3, 4 or 5 amino-acid modifications, preferably substitutions, such as vative substitutions.
In a further embodiment, the dy comprises a VH CDR3 region having the sequence as set forth in SEQ ID NO: 12 or a t thereof, n the variant comprises modification in one or more of the positions 2, 3, 6, 9 and 11, preferably where the modification is a substitution, more preferably where the tution is selected from the group consisting of a. R is substituted with K when in position 2, b. S is substituted with A or T when in position 3, c. G is substituted with T when in position 6, d. L is substituted with F when in position 9, and e. S is substituted with Y when in position 11.
In another ment, the antibody comprises: a) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:10, 11 and 12 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:66, 67 and b) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:14, 15 and 16 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:70, 71 and a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:18, 19, 20 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:74, 75 and 76, a VH region sing the CDR1, 2 and 3 ces of SEQ ID NO:22, 23 and 24 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:78, 79 and a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 26, 27 and 28 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 82, 83 and 84, or f) a variant of any of said antibodies, n 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 a VH having a) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from the group consisting of: SEQ ID NO: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 as compared to a VH region sequence selected from the group consisting of: SEQ ID NO:9, 13, 17, 21, 21 and 25.
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 to a VL region sequence selected from the group consisting of: SEQ ID NO:65, 69, 73, 77 and 81, 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 as compared to a VH region ce selected from the group consisting of: SEQ ID NO:65, 69, 73, 77 and 81.
In a further embodiment, the antibody comprises: a) a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO: 65, b) a VH region comprising the sequence of SEQ ID NO:13 and a VL region comprising the ce of SEQ ID NO:69, C) a VH region comprising the sequence of SEQ ID NO:17 and a VL region comprising the sequence of SEQ ID NO:73, d) a VH region comprising the sequence of SEQ ID NO:21 and a VL region comprising the sequence of SEQ ID NO:77, a VH region comprising the ce of SEQ ID NO:25 and a VL region comprising the ce 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 - es for Tissue Factor binding with an dy comprising a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:65, - competes for Tissue Factor g with an dy comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93.
In a further embodiment, the dy comprises a VH CDR3 region having a) the sequence as set forth in - SEQ ID No: 8, - SEQ ID No: 52, b) a variant of any of said sequences, such as a variant having at most 1,2 or 3 amino- acid modifications, preferably substitutions, such as conservative substitutions.
In a further embodiment, the antibody comprises: a) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:6, 7 and 8 and a VL region comprising the CDR1, 2 and 3 ces of SEQ ID NO:62, 63 and 64, b) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:50, 51 and 52 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:106, 107 and 108, or a t 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 dy comprises a VH having a) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity 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 tutions, such as conservative amino-acid substitutions as compared to a VH region sequence selected from the group consisting of: SEQ ID NO:5 and 49.
In a further embodiment, the antibody ses a VL having C) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VL region sequence selected from the group consisting of: SEQ ID NO: 61 and 105, or d) 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 as compared to a VH region sequence selected from the group consisting of: SEQ ID NO:61 and 105.
In a further embodiment, the antibody comprises: a) a VH region comprising the sequence of SEQ ID NO:5 and a VL region comprising the sequence of SEQ ID NO:61, b) a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:105, or C) a variant of any of said dies, wherein said variant preferably has at most 1,2 or 3 amino-acid modifications, more preferably amino-acid tutions, such as conservative amino-acid substitutions in said sequences.
In a further embodiment, the antibody - does not compete for Tissue Factor binding with an antibody comprising a VH region sing the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:65, and - competes for Tissue Factor binding with an antibody comprising a VH region sing the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93.
In a r ment, the antibody comprises a VH CDR3 region having 6) the sequence as set forth in - SEQ ID No: 32, - SEQ ID No: 36, - SEQ ID No: 40, - SEQ ID No: 56, b) a variant of any of said sequences, such as a variant having at most 1,2 or 3 amino- acid modifications, preferably substitutions, such as conservative substitutions.
In a further embodiment, the antibody comprises: a) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 30, 31 and 32 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 86, 87 and b) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:34, 35 and 36 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:90, 91 and a VH region comprising the CDR1, 2 and 3 ces of SEQ ID NO:38, 39 and 40 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:94, 95 and d) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:54, 55 and 56 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:110, 11 and 112, or a variant of any of said antibodies, wherein said variant preferably has at most 1, 2 or 3 amino-acid cations, more preferably amino-acid substitutions, such as conservative acid substitutions in said sequences.
In a further embodiment, the antibody comprises a VH having a) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity 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 tutions, such as vative amino-acid substitutions as compared to a VH region ce selected from the group consisting of: SEQ ID NO:29, 33, 37 and 53.
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 to a VL region sequence selected from the group consisting of: SEQ ID NO:85, 89, 93 and 109, 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 as compared to a VH region sequence selected from the group ting of: SEQ ID NO:85, 89, 93 and 109.
In a further embodiment, the antibody comprises: a) a VH region comprising the sequence of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID NO:85, b) a VH region comprising the sequence of SEQ ID NO:33 and a VL region comprising the sequence of SEQ ID NO:89, C) a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93, d) a VH region comprising the sequence of SEQ ID NO:53 and a VL region comprising the sequence of SEQ ID NO:109, or 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 ment, the antibody ses antibody competes for Tissue Factor binding with an antibody comprising a VH region sing the ce of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO:97.
In a further ment, the antibody comprises a VH CDR3 region having a) the sequence as set forth in - SEQ ID No: 4, - SEQ ID No: 44, - SEQ ID No: 48, ID) a variant of any of said sequences, such as a variant having at most 1,2 or 3 amino- acid modifications, preferably substitutions, such as conservative substitutions.
In a further embodiment, the antibody comprises: a) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:2, 3 and 4 and a VL region comprising the CDR1, 2 and 3 ces of SEQ ID NO:58, 59 and 60, b) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:42, 43 and 44 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:98, 99 and a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:46, 47 and 48 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:102, 103 and 104, 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 a further embodiment, the antibody comprises a VH having a) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence 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 cations, more preferably amino-acid substitutions, such as conservative amino-acid substitutions as compared to a VH region sequence selected from the group consisting of: SEQ ID NO:1, 41 and 45.
In a further ment, the antibody comprises a VL having C) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VL region ce selected from the group consisting of: SEQ ID NO:57, 97 and 101, or d) at most 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino-acid modifications, more preferably amino-acid tutions, such as conservative amino-acid substitutions as ed to a VH region sequence selected from the group consisting of: SEQ ID NO:57, 97 and 101.
In a further embodiment, the antibody comprises: a) a VH region comprising the sequence of SEQ ID NO:1 and a VL region comprising the sequence of SEQ ID NO:57, b) a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO: 97, C) a VH region comprising the sequence of SEQ ID NO:45 and a VL region sing the ce 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 ably amino-acid substitutions, such as conservative amino-acid substitutions in said sequences.
In an even further embodiment, the antibody of the invention has an affinity to tissue factor which is less than 5 nM, such as less than 3.5 nM, e.g. less than 2 nM when determined by the method described in Example 22 herein.
A particularly interesting group of antibodies of the invention has a binding to Tissue Factor which is characterized by a normal or high avidity and a high te (kd). As demonstrated herein, such antibodies may exhibit tumor specific binding in that they bind cancerous tissue, but do not bind, or bind less to healthy tissues. Without being bound by any specific theory, it is hypothesized that this group of antibodies only binds well to cells that s high levels of TF, because the binding is only efficient if it is bivalent. Examples of these antibodies include antibody O44, 098 and 111, described . ingly, in one embodiment, the antibody of the invention has a kd of more than '3 sec"1 when determined by the affinity method described in Example 22 herein, and an y of less than 5 nM, such as less than 1 nM, e.g. less than 0.2 nM when determined by the avidity method described in Example 22 herein.
In another embodiment, the antibody of the invention has a kd of more than 10'3 sec'l, when determined by the affinity method described in Example 22 herein and/or a ka of more than 5 x 104, Mol '1 '1 when ined sec by the affinity method described in Example 22 herein.
In a further embodiment, the antibody exhibits no binding to healthy tissue, in particular no binding to human glomeruli, e.g. as determined in the assay described in Example 23, but does exhibit binding to pancreatic tumors, e.g. as determined in the assay described in Example 23 herein.
In an even further embodiment, the antibody is effective in inhibiting growth of established BX-PC3 tumors when determined by the method described in Example 26 herein.
In another embodiment, the antibody of the ion has one or more of the ing properties: tion of proliferation, tion of tumor angiogenesis, induction of sis of tumor cells, binding to alternatively d Tissue Factor.
In a further embodiment, the dy of the invention competes for Tissue Factor binding with an antibody comprising a) a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO: 65, b) a VH region comprising the sequence of SEQ ID NO:1 and a VL region comprising the sequence of SEQ ID NO:57, C) a VH region comprising the sequence of SEQ ID NO:5 and a VL region comprising the sequence of SEQ ID NO:61, d) a VH region comprising the sequence of SEQ ID NO:13 and a VL region comprising the ce of SEQ ID NO:69, a VH region comprising the sequence of SEQ ID NO:17 and a VL region comprising the sequence of SEQ ID NO:73, f) a VH region comprising the sequence of SEQ ID NO:21 and a VL region comprising the sequence of SEQ ID NO:77, 9) a VH region comprising the sequence of SEQ ID NO:25 and a VL region comprising the sequence of SEQ ID NO:81, h) a VH region comprising the sequence of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID NO:85, a VH region comprising the sequence of SEQ ID NO:33 and a VL region sing the sequence of SEQ ID NO:89, J') a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the ce of SEQ ID NO:93, k) a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO: 97, I) a VH region comprising the sequence of SEQ ID NO:45 and a VL region comprising the sequence of SEQ ID NO:101, a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:105, or a VH region comprising the sequence of SEQ ID NO:53 and a VL region sing the sequence of SEQ ID NO:109 In a further embodiment, the antibody of the invention binds to the same e on Tissue Factor as an antibody having: a) a VH region sing the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO: 65, b) a VH region comprising the sequence of SEQ ID NO:1 and a VL region comprising the sequence of SEQ ID NO:57, c) a VH region sing the sequence of SEQ ID NO:5 and a VL region comprising the sequence of SEQ ID NO:61, d) a VH region comprising the sequence of SEQ ID NO:13 and a VL region comprising the sequence of SEQ ID NO:69, e) a VH region comprising the sequence of SEQ ID NO:17 and a VL region comprising the sequence of SEQ ID NO:73, f) a VH region sing the sequence of SEQ ID NO:21 and a VL region comprising the sequence of SEQ ID NO:77, g) a VH region comprising the sequence of SEQ ID NO:25 and a VL region comprising the sequence of SEQ ID NO:81, h) a VH region comprising the sequence of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID NO:85, i) a VH region comprising the sequence of SEQ ID NO:33 and a VL region comprising the ce of SEQ ID NO:89, j) a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the ce of SEQ ID NO:93, k) a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the ce of SEQ ID NO: 97, l) a VH region sing the sequence of SEQ ID NO:45 and a VL region comprising the sequence of SEQ ID NO:101, m) a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:105, or n) a VH region comprising the sequence of SEQ ID NO:53 and a VL region comprising the sequence of SEQ ID NO:109.
In a further embodiment, the antibody of the invention comprises: - a heavy chain variable region derived from a human germline VH sequence selected from the group ting of: IGHV1-18*01, IGHV3-23*01, IGHV3-30*01, IGHV3-33*01, IGHV3-33*O3, IGHV1-69*02, IGHV1-69*O4 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, 11*01, and IGKV1D-16*01.
In a further aspect, the invention relates to a monoclonal anti-TF antibody comprising a VH region having the sequence as set forth in seq id no 9, 1, 5, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49 or 53, or a variant of any of said sequences, such as a variant having at most 25 amino acid cations, such 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.
The variant of the ce as set forth 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 said sequences, such as at least 85% ty or 90% identity or 95% ty, such as 96% identity or 97% identity or 98% identity or 99% identity.
In an aspect of the invention the isolated monoclonal anti-TF antibody comprises a VL sequence as set forth in SEQ ID NO: 65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101, or 105 or a variant of any of said ces, such as a variant having at most 25 amino acid modifications, such 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.
The variant of the sequence as set forth in seq id no 65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101, or 105 may have at least 80% identity to any of said sequences, such as at least 85% identity or 90% ty or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity.
In another embodiment, the antibody comprises a) a VL region having the sequence selected from the group consisting of SEQ ID No: 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, wherein said variant preferably only has vative substitutions in said sequences.
In a preferred embodiment the antibody comprises a VL region having the sequence as set forth in SEQ ID No: 65 and a VH region having the sequence as set forth SEQ ID No: 9, or a variant of any of the two sequences, the variants having either a) at most 25 amino acid modifications, such 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 ions, preferably substitutions, such as vative 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% ty 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 as set forth in SEQ ID No: 57 and a VH region having the sequence as set forth SEQ ID No: 1, or a variant of any of the two sequences, the variants having either a) at most 25 amino acid modifications, such 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, or b) at least 80% ty 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 or 99% identity.
In another preferred ment the antibody comprises a VL region having the sequence as set forth in SEQ ID No: 61 and a VH region having the sequence as set forth SEQ ID No: , or a variant of any of the two sequences, the ts having either a) at most 25 amino acid modifications, such 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, 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% ty or 99% identity.
In another preferred embodiment the antibody comprises a VL region having the sequence as set forth in SEQ ID No: 69 and a VH region having the sequence as set forth SEQ ID No: 13, or a variant of any of the two sequences, the variants having either a) at most 25 amino acid modifications, such 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 acid modifications, such as deletions or insertions, preferably substitutions, such as conservative tutions, or b) at least 80% ty to SEQ ID NO: 13 or SEQ ID NO: 69 respectively, such as at least 85% identity or 90% identity or 95% ty, such as 96% identity or 97% identity or 98% ty or 99% identity.
In another preferred embodiment the antibody comprises a VL region having the sequence as set forth in SEQ ID No: 73 and a VH region having the sequence as set forth SEQ ID No: 17, or a variant of any of the two sequences, the ts having either a) at most 25 amino acid modifications, such 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, ably 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, such as 96% identity or 97% identity or 98% ty or 99% ty.
In another preferred embodiment the antibody ses a VL region having the sequence as set forth in SEQ ID No: 77 and a VH region having the sequence as set forth SEQ ID No: 21, or a t of any of the two sequences, the variants having either a) at most 25 amino acid modifications, such 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, 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 as set forth in SEQ ID No: 81 and a VH region having the sequence as set forth SEQ ID No: , or a variant of any of the two sequences, the variants having either a) at most 25 amino acid modifications, such 20, such as at most 15, 14, 13, 12 or 11 amino acid cations, 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, 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, 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 as set forth in SEQ ID No: 85 and a VH region having the ce as set forth SEQ ID No: 29, or a variant of any of the two sequences, the variants having either a) at most 25 amino acid cations, such 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 tutions, 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 red embodiment the antibody ses a VL region having the sequence as set forth in SEQ ID No: 89 and a VH region having the sequence as set forth SEQ ID No: 33, or a variant of any of the two sequences, the variants having either a) at most 25 amino acid modifications, such 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, 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% ty or 98% identity or 99% identity.
In another preferred embodiment the antibody comprises a VL region having the sequence as set forth in SEQ ID No: 93 and a VH region having the sequence as set forth SEQ ID No: 37, or a variant of any of the two sequences, the variants having either a) at most 25 amino acid cations, such 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 ions, 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% ty or 99% identity.
In another preferred embodiment the antibody comprises a VL region having the sequence as set forth in SEQ ID No: 97 and a VH region having the sequence as set forth SEQ ID No: 41, or a variant of any of the two ces, the variants having either a) at most 25 amino acid modifications, such 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, 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% ty or 95% identity, such as 96% identity or 97% identity or 98% ty or 99% identity.
In another preferred embodiment the dy comprises a VL region having the sequence as set forth in SEQ ID No: 101 and a VH region having the sequence as set forth SEQ ID No: 45, or a variant of any of the two sequences, the variants having either a) at most 25 amino acid modifications, such 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, 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% ty or 95% ty, 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 ce as set forth in SEQ ID No: 105 and a VH region having the sequence as set forth SEQ ID No: 49, or a variant of any of the two ces, the variants having either a) at most 25 amino acid modifications, such 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, 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 as set forth in SEQ ID No: 109 and a VH region having the ce as set forth SEQ ID No: 53, or a variant of any of the two sequences, the variants having either a) at most 25 amino acid modifications, such 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, 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.
Monoclonal antibodies of the present invention may e.g. be produced by the oma method first described by Kohler et al., Nature E, 495 (1975), or may be produced by recombinant DNA methods. onal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature fl, 624-628 (1991) and Marks et al., J. Mol. Biol. Q, 581-597 (1991).
Monoclonal dies may be obtained from any suitable . Thus, for e, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for instance in form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of st.
Monoclonal antibodies may also be obtained from omas derived from antibody- expressing cells of immunized humans or non-human mammals such as rats, rabbits, dogs, primates, etc.
In one embodiment, the antibody of the invention is a human antibody. Human onal antibodies directed against tissue factor may be generated using transgenic or hromosomal mice carrying parts of the human immune system rather than the mouse system. Such transgenic and hromosomic mice include mice referred to herein as HuMAb mice and KM mice, tively, and are collectively ed to herein as "transgenic mice".
The HuMAb mouse contains a human immunoglobulin gene miniloci that encodes unrearranged human heavy variable and constant (p and y) and light variable and constant (K) chain immunoglobulin sequences, together with targeted mutations that vate the endogenous u and K chain loci (Lonberg, N. et al., Nature fl, 856-859 (1994)).
Accordingly, the mice exhibit 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 dies (Lonberg, N. et al. , supra; reviewed in g, N. ok of Experimental cology 1_13, 49-101 (1994) N. and Huszar, , Lonberg, D., Intern. Rev.
Immunol. Vol. 1_3 65-93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci M 536-546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research fl, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. i2, 2912-2920 , Taylor, L. et al., ational Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology fl, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US ,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US ,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.
The HCo7 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 1_2, 821-830 (1993)), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 24), a KC05 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology fl, 845-851 (1996)), and a HCo7 human heavy chain transgene (as described in US ,770,429).
The HC012 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 1_2, 821-830 (1993)), a CMD disruption in their endogenous heavy chain genes (as bed in Example 1 of WO 01/14424), a KC05 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology fl, 845-851 (1996)), and a HC012 human heavy chain transgene (as described in Example 2 of wo 01/14424).
In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et a|., EMBO J. 1_2, 811-820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KC05, as described in ld et a|., Nature Biotechnology fl, 1 (1996).
This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 78.
Splenocytes from these transgenic mice may be used to generate hybridomas that secrete human monoclonal antibodies according to well known techniques. Human monoclonal or polyclonal antibodies of the present invention, or dies of the present invention originating from other species may also be generated transgenically through the generation of another non-human mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. In connection with the transgenic production in mammals, antibodies may be produced in, and recovered from, the milk of goats, cows, or other mammals. See for instance US 5,827,690, US 5,756,687, US 5,750,172 and US 5,741,957.
Further, human antibodies of the present ion or dies of the present invention from other species may be generated through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules may be subjected to additional maturation, such as ty maturation, as such techniques are well known in the art (see for instance Hoogenboom et a|., J. Mol. Biol. E, 381 (1991) (phage display), Vaughan et a|., Nature Biotech fi, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA %, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene 7_3, 305-318 (1988) (phage display), Scott TIBS fl, 241-245 (1992), Cwirla et a|., PNAS USA fl, 6378-6382 (1990), Russel et a|., Nucl. Acids ch A, 1081-1085 (1993), Hogenboom et a|., l. Reviews m, 43-68 (1992), ll and McCafferty TIBTECH 1_0, 80-84 , and US 5,733,743). If display technologies are utilized to produce antibodies that are not human, such antibodies may be zed.
The antibody of the invention may be of any isotype. The choice of isotype typically will be guided by the desired or functions, such as ADCC ion. ary isotypes are IgGl, IgGZ, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of an anti-TF antibody of the present invention may be switched by known methods. For example, an antibody of the present invention that was originally IgM may be class switched to an IgG antibody of the present invention. r, class switching ques may be used to convert one IgG subclass to another, for instance from IgGl to IgGZ. Thus, the effector function of the antibodies of the present invention may be changed by isotype switching to, e.g., an IgGl, IgGZ, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In one ment an antibody of the present invention is an IgGl antibody, for instance an IgGl,K.
In one embodiment, the antibody of the ion is a full-length antibody, ably an IgGl dy, in particular an IgGl,K antibody. In another embodiment, the antibody of the invention is an antibody fragment or a single-chain antibody.
Antibodies fragments may e.g. be obtained by fragmentation using conventional techniques, and the fragments screened for utility in the same manner as bed herein for whole antibodies. For example, F(ab')2 fragments may be generated by treating antibody with pepsin. The resulting F(ab')2 fragment may be treated to reduce disulfide bridges to produce Fab' fragments. Fab fragments may be obtained by treating an IgG antibody with papain; Fab' fragments may be obtained with pepsin digestion of IgG antibody. An F(ab') fragment may also be produced by binding Fab' described below via a thioether bond or a disulfide bond. A Fab' fragment is an antibody fragment obtained by g a disulfide bond of the hinge region of the F(ab')2. A Fab' fragment may be obtained by treating an F(ab')2 fragment with a reducing agent, such as threitol. Antibody fragment may also be generated by sion of nucleic acids encoding such fragments in recombinant cells (see for instance Evans et al., J. Immunol. Meth. L84, 123-38 (1995)). For example, a chimeric gene encoding a portion of an F(ab')2 fragment could include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield such a truncated dy fragment molecule.
In one embodiment, the anti-TF antibody is a monovalent antibody, preferably a monovalent dy as described in W02007059782 (Genmab) porated herein by reference) having a deletion of the hinge . Accordingly, in one embodiment, the antibody is a lent antibody, wherein said anti-TF antibody is constructed by a method comprising: i) ing a nucleic acid construct encoding the light chain of said lent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen specific anti-TF antibody and a tide sequence encoding the constant CL region of an Ig, wherein said nucleotide sequence encoding the VL region of a selected antigen specific antibody and said nucleotide sequence encoding the CL region of an Ig are operably linked together, and wherein, in case of an IgGl subtype, the nucleotide sequence encoding the CL region has been modified such that the CL region does not contain any amino acids capable of forming disulfide bonds or covalent bonds with other peptides comprising an identical amino acid ce of the CL region in the presence of polyclonal human IgG or when administered to an animal or human being; ii) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide ce encoding the VH region of a selected antigen specific antibody and a nucleotide sequence encoding a constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that the region corresponding to the hinge region and, as ed by the Ig subtype, other regions of the CH , such as the CH3 region, does not comprise any amino acid residues which ipate in the formation of disulphide bonds or covalent or stable non- covalent inter-heavy chain bonds with other es comprising an identical amino acid sequence of the CH region of the human Ig in the presence of polyclonal human IgG or when administered to an animal human being, wherein said nucleotide sequence encoding the VH region of a selected 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 producing said monovalent antibody; iv) producing said monovalent antibody by co-expressing the c acid constructs of (i) and (ii) in cells of the cell expression system of (iii).
Similarly, in one embodiment, the anti-TF dy is a monovalent antibody, which comprises (i) a variable region of an antibody of the invention as described herein or an antigen binding part of the said region, and (ii) a CH region of an immunoglobulin or a fragment thereof comprising the CH2 and CH3 regions, n the CH region or fragment thereof has been modified such that the region corresponding to the hinge region and, if the immunoglobulin is not an IgG4 subtype, other regions of the CH region, such as the CH3 region, do not comprise any amino acid residues, which are capable of forming disulfide bonds with an identical CH region or other covalent or stable non-covalent inter-heavy chain bonds with an identical CH region in the presence of polyclonal human IgG.
In a further embodiment, the heavy chain of the lent anti-TF antibody has been modified such that the entire hinge has been deleted.
In a r embodiment, said lent dy is of the IgG4 subtype (see SEQ ID NO: 114, a hinge-less variant of SEQ ID NO:113), but the CH3 region has been modified so that one or more of the ing amino acid substitutions have been made: Thr (T) in position 234 has been replaced by Ala (A); Leu (L) in position 236 has been replaced by Ala (A); Leu (L) in position 236 has been ed by Val (V); Phe (F) in position 273 has been replaced by Ala (A); Phe (F) in position 273 has been replaced by Leu (L); Tyr (Y) in position 275 has been replaced by Ala (A).
In another further embodiment, the sequence of said monovalent antibody has been modified so that it does not comprise any acceptor sites for N-linked glycosylation.
Anti-TF antibodies of the invention also include single chain antibodies. Single chain dies are peptides in which the heavy and light chain Fv regions are connected. In one embodiment, the present invention provides a single-chain Fv (scFv) wherein the heavy and light chains in the Fv of an anti-TF dy of the present ion are joined with a flexible peptide linker (typically of about 10, 12, 15 or more amino acid residues) in a single peptide chain. Methods of ing such antibodies are described in for instance US 778, Pluckthun in The Pharmacology of onal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science fl, 423-426 (1988), Huston et al., PNAS USA g, 5879-5883 (1988) and McCafferty et al., Nature fl, 552-554 (1990). The single chain antibody may be monovalent, if only a single VH and VL are 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 the invention is an effector-function- deficient antibody. Such antibodies are particularly useful when the antibody is for use in stimulation of the immune system through blocking of the inhibitory effects of TF. For such applications, it may be advantages that the antibody has no or functions, such as ADCC, as they may lead to undesired cytotoxicity.
In one embodiment, the effector-function-deficient anti-TF antibody is a stabilized IgG4 dy. Examples of suitable stabilized IgG4 antibodies are antibodies, wherein arginine at position 409 in a heavy chain constant region of human IgG4, which is indicated in the EU index as in Kabat et al., is substituted with , threonine, methionine, or leucine, preferably lysine (described in W02006033386 (Kirin)) and/or wherein the hinge region comprises a Cys-Pro-Pro-Cys sequence.
In a further embodiment. the stabilized IgG4 anti-TF dy is an IgG4 antibody comprising a heavy chain and a light chain, n said heavy chain comprises a human IgG4 constant region having a residue selected from the group ting of: Lys, Ala, Thr, Met and Leu at the position corresponding to 409 and/or a residue selected from the group ting of: Ala, Val, Gly, Ile and Leu at the position corresponding to 405, and wherein said antibody optionally comprises one or more further substitutions, deletions and/or insertions, but does not comprise a Cys-Pro-Pro-Cys sequence in the hinge region.
Preferably, said antibody comprises a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CH3 region of human IgGl, of human IgGZ or of human IgG3.
In an even further embodiment. the ized IgG4 anti-TF antibody is an IgG4 dy 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 the position corresponding to 409 and/or a residue selected from the group consisting of: Ala, Val, Gly, Ile and Leu at the position corresponding to 405, and wherein said antibody optionally comprises one or more further substitutions, deletions and/or insertions and wherein said dy comprises a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said antibody ses a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CH3 region of human IgGl, of human IgGZ or of human IgG3.
In a further embodiment, the effector-function-deficient anti-TF dy is an antibody of a G4 type, e.g. IgGl, IgGZ or IgG3 which has been mutated such that the ability to mediate effector ons, such as ADCC, has been reduced or even eliminated. Such mutations have e.g. been described in Dall'Acqua WF et al., J Immunol. fl(2):1129-1138 (2006) and Hezareh M, J Virol. ;fi(24):12161-12168 .
In a further embodiment, the antibody of the invention is conjugated to another moiety, such as a cytotoxic moiety, a radioisotope or a drug. Such antibodies may be produced by chemically ating the other moiety to the inal side or C-terminal side of the anti-TF antibody or nt thereof (e.g., an anti-TF antibody H chain, L chain, or anti-TF specific/selective fragment thereof) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, hed by Chijin Shokan (1994)). Such conjugated antibody derivatives may also be generated by conjugation at internal residues or sugars, where appropriate.
In general, anti-TF antibodies bed herein may be modified by inclusion of any suitable number of such modified amino acids and/or associations with such conjugated tuents. Suitability in this context is generally determined by the ability to at least substantially retain TF selectivity and/or specificity associated with the non-derivatized parent anti-TF antibody. The inclusion of one or more modified amino acids may be advantageous in, for e, increasing polypeptide serum half-life, reducing polypeptide antigenicity, or increasing polypeptide e stability. Amino acid(s) are modified, for example, co-translationally or post-translationally during recombinant production (e. g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means. Non-limiting es of a modified amino acid e a glycosylated amino acid, a sulfated amino acid, a prenylated (e. g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEGyIated amino acid, a biotinylated amino acid, a carboxylated amino acid, a orylated amino acid, and the like. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example ols are found in Walker (1998) Protein Protocols On Cd-Rom, Humana Press, Towata, NJ. The modified amino acid may for instance be selected from a glycosylated amino acid, a ted 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 c derivatizing agent.
Anti-TF antibodies may also be chemically modified by covalent conjugation to a polymer to for instance increase their circulating half-life. Exemplary polymers, and methods to attach them to peptides, are illustrated in for instance US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Additional illustrative rs include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG with a molecular weight of between about 1,000 and about 40,000, such as between about 2,000 and about 20,000, e.g., about 3,000-12,000 g/mol).
In one embodiment, the present 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 , a chemiluminescent marker, a peptide tag, or a magnetic particle. In one ment, an anti-TF antibody may be conjugated to one or more antibody nts, nucleic acids (oligonucleotides), ses, hormones, immunomodulators, chelators, boron compounds, ctive agents, dyes, and the like.
These and other le agents may be coupled either directly or indirectly to an anti-TF antibody of the t invention. One example of indirect ng of a second agent is coupling by a spacer moiety. These spacers, in turn, may be either insoluble or soluble (see for instance Diener et al., Science Q, 148 (1986)) and may be selected to enable drug release from the anti-TF antibody at a target site and/or under particular conditions.
Additional examples of agents that may be coupled to an anti-TF antibody e lectins and fluorescent peptides.
In one embodiment, anti-TF antibodies comprising one or more radiolabeled amino acids are provided. A radiolabeled anti-TF antibody may be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature).
Nonlimiting examples of labels for polypeptides e, but are not limited to 3H, 14C, 15N, 35S, 90Y, 99Tc, and 1251, 1311, and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see for instance Junghans et a|., in Cancer Chemotherapy and Biotherapy 6 (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 RE35,500), US 5,648,471 and US 902. For example, a radioisotope may be conjugated by a chloramine T method.
In one embodiment, an anti-TF antibody of the ion comprises a conjugated nucleic acid or nucleic acid-associated molecule. In one such facet of the present invention, the conjugated nucleic acid is a cytotoxic ribonuclease. In one embodiment, the conjugated nucleic acid is an antisense nucleic acid (for instance a S100A10 targeted antisense molecule, which may also be an independent component in a combination composition or combination administration method of the present invention — see for instance Zhang et a|., J Biol Chem. m8), 2053-62 (2004)). In one embodiment, the conjugated nucleic acid is an inhibitory RNA molecule (e.g., a siRNA molecule). In one embodiment, the ated nucleic acid is an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif- containing DNA molecule). In one embodiment, the conjugated nucleic acid is an expression te coding for expression of a tumor suppressor gene, anti-cancer e, anti-cancer cytokine, or tic agent. Such derivatives also may comprise conjugation of a nucleic acid coding for expression of one or more cytotoxic proteins, such as plant and bacterial toxins.
In one embodiment, an anti-TF antibody is conjugated to a functional nucleic acid molecule. onal nucleic acids include antisense molecules, interfering nucleic acid molecules (e.g., siRNA molecules), aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional nucleic acid molecules may act as ors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the onal nucleic acid molecules may possess a de novo activity independent of any other les.
In another ment, an F antibody of the invention is conjugated to an aptamer.
In r embodiment, the present invention provides an anti-TF antibody which is conjugated to a ribozyme.
Any method known in the art for conjugating the anti-TF antibody to the conjugated mo|ecu|e(s), such as those described above, may be employed, including those methods described by Hunter et a|., Nature m, 945 (1962), David et a|., Biochemistry 1_3, 1014 (1974), Pain et a|., J. l. Meth. 4_0, 219 (1981) and Nygren, J. hem. and Cytochem. E, 407 (1982). us types of cytotoxic compounds may be joined to proteins through the use of a reactive group on the xic compound or through the use of a cross-linking agent. 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 reacts with the s-amine group of lysine. Maleimide is a commonly used ve group to form a stable in vivo covalent bond with the sulfhydryl group on cysteine (Ji., Methods Enzymol fl, 580-609 (1983)). Monoclonal antibodies typically are incapable of forming covalent bonds with radiometal ions, but they may be attached to the dy indirectly through the use of chelating agents that are covalently linked to the antibodies. Chelating agents may be attached h amines (Meares et al., Anal. Biochem. i2, 68-78 (1984)) and sulfhydral groups (Koyama, Chem. Abstr. 120, 217262t (1994)) of amino acid residues and also h carbohydrate groups (Rodwell et al., PNAS USA Q, 636 (1986), Quadri et al., Nucl. Med. Biol. Q, 0 ). Since these chelating agents contain two types of functional groups, one to bind metal ions and the other to joining the chelate to the antibody, they are commonly referred as bifunctional ing agents (Sundberg et al., Nature m, 587-588 (1974)).
In one embodiment, the present invention provides an F 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 which include one or more cytotoxins are referred to as "immunotoxins".
A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. For a description of these classes of drugs which are well known in the art, and their mechanisms of action, see Goodman et al., Goodman and Gilman's The Pharmacological Basis Of eutics, 8th Ed., lan Publishing Co., 1990. Additional techniques relevant to the preparation of antibody immunotoxins are provided in for instance Vitetta, Immunol. Today fi, 252 (1993) and US 5,194,594.
Suitable therapeutic agents for g immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydro- terone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and cin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), ting agents (such as mechlorethamine, thioepa, mbucil, lan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, tin and other platinum derivatives, such as carboplatin), antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and d molecules (such as diphtheria A chain and active fragments thereof and hybrid molecules), ricin toxin (such as ricin A or a deglycosylated ricin A chain , cholera toxin, a Shiga- like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, sarcin, Aleurites fordii proteins, in proteins, Phytolacca americana ns (PAPI, PAPII, and , momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins. Other suitable conjugated molecules e ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, ed antiviral protein, diphtherin toxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell Q, 641 (1986) and berg, Calif. A Cancer Journal for Clinicians fi, 43 (1994). Therapeutic agents, which may be administered in combination with a an anti-TF antibody of the present invention as bed elsewhere herein, may also be candidates for therapeutic moieties useful for conjugation to an anti-TF antibody of the present invention.
In one embodiment, the F antibody of the t invention is attached to a chelator linker, e.g. tiuxetan, which allows for the antibody to be conjugated to a radioisotope.
In a further , the invention relates to a bispecific molecule comprising an anti- TF antibody of the invention as described herein above and a second binding specificity such as a binding specificity for a human or cell, a human Fc receptor or a T cell receptor.
Or a binding specificity for another epitope of TF.
Bispecific molecules of the present ion may further include a third binding specificity, in addition to an anti-TF binding specificity and a binding specificity for a human effector cell, a human Fc receptor or a T cell receptor.
Exemplary bispecific antibody molecules of the invention comprise (i) two antibodies one with a specificity to TF and another to a second target that are conjugated together, (ii) a single antibody that has one chain specific to TF and a second chain specific to a second molecule, and (iii) a single chain antibody that has specificity to TF and a second molecule. lly, the second target/second molecule is a molecule other than TF. In one embodiment, the second molecule is a cancer antigen/tumor-associated antigen such as carcinoembryonic n (CEA), prostate specific antigen (PSA), RAGE (renal antigen), d-fetoprotein, CAMEL (CTL-recognized antigen on ma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-l, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin ns (e.g., MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75, C-myc, Mart1, MelanA, MUM-1, MUM-2, MUM-3, , and Ep-CAM. In one embodiment, the second molecule is a cancer-associated integrin, such as 0583 integrin. In one embodiment, the second molecule is an enic factor or other cancer- associated growth factor, such as a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), angiogenin, and receptors thereof, particularly receptors ated with cancer progression (for instance 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, a bispecific antibody of the present invention is a diabody. ific antibodies also include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in a heteroconjugate may be coupled to avidin and the other to .
Such antibodies have, for example, been proposed to target immune system cells to ed cells (see for instance US 4,676,980). Heteroconjugate antibodies may be made using any convenient cross-linking methods.
In a further aspect, the invention relates to an expression vector encoding an antibody of the invention.
In one embodiment, the expression vector of the invention comprises a tide sequence encoding one or more of the amino acid sequences selected from the group consisting of: SEQ ID NO: 1 — 112.
In r ular embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the 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 a particular embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the VH CDR3 amino acid ces selected from the group consisting of: SEQ ID NO 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52 and 56.
In another particular ment, the expression vector of the invention comprises a nucleotide sequence ng one or more of the VL amino acid sequences selected from the group consisting of: SEQ ID NO: 65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101 and In another embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the 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 a particular embodiment the expression vector of the 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, such 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, ably substitutions, such as conservative substitutions or at least 80% identity to any of said sequences, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% ty to any of the afore mentioned amino acid sequences.
In a r embodiment, the expression vector further comprises a nucleotide sequence encoding the constant region of a light chain, a heavy chain or both light and heavy chains of an antibody, e.g. a human antibody.
Such expression vectors may be used for recombinant production of antibodies of the invention.
An sion vector in the context of the t invention may be any suitable vector, including chromosomal, non-chromosomal, and tic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, 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, an anti-TF antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, ing, for example, a linear expression t (as described in for instance Sykes and Johnston, Nat Biotech fl, 355-59 (1997)), a compacted nucleic acid vector (as described in for instance US 6,077, 835 and/or WO 00/70087), a plasmid vector such as , pUC 19/18, or pUC 118/119, a "midge" lly-sized nucleic acid vector (as described in for ce Schakowski et al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaP04-precipitated uct (as described in for instance WO 00/46147, Benvenisty and Reshef, PNAS USA Q, 9551-55 (1986), Wigler et al., Cell fi, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics Z, 603 (1981)). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance US 5,589,466 and US 972).
In one embodiment, the vector is suitable for expression of the anti-TF antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN s (Van Heeke & er, J Biol Chem M, 5503-5509 (1989), pET vectors (Novagen, Madison WI) and the like).
An sion 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 employed.
Suitable s include, for example, vectors sing tutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: 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 fl, 4 (1987)).
A nucleic acid and/or vector may also comprises a nucleic acid sequence encoding a secretion/localization sequence, which can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides, organelle targeting sequences (e. g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization/anchor sequences (e. g., stop transfer sequences, GPI anchor sequences), and the like.
In an expression vector of the invention, F antibody-encoding nucleic acids may comprise or be associated with any suitable promoter, enhancer, and other expression- facilitating elements. Examples of such ts include strong expression promoters (e. g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as selectable marker, and/or a ient cloning site (e.g., a polylinker). Nucleic acids may also comprise an inducible promoter as opposed to a tutive promoter such as CMV IE (the skilled artisan will recognize that such terms are actually ptors of a degree of gene sion under certain conditions).
In one embodiment, the anti-TF-antibody-encoding expression vector may be positioned in and/or delivered to the host cell or host animal via a viral vector.
In an even further , the invention relates to a recombinant otic or prokaryotic host cell, such as a ectoma, which produces an antibody of the invention as defined herein or a bispecific molecule of the invention as defined herein. Examples of host cells include yeast, bacterial, and mammalian cells, such as CHO or HEK cells. For example, in one embodiment, the present invention provides a cell comprising a nucleic acid stably integrated into the cellular genome that comprises a sequence coding for expression of an anti-TF antibody of the present invention. In another embodiment, the present ion provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of an anti-TF antibody of the invention.
In a further aspect, the invention relates to a hybridoma which es an antibody of the invention as defined herein. In an even further aspect, the ion relates to a transgenic non-human animal comprising nucleic acids encoding a human heavy chain and a human light chain, wherein the animal or plant produces an antibody of the invention of the invention. Generation of such hybridomas and transgenic animals has been described above.
In a further aspect, the invention relates to a method for producing an F antibody of the invention, said method comprising the steps of a) culturing a hybridoma or a host cell of the invention as bed herein above, and b) purifying the antibody of the invention from the culture media.
In a further main aspect, the invention relates to an anti-TF antibody as defined herein or a bispecific molecule as defined herein for use as a medicament.
In an even further , the invention relates to a ceutical ition comprising: - an anti-TF antibody as defined herein or a bispecific molecule as defined herein, and - a pharmaceutically-acceptable carrier.
The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional ques such as those sed in Remington: The Science and Practice of Pharmacy, 19th n, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.
The pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the chosen compound of the present invention and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological ties of the chosen compound or pharmaceutical composition of the present invention (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.)) on n g.
A pharmaceutical ition of the present invention may also include diluents, fillers, salts, buffers, ents (e. g., a nonionic detergent, such as Tween-20 or Tween- 80), izers (e. g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.
It has been reported that in cancer cells, such as human colorectal cancer cells, TF expression is under control of 2 major transforming events driving disease progression (activation of K-ras oncogene and inactivation of the p53 tumor suppressor), in a manner dependent on MEK/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'- kinase (PI3K) (Yu et al. (2005) Blood 105:1734.
Cancer cells overexpressing TF may be particularly good targets for anti-TF antibodies of the invention, since more antibodies may be bound per cell. Thus, in one ment, a cancer patient to be treated with an anti-TF antibody of the invention is a patient, e.g. a pancreatic cancer, lung cancer or colorectal cancer patient who has been diagnosed to have one or more ons 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 an anti-TF antibody of the invention is a patient, e.g. a pancreatic cancer, lung cancer or colorectal cancer patient, who does not have a on in K-Ras. Without being bound by any specific theory, it is le that some tumor cells having K-Ras activation are less susceptible to anti-TF antibody treatment, because the effects of anti-TF dies on intracellular signaling isms may be less effective in cells in which K-Ras is activated.
The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the t. The selected dosage level will depend upon a variety of pharmacokinetic factors including the ty of the particular compositions of the present invention employed, or the amide thereof, the route of administration, the time of stration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, l health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The pharmaceutical composition may be administered by any suitable route and mode. Suitable routes of administering a compound of the present invention in vivo and in vitro are well known in the art and may be selected by those of ordinary skill in the art.
In one embodiment, a pharmaceutical ition of the t invention is administered parenterally.
The phrases "parenteral administration" and administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, rbital, intracardiac, ermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, al and intrasternal injection and infusion.
In one embodiment that pharmaceutical composition is administered by intravenous or aneous injection or infusion.
Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, cterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically ible with a compound of the present ion.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl ose colloidal solutions, tragacanth gum and injectable c , 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 extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated.
Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of tants.
Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (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 yl palmitate, butylated hydroxyanisole (BHA), ted hydroxytoluene (BHT), in, propyl gallate, alphatocopherol , and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid , sorbitol, ic acid, phosphoric acid, and the like.
Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, ol, glycerol or sodium chloride in the compositions.
The pharmaceutical compositions of the present ion may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting , emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the ceutical composition. The compounds of the present invention may be prepared with carriers that will protect the nd against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may e gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, en, polyorthoesters, and polylactic acid alone or with a wax, or other materials well known in the art.. Methods for the preparation of such formulations are generally known to those skilled in the art. See e.g., ned and Controlled Release Drug ry Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
In one embodiment, the compounds of the present ion may be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is atible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated.
Supplementary active compounds may also be incorporated into the compositions.
Pharmaceutical compositions for injection must typically be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered ure suitable to high drug concentration. The carrier may be a aqueous or nonaqueous solvent or dispersion medium containing for instance 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 c esters, such as ethyl oleate. The proper fluidity may be maintained, for example, by the use of a coating such as in, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, , polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the ition. Prolonged tion of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are ed by incorporating the active compound into a sterile e that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile able solutions, examples of s of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a usly sterile-filtered solution thereof.
Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are ed by incorporating the active nd into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of s of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional d ingredient from a previously sterile-filtered solution thereof.
The pharmaceutical composition of the present invention may contain one nd of the present invention or a combination of compounds of the present invention.
As described above, in another aspect, the invention relates to the antibody of the ion as defined herein or a bispecific molecule of the invention as defined herein for use as a medicament.
The anti-TF antibodies of the invention may be used for a number of purposes. In particular, the dies of the invention may be used for the treatment of various forms of cancer. In one aspect the F monoclonal antibodies of the invention are used for the treatment of various solid cancer types such as: tumors of the central nervous system, head and neck , lung cancer (such as non-small cell lung cancer), breast cancer, geal cancer, stomach cancer, liver and biliary cancer, pancreatic cancer, colorectal cancer, r cancer, kidney cancer, prostate cancer, endometrial cancer, ovarian cancer, malignant melanoma, a (soft tissue eg. bone and muscle), tumors of unknown primary origin (i.e. unknown primarys), leukemia, bone marrow cancer (such as multiple myeloma) acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma, skin cancer, glioma, cancer of the brain, uterus, and rectum.
Further autoimmune inflammation, such as myopathies or multiple sclerose may be targeted with the anti-TF monoclonal antibodies of the present invention.
The anti-TF monoclonal antibodies of the present invention may also be useful for the treatment of tatis.
Cancer related hemostatic disorders may also be targeted with the present intervention.
Further diseases with inflammation, such as hies, Rheumatoid Arthritis, osteoarthritis, ankylosing spondylitis, gout, spondyiartbropatbris, ankyiosing spondyiitis, Roiter's syndrome, psoriatio arthropathy, enterabathric spondyiitis, juveniie arthropa‘thy, reactive. arthropatiiy, infantious or post—infectious arthritis, tabercuious artbritis, yirai arthritis, fungal arthritis, sypbiiitio arthritis, glomerulonephritis, end stage renal disease, systemic lupus erythematosus, mb. Crohn, ulcerative colitis, inflammatory bowoi disease, cystic fibrosis, chronic obstructive pulmonary disease (COPD), astma, allergic astma, bronchitis, acuta bronci‘iioiitis, c broncbioiitis, idiopathic pulmonary fibrosis, or multiple sclerose may be targeted with the anti-TF monoclonal antibodies of the present invention.
The anti-TF monoclonal antibodies of the present invention may also be useful for the ent of haemostatis.
Cancer related hemostatic disorders may also be targeted with the present intervention.
Also vascular diseases such as ar rastemosis, myocardial vascular disease, cerebral vascular disease, retinopathia and macular degeneration, including but not limited to wet AMD can be d with anti-TF monoclonal antibodies.
The anti-TF monoclonal antibodies of the present invention may also be useful for the treatment of patients with cardiovascular risk, such as atherosclerosis, hypertension, diabetis, dyslipidemia, and acute coronary syndrome, including but not limited to Acute Myocardial t, stroke.
The anti-TF monoclonal antibodies of the present invention may also be useful for inhibition of thrombosis, such as DVT, renal embolism, lung embolism, arterial thrombosis, or to treat thrombosis ocouring ing al surgiaai, poripharai ‘v’aSCLiiai‘ bypass grafts or coronary arteryi bypass grafts, arterio-venous shunts, removal of an entation, such as a stent or er The anti-TF monoclonal antibodies of the present invention may also be useful for inhibition of renal isohamir: usiori injury The anti-TF monoclonal antibodies of the t invention may also be useful for treatment of iwpariipoproteineimia, hyperparatbyroiciism, The anti-TF onal antibodies of the present invention may also be useful for treatment of vascuiitis, ositiye VBSCLilitiS, Beta-cat’s diseasa The anti-TF monoclonal antibodies of the present invention may also be useful for blocking traume-induced respiratory failure, such as Acute Respitory Distress Syndrome, Acute lung Injury.
The anti-TF monoclonal antibodies of the present invention may also be useful for blocking infection-induced organ dysfunction, such as renal failure, Acute Respiratory Distress Syndrome, Acute Lung Injury The anti-TF onal antibodies of the present invention may also be useful to treat 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 in a prophylactic setting to treat TF-mediated complications to systemic infections, such as sepsis or pneumonia.
The F onal antibodies of the present invention may also be useful as prophylactic treatment of ts with atherosclemtic vessels at risk for thrombosis The anti-TF monoclonal antibodies of the present ion may also be useful for treatment of Graft-versus-host disease.
The anti-TF monoclonal antibodies of the present invention may also be useful for sing beta cell engraftment in islet transplantation, to prevent cardiac allograft vasculopathy (CAV), to prevent acute graft ion The anti-TF monoclonal antibodies of the present ion may also be useful for treatment of diseases where circulating tissue-factor exposing microparticles are present, such as but not limited to vascular thrombosis, type II diabetis, AMI, pulmonary arterial hypertension Similarly, the invention relates to a method for inhibiting growth and/or proliferation of a tumor cell expressing TF, comprising administration, to an individual in need thereof, of an antibody or a bispecific molecule of the ion. In one embodiment, said tumor cell is involved in 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, endometrial cancer, n cancer, cutaneous melanoma, leukemia bone marrow cancer (such as multiple myeloma), acute lymphoblastic leukemia, chronic blastic leukemia and non-Hodgkin lymphoma, skin cancer, prostate cancer, glioma, cancer of the brain, kidneys, uterus, bladder, and .
Also, the invention relates to the use of a monoclonal antibody that binds to human TF for the preparation of a medicament for the treatment of , such as one of the specific cancer indications mentioned above.
In an embodiment selection of patients to be treated with anti-TF antibody is based on the level of tissue factor (TF) in their urine and/or blood. In a particular embodiment the patient to be treated has a vely high level of TF in urine and/or blood. For example, the patient to be treated may have a TF level in urine of more than 20 ng/ml, such as more than 40 ng/ml. e.g. more than 100 ng/ml, such as more than 200 ng/ml. Alternatively, or in addition, the TF level in serum of the patients may be more than 100 pg/ml, such as more than 200 pg/ml. This may e.g. be determined using an ELISA.
In a further embodiment of the methods of ent of the present invention, the efficacy of the treatment is being monitored during the therapy, e.g. at predefined points in time. In one ment, the efficacy may be monitored by measuring the level of TF in urine or blood, for example by ELISA. In another embodiment, the efficacy may be determined by visualization of the disease area, e.g. by performing one or more PET-CT scans, for example using a labeled anti-TF antibody, such as a labeled anti-TF antibody of the present invention. Furthermore, labeled anti-TF antibodies, such as labeled anti-TF antibodies of the invention, could be used to detect ducing tumors e.g. using a PET- CT scan.
Dosage regimens in the above methods of treatment and uses are adjusted to provide the m desired response (e.g., a therapeutic response). For example, a single bolus may be administered, l divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of stration and uniformity of . Dosage unit form as used herein refers to physically discrete units suited as y dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the t invention are ed by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active nd for the treatment of sensitivity in individuals.
The efficient s and the dosage regimens for the anti-TF antibodies depend on the disease or condition to be treated and may be determined by the persons skilled in the art. An ary, non-limiting range for a therapeutically effective amount of a compound of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, or about 3 mg/kg.
A physician or veterinarian having ordinary skill in the art may readily determine and prescribe the effective amount of the ceutical composition required. For example, the physician or veterinarian could start doses of the anti-TF antibody employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the present invention will be that amount of the nd which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. If desired, the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition as bed above.
In one embodiment, the anti-TF antibodies may be administered by infusion in a weekly dosage of from 10 to 500 mg/mZ, such as of from 200 to 400 mg/mZ. Such administration may be repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours.
In one embodiment, the anti-TF antibodies may be stered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.
In one embodiment the anti-TF antibodies may be administered in a weekly dosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4 to 6 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. Such n may be repeated one or more times as necessary, for example, after 6 months or 12 . The dosage may be determined or adjusted by measuring the amount of compound of the t invention in the blood upon administration by for instance taking out a biological sample and using anti-idiotypic antibodies which target the antigen binding region of the anti-TF antibodies of the t invention.
In one embodiment, the anti-TF antibodies may be administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.
In one ment, the anti-TF antibodies may be stered by a regimen including one infusion of an anti-TF antibody of the present invention followed by an infusion of an F antibody of the present invention conjugated to a radioisotope. The regimen may be repeated, e.g., 7 to 9 days later.
As non-limiting examples, ent according to the t invention may be provided as a daily dosage of a compound of the present 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, per day, on at least one of day 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 week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
An tive amount" for tumor therapy may also be measured by its ability to stabilize the progression of disease. The ability of a compound to inhibit cancer may be evaluated in an animal model system tive of efficacy in human tumors. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to inhibit cell growth or to induce apoptosis by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a eutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such s as the t's size, the severity of the subject's symptoms, and the ular composition or route of administration selected.
An F antibody may also be stered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in ts wherein it is difficult to locate a tumor that is known to be present due to other biological factors.
Anti-TF antibodies may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the e or ion to be treated.
Accordingly, in one embodiment, the antibody-containing medicament is for combination with one or more further therapeutic agent, such as a cytotoxic, chemotherapeutic or anti- angiogenic agent.
Such combined administration may be aneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. The present invention thus also provides methods for treating a disorder involving cells expressing TF as described above, which methods comprise administration of an anti-TF dy of the present invention combined with one or more additional therapeutic agents as described below.
In one embodiment, the present invention provides a method for treating a disorder involving cells expressing TF in a subject, which method comprises administration of a eutically effective amount of an anti-TF dy of the present invention and at least one chemotherapeutic agent to a t in need thereof.
In one embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of an anti-TF antibody of the present invention and at least one chemotherapeutic agent to a t in need f.
In one embodiment, the present invention provides the use of an anti-TF antibody of the present ion for the preparation of a pharmaceutical composition to be administered with at least one chemotherapeutic agent for treating cancer.
In one embodiment, such a chemotherapeutic agent may be selected from an antimetabolite, such as rexate, 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 , lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, ozotocin, 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 anti- c agent, such as taxanes, for ce docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, stine, vinblastine, and vinorelbine.
In one embodiment, such a chemotherapeutic agent may be selected from a omerase inhibitor, such as topotecan or irinotecan.
In one embodiment, such a chemotherapeutic agent may be selected from a cytostatic drug, such as etoposide and teniposide.
In one embodiment, such a chemotherapeutic agent may be selected from a growth factor inhibitor, such as an inhibitor of ErbBl (EGFR) (such as Iressa, x (cetuximab), tarceva and similar agents), an inhibitor of ErbBZ (Her2/neu) (such as herceptin and similar agents) and similar agents.
In one embodiment, such a chemotherapeutic agent may be selected from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STIS71), lapatinib, PTK787/ZK222584 and similar agents.
In one embodiment, the present invention provides a method for treating a disorder involving cells sing TF in a subject, which method comprises administration of a therapeutically effective amount of an F antibody of the present invention and at least one inhibitor of angiogenesis, neovascularization, and/or other arization to a subject in need thereof Examples of such enesis inhibitors are urokinase inhibitors, matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY 12-9566, AG 3340, EMS-275291 and similar agents), inhibitors of endothelial cell migration and proliferation (such as TNP-470, squalamine, 2-methoxyestradiol, combretastatins, endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison, NJ), R115777 (Janssen Pharmaceutica, Inc, Titusville, NJ) and similar agents), nists of angiogenic growth factors (such as such as ZD6474, SU6668, dies against angiogenic agents and/or their receptors (such as VEGF, bFGF, and oietin-1), thalidomide, thalidomide analogs (such as CC-5013), Sugen 5416, SU5402, antiangiogenic ribozyme (such as yme), eron Cl (such as interferon 02a), suramin and similar agents), VEGF-R kinase inhibitors and other ngiogenic tyrosine kinase inhibitors (such as SU011248), inhibitors of endothelial-specific integrin/survival signaling (such as vitaxin and similar agents), copper antagonists/chelators (such as tetrathiomolybdate, captopril and similar agents), carboxyamido-triazole (CAI), ABT-627, CM101, eukin-12 (IL-12), IM862, PNU145156E as well as nucleotide les inhibiting angiogenesis (such as antisense- VEGF-cDNA, cDNA coding for angiostatin, cDNA coding for p53 and cDNA coding for deficient VEGF receptor-2) and similar agents.
Other examples of such inhibitors of angiogenesis, neovascularization, and/or other vascularization are anti-angiogenic heparin derivatives and related les (e.g., heperinase III), lomide, NK4, macrophage migration inhibitory factor (MIF), xygenase-2 inhibitors, inhibitors of hypoxia-inducible factor 1, anti-angiogenic soy isoflavones, oltipraz, fumagillin and analogs thereof, somatostatin analogues, pentosan polysulfate, tecogalan sodium, dalteparin, tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin, antibodies against other relevant targets (such as anti-alpha-v/beta-3 integrin and ininostatin mAbs) and similar agents.
In one embodiment, a therapeutic agent for use in combination with an anti-TF dy for treating the disorders as described above may be an anti-cancer immunogen, such as a cancer antigen/tumor-associated antigen (e.g., epithelial cell adhesion molecule (EpCAM/TACSTDl), mucin 1 (MUCl), carcinoembryonic antigen (CEA), tumor-associated rotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCASl, MDA7, cancer-associated viral vaccines (e.g., human papillomavirus es), tumor-derived heat shock ns, 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 embodiment. Anti-cancer immunogenic peptides also include anti-idiotypic "vaccines" such as BEC2 anti-idiotypic antibodies, Mitumomab, CeaVac and related anti- idiotypic antibodies, diotypic antibody to MG7 antibody, and other anti-cancer anti- idiotypic antibodies (see for instance nt et a|., Vaccine. Q(15), 1601-12 (2003), Li et a|., Chin Med J . MG), 962-6 (2001), Schmitt et a|., Hybridoma. 1_3(5), 389-96 (1994), Maloney et a|., Hybridoma. 4(3), 191-209 , Raychardhuri et a|., J Immunol. m6), 1743-9 , Pohl et a|., Int J Cancer. 5_0(6), 958-67 (1992), Bohlen et a|., Cytokines Mol Ther. 2(4), 231-8 (1996) and Maruyama, J Immunol Methods. Mfl-Z), 121-33 (2002)). Such anti-idiotypic Abs may optionally be conjugated to a carrier, which may be a synthetic (typically inert) molecule carrier, a protein (for instance keyhole limpet hemocyanin (KLH) (see for instance Ochi et a|., EurJ Immunol.fi(11), 1645-8 (1987)), or a cell (for instance a red blood cell — see for ce Wi et a|., J Immunol Methods. QQ), 227-34 (1989)).
In one embodiment, a therapeutic agent for use in combination with an anti-TF antibody for treating the disorders as described above may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth s 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, IFNd (e.g., , IFNB, GM-CSF, CD40L, Flt3 ligand, stem cell factor, im, and TNFd. le chemokines may include Glu-Leu-Arg (ELR)- negative chemokines such as IP-10, MCP-3, MIG, and SDF-1d from the human CXC and C-C chemokine es. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins. These and other methods or uses involving naturally occurring peptide-encoding c acids herein may alternatively or additionally be performed by "gene activation" and homologous ination gene upregulation techniques, such as are described in US 5,968,502, US 6,063,630 and US 6,187,305 and EP 0505500.
In one embodiment, a eutic agent for use in combination with an anti-TF antibody for treating the disorders as described above may be a cell cycle control/apoptosis regulator (or ating agent"). A cell cycle control/apoptosis regulator may include molecules that target and modulate cell cycle l/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), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase tors (such as BIBR1532, SOT-095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055). Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs,_ and anti-sense Bcl-2.
In one ment, a therapeutic agent for use in combination with an anti-TF antibody for treating the disorders as described above may be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene (such as flutaminde/eulexin), a progestin (such as such as hydroxyprogesterone te, medroxyprogesterone /provera, megestrol acepate/megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs f and other LHRH agonists such as lin and goserelin), an aromatase inhibitor (such as anastrazole/arimidex, aminoglutethimide/cytraden, exemestane), a hormone inhibitor (such as octreotide/sandostatin) and similar agents.
In one embodiment, a therapeutic agent for use in combination with an anti-TF antibody for treating the disorders as described above may be an anti-anergic agent (for instance small molecule compounds, proteins, glycoproteins, or antibodies that break tolerance to tumor and cancer antigens). Examples of such compounds are molecules that block the activity of CTLA-4, such as MDX-010 (ipilimumab) (Phan et al., PNAS USA 1_00, 8372 (2003)).
In one embodiment, a therapeutic agent for use in combination with an anti-TF antibody for treating the disorders as described above may be a tumor suppressor gene- containing nucleic acid or vector such as a replication-deficient adenovirus encoding human recombinant wild-type p53/SCH58500, etc.; nse nucleic acids targeted to oncogenes, mutated, or deregulated genes; or siRNA targeted to mutated or deregulated genes.
Examples of tumor suppressor targets include, for example, BRCA1, RBl, BRCA2, DPC4 (Smad4), MSH2, MLH1, and DCC.
In one embodiment, a therapeutic agent for use in ation with an anti-TF antibody for treating the ers as described above may be an anti-cancer nucleic acid, such as genasense (augmerosen/G3139), 03 (ISIS 3521), ISIS 2503, OGX-011 (ISIS ), LE-AON/LEraf-AON (liposome encapsulated c-raf antisense ucleotide/ISIS-5132), MG98, and other antisense nucleic acids that target PKCd, clusterin, IGFBPs, protein kinase A, cyclin D1, or Bcl-2h.
In one embodiment, a therapeutic agent for use in combination with an anti-TF antibody for treating the disorders as bed above may be an anti-cancer inhibitory RNA le (see for instance Lin et al., Curr Cancer Drug Targets. 1(3), 241-7 , Erratum in: Curr Cancer Drug Targets. 3(3), 237 (2003), Lima et al., Cancer Gene Ther.
Q6), 309-16 , Grzmil et al., Int J Oncol. fl(1), 97-105 (2004), Collis et al., Int J Radiat Oncol Biol Phys. 3(2 Suppl), S144 (2003), Yang et al., Oncogene. Q66), 5694-701 (2003) and Zhang et al., Biochem Biophys Res Commun. fifl), 1169-78 (2003)).
Compositions and combination administration s of the t invention also include the administration of nucleic acid vaccines, such as naked DNA vaccines encoding such cancer antigens/tumor-associated antigens (see for instance US 5,589,466, US ,593,972, US 5,703,057, US 687, US 523, and US 6,387,888). In one embodiment, the combination stration method and/or combination composition comprises an gous e composition. In one embodiment, the combination composition and/or combination administration method comprises a whole cell vaccine or cytokine-expressing cell (for instance a recombinant IL-2 expressing fibroblast, recombinant cytokine-expressing dendritic cell, and the like) (see for instance Kowalczyk et al., Acta Biochim Pol. 5_0(3), 613-24 (2003), Reilly et al., Methods Mol Med. Q, 233-57 (2002) and Tirapu et al., Curr Gene Ther. 2(1), 79-89 (2002). Another example of such an autologous cell approach that may be useful in ation methods of the present invention is the MyVax® Personalized Immunotherapy method (previously referred to as GTOP-99) (Genitope ation — Redwood City, CA, USA).
In one embodiment, the present invention provides combination compositions and combination administration methods wherein an anti-TF antibody is combined or co- administered with a virus, viral proteins, and the like. Replication-deficient viruses, that generally are capable of one or only a few rounds of replication in vivo, and that are targeted to tumor cells, may for instance be useful components of such compositions and methods. Such viral agents may comprise or be associated with c acids encoding immunostimulants, such as GM-CSF and/or IL-2. Both lly oncolytic and such inant oncolytic viruses (for instance HSV-1 viruses, reoviruses, replication-deficient and replication-sensitive irus, etc.) may be useful components of such methods and compositions. Accordingly, in one embodiment, the present invention provides combination compositions and combination administration methods wherein an anti-TF antibody is combined or co-administered with an tic virus. Examples of such viruses include oncolytic adenoviruses and herpes viruses, which may or may not be modified viruses (see for instance Shah et al., J Neurooncol. QB), 203-26 , Stiles et al., Surgery. ma), 357-64 (2003), Sunarmura et al., Pancreas. EB), 326-9 (2004), Teshigahara et al., J Surg Oncol. gm), 42-7 (2004), Varghese et al., Cancer Gene Ther. 9(12), 967-78 (2002), Wildner et al., Cancer Res. 3(2), 410-3 (1999), Yamanaka, Int J Oncol. EM), 919-23 (2004) and Zwiebel et al., Semin Oncol. EM), 336-43 (2001).
Combination compositions and combination administration methods of the present invention may also involve "whole cell and ive" immunotherapy methods. For instance, such methods may comprise infusion or re-infusion of immune system cells (for instance tumor-infiltrating lymphocytes (TILs), such as CD44r and/or CD84r T cells (for instance T cells ed with tumor-specific antigens and/or genetic enhancements), antibody-expressing B cells or other antibody producing/presenting cells, dendritic cells (e.g., anti-cytokine expressing recombinant dendritic cells, dendritic cells cultured with a DC-expanding agent such as GM-CSF and/or F|t3-L, and/or tumor-associated antigen- loaded dendritic cells), anti-tumor NK cells, led hybrid cells, or combinations thereof.
Cell |ysates may also be useful in such methods and compositions. Cellular "vaccines" in clinical trials that may be useful in such aspects include CanvaxinTM, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and ne® cell |ysates. Antigens shed from cancer cells, and mixtures thereof (see for instance n et al., al Cancer Research Vol. 7, 1882-1887, July 2001), ally admixed with nts such as alum, may also be components in such methods and combination compositions.
In one embodiment, an anti-TF antibody may be delivered to a patient in combination with the application of an internal vaccination method. Internal vaccination refers to induced tumor or cancer cell death, such as drug-induced or radiation-induced cell death of tumor cells, in a patient, that typically leads to elicitation of an immune response directed s (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 roteins or other components associated with or inserted in membranes, and/or (c) intracellular proteins or other intracellular components. An al ation-induced immune response may be humoral (i.e. antibody — ment-mediated) or cell-mediated (e.g., the pment and/or increase of endogenous cytotoxic T lymphocytes that recognize the internally killed tumor cells or parts thereof). In addition to radiotherapy, non- limiting examples of drugs and agents that may be used to induce said tumor cell-death and internal ation are conventional chemotherapeutic agents, cell-cycle inhibitors, anti- angiogenesis drugs, monoclonal antibodies, apoptosis-inducing , and signal transduction inhibitors.
Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with an anti-TF antibody for treating the disorders as described above are differentiation inducing agents, retinoic acid analogues (such as all trans retinoic acid, 13-cis retinoic acid and similar agents), vitamin D analogues (such as seocalcitol and similar agents), inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2, FIt4, FGFRl, FGFRZ, FGFR3, FGFR4, TRKA, TRKC, c-met, Ron, Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7 and similar agents.
Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with an anti-TF antibody for treating the disorders as bed above are cathepsin B, modulators of cathepsin D dehydrogenase activity, g|utathione-S- transferase (such as g|utacy|cysteine synthetase and lactate dehydrogenase), and similar agents.
Examples of other anti-cancer agents, which may be nt as therapeutic agents for use in combination with an anti-TF antibody for treating the disorders as described above are estramustine and icin.
Examples of other anti-cancer agents, which may be nt as therapeutic agents for use in ation with an anti-TF antibody for treating the disorders as described above are a HSP9O inhibitor like 17-allyl amino geld-anamycin, antibodies directed against a tumor antigen such as PSA, CA125, KSA, etc., integrins like in [31, inhibitors of VCAM and similar agents.
Examples of other ancer agents, which may be relevant as eutic agents for use in combination with an anti-TF antibody for treating the disorders as described above are calcineurin-inhibitors (such as valspodar, PSC 833 and other MDR-l or p-glycoprotein inhibitors), TOR-inhibitors (such as sirolimus, everolimus and rapamcyin). and inhibitors of "lymphocyte " mechanisms (such as ), and agents with effects on cell signaling such as adhesion molecule inhibitors (for instance anti-LFA, etc.).
In one embodiment, the anti-TF antibody of the invention is for use in combination with one or more other therapeutic antibodies, such as bevacizumab (Avastin®), zalutumumab, cetuximab (Erbitux®), panitumumab (VectibixTM), ofatumumab, mumab, daratumumab, ranibizumab (Lucentis®), x, Simulect, Remicade, Humira, Tysabri, Xolair, raptiva, nimotuzumab, rituximab and/or trastuzumab (Herceptin®).
Other therapeutic antibodies which may be used in combination with the antibody of the t invention are those disclosed in WO98/40408 (antibodies that can bind native human TF), WOO4/O94475 (antibodies capable of binding to human tissue factor, which do not inhibit factor ed blood coagulation ed to a normal plasma control), WOO3/O93422 (antibodies that bind with greater affinity to the TF:VIIa x than to TF , or WOO3/O37361 (TF agonist or antagonist for treatment related to apoptosis).
In another embodiment, two or more different antibodies of the invention as described herein are used in combination for the treatment of disease. Particularly interesting combinations include two or more non-competing antibodies. Thus, in one embodiment, a patient is treated with a combination of an antibody of cross-block Group I defined herein with an antibody of Group II or III, as defined herein. In another embodiment, a patient is d with a combination of an antibody of Group II as defined herein below, with an antibody of Group III. Such combination therapy may lead to binding of an increased number of antibody molecules per cell, which may give increase cy, e.g. via activation of ment-mediated lysis.
In one embodiment, an anti-TF antibody may be stered in connection with the delivery of one or more agents that promote access of the anti-TF dy or combination composition to the interior of a tumor. Such methods may for example be performed in association with the delivery of a n, which is capable of relaxing a tumor (see for instance US 6,719,977). In one embodiment, an anti-TF dy of the present invention may be bonded to a cell ating peptide (CPP). Cell penetrating peptides and related peptides (such as engineered ce|| penetrating antibodies) are described in for instance Zhao et al., J l Methods. flfl-Z), 137-45 (2001), Hong et al., Cancer Res. @(23), 6551-6 (2000). Lindgren et al., Biochem J. fl(Pt 1), 69-76 (2004), Buerger et al., J Cancer Res C|in Oncol.m(12), 669-75 (2003), Pooga et aI., FASEB J. 1_2(1), 67-77 (1998) and Tseng et al., Mol Pharmacol. QM), 864-72 .
In one embodiment, the present invention provides a method for treating a disorder involving cells expressing TF in a subject, which method comprises administration of a therapeutically effective amount of an anti-TF antibody and at least one nflammatory agent to a subject in need thereof In one embodiment such an anti-inflammatory agent may be selected from aspirin and other salicylates, Cox-2 tors (such as rofecoxib and celecoxib), NSAIDs (such as ibuprofen, fenoprofen, naproxen, sulindac, enac, piroxicam, ketoprofen, isal, nabumetone, etodolac, oxaprozin, and indomethacin), anti-IL6R antibodies, anti-IL8 antibodies (e.g. des described in W02004058797, e.g. 10F8), anti-IL15 antibodies (e.g. antibodies described in WOO3017935 and W02004076620), anti-IL15R antibodies, anti-CD4 antibodies (e.g. zanolimumab), anti-CD11a antibodies (e.g., efalizumab), anti- alpha-4/beta-1 integrin (VLA4) antibodies (e.g. natalizumab), CTLA4-Ig for the treatment of inflammatory diseases, prednisolone, prednisone, disease modifying antirheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine, sulfasalazine, dine synthesis inhibitors (such as leflunomide), IL-1 receptor blocking agents (such as anakinra), TNF-d blocking agents (such as cept, 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, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentin, thymosin-d and similar agents.
In one embodiment, such an immunosuppressive and/or immunomodulatory agent may be selected from immunosuppressive antibodies, such as antibodies binding to p75 of the IL-2 receptor, antibodies against CD25 (e.g. those bed in W02004045512, such as AB1, AB7, A811, and AB12), or antibodies binding to for instance MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFNv, TNF-CI, IL-4, IL-5, IL-6R, IL-7, IL-8, IL-10, CDlla, or CD58, or antibodies binding to their ligands.
In one embodiment, such an suppressive and/or immunomodulatory agent may be selected from soluble IL-15R, IL-10, B7 molecules (87-1, 87-2, variants thereof, and fragments thereof), ICOS, and 0X40, an inhibitor of a negative T cell regulator (such as an antibody t CTLA4) and similar agents.
In one embodiment, the present invention provides a method for treating a disorder involving cells expressing TF in a subject, which method comprises administration of a therapeutically effective amount of an anti-TF antibody and an anti-C3b(i) antibody to a t in need thereof In one embodiment, a therapeutic agent for use in combination with F antibodies for treating the disorders as described above may be selected from histone deacetylase inhibitors (for instance phenylbutyrate) and/or DNA repair agents (for instance DNA repair enzymes and related compositions such as dimericine).
Methods of the present invention for ng a disorder as bed above comprising administration of a therapeutically effective amount of an F antibody may also comprise anti-cancer directed photodynamic therapy (for instance anti-cancer laser therapy — which optionally may be practiced with the use of photosensitizing agent, see, for instance Zhang et al., J Control e. 3(2), 141-50 (2003)), anti-cancer sound-wave and shock-wave therapies (see for instance Kambe et al., Hum Cell. 1_O(1), 87-94 (1997)), and/or anti-cancer nutraceutical therapy (see for instance Roudebush et al., Vet Clin North Am Small Anim Pract. 3_4(1), , viii (2004) and Rafi, Nutrition. flu), 78-82 (2004).
Likewise, an anti-TF antibody may be used for the preparation of a pharmaceutical composition for treating a disorder as described above to be administered with anti-cancer directed ynamic therapy (for ce anti-cancer laser therapy — which optionally may be practiced with the use of photosensitizing agent, anti-cancer sound-wave and shock-wave therapies, and/or anti-cancer nutraceutical y.
In one embodiment, the present ion provides a method for treating a disorder involving cells expressing TF in a subject, which method ses administration of a therapeutically effective amount of an anti-TF antibody, such as an anti-TF dy of the present invention, and radiotherapy to a subject in need thereof.
In one embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of an anti-TF antibody, such as an anti-TF antibody of the present invention, and radiotherapy to a subject in need thereof.
In one embodiment, the present invention provides 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 treating cancer to be administered in combination with radiotherapy.
Radiotherapy may comprise radiation or associated administration of harmaceuticals to a patient is provided. The source of radiation may be either external or internal to the patient being d tion treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in cing such methods include, e.g., radium, -137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, tium-99, iodide-123, iodide-131, and indium-111.
In a further embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration to a subject in need thereof of a therapeutically ive amount of an anti-TF antibody, such as an anti-TF antibody of the present ion, in ation with surgery.
As described above, a pharmaceutical composition of the present invention may be administered in combination therapy, i.e., combined with one or more agents relevant for the disease or ion to be treated either as separate pharmaceutical itions or with a compound of the present invention coformulated with one or more additional therapeutic agents as described above. Such combination therapies may require lower dosages of the compound of the present invention and/or the co-administered agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
In addition to the above, other interesting combination therapies include the . For the treatment of pancreatic cancer an anti-TF antibody in ation with an antimetabolite, such as 5-fluorouracil and/or gemcitabine, possibly in combination with one or more compounds selected from: 9OY-hPAM4, ARC-100, ARQ-197, AZD- 6244, bardoxolone methyl, cixutumumab, (IMC-A12), folitixorin calcium, GVAX, ipilimumab, KRX-O601, merbarone, 103, MORAb-OO9, PX-12, Rh-ApoZL, TLN-4601, trabedersen, ximab (M200), WX-671, pemetrexed, can, ilone, 91Vion, 2165868, Iapatinib, matuzumab, imatinib, sorafinib, trastuzumab, exabepilone, erlotinib, avastin and cetuximab o For the treatment of colorectal cancer an anti-TF antibody in combination with one or more compounds selected from: gemcitabine, bevacizumab, FOLFOX, I, 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 nib. o For the treatment of breast cancer an anti-TF antibody in combination with one or more compounds selected from: antimetabolites, anthracyclines, taxanes, alkylating agents, epothilones ormonal (femar, fen etc), inhibitors of ErbBZ (Her2/neu) (such as herceptin and similar agents),CAF/FAC (cyclofosfamide, bicine, 5FU) AC (cyclo, doxo), CMF (cyclo, methotrexate, 5FU), Docetaxel + capecitabine, GT (paclitaxel, abine) FEC (cyclo, epi, 5FU) in combination with herceptine: Paclitaxel +/- carboplatin, Vinorelbine, Docetaxel, CT in combination with lapatinib; Capecitabine o For the treatment of bladder an anti-TF antibody in combination with one or more compounds selected from: antimetabolites (gemcitabine, alimta, methotrexate), platinum analogues (cisplatin, carboplatin), EGFr inhibitors (such as cetuximab or zalutumumab), VEGF inhibitors (such as Avastin) doxorubicin, tyrosine kinase inhibitors such as gefitinib, trastuzumab, anti-mitotic agent, such as s, for instance paclitaxel, and vinca alkaloids, for instance vinblastine. o For the treatment of prostate cancer an anti-TF antibody in combination with one or more nds ed from: al/antihormonal therapies; such as antiandrogens , Luteinizing hormone releasing hormone (LHRH) agonists, and chemotherapeutics such as taxanes, mitoxantrone, estramustine, 5FU, vinblastine, ixabepHone, o For the treatment of ovarian cancer an anti-TF antibody in combination with one or more compounds selected from: an anti-mitotic agent, such as taxanes, and vinca alkaloids, caelyx, topotecan.
Diagnostic uses The anti-TF antibodies of the invention may also be used for diagnostic es.
Thus, in a further aspect, the invention s to a diagnostic composition comprising an anti-TF antibody as defined herein.
In one embodiment, the anti-TF antibodies of the present invention may be used in vivo or in vitro for diagnosing diseases wherein activated cells expressing TF play an active role in the pathogenesis, by detecting levels of TF, or levels of cells which contain TF on their membrane surface. This may be achieved, for example, by contacting a sample to be tested, optionally along with a control sample, with the F antibody under conditions that allow for formation of a complex between the antibody and TF. Complex formation is then detected (e.g., using an ELISA). When using a control sample along with the test sample, complex is detected in both samples and any statistically significant difference in the formation of complexes between the samples is indicative of the presence of TF in the test sample.
Thus, in a further aspect, the invention relates to a method for detecting the presence of TF antigen, or a cell expressing TF, in a sample sing: - contacting the sample with an anti-TF antibody of the ion or a bispecific molecule of the invention, under conditions that allow for formation of a complex between the antibody and TF; and - ing whether a complex has been formed.
In one embodiment, the method is performed in vitro.
More specifically, the present invention provides methods for the identification of, and sis of invasive cells and tissues, and other cells targeted by anti-TF antibodies of the present invention, and for the monitoring of the ss of therapeutic treatments, status after treatment, risk of developing cancer, cancer progression, and the like.
In one e of such a diagnostic assay, the t ion provides a method of diagnosing the level of invasive cells in a tissue sing g an immunocomplex between an anti-TF antibody and potential TF-containing tissues, and detecting formation of the immunocomplex, n the formation of the immunocomplex correlates with the presence of ve cells in the tissue. The contacting may be performed in vivo, using labeled isolated antibodies and standard imaging techniques, or may be performed in vitro on tissue samples.
Anti-TF antibodies may be used to detect TF-containing es and peptide fragments in any suitable biological sample by any suitable que. Examples of conventional immunoassays provided by the present ion include, without limitation, an ELISA, an RIA, FACS assays, plasmon resonance assays, chromatographic assays, tissue immunohistochemistry, Western blot, and/or immunoprecipitation using an anti-TF antibody. Anti-TF antibodies of the present invention may be used to detect TF and TF- fragments from humans. Suitable labels for the anti-TF antibody and/or secondary antibodies used in such techniques include, without limitation, s enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish dase, alkaline phosphatase, B-galactosidase, or acetylcholinesterase; es of suitable prosthetic group xes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include iferone, fluorescein, fluorescein isothiocyanate, ine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 1251, 1311, 35S, and 3H.
F antibodies may also be assayed in a biological sample by a competition immunoassay utilizing TF peptide standards labeled with a detectable substance and an unlabeled F antibody. In such an assay, the biological sample, the d TF peptide standard(s) and the anti-TF antibodies are combined and the amount of labeled TF standard bound to the unlabeled anti-TF antibody is determined. The amount of TF peptide in the biological sample is inversely proportional to the amount of labeled TF standard bound to the F dy.
The anti-TF antibodies are particularly useful in the in vivo imaging of tumors. In vivo imaging of tumors associated with TF may be performed by any suitable technique. For example, 99Tc-labeling or labeling with another gamma-ray emitting isotope may be used to label anti-TF antibodies in tumors or secondary labeled (e.g., FITC labeled) anti-TF antibody:TF complexes from tumors and imaged with a gamma scintillation camera (e.g., an Elscint Apex 409ECT device), lly using low-energy, high resolution collimator or a low-energy rpose collimator. Stained tissues may then be assessed for radioactivity ng as an indicator of the amount of TF-associated peptides in the tumor. The images ed by the use of such techniques may be used to assess 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 ce of invasive cancer cells. Variations on this technique may include the use of magnetic resonance imaging (MRI) to improve imaging over gamma camera techniques. Similar immunoscintigraphy methods and principles are described in, e.g., Srivastava (ed.), Radiolabeled Monoclonal dies For Imaging And Therapy (Plenum Press 1988), Chase, al 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 , Pezzuto et al., (eds.) (Chapman & Hall 1993). Such images may also be used for targeted delivery of other anti-cancer agents, examples of which are described herein (e.g., apoptotic agents, toxins, or CHOP chemotherapy compositions). Moreover, such images may also or alternatively serve as the basis for surgical ques to remove tumors. Furthermore, such in vivo imaging techniques may allow for the identification and localization of a tumor in a situation where a patient is fied as having a tumor (due to the presence of other biomarkers, metastases, etc.), but the tumor cannot be identified by traditional analytical ques. All of these s are features of the present invention.
The in vivo imaging and other diagnostic methods provided by the present invention are particularly useful in the detection of micrometastases in a human patient (e.g., a patient not previously diagnosed with cancer or a patient in a period of recovery/remission from a cancer). Carcinoma cancer cells, which may make up to 90% of all cancer cells, for example, have been demonstrated to stain very well with anti-TF antibody conjugate compositions. Detection with monoclonal anti-TF antibodies described herein may be indicative of the presence of carcinomas that are aggressive/invasive and also or alternatively provide an tion of the feasibility of using related monoclonal anti-TF antibody against such micrometastases.
In one embodiment, the present invention provides an in vivo imaging method wherein an anti-TF antibody of the present invention is conjugated to a detection-promoting radio-opaque agent, the conjugated antibody is stered to a host, such as by injection into the bloodstream, and the presence and location of the labeled dy in the host is assayed. Through this technique and any other diagnostic method provided , the present invention provides a method for screening for the presence of disease-related cells in a human patient or a biological sample taken from a human patient.
For stic g, radioisotopes may be bound to a anti-TF antibody either directly, or indirectly by using an intermediary functional group. Useful intermediary functional groups include chelators, such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see for instance US 5,057,313).
In addition to radioisotopes and radio-opaque agents, diagnostic methods may be performed using anti-TF antibodies that are conjugated to dyes (such as with the biotin- avidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g. paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g., US Pat.
No. 6,331,175, which describes MRI techniques and the preparation of dies conjugated to a MRI enhancing . Such diagnostic/detection agents may be selected from agents for use in magnetic resonance imaging, and fluorescent compounds. In order to load an anti-TF dy with radioactive metals or paramagnetic ions, it may be necessary to react it with a t having a long tail to which are attached a licity of chelating groups for binding the ions. Such a tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which may be bound chelating groups such as, e.g., porphyrins, polyamines, crown ethers, bisthiosemicarbazones, imes, and like groups known to be useful for this purpose.
Chelates may be d to anti-TF antibodies using standard chemistries.
Thus, the present invention provides diagnostic anti-TF antibody conjugates, wherein the anti-TF antibody is conjugated to a contrast agent (such as for magnetic resonance imaging, computed tomography, or ultrasound contrast-enhancing agent) or a radionuclide that may be, for example, a gamma-, beta-, alpha-, Auger electron-, or positron-emitting In a r aspect, the invention relates to a kit for detecting the ce of TF antigen, or a cell expressing TF, in a sample comprising - an anti-TF antibody of the ion or a bispecific molecule of the invention; and - instructions for use of the kit.
In one embodiment, the present invention provides a kit for diagnosis of cancer comprising a container comprising an anti-TF antibody, and one or more ts for detecting binding of the anti-TF antibody to a TF peptide. Reagents may include, for example, fluorescent tags, enzymatic tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions, wherein the tic reactions produce a product that may be visualized. In one embodiment, the present invention provides a diagnostic kit comprising one or more anti-TF dies, of the present invention in labeled or unlabeled form in suitable container(s), reagents for the incubations for an indirect assay, and substrates or derivatizing agents for detection in such an assay, depending on the nature of the label. Control reagent(s) and instructions for use also may be included.
Diagnostic kits may also be supplied for use with an anti-TF antibody, such as a conjugated/labeled anti-TF antibody, for the ion of a cellular activity or for detecting the presence of TF peptides in a tissue sample or host. In such diagnostic kits, as well as in kits for therapeutic uses bed ere herein, an anti-TF antibody typically may be provided in a lyophilized form in a container, either alone or in conjunction with additional antibodies specific for a target cell or peptide. Typically, a ceutical able carrier (e.g., an inert diluent) and/or components thereof, such as a Tris, ate, or carbonate buffer, stabilizers, preservatives, biocides, biocides, inert proteins, e.g., serum n, or the like, also are included (typically in a separate container for mixing) and additional reagents (also typically in te container(s)). In certain kits, a secondary antibody capable of binding to the anti-TF antibody, which typically is present in a separate container, is also included. The second antibody is typically conjugated to a label and formulated in manner similar to the anti-TF antibody the present invention. Using the methods described above and ere herein F antibodies may be used to define subsets of /tumor cells and characterize such cells and related tissues/growths.
In situ detection may be accomplished by removing a histological specimen from a patient, and ing the ation of labeled anti-TF antibodies, of the present ion to such a specimen. The anti-TF antibody of the present invention may be provided by applying or by overlaying the labeled anti-TF antibody of the present invention to 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 es in the examined tissue (e.g., in the context of assessing the spread of cancer cells). Using the present ion, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) may be modified in order to achieve such in situ detection.
In a further aspect, the ion relates to an anti-idiotypic antibody which binds to an anti-TF antibody of the invention as described herein.
An anti-idiotypic (Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody may be prepared by immunizing an animal of the same species and genetic type as the source of an anti-TF mAb with the mAb to which an d is being prepared. The immunized animal typically can recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody).
Such antibodies are described in for instance US 4,699,880. Such antibodies are r features of the present invention.
An anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. An anti-anti-Id may be epitopically identical to the original mAb, which d the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of cal specificity. Anti-Id antibodies may be varied (thereby producing anti-Id antibody variants) and/or tized by any suitable que, such as those described elsewhere herein with respect to anti-TF dies of the present invention. For example, anti-Id mAbs may be coupled to a carrier such as e limpet hemocyanin (KLH) and used to immunize BALB/c mice. Sera from these mice typically will contain anti-anti-Id dies that have the binding properties similar if not identical to an original/parent TF antibody.
The present invention is further illustrated by the following examples which should not be construed as further limiting.
EXAMPLES Example 1 Ex ression constructs for tissue factor TF Fully codon-optimized constructs for expression of TF or its extracellular domains in HEK, N80 or CHO cells, were generated. The proteins encoded by these constructs are identical to Genbank ion NP_001984 for TF. The constructs ned suitable restriction sites for cloning and an optimal Kozak sequence (Kozak, 1987). The constructs were cloned in the mammalian expression vector pEE13.4 (Lonza ics) (Bebbington, Renner et al. 1992), obtaining pEE13.4TF. PCR was used to amplify the part, ng the extracellular domain (ECD) (amino acid 1-251) of TF, from the synthetic construct, adding a C-terminal His tag containing 6 His residues (TFECDHis). The uct was cloned in pEE13.4 and fully sequenced to confirm the tness of the construct.
Example 2 Transient expression in HEK-293F cells FreestyleTM 293-F (a HEK-293 subclone adapted to suspension growth and chemically defined Freestyle medium, (HEK-293F)) cells were ed from Invitrogen and transfected with the appropriate plasmid DNA, using 293fectin (Invitrogen) according to the manufacturer’s instructions. In the case of antibody expression, the appropriate heavy chain and light chain vectors, as described in Example 10, were co-expressed.
Semi-stable expression in N80 cells pEE13.4TF was stably transfected in N80 cells and stable clones were selected on growth in the absence of glutamine and in the presence of 7.5 uM of methylsulphoximine (MSX). A pool of clones was grown in sion culture while maintaing selection pressure. Pools were tested for TF expression by FACS analysis and secured for further use.
Example 4 Stable expression in CHO cells pEE13.4TF was stably transfected in CHO-KlSV (Lonza Biologics) cells and stable clones were selected on growth in the absence of glutamine and in the presence of 50 uM MSX.
Single clones were picked and expanded and tested for TF expression by FACS analysis as described below. High expressing clones were chosen and secured for further use.
Example 5 Purification of His-tagged TF TFECDhis was expressed I 3F cells. The his-tag in TFECDHis enables purification with immobilized metal affinity chromatography. In this process, a or fixed onto the tographic resin is charged with Co2+ cations. TFECDHis-containing atant is incubated with the resin in batch mode (i.e. solution). The His-tagged protein binds strongly to the resin beads, while other proteins present in the culture supernatant do not bind strongly. After incubation the beads are retrieved from the supernatant and packed into a column. The column is washed in order to remove weakly bound proteins. The strongly bound TFECDHis ns are then eluted with a buffer containing imidazole, which competes with the binding of His to Co". The eluent is removed from the protein by buffer exchange on a desalting column.
Example 6 Immunization procedure of transgenic mice HuMab mice were immunized every fortnight alternating with 5x106 semi-stable transfected NSO-TF cells, or with 20 pg of is protein. Eight immunizations were performed in total, four intraperitoneal (IP) and four subcutaneous (SC) immunizations at the tail base.
The first zation with cells was done in te Freunds’ adjuvant (CFA; Difco Laboratories, Detroit, MI, USA). For all other zations, cells were injected IP in PBS and TFECDHis was injected SC using incomplete Freunds’ nt (IFA; Difco Laboratories, Detroit, MI, USA). When serum titers were found to be sufficient (dilution of serum of 1/50 or lower found positive in antigen specific screening assay as described in Example 7 on at least 2 sequential, biweekly screening ), mice were additionally boosted twice intravenously (IV) with 10 pg TFECDHis protein in 100 pl PBS, 4 and 3 days before fusion.
The first immunization with cells was done in CFA, for all other (7) immunizations cells were ed IP in PBS. When serum titers were found to be sufficient, mice were additionally boosted twice IV with 1x106 transiently semi-stable transfected NSO-TF cells in 100 pl PBS, 4 and 3 days before fusion.
When serum titers were found to be sufficient (defined as above), mice were additionally boosted twice intravenously (IV) with 10 pg TFECDHis protein in 100 pl PBS, 4 and 3 days before fusion.
Example 7 Homogeneous antigen specific screening assay The presence of anti-TF antibodies in sera of immunized mice or HuMab (human monoclonal antibody) hybridoma or transfectoma culture supernatant was determined by homogeneous antigen specific screening assays (four quadrant) using Fluorometric Micro volume Assay Technology (FMAT; Applied Biosystems, Foster City, CA, USA).
For this, a combination of 3 cell based assays and one bead based assay was used. In the cell based assays, binding to TH1015-TF (HEK-293F cells transiently expressing TF; produced as described above) and A431 (which express TF at the cell surface) as well as HEK293 wild type cells (do not express TF, negative control) was determined. In the bead based assay, g to ylated TF coupled on a streptavidin bead (SBlOl5-TF) was determined.
Samples were added to the beads to allow binding to TF. Subsequently, binding of HuMab was detected using a fluorescent conjugate (Goat anti-Human IgG-Cy5; Jackson ImmunoResearch). Mouse anti-human TF antibody (ERL; coupled to Alexa-647 at Genmab) was used as positive l, HuMAb-mouse pooled serum and mouse-chrompure-Alexa647 antibody were used as negative controls. The samples were scanned using an Applied Biosystems 8200 Cellular ion System (8200 CBS) and s x fluorescence’ was used as read-out.
Example 8 HuMab hybridoma generation HuMab mice with sufficient antigen-specific titer pment (defined as above) were euthanized and the spleen and lymph nodes flanking the abdominal aorta and vena cava were collected. Fusion of splenocytes and lymph node cells to a mouse myeloma cell line was done by electrofusion using a CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie, MD, USA), essentially ing to the manufacturer’s instructions. Selection and ing of the resulting HuMab hybridomas was done based upon standard protocols (e.g. as described in Coligan J.E., Bierer, B.E., Margulies, D.H., Shevach, EM. 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 6-well or Hyperflask stage were purified using PhyTip columns containing Protein G resin (PhyNexus Inc., San Jose, USA ) on a Sciclone ALH 3000 workstation (Caliper Lifesciences, Hopkinton, USA). The PhyTtip columns were used according to manufacturers ctions, but buffers were replaced by: Binding Buffer PBS (B.Braun, Medical B.V., Oss, Netherlands) and Elution Buffer 0.1M Glycine-HCI pH 2.7 (Fluka Riedel-de Ha'en, Buchs, Germany). After cation, samples were neutralized with 2M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands). Alternatively, in some cases larger volumes of culture supernatant were ed using Protein A affinity column chromatography.
After purification, the samples were placed in a ll plate (Waters, 100 ul square well plate, part# 186002631). Samples were deglycosylated ght at 37°C with N- idase F (Roche cat no 11365177001. D'I'I' (15 mg/ml) was added (1 pl / well) and incubated for 1 h at 37°C. Samples (5 or 6 ul) were desalted on an Acquity UPLCTM (Waters, Milford, USA) with a BEH300 C18, 1.7um, 2.1x 50 mm column at 60 oC. MQ water and LC- MS grade acetonitrile (Biosolve, cat no 01204101,Va|kenswaard, The Netherlands) with both 0.1% formic acid (Fluka, cat no 56302, Buchs, Germany), were used as Eluens A and B, respectively. Time-of-flight ospray ionization mass spectra were recorded on-line on a micrOTOFTM mass ometer (Bruker, Bremen, Germany) operating in the positive ion mode. Prior to analysis, a 900-3000 m/z scale was calibrated with ES tuning mix (Agilent Technologies, Santa Clara, USA). Mass spectra were oluted with DataAnalysisTM software v. 3.4 (Bruker) using the Maximal Entropy algorithm searching for molecular weights between 5 and 80 kDa.
After olution the resulting heavy and light chain masses for all samples were compared in order to find duplicate antibodies. In the comparison of the heavy chains the possible presence of C-terminal lysine variants was taken into account. This resulted in a list of unique antibodies, where unique is defined as a unique combination of heavy and light chains. In case duplicate antibodies were found, the results from other tests were used to decide which was the best material to continue experiments with.
MS analysis of the molecular weights of heavy and light chains of 118 TF ic hybridomas yielded 70 unique antibodies (unique heavy chain/light chain combination).
These were characterized in a number of functional assays, identifying 14 lead candidates, TF specific antibodies.
Example 10 Sequence analysis of the anti-TF HuMab variable domains and cloning in expression s Total RNA of the anti-TF HuMabs was prepared from 5x106 hybridoma cells and 5’-RACE- Complementary DNA (cDNA) was prepared from 100 ng total RNA, using the SMART RACE cDNA ication kit (Clontech), according to the manufacturer’s instructions. VH ble region of heavy chain) and VL (variable region of light chain) 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.
The sequences are given in the Sequence Listing and Figure 1 herein.
Table 1A and Table 13 (below) give an overview of the dy sequences information and most homologous germline sequences.
Table 1A Heavy chain homologies J-GENE D-GENE CDR-IMGT Ab V-GENE and allele V-REGION Identity, % and allele and allele lengths 003 lGHV1-69*02, or 69*04 97.57% (281/288 nt) IGHJ4*02 13*01 [8,8,11] 098 lGHV1-69*04 95.49% (275/288 nt) IGHJ3*02 lGHD2-21*02 [8,8,11] 011 lGHV3-23*01 96.53% (278/288 nt) IGHJ4*02 lGHD1-26*01 [8,8,11] 017 lGHV3-23*01 98.26% (283/288 nt) IGHJ2*01 lGHD2-15*01 [8,8,13] 092 lGHV3-23*01 97.92% (282/288 nt) IGHJ4*02 lGHD7-27*01 [8,8,11] 101 lGHV3-23*01 95.83% (276/288 nt) IGHJ4*02 27*01 [8,8,11] 025 lGHV33*01 97.57% (281/288 nt) IGHJ4*02 27*01 [8,8,13] 109 lGHV33*01 96.18% (277/288 nt) IGHJ4*02 lGHD7-27*01 [8,8,13] 111 lGHV33*01 97.57% (281/288 nt) IGHJ4*02 10*01 [8,8,13] 114 lGHV3-33*01, or lGHV3-33*03 94.44% (272/288 nt) IGHJ6*02 lGHD3-10*01 [8,8,12] 013 lGHV5-51*01 99.65% (287/288 nt) IGHJ3*02 lGHD6-13*01 [8,8,19] Table 13 Light chain homologies J-GENE CDR-IMGT Ab V-GENE and allele V-REGION identity % (nt) and allele lengths 003 lGKV1-13*02 99.28% (277/279 nt) IGKJ4*01 [6.3.9] 011 lGKV1D-16*01 98.57% (275/279 nt) IGKJ2*01 [6.3.9] 013 lGKV1D-16*01 98.57% (275/279 nt) IGKJ5*01 [6.3.9] 092 lGKV1D-16*01 99.28% (277/279 nt) IGKJ2*01 [6.3.10] 098 lGKV1D-16*01 % (279/279 nt) IGKJ2*01 [6.3.9] 101 lGKV1D-16*01 100.00% (279/279 nt) 01 [6.3.10] 025 lGKV3-11*01 100.00% (279/279 nt) IGKJ4*01 [6.3.9] 109 lGKV3-11*01 99.64% (278/279 nt) IGKJ4*01 [6.3.9] 017 lGKV3-20*01 99.29% (280/282 nt) 01 ] 114 lGKV3-20*01 99.65% (281/282 nt) 01 [7.3.8] References to the sequence listing: VH-region SEQ ID No: 1 VH 013 SEQ ID No: 2 VH 013 CDRl SEQ ID No: 3 CDR2 SEQ ID No: 4: VH013,CDR3 SEQ ID No: VH 114 SEQ ID No: VH 114 CDR1 SEQ ID No: CDRZ SEQ ID No: (1)\IO\U'I VH 114 VH 114 CDR3 SEQ ID No: VH 011 SEQ ID No: 10 VH011,CDR1 SEQ ID No: 11 VH011,CDR2 SEQ ID No: 12 VH011,CDR3 SEQ ID No: 13 VH 017-D12 SEQ ID No: 14 VH 017-D12 CDR1 SEQ ID No: 15 VH 017-D12 CDRZ SEQ ID No: 16 VH 017-D12 CDR3 SEQ ID No: 17 VH 042 SEQ ID No: 18 VHO42,CDR1 SEQ ID No: 19 VH042,CDR2 SEQ ID No: 20 VHO42,CDR3 SEQ ID No: 21 VH 092-A09 SEQ ID No: 22 VH 092-A09, CDR1 SEQ ID No: 23 VH 092-A09, CDRZ 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 CDRZ SEQ ID No: 28 VH 101 CDR3 SEQ ID No: 29 VH 003 SEQ ID No: 30 VH 003 CDR1 SEQ ID No: 31 VH 003 CDRZ SEQ ID No: 32 VH 003 CDR3 SEQ ID No: 33 VH 025 SEQ ID No: 34 VH 025 CDR1 SEQ ID No: 35 VH 025 CDRZ SEQ ID No: 36 VH 025 CDR3 SEQ ID No: 37 VH 109 SEQ ID No: 38 VH 109 CDRl SEQ ID No: 39 VH 109 CDRZ SEQ ID No: 40 VH 109 CDR3 SEQ ID No: 41 VH 044 SEQ ID No: 42 VH 044 CDRl SEQ ID No: 43 VH 044 CDRZ SEQ ID No: 44 VH 044 CDR3 SEQ ID No: 45 VH 6 SEQ ID No: 46 VH 087-Lg6, CDRl SEQ ID No: 47 VH 087-Lg6, CDRZ SEQ ID No: 48 VH 087-Lg6, CDR3 SEQ ID No: 49 VH 098 SEQ ID No: 50 VH 098 CDR1 SEQ ID No: 51 VH 098 CDRZ SEQ ID No: 52 VH 098 CDR3 SEQ ID No: 53 VH 111 SEQ ID No: 54 VH 111 CDRl SEQ ID No: 55 VH 111 CDRZ SEQ ID No: 56 VH 111 CDR3 VL-region SEQ ID No: 57 VL 013 SEQ ID No: 58 VL 013 CDRl SEQ ID No: 59 VL 013 CDRZ SEQ ID No: 60 VL 013 CDR3 SEQ ID No: 61 VL 114 SEQ ID No: 62 VL 114 CDRl SEQ ID No: 63 VL 114 CDRZ 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 CDR3 SEQ ID No: 69 VL 017-D12 SEQ ID No: 70 VL 017-D12 CDRl SEQ ID No: 71 VL 017-D12 CDRZ SEQ ID No: 72 VL 017-D12 CDR3 SEQ ID No: 73 VL 042 SEQ ID No: 74 VL 042 CDRl SEQ ID No: 75 VL 042 CDRZ SEQ ID No: 76 VL 042 CDR3 SEQ ID No: 77 VL 092-A09 SEQ ID No: 78 VL 092-A09, CDRl SEQ ID No: 79 VL 092-A09, CDRZ SEQ ID No: 80 VL 092-A09, CDR3 SEQ ID No: 81 VL101 SEQ ID No: 82 VL101,CDR1 SEQ ID No: 83 VL101,CDR2 SEQ ID No: 84 VL101,CDR3 SEQ ID No: 85 VL 003 SEQ ID No: 86 VL 003 CDRl SEQ ID No: 87 VL 003 CDRZ SEQ ID No: 88 VL 003 CDR3 SEQ ID No: 89 VL 025 SEQ ID No: 90 VL 025 CDR1 SEQ ID No: 91 VL 025 CDRZ SEQ ID No: 92 VL 025 CDR3 SEQ ID No: 93 VL 109 SEQ ID No: 94 VL 109 CDR1 SEQ ID No: 95 VL 109 CDRZ SEQ ID No: 96 VL 109 CDR3 SEQ ID No: 97 VL 044 SEQ ID No: 98 VL 044 CDR1 SEQ ID No: 99 VL 044 CDRZ SEQ ID No: 100 VL 044 CDR3 SEQ ID No: 101 VL 087 SEQ ID No: 102 VL 087 CDR1 SEQ ID No: 103 VL 087 CDRZ SEQ ID No: 104 VL 087 CDR3 SEQ ID No: 105 VL 098 SEQ ID No: 106 VL 098 CDR1 SEQ ID No: 107 VL 098 CDRZ SEQ ID No: 108 VL 098 CDR3 SEQ ID No: 109 VL 111 SEQ ID No: 110 VL 111 CDR1 SEQ ID No: 111 VL111,CDR2 SEQ ID No: 112 VL 111 CDR3 Example 11 Purification of dies Culture supernatant was filtered over 0.2 pm dead-end filters and loaded on 5 ml Protein A columns (rProtein A FF, Amersham Bioscience) and eluted with 0.1 M citric acid-NaOH, pH 3. The eluate was ately neutralized with 2M Tris-HCI, pH 9 and dialyzed overnight to 12.6 mM NaHZPO4, 140 mM NaCl, pH 7.4 (B.Braun). After dialysis samples were sterile filtered over 0.2 pm dead-end filters. Purity was determined by SDS-PAGE and concentration was ed by nephelometry and absorbance at 280nm. Purified antibodies were aliquoted and stored at -80°C. Once thawed, purified antibody aliquots were kept at 4°C. Mass ometry was performed to fy the molecular mass of the antibody heavy and light chains expressed by the hybridomas as described in Example 9.
Example 12 Antibody cross-com petition studies using sandwich-ELI SA ELISA plate wells were coated overnight at +4°C with each of the anti-TF HuMabs (0.5 or 2 ug/ml 100 uL/well) diluted in PBS. The ELISA wells were washed with PBS, blocked for one hour at room temperature with 2% (v/v) chicken serum (Gibco, Paisley, Scotland) in PBS and washed again with PBS. Subsequently, 50 uL anti-TF HuMab (10 ug/mL) followed by 50 uL TFECDHis (0.5 or 1 pg/ml) (generated at Genmab; Example 5) was added, and incubated for 1 hour at RT (while shaking). Plates were washed 3 times with PBST (PBS+0.05°/otween), and incubated with 1:2000 d anti-his biotin BAM050 for one hour at RT (while shaking). Plates were washed and incubated with Streptavidin-poly-HRP (Sanquin, Amsterdam, The Netherlands) for 20 minutes at RT, and washed again. The reaction was further developed with ABTS (Roche Diagnostics) at RT in the dark, stopped after 15 minutes by adding 2% (w/v) oxalic acid and the absorbance at 405 nm was measured.
Table 2 shows that 3 cross-block groups (groups of antibodies competing with each other for TFECDHis binding) could be fied, with dies 013, 044 and 087-Lg6 belonging to one cross-block group (group I), antibodies 011, 017-D12, 42, 092-A09 and 101 belonging to another cross-block group (group II), and antibodies 003, 025, 109 and 111 ing to a third cross-block group (group III). dy 114 was found to compete for TFECDHis binding with antibodies from both block group II and III. Antibody 098 binding to TFECDHis could be competed for by antibodies from both cross-block group II and III. -0.5ugcoat 0.5ugcoat 0.5ugcoat 0.5ugcoat 0.5ugcoat at \\\\%\%\X\ \\\\\\‘k\\\\\i mm W_ ‘mm— \\ \_ _3_ \\\\\\\\\\\\\\\\\\-=E \ \\\\\\\\Q\\\\\\\\ :\\\\\ \\\\\\\\\\\ \" \\\\\\\ \\\\\\\\\___x\\\\\\\\>;\ \\\\\\\\§\&\\\\\\" \\\\\\\\\\\\\\\_m___-E \\\\\\\\\\\\\\\\———m— \\\\\\\\\\\\\\mm 0.5ugcoat 0.5ugcoat 0.5ugcoat 0.5ugcoat_ \\\\\\§\\\\\\\\\\\\\\\_-E \\\\\\\\\\R\\\\-E_ &\\\ \\\\\\\\\\\\\\\\\\\\\\\\\‘_ WW§K\\\\\\SE_m \\\\\\\\\\\\\\\:\\\\\\\‘ WWmem \\\\\\\\\\\\\\\_\\\\\" \\\\\\\\\\\\\\\\\\\\\\\"\W \\\\\\\\\\\\\ \\\\\\\ \\\\\\\ \\\\\\\\ Table 2 — Com petition of anti-TF antibodies for g to TFECDHis.
White boxes indicate no competition for binding, light grey boxes indicate partial competition for binding, and dark grey boxes indicate ition for binding to TFECDHis.
Example 13 Binding of anti-TF HuMabs to the extracellular domain of TF in ELISA The specificity of the obtained anti-TF HuMabs was evaluated by ELISA. ELISA plates (Microlon; Greiner Bio-One) were coated overnight at +4°C with 0.5 ug/mL of 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 with PBS containing 0.05% Tween 20 (PBST). Subsequently, HuMabs, serially diluted in PBSTC (PBS supplemented with 2% (v/v) chicken serum and 0.05% (v/v) Tween-20), were incubated for 1 hr at RT under shaking conditions (300 rpm). Bound HuMabs were detected using HRP-conjugated goat-anti-human IgG dies (Jackson Research) diluted 1:5,000 in PBSTC, which were incubated for 1 hr at RT uncler shaking conditions (300 rpm).
The reaction was further developed with ABTS (Roche Diagnostics) at RT in the dark, d after 15-30 minutes by adding 2% (w/v) oxalic acid and then the absorbance at 405 nm was measured. HuMab-KLH (a human monoclonal antibody against KLH (keyhole limpet haemocyanin)), was used as a negative l. Mouse anti-human TF (ERL) was used as positive control (HRP d anti-mouse IgG as conjugate). g curves were analyzed using non-linear regression (sigmoidal dose-response with le slope) using GraphPad Prism V4.03 software.
As can been seen in Figure 3, all of the anti-TF antibodies bound TFECDHis. The EC50 values for the HuMabs are the mean of 3 ments and varied between 0.09 and 0.46 nM (Table 3 below).
Table 3: Example 14 Binding of anti-TF HuMabs to membrane-bound TF Binding of anti-TF HuMabs to membrane-bound TF was determined by FACS analysis, using TF transfected CHO cells, or TF expressing tumor cell lines MDA-MB-231, (luciferase transfected) A431 and Bx-PC3.
Cells were resuspended in PBS (2 X 106 ml), put in l V-bottom plates (50 ul/well). 50 ul of serially diluted HuMab in FACS buffer (PBS supplemented with 0.1% BSA and 0.02% Na-azide) was added to the cells and incubated for 30 minutes on ice. After g three times with FACS buffer, 50 ul of phycoerythrin (PE)-conjugated goat anti- human IgGFc on ImmunoResearch), diluted 1:100 in FACS buffer, was added. After s on ice (in the dark), cells were washed three times, and specific binding of the HuMabs was detected by flow cytometry on a FACSCalibur (BD Biosciences). HuMab-KLH was used as a negative control. Mouse anti-TF followed by PE-conjugated anti-mouse IgGFc was used as positive control. Binding curves were analyzed using non-linear sion (sigmoidal dose-response with variable slope) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, CA, USA).
Figure 4 shows an example of binding curves of TF-specific HuMabs to MDA-MB-231 cells.
Table 4 gives an overview of EC50 values of binding of TF-specific HuMabs to TF transfected CHO cells (S1015-TF), MDA-MB-231, A431 and Bx-PC3 cells.
-————————— -————————— --———————— WI" "-393 1956 1151 2755 In 5229 WI" 2438 1407 IE!" 6095 1798 1106 HE. 2530 4247 4 1320 3170 5808 "——2052 1324 3124 5629 ——1774 1128 5353 Table 4 - Overview of EC50 and maximum mean fluorescence index (max MFI) values determined by FACS analysis of binding of TF-specific HuMabs to different cell types.
EC50 values are in nM. Max MFI for MDA-MB-231, BxPC3 and A431 cells at 30 ug/mL antibody, for S1015-TF at 7.5 ug/mL antibody.
Inhibition of FVIIa binding to TF Inhibition of binding of FVIIa to TFECDHis by TF-HuMabs was measured by ELISA. ELISA plates were coated overnight with TFECDHis (0.5 ug/mL, 100 uL per well). Plates were emptied, blocked with PBS containing 2% (v/v) chicken serum (1 hour, RT), and emptied again. 4-fold serial dilutions of TF-HuMabs or HuMab-KLH (negative control) were added to the wells followed by FVIIa at EC50 concentration (100 nM), and plates ted for 1 hour at RT (while shaking, 300 rpm). Plates were washed and incubated with rabbit-anti-FVIIa (2.5 ug/mL; Abcam) as above. Plates were washed and ted with swine-anti-rabbit IgG-HRP dy (1:2,500; DAKO). After washing, the immune complexes were visualized using ABTS as a substrate. The reaction was stopped by the addition of 2% v/v oxalic acid followed by optical density measurement at 405 nm using an ELISA reader. The concentration of antibody needed to obtain 50% tion (IC50) was calculated using GraphPad prism (non linear regression analysis).
Figure 5 shows that antibodies from cross-block groups II and III efficiently inhibited FVIIa binding to TF, while antibodies from cross-block group I did not (or to a much lesser extent) inhibited FVIIa binding.
Table 5 shows IC50 values and maximum inhibition values (percentage) of inhibition of FVIIabinding to TF\by TF-specific HuMabs.
A HA87 L6 -iE-— —017 D12 —m_19 ——27 CD CD —092 A09 —.1_15 —101 "— Will mu.3.— ||/||| _114 —‘1_ _109 —.1_ —111 ——\l«3 Table 5 - IC50 values and maximum inhibition values (percentage) of inhibition of FVI I a g to TF by TF-specific HuMabs Example 16 Inhibition of FVIIa induced ERK hos hor lation Upon binding of coagulation factor VIIa (FVIIa) to TF, orylation of mitogen activated kinase (p42 and p44 MAPK or ERK1 and ERK2) is triggered. The epidermoid carcinoma cell line A431 ses high levels of TF, and after stimulation with FVIIa an optimal (3 to 5 fold) ERK phosphorylation (ERK-P), measured using the AlphaScreen Surefire ERK assay (Perkin Elmer), is induced within 10 minutes.
A431 cells (30,000 cells per well) were seeded in 96 well TC , and ed O/N (37°C, % C02, 85% humidity) in serum-free medium (RPMI containing 20% HSA and penicillin/streptomycin). Medium was then replaced by DMEM (without additives) and cells were incubated for 1.5 hours. 3 fold serial dilutions of TF-HuMabs or HuMab-KLH were added and cells incubated for 0.5 hours. Cells were then stimulated with FVIIa at EC80 concentration (50 nM; 10 minutes; 37°C, 5% C02, 85% humidity). Cells were washed once with PBS, and lysed using 25 uL lysis buffer (Perkin Elmer, Surefire kit). Lysates were centrifuged (3 minutes, 330 X g, RT). Four uL of supernatant was transferred to 384 well Proxiplates (Perkin Elmer). 7 ul Reaction buffer/Activation buffer mix containing AlphaScreen beads (Perkin Elmer Surefire kit) was added, and plates were incubated in the dark for 2 hours at RT. Plates were read using the "Surefire Plus" protocol from EnVision technology.
Figure 6 shows that, ed using the AlphaScreen Surefire ERK assay, antibody 013 does not inhibit FVIIa induced ERK orylation, 044 and 111 moderately inhibit ERK phosphorylation, and all other antibodies efficiently block ERK phosphorylation.
Table 6 shows IC50 values and maximum inhibition values (percentage) of inhibition of FVIIa induced ERK phosphorylation by cific HuMabs, measured using the AlphaScreen Surefire ERK assay.
WWW"~21 : 874L6 > 66. 6 -l> 0171-D12 0.79 2.01 CD AN l-P 092-A09 1.27 101 1.05 01 189 CD Willm.
Table 6 - |C50 values and maximum tion values (percentage) of inhibition of FVlla induced ERK phosphorylation (measured using the AlphaScreen Surefire ERK assay) by TF-specific HuMabs.
The results obtained in the AlphaScreen re ERK assay were confirmed by Western Blot analysis, using HaCaT and BXPC3 cell lines. 30,000 well were seeded in DMEM containing minimal concentrations of serum (starvation medium), and cultured overnight.
Cells were further ed for 2 hours in DMEM without serum, anti-TF antibodies were added during the final 30 minutes of culture. Cell were stimulated with 0, 10 or 50 nM FVIIa for 10 minutes , and subsequently lysed in cell lysis buffer (50 uL lysis buffer per well, 30-60 minutes lysis under shaking condition, RT). 25 (JL SDS containing sample buffer was added to each sample. s were loaded onto SDS-PAGE gels, run and blotted using standard procedures for Western blotting. Blots were blocked with TBST1x containing % irrelevant protein (ELK) for 1 hour at RT. Blots were incubated with rabbit anti-ERK-P antibody (O/N, 4°C). Blots were washed with TBST1X, and incubated with anti-rabbit IgG HRP (1 hour, RT), washed, ped using HRP substrate and imaged using Optigo Ultima Imaging system (Isogen Life Sciences).
Figure 6A shows the results in BXPC3 cells for a sub-panel of antibodies. ERK phosphorylation induced by 10 nM of FVIIa was not inhibited by antibody 013, while it was efficiently inhibited by antibodies 111, 044 and 025 (the latter as an example for all other TF-specific HuMabs described here). Stronger induced ERK phosphorylation (50 nM FVIIa) was not inhibited by antibodies 013, 111 and 044, but was inhibited by antibody 025. tion of FVlla d lL-8 release The y of TF ic HuMabs to inhibit FVIIa induced release of IL-8 was tested using MDA-MB-231 cells. Cells were seeded into 96 well plates (60,000 cells/well) and cultured (O/N, 37°C, 5% C02) in DMEM containing CS, sodium pyruvate, l-glutamine, MEM NEAA and penicillin/streptomycin. Tissue culture medium was removed, cells were washed twice in serum free, high calcium medium (DMEM containing penicillin/streptomycin), and cultured in this medium for an additional 105 minutes. Serial dilutions of antibodies were added, and cells ed for 15 minutes. FVIIa (Novo Nordisk; final concentration 10 nM) was added and cells were cultured for 5 hours. Supernatant was removed and centrifuged (300 X g, RT). IL-8 concentrations in the supernatant were measured using an IL-8 ELISA kit according to the manufacturer’s protocol (Sanquin).
Figure 7 shows that antibodies from cross-block groups II and III efficiently inhibited FVIIa induced IL-8 release 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 IL-8 release.
Table 7 shows IC50 values and maximum tion values (percentage) of inhibition of FVIIa induced IL-8 release by TF-specific HuMabs.
Table 7 - IC50 values and maximum inhibition values (percentage) of inhibition of FVlla induced lL-8 release by TF-specific HuMabs.
Example 18 Inhibition of FXa generation The ability of TF specific HuMabs to inhibit FXa generation was tested in an assay in which conversion of FX into FXa by the TF/FVIIa complex is measured using a colometric FXa specific substrate. TF (Innovin) was added to flatbottom 96 well plates, together with a serial dilution of TF ic HuMabs, positive control (mouse anti-TF) of negative control (HuMab-KLH)(a|I diluted in Hepes buffer containing 3 mM CaCIZ. Plates were incubated for minutes at RT, and FVIIa (final tration 1 nM) and FX (ERL; final tration 200 nM) was added. Plates were incubated 30 minutes at 37°C. 50 ul from each well was transferred to a 96 well plate containing (pre-heated, 37°C) stop-buffer (5 mM EDTA in 100 ml Hepes buffer). FXa specific substrate Chromogenix-2765 (Instrumation Laboratory Company) was added, plates incubated for 60 minutes at 37°C and OD405 nm at 37°C was measured.
Figure 8 shows that dy 017-D12 strongly ted FXa generation, 013 demonstrated intermediate inhibition and other antibodies showed low to no inhibition of FXa generation.
Table 8 shows IC50 values and maximum inhibition values (percentage) of inhibition of FXa generation by TF-specific HuMabs.
I-__— I-__— I--_— Il-__— Il-__— Il-__— Il-__— Il-__— -E__— Ill-__— Ill-__— Ill-__— Table 8 - IC50 values and maximum tion values (percentage) of inhibition of FXa generation by TF-specific HuMabs.
Example 19 Inhibition of blood coagulation Inhibition of blood coagulation by TF-HuMabs was measured in an assay determining TF induced clotting time. Mixtures of 17 pl 100 mM CaClZ (final conc. 17 mM), 10 pl 1:100 innovin (final conc. 1:1000), 23 pl 1x HEPES-buffer and 50 pl serially diluted dy were prepared in 96 well plates. Fifty pl pooled human plasma was added to wells of Immulon ZB plates (Thermo Electron). Fifty pl of the prepared antibody mixtures was added to the Immulon 2b , and coagulation development at 405 nm was measured every 15 sec for min using a kinetic plate reader. The increase in optical density was plotted in time and clotting time (t1/2) was calculated. Clotting time was plotted against antibody tration. IC50 of dy induced tion of coagulation was calculated from this by non linear regression analysis using GraphPad Prism.
Figure 9 shows that antibody 044, 087 and 111 did not inhibit TF induced blood coagulation, whereas all other antibodies did.
Table 9 shows IC50 values of inhibition of blood coagulation by TF-specific HuMabs.
Table 9 - IC50 values of inhibition of blood coagulation by TF-specific HuMabs.
Example 20 Antibod -de endent cell-mediated c totoxicit Preparation of target cells: TF expressing target cells (5x106 Bx-PC3 cells, MDA—MB-231 cells or A431 cells) were harvested, washed (twice in PBS, 1500 rpm, 5 min) and collected in 1 ml RPMI 1640 culture medium supplemented with Cosmic Calf Serum, Sodium te, L-Glutamine, MEM NEAA and llin/Streptomycin, to which 100 pCi 51Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands) was added. The e was incubated in a shaking water bath for 1 hr at 37°C. After washing of the cells (twice in PBS, 1500 rpm, 5 min), the cells were resuspended in culture medium and viable cells counted by trypan blue exclusion. Viable cells were brought to a concentration of 1x105 cells/ml.
Preparation of effector cells: Peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats (Sanquin, Amsterdam, The Netherlands) using standard Ficoll density centrifugation according to the manufacturer’s instructions (lymphocyte separation ; Lonza, Verviers, France). After resuspension of cells in culture medium, cells were counted by trypan blue exclusion and brought to a concentration of 1x107 cells/ml.
ADCC set up: 50 ul of 51Cr-labeled targets cells were transferred to microtiter wells, and 50 ul of serially diluted antibody was added, diluted in culture medium. Cells were ted (RT, 15 min), and 50 ul effector cells were added, resulting in an effector to target ratio of 100:1. To determine the maximum level of |ysis, 100 pl 5% Triton-X100 was added instead of effector cells; to determine the neous level of |ysis, 100 pl culture medium was added; to determine the level of antibody independent |ysis, 50 ul effector cells and 50 ul e medium was added). Subsequently, cells were incubated O/N at 37°C, 5% C02. After ng down the cells (1200 rpm, 3 min), 75 ul of supernatant was erred to micronic tubes. The ed 51Cr was counted in a gamma counter and the percentage of antibody mediated |ysis was calculated as follows: ((cpm sample - cpm antibody independent |ysis)/(cpm maximal |ysis - cpm spontaneous |ysis)) x 100% wherein cpm is counts per minute.
Figure 10 shows that all tested TF-HuMabs induced |ysis of Bx-PC3 cells by ADCC, albeit with different efficiencies (EC50).
Table 10 shows EC50 values (nM) of ADCC of ent cell lines by TF-specific HuMabs.
WMWWW =_———m_07_.11 _—___0.) _____ Table 10 - EC50 values (nM) of ADCC of different cell lines by TF-specific HuMabs.
Example 21 Complement deposition Deposition of complement fragments C3c and C4c to TF-HuMab ted target cells was ed by FACS analysis. TF expressing target cells 3 or MDA-MB-231 cells) were plated in 96 well round bottom plates (1x10e5 cells/well) in RPMI containing 1% BSA.
Antibody (30 ug/mL) was added and cells incubated at RT for 15 minutes. Twenty-five uL pooled human serum was added as a source of complement, heat inactivated human serum was used to ine spontaneous complement binding. Cells were incubated at 37°C for 45 minutes. Cells were washed once, and incubated with anti-human C3c FITC or anti- human C4c FITC (DAKO) in FACS buffer, and incubated for 30 minutes on ice. Samples were analyzed using FACS Canto.
Figure 11 shows that antibodies from cross-block group I did not induce C3c or C4c deposition on either BxPC3 or MDA-MB-231 cells. All tested antibodies from cross-block group II did induce C3c and C4c deposition, as did antibodies from cross-block group III, with the exception of antibody 003.
Example 22: Avidity/ Affinity studies Determination of affinity: Antibody binding to TF was analyzed by surface n resonance in a BIAcore 3000 (GE Healthcare). is was used for the is. HuMab antibodies (500 resonance units) were lized on the CM-5 sensor chip according to the procedure recommended by the cturer. Briefly, after surface activation by EDC and NHS HuMab antibody was injected over the activated CM-5 surface in 10 mM sodium-acetate, pH ranging from 4.0 to .5 at 5 uI/min. followed by 1 M Ethanolamine for deactivation. Concentration series of TFECDHis in HBS-EP buffer were injected over the immobilized antibodies at a flow rate of uI/min for 180 sec. Regeneration of the HuMab surface was performed by injection of 10 mM Gchine-HCI pH 2.0 or 10 mM sodium acetate pH 3.0. Kinetic analysis was performed using double reference subtraction and model 1:1 (Iangmuir) binding analysis.
Table 11 shows for most HuMabs the determined affinity in (sub) nanomolar range. Not from all antibodies the kinetic parameters could be determined. 044 did give a high variation in off-rates (kd) and had high residuals, which means that the fitting of the curves was not well. 098, 111 and 087-Lg6 had off-rates which where too high for the e 3000 to e.
W\WWW\ \\ fl—m————_—nm—m ||/||| mum—m ||/||| n.a. not assessable = > 10'3 sec"1 Table 11. Kinetic constants of TF-HuMabs for reactivity with TFECDHis — affinity measurements. ination of y: TF (TFECDHis) binding to TF-specific HuMabs was determined essentially as described above, with TFECDHis being immobilized on the CM-5 sensor chip (300 resonance , and concentration series of Humab antibodies used for kinetic analysis. Kinetic analysis was performed using double reference subtraction and model 1:1 (langmuir) binding analysis.
Table 12 shows avidity ements for antibodies 11, 98, 109 and 111. Whereas affinity measurements for 98 and 111 indicated high-off rates (beyond the limits of determination by Biacore (i. e. >10'3)), avidity determination revealed ction in the nanomolar range.
III-_— III-_— Table 12. Kinetic constants of TFECDHIS for reactivity with TF-HuMabs — avidity measurements.
Example 23: lmmunohistochemical analysis of binding to normal human tissues and pancreatic tumors Binding of TF-HuMabs 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 pm thickness) and fixated in acetone. nous tissue peroxidase (P0) was blocked and tissue slides were pre-incubated with normal human serum to prevent aspecific binding of later applied antibodies to endogenous Fc receptors. Mouse-Ab directed t human TF (and negative control mouse Ab) was applied at the tissues at optimal dilution and subsequently detected with Powervision-PO (Goat anti -mouse/-rabbit IgG)-PO. TF-specific HuMabs were coupled to Fab' goat anti- human IgG ITC and thereafter applied to the frozen tissue slides at 3 dilutions, including a pre-determined optimal dilution. Subsequently the HuMab - Fab-FITC complex was detected by rabbit anti-FITC and Powervision-PO. PO activity was ized with AEC as substrate and nuclei were visualized with hematoxylin. Staining was ed by brightfield microscope.
IHC with mouse Ab on formalin fixated and paraffin embedded (FFPE) tissue FFPE tissue biopsies were cut at 4 pm, de-paraffinized, d for nous tissue peroxidase and subjected to antigen retrieval (pH6, citrate buffer). Prior to the incubation with the mouse-Ab tissue slides were preincubated in normal human serum to prevent aspecific g to endogenous Fc receptors. Mouse Ab directed against human TF (and negative control mouse Ab) was applied to the tissue slides at l dilution and subsequently detected with Powervision-PO (Goat anti -mouse/—rabbit IgG)-PO. PO activity was visualized with AEC as substrate and nuclei were visualized with hematoxylin. Staining was analyzed by field microscope.
Figure 12 shows an example of binding of antibody 013 (positive staining), 011 (positive ng), 114 (positive staining) and 111 (intermediate staining) to kidney uli.
Antibody 098 and 044 did not bind glomeruli.
Table 13 gives an overview of staining results for all TF-HuMabs in human kidney all tissues examined.
||/||| ||/||| Table 13. IHC staining of human glomeruli Table 14 gives an overview of staining results of selcted TF-specific HuMabs in human , colon, heart, cerebrum and skin as well as in human pancreatic tumors. _—————— _—-——-_—— _—-——_I-—— m————— Table 14. IHC staining of normal human tissue and pancreatic tumors.
IHC analysis of binding of TF-HuMabs to human pancreatic tumors revealed positive staining for all TF-HuMabs (exemplified in figure 13).
Example 24: Treatment of established MDA-MB-231 tumor aft in y fat pads of SCI D mice The in vivo efficacy of TF-HuMabs was determined in established orthotopic MDA-MB-231 xenograft tumors in SCID mice. 2X106 tumor cells in PBS were injected s.c. in the 2nd y fat pad of female SCID mice, followed by treatment with TF-HuMabs or control mAb (HuMab-KLH), starting at a moment that tumor sizes became measurable. Antibodies were injected on day 21 (260 ug/mouse), day 28 (130 ug/mouse) and day 42 (130 ug/mouse). Tumor volume was determined at least 2x /week. Volumes (mm3) were calculated from caliper (PLEXX) measurements as 0.52 x (length) x (width)2.
Figure 14 shows that antibodies 114, 111, 013, 098, 011 and 044 were all effective in inhibiting growth of ished orthotopic MDA-MB-231 tumors.
Example 25: Pilot repeat dosing of a TF-specific HuMab in cynomolgus monkeys To obtain initial ation on the toxicology of TF-specific HuMabs, including an assessment of the ability of the antibodies to interfere with the coagulation cascade and hence potentially increase the bleeding risk in exposed animals, a pilot repeat dosing study in cynomolgus monkeys was performed.
Two male and two female cynomolgus monkeys (Macaca fascicu/aris), age approximately 2 years, received intravenous injections of antibody 011: -day 1 of study: 0 mg/kg (vehicle only) -day 8: 1 mg/kg; 1 mL/minute -day 15: 10 mg/kg; 1 mL/minute -day 22: 100 mg/kg; 1 ute The animals were followed until day 27, at which time point the animals were euthanized for necropsy and histological evaluation of organs.
The main end point of the study were: ca| observations: determined daily, signs of bleeding from gums, eyes. -functiona| bleeding time and blood loss: determined on days 1, 8, 15 and 22 (1, 24 and 120 h post dosing) and at two pre-trial time points. -b|ood/traces of clots: HE stain of all tissues (determined at s obtained at final sacrifice) -b|ood in urine, feces, vomit: determined daily/weekly.
No apparent ty of repeated, increasing dosing of antibody 011 was observed. The animals showed no clinical signs and there was no indication of cytokine release. In addition, there were no apparent clinical signs of a compromised coagulation system or ic bleedings. At the 1 h post close time-point, the mean bleeding time on Day 22 was significantly higher than that seen on Day 1 12). There were no other statistically significant differences between Days 8, 15 and 22 compared with Day 1. rmore, it was found that there was no apparent toxicity to major organs and no adverse hematological effects. The preliminary conclusion on the histological evaluation of tissues from this study is that there were no histology findings in the four treated animals that could be attributed to treatment with the test item.
Figure 15 shows the individual data points for each animal cate samples) as a function of time. Bleeding time for 4 animals were determined on days 1, 8, 15 and 22 (1, 24 and 120 h) and at two pre-trial time points..
Example 26 Preventive and therapeutic treatment of BxPCS tumor afts in SCI D mice The in vivo efficacy of TF-HuMabs in preventive or therapeutic treatment of BXPC3 cell xenografts in SCID mice was determined. 10x106 BXPC3 tumor cells in PBS were injected s.c. in female SCID mice, followed by treatment with TF-HuMabs or control mAb (HuMab- KLH). For preventive treatment, antibodies (400 pg/mouse) were injected i.p. 1 hour after tumor induction. For therapeutic ent, antibody injection (300 pg/mouse) was started on day 8 after tumor induction, followed by weekly antibody injections (150 pg/mouse).
Tumor volume was determined at least 2x /week. Volumes (mm3) were calculated from caliper (PLEXX) measurements as 0.52 X h) X (width)2.
Figure 16 shows that TF-specific HuMabs are capable of preventive as well therapeutic ent of BXPC3 xenograft tumors.
Example 27 DNA ing between murine and human TF to determine domains important for binding of anti-TF HuMabs To determine domains important for binding of anti-TF HuMabs to human TF, DNA shuffling was med between human and murine TF. Shuffle constructs were prepared from DNA ng human TF, by replacing human domains with murine domains and from DNA encoding murine TF by replacing murine domains with human domains. If a domain in human TF is important for binding of an anti-TF HuMab, binding will be lost upon replacement of that domain with the murine domain. Human and murine TF are 57% homologous on n level. Figure 17 A and 17 B show the constructs for human TF ning murine TF domains (TFhs, containing Tme domains) and for murine TF containing human TF domains. HEK293F cells were transiently transfected with the constructs or with the vector alone (pcDNA3.3SP; mock). FACS analysis was performed essentially as described supra, with 30 pg/mL purified parental material. HuMab-KLH was used as a control Ab.
Figure 17 shows that all but one anti-TF HuMabs bind solely to human TF and not to murine TF. HuMab-TF-OO3 shows some binding to murine TF.
Figure 18 A to 0 shows the results for binding of the ent anti-TF HuMabs to the constructs expressed on HEK293F cells. These s are summarized in Table 15. In this table the anti-TF HuMabs are classified in groups, based on the domains on human TF that are important for binding of these HuMabs.
Shuffle constructs: HuMabs that show decreased 1-41 mm None 85-122 mm 25, 42, 98, 109, 111 Groups based on binding to shuffle constructs 1. 42-84 11, 17, 92, 101 4. 123-137 114 6. 7 + 185-225 + 226-250 Table 15 Example 28 Binding of Fab fragments of anti-TF HuMabs to the extracellular domain of TFI determined by ELISAI and to cellular TF on BxPC3 cellsI determined by FACS Binding of Fab nts of anti-TF HuMabs to TF was measured by ELISA (coated extracellular domain of TF) and by FACS (TF on BXPC3 cells). ELISA was performed essentially as described supra. Bound Fab fragments were detected using HRP-conjugated donkey-anti human H+L. FACS is was performed essentially as described supra.
FITC-conjugated goat anti-human IgG (H+L) (Jackson) was used to detect bound lead candidates. Fluorescence was measured on a FACSCantoII. Binding curves were analyzed as described supra, using GraphPad Prism 5 software.
Figure 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.
Figure 20 shows less binding of HuMab-TF-098 and -111 Fab nts to cellular TF, compared to -011 Fab fragments, measured by FACS on BXPC3 cells.
Table 16 shows EC50 values of TF Fab nts for g to the extracellular domain of TF by ELISA and to cellular TF by FACS on BXPC3 cells.
HuMab-TF EC50 (ELISA) EC50 (FACS) 011 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 na Table 16 — Overview of EC50 values for binding of HuMab-TF Fab fragments to the extracellular domain of TF, determined by ELISA, and to cellular TF on BxPC3 cells, determined by FACS.
EC50 values are in pg/mL. na — could not be calculated.
Example 29 Binding of anti-TF HuMabs to cell lines expressing different levels of TF Binding of anti-TF HuMabs to membrane-bound TF on cell lines sing different levels of TF was determined by FACS analysis, essentially as described supra. Mouse anti-TF antibody followed by PE-conjugated anti-mouse IgGFc was used as a positive control.
Fluorescence was measured on a FACSCantoII. Binding curves were analyzed essentially as described supra, using GraphPad Prism 5 software. The amount of TF les on cell lines was determined by Qifi kit (Dako, Glostrup, Denmark), according to the manufacturer’s ctions. It was determined that SW480 cells express ~ 20,000 molecules of TF per cell, SK-OV-3 cells express ~ 60,000 molecules per cell, AsPC-1 cells s ~ 175,000 molecules per cell and MDA-MB-231 cells express ~ 900,000 les per cell.
Figure 21 HuMab-TF-98 and -111 display similar binding characteristics as HuMab-TF-11, - 13 and 109 in the high TF sing cell line MDA-MD-231. In the cell lines with lower TF molecules per cell, for example the SK-OV-3 and SW480 cell lines, HuMab-TF-98 and 111 display different binding characteristics as compared to the other HuMab-TF antibodies.

Claims (43)

1. A human antibody which binds human Tissue Factor, wherein the antibody comprises: a) a variable heavy (VH) region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:10, 11 and 12 and a variable light (VL) region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:66, 67 and 68.
2. The antibody of claim 1, comprising a VH region having a) at least 80% ty, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region comprising the sequence of SEQ ID NO:9 or b) at most 20, such as 15, or 10, or less amino-acid modifications, more preferably amino-acid substitutions, such as vative amino-acid substitutions as compared to a VH region comprising the sequence of SEQ ID NO:9.
3. The antibody of claim 1 or 2, sing a VL region having a) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% ty to a VL region comprising the sequence of SEQ ID NO:65 or b) at most 20, such as 15, or 10, or less amino-acid modifications, more preferably amino-acid substitutions, such as conservative amino-acid substitutions as compared to a VL region comprising the sequence of SEQ ID NO:65.
4. The antibody according to any one of claims 1 to 3, comprising a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO: 65.
5. The antibody of any one of the ing claims, wherein the antibody binds to the extracellular domain of Tissue Factor with an apparent affinity (EC50) of 3 nM or less, such as 0.50 nM or less, or 0.35 nM or less, or 0.20 nM or less, or 0.1 nM or less.
6. The antibody of any one of the preceding claims, wherein the antibody binds to mammalian cells expressing Tissue Factor, such as A431 cells transfected with a construct ng Tissue Factor, preferably with an apparent affinity (EC50) of 10 nM or less, such as 8 nM or less, or 5 nM or less, such as 2 nM or less, or 1 nM or less, such as 0.5 nM or less, or 0.3 nM or less. 18563328_1 (GHMatters) P34563NZ04
7. The antibody of any one of the preceding claims, wherein the antibody is capable of ng antibody-dependent cellular cytotoxicity in A431 cells, preferably with an EC50 value of 2 nM or less, such as 1 nM or less, or 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.
8. The dy of any one of the preceding claims, wherein the antibody is effective in inhibiting growth of established MDA-MB-231 tumors.
9. The antibody of any one of the preceding claims, wherein the antibody inhibits Tissue Factor induced blood coagulation, preferably with a median tion concentration of less than 10 nM, such as less than 5 nM, or less than 2 nM, such as less than 1 nM.
10. The antibody of any one of the preceding claims, wherein the antibody inhibits FVIIa binding to Tissue Factor, preferably with a maximum inhibition value of inhibition of more than 80%, such as more than 90%.
11. The antibody of any one of the preceding claims, wherein the antibody inhibits FVIIa-induced IL-8 release by MDA-MB-231 cells, preferably with a m inhibition value of inhibition of more than 40%, such as more than 50%, or more than 60%.
12. The antibody of any one of the preceding claims, wherein the antibody inhibits conversion of FX into FXa by the TF/FVIIa complex, preferably by less than 50%, such as less than 40%, or in the range of 1-30%.
13. The antibody of any one of the preceding claims, wherein binding to Tissue Factor does not involve any one of the amino acids W in on 45, K in position 46 or Y in position 94 of Tissue Factor.
14. The antibody of any one of the preceding claims, wherein said antibody inhibits FVIIa d ERK phosphorylation, preferably with a median inhibition concentration of less than 10 nM, such as less than 5 nM, or less than 2 nM.
15. The antibody of any one of the ing claims, n the antibody is capable of inducing C3c and C4c tion. 18563328_1 (GHMatters) P34563NZ04
16. The antibody of any one of the preceding claims, wherein the antibody Fab fragments binds to the extracellular domain of Tissue Factor with an EC50 value of below 0.1 μg/mL, such as below 0.05 μg/mL, or below 0.04 μg/mL measured by ELISA.
17. The dy of any one of the preceding claims, wherein the antibody Fab fragments binds to the extracellular domain of Tissue Factor with an EC50 value of above 1.0 μg/mL as measured by ELISA.
18. The antibody of any one of the preceding claims, wherein the antibody Fab fragments binds to the extracellular domain of Tissue Factor with an EC50 value of below 10g/mL, such as below 1 g/mL, or below 0.5 g/mL, or below 0.2g/mL.
19. The antibody of any one of the preceding claims, wherein the antibody binds to human Tissue Factor and not murine Tissue Factor and shows reduced binding as compared to binding to human Tissue Factor to the shuffle construct 42-84 mm, containing the human sequence for Tissue Factor except for amino acid 42-84, which has been replaced with a mouse sequence.
20. The antibody of any one of the preceding claims n the antibody has an affinity to Tissue Factor which is less than 5 nM, such as less than 3.5 nM, or less than 2 nM.
21. The dy of any one of the preceding claims wherein the antibody has a kd of more than 10-3 sec-1, and/or a ka of more than 5x104,mol-1 sec-1.
22. The antibody of any one of the preceding claims wherein the antibody has a kd of more than 10-3 sec-1 and an y of less than 5 nM, such as less than 1 nM, or less than 0.2 nM.
23. The antibody of any one of the preceding claims which binds to the same epitope on Tissue Factor as an antibody having a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO: 65.
24. The antibody of any one of the preceding , n the antibody is a full- length antibody, preferably an IgG1 antibody, in particular an IgG1,κ antibody.
25. The antibody of any one of the preceding claims, wherein the antibody is conjugated to another , such as a cytotoxic moiety, a radioisotope or a drug. 18563328_1 (GHMatters) P34563NZ04
26. The antibody of any one of the preceding claims, wherein the dy is a lent antibody.
27. The dy of claim 26, wherein said monovalent antibody is constructed by a method sing: i) providing a nucleic acid construct encoding the light chain of said lent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen specific antibody and a nucleotide sequence encoding the nt CL region of an Ig, wherein said nucleotide sequence encoding the VL region of a selected antigen ic antibody and said nucleotide sequence encoding the CL region of an Ig are operably linked together, and wherein, in case of an IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that the CL region does not contain any amino acids capable of forming disulfide bonds or nt bonds with other peptides comprising an identical amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human being; ii) providing a nucleic acid construct encoding the heavy chain of said lent dy, said construct comprising a nucleotide sequence encoding the VH region of a selected antigen specific antibody and a nucleotide sequence encoding a constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, does not comprise any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the CH region of the human Ig in the presence of polyclonal human IgG or when administered to an animal human being, wherein said nucleotide sequence encoding the VH region of a selected antigen specific dy and said nucleotide sequence encoding the CH region of said Ig are operably linked together; iii) providing a cell expression system for ing said monovalent antibody; iv) producing said monovalent antibody by co-expressing the c acid ucts of (i) and (ii) in cells of the cell expression system of (iii).
28. The antibody of any one of claims 26 to 27, wherein the heavy chain has been modified such that the entire hinge has been deleted. 18563328_1 (GHMatters) P34563NZ04
29. A ific molecule comprising an antibody of any of claims 1 to 25 and a second binding icity, such as a binding specificity for a human effector cell, a human Fc receptor or a T cell receptor.
30. An sion vector comprising a tide sequence encoding an antibody according to claims 1 to 4 comprising one or more of the amino acid sequences selected from the group consisting of SEQ ID NO:9, SEQ ID NO: 12, and SEQ ID NO: 65-68.
31. An expression vector according to claim 30, further comprising a nucleotide sequence encoding the constant region of a light chain, a heavy chain or both light and heavy chains of a human antibody.
32. An ex vivo recombinant eukaryotic or prokaryotic host cell which produces an antibody as defined in any one of claims 1 to 24 or 26 to 28, wherein a cell capable of generating a human being is ed.
33. A pharmaceutical composition comprising an antibody as defined in any one of claims 1 to 28 or a bispecific le as defined in claim 29, and a pharmaceutically acceptable carrier.
34. The dy as defined in any of claims 1 to 28 or a bispecific molecule as defined in claim 28 for use as a medicament.
35. The antibody or the bispecific molecule of claim 34, wherein the use is for the treatment of cancer.
36. Use of an antibody as defined in any of claims 1 to 28 or a bispecific molecule as defined in claim 29 in the manufacture of a medicament for treating cancer.
37. The dy or the bispecific molecule of claim 35, or the use of claim 36, wherein the cancer is selected from the group consisting of: tumors of the central nervous system, head and neck cancer, lung cancer. breast cancer, esophageal cancer, stomach cancer, liver and biliary cancer, pancreatic , colorectal cancer, bladder cancer, kidney , prostate cancer, endometrial cancer, ovarian cancer, malignant melanoma, sarcoma, tumors of n primary origin, bone marrow cancer, acute lymphoblastic leukemia, c lymphoblastic leukemia, non-Hodgkin lymphoma, skin cancer, glioma, and cancer of the brain, uterus, and rectum. 18563328_1 (GHMatters) P34563NZ04
38. The antibody or the bispecific molecule, or the use, of claim 36, wherein the cancer is selected from the group consisting of: pancreatic cancer, colorectal cancer, ovarian cancer, breast cancer, prostate cancer and bladder cancer.
39. The antibody or the bispecific molecule of any one of claims 34, 35, 37 or 38, or the use of any one of claims 36 to 38, wherein the medicament is for use in combination with one or more further therapeutic agents, such as a chemotherapeutic agent.
40. A method for producing an antibody of any of claims 1 to 24 or 26 to 28, said method sing the steps of a) culturing a host cell of claim 32, and b) ing the antibody from the culture media.
41. A stic composition comprising an antibody as defined in any of claims 1 to
42. A method for detecting the presence of Tissue Factor in a sample, comprising: - contacting the sample with an antibody of any of claims 1 to 28 or a ific molecule of claim 29 under conditions that allow for formation of a complex between the dy or bispecific molecules and Tissue Factor; and - analyzing whether a complex has been .
43. A kit for detecting the presence of Tissue Factor in a sample comprising - an antibody of any of claims 1 to 28 or a bispecific molecule of claim 29; - instructions for use of the kit. 18563328_1 (GHMatters) P34563NZ04 ImHOHm> > ImfiOHm> Imaoam> 1m 1m 1m [m Im 1m cam> 0Hm> oam> OHI> OHm> QHm> mmucmzcwm mm>a>zewowz mm>e>qeoom _ mm>H>aeooozw umcEmuc: ......... \\\\
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