WO2011040973A2

WO2011040973A2 - Tnf-immunoconjugates with fibroblast activation protein antibodies and methods and uses thereof

Info

Publication number: WO2011040973A2
Application number: PCT/US2010/002661
Authority: WO
Inventors: Stefan Bauer; Thomas Wuest; Christoph Renner
Original assignee: Ludwig Institute For Cancer Research Ltd.
Priority date: 2009-10-02
Filing date: 2010-10-01
Publication date: 2011-04-07
Also published as: WO2011040973A3

Abstract

Protein-immunocoηjugates are provided, particularly TNF conjugates to antibodies and fragments thereof which bind to Fibroblast Activation Protein (FAP). TNF-FAP antibody conjugates are provided wherein the FAP antibody recognizes both human and mouse FAP. These immunoconjugates are useful in the diagnosis and treatment of conditions associated with activated stroma, including wound healing, epithelial cancers, osteoarthritis, rheumatoid arthritis, cirrhosis and pulmonary fibrosis, and more broadly in TNF responsive conditions and disorders, including cancer and hyperproliferative diseases. In addition, the FAP-immunoconjugates can be used for the diagnosis and treatment of FAP positive tumors such as pancreatic cancer, melanoma and sarcomas. The immunoconjugates, antibodies, variable regions or CDR domain sequences thereof, and fragments thereof of the present invention may also be used in therapy in combination with chemotherapeutics, immune modulators, or anti-cancer agents and/or with other antibodies or fragments thereof.

Description

TNF-EMMUNOCONJUGATES WITH FIBROBLAST ACTIVATION PROTEIN

ANTIBODIES AND METHODS AND USES THEREOF

FIELD OF THE INVENTION

[0001] The present invention relates to protein-immunoconjugates, particularly TNF conjugates to antibodies and fragments thereof which bind to Fibroblast Activation Protein (FAP). Particular TNF-FAP antibody conjugates are provided wherein the FAP antibody recognizes both human and mouse FAP. These immunoconjugates are useful in the diagnosis and treatment of conditions associated with activated stroma, including wound healing, epithelial cancers, osteoarthritis, rheumatoid arthritis, cirrhosis and pulmonary fibrosis, and more broadly in TNF responsive conditions and disorders, including cancer and hyperproliferative diseases. In addition, the FAP-immunoconjugates can be used for the diagnosis and treatment of FAP positive tumors such as pancreatic cancer, melanoma and sarcomas. The immunoconjugates, antibodies, variable regions or CDR domain sequences thereof, and fragments thereof of the present invention may also be used in therapy in combination with chemotherapeutics, immune modulators, or anti-cancer agents and/or with other antibodies or fragments thereof.

BACKGROUND OF THE INVENTION

[0002] Fibroblast activation protein (FAP) was originally identified as a serine protease on reactive stromal fibroblasts [1, 2]. Subsequent molecular cloning revealed that FAP is identical to seprase, a 170 kDa membrane associated gelatinase that is expressed by melanoma cell lines [3, 4]. Full length cDNA encoded a type II transmembrane protease of 760 amino acids (aa) highly homologous to dipeptidyl peptidase IV (DPPIV) with a 52% aa identity over the entire sequence and almost 70% identity in the catalytic domain [3, 5]. U.S. Patent 5,587,299, incorporated herein by reference, describes nucleic acid molecules encoding FAP and applications thereof.

[0003] FAP and DPPIV have similar gene sizes and are chromosomally adjacent to each other at

2q24, suggesting a gene duplication event (Genebank accession number U09278). Both proteins are members of the prolyl peptidase family [1 , 6]. This class of enzymes is inducible, active on the cell surface or in extracellular fluids, and uniquely capable of cleaving N-terminal dipeptides from polypeptides with proline or alanine in the penultimate position [7]. DPPIV, also termed CD26, is constitutively expressed by several cell types including fibroblasts, endothelial and epithelial cells, leukocyte subsets like NK-cells, T- lymphocytes and macrophages. A small proportion of DPPIV circulates as soluble protein in the blood. In contrast to DPPIV, FAP is typically not expressed in normal adult tissue [1] and its proteolytically active soluble form is termed a2-Antiplasmin Cleaving Enzyme (APCE) [8]. Marked FAP expression occurs in conditions associated with activated stroma, including wound healing, epithelial cancers, osteoarthritis, rheumatoid arthritis, cirrhosis and pulmonary fibrosis [4, 9-1 1].

[0004] The FAP structure has been solved (PDB ID 1Z68) and is very similar to that of DPPIV

[12]. FAP is anchored in the plasma membrane by an uncleaved signal sequence of approximately 20 amino acids and has a short, amino terminal, cytoplasmic domain of six amino acids [3-5]. The major part of the protein, including the catalytic domain, is exposed to the extracellular environment [13]. The FAP glycoprotein is a homodimer consisting of two identical 97-kDa subunits. Each FAP-monomer subunit consists of two domains, an αβ hydrolase domain (aa 27-53 and 493-760) and an eight-blade β propeller domain (aa 54-492) that enclose a large cavity. A small pocket within this cavity at the interface of both domains contains the catalytic triad (Ser624, Asp702 and His734) [12]. FAP gains its enzymatic activity upon homodimerization of the subunits [14] and beside its dipeptidyl peptidase activity, FAP also has collagen type I specific gelatinase [15] and endopeptidase activity [16]. The β propeller acts as scaffolding for protein-protein interactions and determines substrate and extracellular matrix (ECM) binding [17]. Furthermore, the β propeller is involved in forming supra-molecular complexes of FAP with other prolyl peptidases or with other membrane-bound molecules [18, 19]. The formation of heteromeric or tetrameric complexes of FAP and DPPrV were found to be associated with invadopodia of migrating cells on a collagen substrate [20]. Type I collagen induces a close association of FAP with βΐ integrins, thereby playing major organizational roles in the formation and adhesion of invadopodia [21]. Although the involved mechanisms are not understood in detail, the formation of such proteinase-rich membrane domains at the cellular invasion front contributes to directed pericellular ECM degradation [22]. This indicates that FAP and ECM interactions may be closely related to invasive cell behaviour by influencing cell adhesion, migration, proliferation and apoptosis through integrin pathways [19, 21, 23] and supports o role of FAP in disease pathogenesis and progression [24]. In summary, FAP is recognized as a multifunctional protein that executes its biological functions in a cell dependent manner through a combination of its protease activity and its ability to form complexes with other cell-surface molecules. Over-expression of FAP in epithelial and fibroblastic cell lines promotes malignant behaviour [22], pointing to the clinical situation, where cellular expression levels of FAP are correlated with worse clinical outcome [25, 26]. [0005] Through paracrine signaling molecules, cancer cells activate stromal fibroblasts and induce the expression of FAP, which in turn, affects the proliferation, invasion and migration of the cancer cells. Recent studies have demonstrated that TGF-β is the dominant factor in promoting FAP protein expression (Chen, H et al (2009) Exp and Molec Pathology, doi: 10.1016/j.yexmp. 2009.09.001). FAP is heavily expressed on reactive stromal fibroblasts in 90% of human epithelial carcinomas, including those of the breast, lung, colorectum and ovary (Garin-Chesa, P et al (1990) PNAS USA 87: 7236-7239). Chen et al have recently shown that FAPa influences the invasion, proliferation and migration of HO-8910PM ovarian cancer cells (Chen, H et al (2009) Exp and Molec Pathology, doi: 10.1016/j.yexmp. 2009.09.001).

[0006] The morphological and functional properties of FAP have promoted the investigation of

FAP as a therapeutic target. The disease related and cell surface bound expression pattern especially qualifies FAP for antibody targeting. With regard to the pathophysiological involvement in ECM remodelling, targeting strategies should aim at the disruption of the signalling supra-molecular FAP complexes. Although FAP has attracted increased interest as a target for antibody based immunotherapy, data of therapeutically active native FAP-specific antibodies are missing to date. The monoclonal antibody F19 was the first antibody investigated in a phase I clinical trial targeting metastatic colorectal cancer [30]. This trial served as a proof of principle for anti-FAP based tumor stroma targeting [1]. Although patients included in the trial had extensive scarring due to surgery, no specific enrichment of ¹³¹I-F19 could be detected at these sites. There were no toxic side effects associated with intravenous administration of iodine¹³¹ labelled F19 and carcinoma lesions were specifically detected by imaging down to a size of 1 cm in diameter. With regard to the immunogenicity of murine antibodies in humans, recent phase I and phase II clinical trials were conducted using the humanized version of F19, called Sibrotuzumab [31, 32]. Results from these trials demonstrated the safe and well tolerated administration of Sibrotuzumab. Similar to the results obtained in the pivotal phase I trial [30], trace-labelling with ¹³¹I and imaging analysis revealed the specific accumulation of Sibrotuzumab at the tumor area. Unfortunately, unconjugated Sibrotuzumab demonstrated no anti-tumor or any therapeutic activity, respectively [32]. Although the biologic function of FAP is still not known in detail, its dipeptidyl peptidase activity was postulated to be involved in tumor progression and metastasis [15, 33]. The lack of Sibrotuzumab to affect FAP enzymatic function was suggested to be the reason for the lack of therapeutic efficacy [34]. In consequence, anti-FAP directed polyclonal antibodies have been raised in order to inhibit the catalytic activity in-vitro. Indeed, treatment of FAP-positive xenografts with anti-FAP anti-sera attenuated tumor growth [13]. However, since polyclonal sera were raised by immunization of rabbits with murine FAP, it is most likely that additional epitopes, different from the catalytic domain, have also been targeted. Therefore, it is difficult to conclude from this study that anti-tumor effects seen really depended on dipeptidyl-peptidase inhibition. [0007] TNF is a pleiotropic cytokine with a wide variety of biological activities and immunomodulatory properties (Locksley, R. M. et al (2001) Cell 104: 487-501). The soluble form of the cytokine occurs as a trimer of three identical 17-kDa subunits. Because systemic administration of TNF mediates regression of murine and xenotransplanted human tumors, TNF has attracted attention as a potent antitumor agent (Carswell, E. A. et al (1975) Proc. Natl. Acad. Sci. USA 72: 3666-3670; Asher, A. et al

(1987) J. Immunol. 138:963-974; Creasey, A. A. et al (1986) Cancer Res. 46: 5687-5690). However, the systemic use of the cytokine for cancer therapy in humans is restricted by its very short circulatory half-life and its dose-limiting toxicity (Blick, M. et al (1987) Cancer Res. 47:2986-2989; Sherman, M. L. et al

(1988) J. Clin. Oncol. 6: 344-350; Spriggs, D. R. et al (1998) J. Natl. Cancer Inst. 80: 1039-1044).

[0008] Like other cytokines, TNF rapidly binds to its ubiquitously expressed receptor type 1

(TNFRSFIA (TNFR superfamily, member 1A; p60; TNF-Rl)) in various normal tissues after i.v. application and thereby exerts multiple systemic side effects, such as activation of coagulation and inflammatory cascades, before achieving a therapeutic dose at the tumor site (Manusama, E. R. et al (1998) Semin. Surg. Oncol. 14: 232-237). However, TNF-mediated antitumor activity depends more on triggering indirect tumoricidal mechanisms than on direct induction of death signaling pathways in malignant cells themselves. Therefore, local exposure of high dose TNF is an obligate prerequisite to induce sufficient strength in TNFR triggering with subsequent recruitment of cellular and humoral effector mechanisms for successful tumor immunotherapy (Clauss, M. et al (1990) J. Biol. Chem. 265: 7078-7083; Nawroth, P. P. et al (1986) J. Exp. Med 163: 740-745; Nawroth, P., D. (1988) J. Exp. Med. 168: 637-647). Until recently, isolated limb perfusion was the only accepted clinical method to obtain high cytokine concentrations in more localized areas (Eggermont, A. M., and T. L. ten Hagen (2001) Curr. Oncol. Rep. 3: 359-367; Lejeune, F. J. et al (1998) Curr. Opin. Immunol. 10: 573-580).

[0009] To overcome the limitations of TNF's severe side effects, genetic fusion of TNF to tumor- selective Abs is a strategy to increase site-specific cytokine targeting. To gain high antitumor activity by local enrichment of TNF, many investigations have aimed at decreasing its unwanted toxicity to facilitate systemic application at therapeutic doses needed. Most strategies focused on Ab-directed targeting of TNF to a tumor-associated Ag by genetically engineered fusion proteins (Xiang, J. (1999) Hum. Antibodies 9: 23-36). Because wild-type TNF exerts its bioactivity as a homotrimeric protein, structural prerequisites for assembly of monomeric subunits have been addressed by designing single-chain derived immunocytokines (Liu, Y. et al (2004) Int. J. Cancer 108: 549-557; Scherf, U. et al (1996) Clin. Cancer Res. 2: 1523-1531; Yang, J. et al (1995) Mol. Immunol. 32: 873-881 ). However, trimerized TNF fusion proteins carry a fully active TNF molecule with high affinity to TNF-Rl and capability of signaling before the target Ag expressed by the tumor cell has been reached. As a result, similar levels of TNF-R1 -mediated cytotoxicity are observed, compared with wildtype soluble TNF (Curnis, F. et al (2000) Nat. Biotechnol. 18: 1185- 1190; Halin, C. et al (2003) Cancer Res. 63:3202-3210).

[00010] More recently, targeted bioactivity of TNF has been described by an Ab-derived fusion protein, based on an IgGl-format (Bauer S et al (2004) J Immunol 172:3930-3939). This fusion protein consists of a humanized anti-fibroblast-activating protein (FAP)3 Ab and human (hu)TNF replacing the IgGl CH2/CH3 Fc domain. In contrast with trimerized wild-type TNF, this construct preserved its IgGl- derived dimeric structure with the TNF molecule forced to form a dimer. Avoiding the 3-fold symmetry of TNF resulted in a significantly reduced TNF-R1 -mediated toxicity in vitro and in vivo, compared with trimerized TNF at equimolar concentrations (Bauer, S. et al (2004) J. Immunol. 172: 3930-3939). Further studies analyzing the structure-activity relationship of trimerized (single-chain variable fragment (scFv) format) and dimerized (truncated IgGl format) Ab-TNF conjugates were undertaken (Bauer S et al (2006) J Immunol 177:2423-2430). Bioactivity profiles, including TNF-R1 -mediated dose-limiting toxicity and therapeutic potency, differ significantly between rTNF-conjugated Ab formats when analyzed in vitro and in vivo with a clear disadvantage for the trimeric scFv-TNF construct.

[00011] Thus, while the extant evidence of activity of TNF-antibody constrcts, including TNF-FAP antibody conjugates is encouraging, limitations on efficacy, anti-tumor activity, and the lack of cross- reactivity of the known FAP antibodies against mouse FAP remain. Accordingly, it would be desirable to develop TNF-antibody constructs with anhanced activity and using novel antibodies, including FAP antibodies, particularly antibodies which can be utilized in mouse animal models and which demonstrate increased efficacy and applicability in diagnosis and therapy, and it is toward the achievement of that objective that the present invention is directed.

[00012] The citation of references herein shall not be construed as an admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

[00013] The present invention generally provides immunoconjugates, comprising an antibody or antibody-like moiety and an active molecule moiety, which are, in part, mediated by the uniqueness and capabilities of the antibodies or fragments thereof which are conjugated to the active molecule moiety. In one particular aspect, novel anti-FAP antibodies or portions thereof are conjugated to one or more active molecule moiety. In a particular aspect, novel anti-FAP antibodies or portions thereof are conjugated to one or more active molecule moiety which is an anti-cancer agent, a cell toxicity mediator, a ligand, an immunomodulatory molecule, a pro-apoptotic molecule. In a further a particular aspect, novel anti-FAP antibodies or portions thereof are conjugated to one or more active molecule moiety which is tumor necrosis factor.

[00014] The invention provides antibody immunoconjugates utilizing novel antibodies directed against Fibroblast activation protein (FAP) for diagnostic and therapeutic purposes. In particular, the antibodies specific for FAP provided and utilized herein recognize and are capable of binding human and mouse FAP. In addition, the novel FAP antibodies utilized in the immunoconjugates do not cross react/bind to CD26 (dipeptidyl peptidase IV (DPPIV)). In a further aspect, the antibody or antibody molecules of the present immunoconjugates do not directly effect dipetidyl peptidase activity of FAP. In a still further aspect, the antibody or fragment mediates down regulation of FAP expression. In another aspect the antibody or fragment induces or mediates apoptosis in FAP expressing cells. In a still additional aspect the antibody or fragment inhibits cellular adhesion to ECM proteins. Thus, the anti-FAP antibody(ies) or active fragment(s) thereof utilized in the immunoconjugates of the invention have at least two of the following characteristics: reactive with human and mouse FAP; do not react with or bind to CD26 (DPPrV); do not directly affect dipeptidyl peptidase activity of FAP; mediate down regulation of FAP expression; induce or otherwise mediate apoptosis in FAP expressing cells; and inhibit cellular adhesion to ECM proteins.

[00015] The active molecule moiety of the immunoconjugate of the present invention may comprise or consist of a molecule, compound, agent, peptide which is an anti-cancer agent, a cell toxicity mediator, a ligand, an immunomodulatory molecule, a pro-apoptotic molecule which confers a therapeuticall relevant activity to the immunoconjugate. In one aspect, the one or more active molecule moiety is a growth factor or an anti cancer agent. In a further a particular aspect, the one or more active molecule moiety is tumor necrosis factor. Any of the known sequences or portions or homologs of tumor necrosis factor may be utilized which have an effective, desired, and useful activity. Therefore, a mouse, human or other mammalian sequence or portion thereof of TNF may be utilized.

[00016] The immmunoconjugates of the present invention have diagnostic and therapeutic use in conditions associated with activated stroma, including wound healing, epithelial cancers, osteoarthritis, rheumatoid arthritis, cirrhosis and pulmonary fibrosis, and more broadly in TNF responsive conditions and disorders, including cancer and hyperproliferative diseases. In a particular aspect the immunoconjugates of the invention are applicable in cancers. In a particular aspect immunoconjugates of the invention are applicable in epithelial cancers, including breast, lung, colorectal and ovarian cancers.

[00017] In a particular aspect, the immunoconjugates of the present invention comprise FAP antibodies directed against human and mouse FAP and which do not cross react/bind to CD26 (dipeptidyl peptidase IV (DPPIV)). The present invention provides immunoconjugates utilizing or comprising an antibody or fragment thereof, including a Fab fragment and a single chain or domain antibody, which recognizes human FAP. In a further aspect, the immunoconjugates comprise an antibody or fragment thereof which recognizes human FAP and comprises the amino acid sequence of ESC 1 1 or ESC 14 including as set out in Figure 1 and/or Figure 2. In a further preferred aspect the immunoconjugates comprise an antibody or fragment thereof which recognizes human FAP and comprises the VH and VL amino acid sequences depicted in Figures 1 and 2 (VH sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and VL sequences SEQ ID NO: 3, SEQ ID NO: 8). In one such aspect, the invention provides an anti-human FAP antibody comprising the variable region CDR sequences set out in Figure 2 (heavy chain CDR 1 (SEQ ID NO: 1 1), CDR2 (SEQ ID NO: 12), CDR3 (SEQ ID NO: 13); light chain CDR 1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 18), CDR3 (SEQ ID NO: 19)).

[00018] In a further aspect, the immunoconjugates comprise anti-FAP antibodies covalently attached to murine or human TNF. Particular exemplary immunoconjugates based on antibodies ESC 1 1 and ESC 14 are provided in Figures 7 and 8. Sequences for the exemplary murine TNF immunoconjugates of ESC1 1 and ESC 14 are depicted in Figure 7 (ESC1 lmuTNF (heavy chain DNA SEQ ID NO: 23, peptide SEQ ID NO: 24; light chain DNA SEQ ID NO: 25, peptide SEQ ID NO: 26) and ESC14muTNF (heavy chain DNA SEQ ID NO: 27, peptide SEQ ID NO: 28; light chain DNA SEQ ID NO: 29, peptide SEQ ID NO: 30). Sequences for the exemplary human TNF immunoconjugates of ESC 1 1 and ESC 14 are depicted in Figure 8 (ESC1 lhuTNF (heavy chain DNA SEQ ID NO: 31 , peptide SEQ ID NO: 32; light chain DNA SEQ ID NO: 33, peptide SEQ ID NO: 34) and ESC14huTNF (heavy chain DNA SEQ ID NO: 35, peptide SEQ ID NO: 36; light chain DNA SEQ ID NO: 37, peptide SEQ ID NO: 38).

[00019] In a particular aspect, the antibody or fragment of use the invention is reactive with, capable of binding human and mouse FAP. In a further aspect the antibody or fragment does not react with, does not bind to CD26 (DPPIV). In an aspect, binding of the antibody or fragment ocomprising the invention does not directly affect dipeptidyl peptidase activity. In an additional aspect, the antibody or fragment down regulates the expression of FAP and therefore reduces the number or amount of active dipetidyl peptidase enzyme activity on a cells surface. By reducing FAP expression on the cell surface, the antibody(ies) of the invention indirectly impact on dipeptidyl peptidase activity. In a still further aspect, the antibody or fragment mediates down regulation of FAP expression. In another aspect the antibody or fragment induces, mediates apoptosis in FAP expressing cells. In a still additional aspect the antibody or fragment inhibits cellular adhesion to ECM proteins. Thus, the anti-FAP antibody(ies) or active fragment(s) thereof of use in or comprising the immunoconjugates of the invention has at least two of the following characteristics: is reactive with human and mouse FAP; does not react with or bind to CD26 (DPPrV); does not directly affect dipeptidyl peptidase activity of FAP; mediates down regulation of FAP expression; induces or otherwise mediates apoptosis in FAP expressing cells; and inhibits cellular adhesion to ECM proteins.

[00020] In further aspects, the invention provides an isolated nucleic acid which comprises a sequence encoding a immunoconjugate comprising an immune molecule component and an active moiety component and methods of preparing immunoconjugates of the invention which comprise expressing said nucleic acids under conditions to bring about expression of saidimmunoconjugate, and recovering the immunoconjugate. In one aspect the immune molecule component comprises an antibody or fragment comprising a nucleic acid encoding antibody variable region sequence having the amino acid sequences as set out in Figures 1 or 2 is provided or an antibody having CDR domain sequences as set out in Figure 2 is provided. In one aspect, a nucleic acid comprising the antibody variable region encoding sequence of Figure 1 is provided. In a further aspect, the invention provides an isolated nucleic acid comprising a nucleic acid sequence set out in Figure 7 or Figurre 8. In another aspect an isolated nucleic acid of the invention is capable of encoding an immunoconjugate compsising an amono acid sequence set out in Figure 7 or Figure 8. The present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes an immunoconjugate of the present invention; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding an active moiety component and an antibody component, wherein the antibody component comprises the antibody VH and VL, particularly the CDR region sequences, which has a sequence or is capable of encoding a sequence shown in Figure 1 or 2.

[00021] The antibodies, fragments thereof and recombinant antibodies comprising the CDR domains according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment of a tumor in a human patient which comprises administering to said patient an effective amount of the antibodies, fragments thereof and recombinant antibodies of the invention. [00022] The diagnostic utility of the present invention extends to the use of the immunoconjugates of the present invention in assays to characterize tumors or cellular samples or to screen for tumors or cancer, including in vitro and in vivo diagnostic assays. In an immunoassay, a control quantity of the immunoconjugate, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.

[00023] Immunoconjugates, specific binding members, and/or antibodies of the invention may carry a detectable or functional label. The specific binding members may carry a radioactive label, such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ^5,Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²¹1, ¹²⁴I, ^,25I, ^,3,I, ^mIn, ¹¹⁷Lu, ^{21 ,}At, ^,98Au, ⁶⁷Cu, ²²⁵ Ac, ²¹³Bi, ⁹⁹Tc and ¹⁸⁶Re. When radioactive labels are used, known currently available counting procedures may be utilized to identify and quantitate the specific binding members. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.

[00024] In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the immunoconjugate and its one or more components, including the active moiety molecule component and/or the immune molecule component. A first therapeutic method is associated with the prevention or treatment of cancer, including but not limited to melanoma, lung, esophageal, liver, gastric, prostate, ovarian, bladder and synovial sarcoma. In a further method, the immunoconjugate is utilized in generating resistance to tumor cells, particularly in protection from a re- challenge of cancer cells. Thus, the immunoconjugates, including FAP-TNF immunoconjugates may be administered in combination with or subsequent to tumor cells or tumor antigens and utilized in generating a response or preventative effect such that the animal or human will demonstrate prolonged survival and enhanced resistance to rechallenge or metastatic disease.

[00025] The immunoconjugates of the present invention, and in particular embodiments those comprising the amino acid sequences set out in Figure 7 or Figure 8, or those comprising an immune molecule or antibody whose sequences are presented in Figure 1 and 2 herein, or active fragments thereof, and single chain, recombinant or synthetic antibodies derived therefrom, particularly comprising the CDR region sequences depicted in Figure 2, can be prepared in pharmaceutical compositions, including a suitable vehicle, carrier or diluent, for administration in instances wherein therapy is appropriate, such as to treat cancer. Thus, pharmaceutical compositions of one or more immunoconjugates comprising an amino acid sequence set out in Figure 7 or Figure 8, including a suitable vehicle, carrier or diluent are provided herein. Such pharmaceutical compositions may also include methods of modulating the half-life of the conjugates, binding members, antibodies or fragments by methods known in the art such as pegylation. Such pharmaceutical compositions may further comprise additional antibodies or therapeutic agents.

[00026] A composition of the present invention may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anti-cancer agents or therapeutics, anti-mitotic agents, apoptotic agents or antibodies, or immune modulators. More generally these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), inhibitors or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal antiinflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. In addition, the composition may be administered with immune modulators, such as interleukins, or other growth factors, colony stimulating factors, cytokines or hormones such as dexamethasone which stimulate the immune response and reduction or elimination of cancer cells or tumors. The composition may also be administered with, or may include combinations along with other anti-FAP antibodies or other anti-tunor antigen antibodies.

[00027] Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00028] FIGURE 1: ESC1 1 (A) and ESC 14 (B) antibody sequences. The DNA sequences of heavy and light chains of both antibodies are depicted. ESC 1 1 heavy and light chain DNA sequences correspond to SEQ ID NO: 4 or 5 and SEQ ID NO: 6 respectively. ESC 14 heavy and light chain DNA sequences correspond to SEQ ID NO: 9 and SEQ ID NO: 10 respectively. The heavy chain amino acid sequence of ESC1 1 (SEQ ID NO: 1) was mutated (highlighted in red) from Histidine (H) to Glutamine (Q) at amino- acid position 1 to allow for expression in eukaryotic cells (SEQ ID NO: 2). The light chain peptide sequence of ESC 1 1 coresponds to SEQ ID NO: 3. The heavy chain amino acid sequence of ESC 14 corresponds to SEQ ID NO: 7 and the light chain to SEQ ID NO: 8.

[00029] FIGURE 2A and 2B depicts an alignment of the ESC11 and ESC 14 antibody variable region Heavy chain (A) and Light chain (B) sequences. The CDR I, II and III region sequences are highlighted. Identical amino acids are noted, conserved amino acids are shown as a +, and gaps in the sequence are noted as dashes (-). Heavy chain CDR I, II and III of ESC 1 1 correspond to SEQ ID NOS: 11, 12 and 13, and light chain CDR I, II and II to SEQ ID NO: 17, 18 and 19. Heavy chain CDR I, II and III of ESC 14 correspond to SEQ ID NOS: 14, 15 and 16, and light chain CDR I, II and II to SEQ ID NO: 20, 21 and 22.

[000301 FIGURE 3A and 3B: Specificity of Fabs ESCl 1 and ESC14. A. ELISA of Fabs (100 nM) binding to cell-lysate-coated plates. HT1080 FAP⁺ (black bars), HT1080 (white bars), HEK293 huCD26⁺ (striped bars), HEK293 muFAP⁺ (grey bars), HE 293 mock transfected line (hatched bars). B. FACS analysis of Fab ESCl 1 (black line), ESC 14 (grey line), and a non-binding control Fab (filled) on HT1080 FAP⁺ cells (left) and 293 muFAP⁺ cells (right)

[00031] FIGURE 4A and 4B. A depicts cell killing of HT1080 and HT1080 HuFAP by muTNF. B depicts cell killing of HT1080 and HT1080 HuFAP by TNF-immunoconjugate construct ESCl lQmuTNF. The ESCl l-muTNF construct has a higher killing activity on HT1080FAP+ cells when compared to HT1080 wt cells due to rapid internalisation of the FAP-TNF.

[00032] FIGURE 5A and 5B: ESC14muTNF was tested on HT1080 and HT1080FAP. The cytotoxic activity of mu-TNF and the FAP-TNF construct is shown for HT1080 wt (A) or HT1080FAP+ (B) cells. Viability was measured with Cristal violet staining. The FAP positive HT1080FAP cell line led to internalisation of ESC14-muTNF constructs which increased cytotoxic activity significantly as shown (B).

[00033] FIGURE 6A and 6B depict studies of the effects of FAP antibody-TNF constructs on tumors in animals. In A, survival of tumor-bearing animals was determined after inoculation with mouse colon cancer cell line CT26 and treatment with control PBS or ESCl ImuTNF on day 8, 9 and 10. Only oneof 5 animals died with ESCl ImuTNF treatment, while only one animal survived with PBS treatment. In B, surviving animals were re-challenged with lxlO⁶ CT26 tumor cells. All naive mice developed tumors, however ESC1 lmuTNF treated animals were resistant against re-challenge.

[00034] FIGURE 7A and B provides DNA and amino acid sequences of the (A) ESC1 lmuTNF and (B) ESC14muTNF immunoconjugate constructs.

[00035] FIGURE 8A and B provides DNA and amino acid sequences of the (A) ESC1 lhuTNF and (B) ESC14huTNF immunoconjugate constructs.

DETAILED DESCRIPTION

[00036] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan, J. E., ed. (1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

[00037] Therefore, if appearing herein, the following terms shall have the definitions set out below. A. TERMINOLOGY

[00038] The term "specific binding member"describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application is concerned with antigen-antibody type reactions. [00039] The term "immunoconjugate" describes a covalent conjugate or fusion, preferably recombinantly generated, between an antibody or immunoglobulin component and an active molecule moiety. The antibody or immunoglobulin component may include any antibody, antibody molecule, domain thereof, chain thereof, fragment thereof, or portion thereof which confers an antibody-like antigen recognition and antigen binding capacity. The active molecule moiety may be a molecule, compound, agent or peptide, which can be recombinantly attached or generated as a fusion with the antibody or immunoglobulin component and which confers an additional activity thereto. For instance but not by limitation, the active molecule moiety may include a therapeutic and/or recognition capacity. The therapeutic capacity may be an anti-tumor, anti-cancer, anti-mitotic, pro-apoptotic, immunomodulatory, etc activity.

[00040] The term "antibody" describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567. The term "antibody(ies)" includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl , IgG2, IgG3, IgG4, IgAl, and IgA2). Also included within the meaning of the term "antibody" are any "antibody fragment".

[00041] An "antibody fragment" means a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CHI domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward,

E.S. et al., Nature 341 , 544-546 (1989)); (vi) a camelid antibody; (vii) an isolated complementarity determining region (CDR); (viii) a Single Chain Fv Fragment wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) a diabody, which is a bivalent, bispecific antibody in which 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 the complementarity domains of another chain and creating two antigen binding sites (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)); and (x) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J Immunol. Methods 242: 193-204 9 (2000)); and (xii) other non-full length portions of heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

[00042] As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U.S. Patent Nos. 4,816,397 and 4,816,567.

[00043] An "antibody combining site" is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.

[00044] The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

[00045] Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab')₂ and F(v), which portions are preferred for use in the therapeutic methods described herein. [00046] Antibodies may also be bispecific, wherein one binding domain of the antibody is a specific binding member of the invention, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like. Bispecific antibodies of the present invention include wherein one binding domain of the antibody is a specific binding member of the present invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct anticancer or anti-tumor specific antibody. The other binding domain may be an antibody that recognizes or targets a particular cell type, as in a neural or glial cell-specific antibody. In the bispecific antibodies of the present invention the one binding domain of the antibody of the invention may be combined with other binding domains or molecules which recognize particular cell receptors and/or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine (e.g. tumor necrosis factor (TNF), and particularly, the TNF bispecific modality demonstrated in U.S.S.N. 60/355,838 filed February 13, 2002 incorporated herein in its entirely) or a toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor. Thus, the anti-FAP antibodies of the invention may be utilized to direct or target agents, labels, other molecules or compounds or antibodies to stromal sites, particular areas of wound healing, inflammation, cancer or tumors.

[00047] The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may also contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.

[00048] The term "antigen binding domain" describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[00049] Immunoconjugates or antibody fusion proteins of the present invention, wherein the antibodies, antibody molecules, or fragments thereof, of use in the present invention are conjugated or attached to other molecules or agents further include, but are not limited to such antibodies, molecules, or fragments conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, antimicrobial agent or peptide, cell wall and/or cell membrane disrupter, or drug.

[00050] The term "specific" may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

[00051J The term "comprise"generally used in the sense of include, that is to say permitting the presence of one or more features or components.

[00052] The term "consisting essentially of refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product. In the case of the peptide of the invention referred to above, those of skill in the art will appreciate that minor modifications to the N- or C- terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.

[00053] The term "isolated" refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members will be, in accordance with the present invention. Members and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.

[00054] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μ§," mean microgram, "mg" means milligram, "ul" or "μΐ" mean microliter, "ml" means milliliter, "1" means liter.

[00055] The terms "antibody", "anti-FAP antibody", "FAP antibody", "human/mouse FAP antibody", "antibody ESCU", "antibody ESC 14" and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in Figures 1 and 2 and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms "antibody", "anti-FAP antibody", "FAP antibody", "human/mouse FAP antibody", "antibody ESC 1 1", "antibody ESC 14" are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.

[00056] The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide. NH₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

SYMBOL AMINO ACID

1 -Letter 3-Letter

Y Tyr tyrosine

G Gly glycine

F Phe phenylalanine

M Met methionine

A Ala alanine

S Ser serine

I He isoleucine

L Leu leucine

T Thr threonine

V Val valine

P Pro proline

K Lys lysine

H His histidine Q Gin glutamine

E Glu glutamic acid

W Tip tryptophan

R Arg arginine

D Asp aspartic acid

N Asn asparagine

C Cys cysteine

[00057] It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

[00058] A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.

[00059] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

[00060] A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

[00061] An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

[00062] A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[00063] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

[00064] A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S I), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

[00065] An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

[00066] A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

[00067] The term "oligonucleotide," as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. [00068] The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single- stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

[00069] The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

[00070] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

[00071] A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. [00072] Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.

[00073] It should be appreciated that also within the scope of the present invention are DNA sequences encoding specific binding members (antibodies) of the invention which code for e.g. an antibody having the same amino, acid sequence as provided in Figure 1 or 2, or comprising the CDR domain region sequences set out herein or in Figure 2 but which are degenerate thereto. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) uuu or UUC

Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG

Isoleucine (He or I) AUU or AUC or AUA

Methionine (Met or M) AUG

Valine (Val or V) GUU or GUC of GUA or GUG

Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC

Proline (Pro or P) CCU or CCC or CCA or CCG

Threonine (Thr or T) ACU or ACC or ACA or ACG

Alanine (Ala or A) GCU or GCG or GCA or GCG

Tyrosine (Tyr or Y) UAU or UAC

Histidine (His or H) CAU or CAC

Glutamine (Gin or Q) CAA or CAG

Asparagine (Asn or N) AAU or AAC

Lysine (Lys or K) AAA or AAG

Aspartic Acid (Asp or D) GAU or GAC

Glutamic Acid (Glu or E) GAA or GAG

Cysteine (Cys or C) UGU or UGC

Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine (Gly or G) GGU or GGC or GGA or GGG

Tryptophan (Trp or W) UGG

Termination codon UAA (ochre) or UAG (amber) or UGA

[00074] It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U.

[00075] Mutations can be made in the sequences encoding the amino acids, immunoconjugates, antibodies, antibody fragments, variable regions, domain sequences, CDR region sequences set out in Figures 1 , 2, 7 or 8, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.

[00076] The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine

Amino acids with uncharged polar R groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino acids with charged polar R groups (negatively charged at Ph 6.0)

Aspartic acid, Glutamic acid

Basic amino acids (positively charged at pH 6.0)

Lysine, Arginine, Histidine (at pH 6.0)

[00077] Another grouping may be those amino acids with phenyl groups:

Phenylalanine, Tryptophan, Tyrosine [00078] Another grouping may be according to molecular weight (i.e., size of R groups):

Glycine 75 Alanine 89

Serine 105 Proline 1 15

Valine 1 17 Threonine 1 19

Cysteine 121 Leucine 131

Isoleucine 131 Asparagine 132

Aspartic acid 133 Glutamine 146

Lysine 146 Glutamic acid 147

Methionine 149 Histidine (at pH 6.0) 155

Phenylalanine 165 Arginine 174

Tyrosine 181 Tryptophan 204

[00079] Particularly preferred substitutions are:

- Lys for Arg and vice versa such that a positive charge may be maintained;

- Glu for Asp and vice versa such that a negative charge may be maintained;

- Ser for Thr such that a free -OH can be maintained; and

- Gin for Asn such that a free NFL. can be maintained.

[00080] Exemplary and preferred conservative amino acid substitutions include any of:

glutamine (Q) for glutamic acid (E) and vice versa; leucine (L) for valine (V) and vice versa; serine (S) for threonine (T) and vice versa; isoleucine (I) for valine (V) and vice versa; lysine (K) for glutamine (Q) and vice versa; isoleucine (I) for methionine (M) and vice versa; serine (S) for asparagine (N) and vice versa; leucine (L) for methionine (M) and vice versa; lysine (L) for glutamic acid (E) and vice versa; alanine (A) for serine (S) and vice versa; tyrosine (Y) for phenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid (D) and vice versa; leucine (L) for isoleucine (I) and vice versa; lysine (K) for arginine (R) and vice versa.

[00081] Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces β-turns in the protein's structure. [00082] Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions. The CDR regions of two antibodies are substantially homologous when one or mre amino acids are substituted with a similar or conservative amino acid substitution, and wherein the antibody/antibodies have the profile of binding and activities of one or more of ESC 1 1 or ESC 14 disclosed herein.

[00083] A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

[00084] A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

[00085] The term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65°C for both hybridization and wash. However, one skilled in the art will appreciate that such "standard hybridization conditions" are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of "standard hybridization conditions" is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20°C below the predicted or determined T_m with washes of higher stringency, if desired. [00086] The term 'agent' means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules. In particular the term agent includes compounds such as test compounds or drug candidate compounds.

[00087] The term 'agonist' refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.

[00088] The term 'assay' means any process used to measure a specific property of a compound. A 'screening assay' means a process used to characterize or select compounds based upon their activity from a collection of compounds.

[00089] The term 'preventing' or 'prevention' refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.

[00090] The term 'prophylaxis' is related to and encompassed in the term 'prevention', and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non- limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.

[00091] 'Therapeutically effective amount' means that amount of a drug, compound, antimicrobial, antibody, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. In particular, with regard to gram-positive bacterial infections and growth of gram-positive bacteria, the term "effective amount" is intended to include an effective amount of a compound or agent that will bring about a biologically meaningful decrease in the amount of or extent of infection of gram-positive bacteria, including having a bacteriocidal and/or bacteriostatic effect. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the growth or amount of infectious bacteria, or other feature of pathology such as for example, elevated fever or white cell count as may attend its presence and activity. [00092] The term 'treating' or 'treatment' of any disease or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., arresting the disease or growth of the infectious agent or bacteria or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment 'treating' or 'treatment' refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, 'treating' or 'treatment' refers to modulating the disease or infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, 'treating' or 'treatment' relates to slowing the progression of a disease or reducing an infection.

[00093] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

[00094] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or '^g" mean microgram, "mg" means milligram, "ul" or "μ mean microliter, "ml" means milliliter, "1" means liter.

B. DETAILED DISCLOSURE.

[00095] The development and application of antibody fusion molecules and immunoconjugates shows promise as an alternative to solely antibody based therapy. Thus, antibody molecules with enhanced, specific or targeted activities can be generated by fusing an antibody molecule, domain thereof, chain thereof, fragment thereof or any active portion thereof to another molecule, compound or peptide to provide a new and unique fusion protein. Methods and constructs for generating antibodies or antibody molecule fusion proteins are known in the art. Certain methods and constructs, for example, are described in US 2006/0045876 Al and WO 03/068924 A2, which are incorporated herein by reference.

[00096] For example, and not by limitation, antibodies and antibody molecules have been fused to tumor necrosis factor (TNF) (US 2006/0045876; Bauer, S. et al (2004) J. Immunol 172:3930-3939; Bauer, S. et al (2006) J. Immunol 177:2423-2430). Various other fusion molecules including other anti-tumor, anti-mitotic, pro-apoptotic, immuno-modulatory, receptor, ligand, etc. molecules may be fused to the antibodies or antibody molecules to generate novel and active immunoconjugates. [00097] In epithelial cancers, the tumor compartment consists of two discrete, but interdependent, components, the malignant cells and the tumor stroma. The predominant cells in the tumor stroma are nontransformed fibroblasts, expressing the genetically stable fibroblast-activating protein (FAP) in >90% of all cases (Garin-Chesa, P. et al (1990) Proc. Natl. Acad. Sci. USA 87: 7235). The FAP-positive stromal cells commonly contribute 50-90% of the tumor mass and are located in close vicinity to the endothelial cells of the tumor capillaries and surround the tumor nodules. Focusing the bioactivity of TNF to FAP-positive tumor stroma is predicted to promote the antitumor efficacy of the cytokine at several levels. 1) Reflecting the fact that endothelial cells are the major target for TNF-mediated hemorrhagic tumor necrosis, the FAPdependent enrichment of TNF in a memTNF-like manner should initiate enhancement of endothelial tissue factor production by cooperative TNF-R signaling. 2) The TNF-initiated induction of adhesion molecules on the luminal surface of the tumor vessels should favor Ag-neighbored adherence of PMNs and trigger the release of reactive oxygen species, causing stromal tissue damage. 3) Furthermore, not only FAP-positive cells, but also the malignant cells in their proximity, could activate autoproteolytic programs, even under conditions where a phenotypic switch of the cellular response pattern should have to be induced to become susceptible. To direct TNF-induced cellular response programs to the FAP-positive tumor stroma, Ab fusion protein was generated and tested that consisted of humanized anti-FAP Ab and human TNF (Scott, A. M. et al (2001) Proc. Am. Soc. Clin. Oncol. 20: 258a). In vitro and in vivo studies with this Ab fusion protein demonstrated that it exhibited an effector profile identical with that of memTNF, associated with up to 10-fpld reduced toxicity compared with sTNF, showing that the Ab-linked TNF dimer is an optimal transmitter of TNF bioactivity Bauer S et al (2004) J Immunol 172:3930-3939). However, these studies were limited by the use of anti huFAP antibody and rhuTNF, therefore the animal model was not designed to demonstrate antitumor efficacy because rhuTNF fails to induce murine TNF-R2 signaling, and the HT1080 FAP+ cell line utilized is not TNF sensitive therefore induction of tumor cell apoptosis could not been induced. Limited conclusions and limited results were therefore available from these prior studies.

[00098] The use and application of the immunoconjugates of the present invention, comprising an antibody or antibody-like moiety and an active molecule moiety, are, in part, mediated by the uniqueness and capabilities of the antibodies or fragments thereof which are conjugated to the active molecule moiety. In one particular aspect, novel anti-FAP antibodies or portions thereof are conjugated to one or more active, molecule moiety. In a particular aspect, novel anti-FAP antibodies or portions thereof are conjugated to one or more active molecule moiety which is an anti-cancer agent, a cell toxicity mediator, a ligand, an immunomodulatory molecule, a pro-apoptotic molecule. In a further a particular aspect, novel anti-FAP antibodies or portions thereof are conjugated to one or more active molecule moiety which is tumor necrosis factor.

[00099] The invention provides exemplary murine and human TNF immunoconjugates, particularly ESC1 1 and ESC 14 antibody-TNF conjugates and their activities, capabilities and sequences. Exemplary nucleic acid and amino acid sequences of ESC 1 1 and ESC 14 TNF immunoconjugates are provided herein and in Figures 7 and 8.

[000100] The invention provides antibody immunoconjugates utilizing novel antibodies directed against Fibroblast activation protein (FAP) for diagnostic and therapeutic purposes. In particular, the antibodies specific for FAP provided and utilized herein recognize and are capable of binding human and mouse FAP. In addition, the novel FAP antibodies utilized in the immunoconjugates do not cross react/bind to CD26 (dipeptidyl peptidase rv (DPPrV)). In a further aspect, the antibody or antibody molecules of the present immunoconjugates do not directly effect dipetidyl peptidase activity of FAP. In a still further aspect, the antibody or fragment mediates down regulation of FAP expression. In another aspect the antibody or fragment induces or mediates apoptosis in FAP expressing cells. In a still additional aspect the antibody or fragment inhibits cellular adhesion to ECM proteins. Thus, the anti-FAP antibody(ies) or active fragment(s) thereof utilized in the immunoconjugates of the invention have at least two of the following characteristics: reactive with human and mouse FAP; do not react with or bind to CD26 (DPPrV); do not directly affect dipeptidyl peptidase activity of FAP; mediate down regulation of FAP expression; induce or otherwise mediate apoptosis in FAP expressing cells; and inhibit cellular adhesion to ECM proteins.

[000101] The active molecule moiety of the immunoconjugate of the present invention may comprise or consist of a molecule, compound, agent, peptide which is an anti-cancer agent, a cell toxicity mediator, a ligand, an immunomodulatory molecule, a pro-apoptotic molecule which confers a therapeuticall relevant activity to the immunoconjugate. In one aspect, the one or more active molecule moiety is a growth factor or an anti cancer agent. In a further a particular aspect, the one or more active molecule moiety is tumor necrosis factor. Any of the known sequences or portions or homologs of tumor necrosis factor may be utilized which have an effective, desired, and useful activity. Therefore, a mouse, human or other mammalian sequence or portion thereof of TNF may be utilized. One skilled in the art will have publicly available TNF sequences, fragments, etc and will be able to generate suitable immunoconjugates based thereon. [000102] The immmunoconjugates of the present invention have diagnostic and therapeutic use in conditions associated with activated stroma, including wound healing, epithelial cancers, osteoarthritis, rheumatoid arthritis, cirrhosis and pulmonary fibrosis, and more broadly in TNF responsive conditions and disorders, including cancer and hyperproliferative diseases. In a particular aspect the antibodies of the invention are applicable in cancers, including epithelial cancers, including breast, lung, colorectal and ovarian cancers.

[000103] In a general aspect, the immunoconjugates of the present invention utilize FAP antibodies directed against human and mouse FAP and which do not cross react/bind to CD26 (dipeptidyl peptidase rV (DPPIV)). In a broad aspect, the present invention provides an immunoconjugate ulitizing or comprising an antibody, antibody molecule or fragment thereof, including an Fab fragment and a single chain or domain antibody, which recognizes human FAP. In a further aspect, the present invention provides an immunoconjugate utilizing or comprising an antibody or fragment thereof, which recognizes human FAP and comprises the amino acid sequence of ESC 1 1 or ESC 14 including as set out in Figure 1 and/or Figure 2. In one such aspect, the invention provides an anti-human FAP antibody comprising the variable region CDR sequences set out in Figure 2.

[000104] Panels of monoclonal antibodies recognizing human and murine FAP can be screened for various properties; i.e., isotype, epitope, affinity, etc. Of particular interest are antibodies that mimic the activity of exemplary antibodies ESC1 1 and ESC 14, and have affinity for human and mouse FAP, do not react with CD26, and do not directly effect the dipeptidyl peptidase activity of FAP. Such antibodies can be readily identified and/or screened in specific binding member activity assays.

[000105] In general, the CDR regions, comprising amino acid sequences substantially as set out as the CDR regions of Figure 2 will be carried in a structure which allows for binding of the CDR regions to the stromal protein FAP, and particularly to human and mouse FAP.

[000106] By "substantially as set out" it is meant that that sequences of the invention, including the antibody molecule portion or active moiety portion of the immunoconjugate will be either identical or highly homologous to the sequences set out and provided herein. Thus, the antibody portion, particularly the variable region sequences, and/or particularly the CDR sequences, of the immunoconjugate of the invention may be either identical or highly homologous to the specified regions of Figure 1 or Figure 2.

The active moiety portion, particularly tumor necrosis factor, and or particularly murine or human TNF, of the immunoconjugate of the invention may be either identical or highly homologous to the specified regions of Figure 7 or Figure 8. By "highly homologous" it is contemplated that only a few substitutions, preferably from 1 to 8, preferably from 1 to 5, preferably from 1 to 4, or from 1 to 3, or 1 or 2 substitutions may be made in the tumor necrosis vactor sequence, the variable region sequence and/or in the CDR sequences. The term substantially set out as includes particularly conservative amino acid substitutions which do not materially or significantly affect the specificity and/or activity of the instant immunoconjugates, or the antibodies, or active moieties. Conservative amino acid substitutions are exemplified herein and also in Figure 2 for the CDR region sequences of the immunoglobulin molecules.

[000107] Substitutions may be made in the variable region sequence outside of the CDRs so as to retain the CDR sequences. Thus, changes in the variable region sequence or alternative non-homologous or veneered variable region sequences may be introduced or utilized, such that the CDR sequences are maintained and the remainder of the variable region sesuence may be substituted.

[000108] Alternatively, substitutions may be made particularly in the CDRs. CDR sequences for the antibodies of the present invention are set out and described herein including in Figure 2. Antibody ESC 1 1 comprises heavy chain CDR sequences GGSISSNNYYWG, SIYYSGSTNYNPSLKS and GARWQARPATR1DG V AFD I, and light chain CDR sequences RASQTVTRNYLA, GASNRAA and QQFGSPYT, as set out in Figure 2. Antibody ESC 14 comprises heavy chain CDR sequences GYTFTSYGIS, WISAYNGNTNYAQKLQG and DWSRSGYYLPDY and light chain CDR sequences RSSQSLLHSNGYNYLD, LGSNRAS and MQALQTPPT, as set out in Figure 2. Core CDR sequences based on the homology and similarity of the ESC 1 1 and ESC 14 antibody CDR sequences include for the heavy chain, CDR I of G G/Y S T I/F S/T S N/- N/- Y Y/G W/I G/S , CDRII of S/W I S/- Y/A Y S/N G S/N T N Y N/A P/Q S/K L K Q S/G, and CDRIII of G/D A/- RJ- W Q/- A/- R - P/- A/- T/S R I/S D/G G/Y V/Y A/L F/P D I/Y. Core CDR sequences based on the homology and similarity of the ESC 1 1 and ESC 14 antibody CDR sequences include for the light chain, CDR I of R A/S S Q T/S V/L T/L R/H S/- N/- G/- Y/- N Y L A/D, CDRII of G/L A/G S N R A A S, and CDRIII of Q/M Q A/- F/L G/Q S/T P Y/P T.

[000109] Antibodies of use in the immunoconjugates of the invention having substitutions as above described and contemplated are selected to maintain the activities and specifity commensurate with the exemplary antibodies, including antibodies ESC 1 1 and ESC 14 and having the characteristics as set out herein and in the claims.

[000110] The structure for carrying the CDRs of the invention will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR regions are located at locations corresponding to the CDR region of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to abat, E.A. et al, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (http://immuno.bme.nwu.edu)).

[000111] The variable domains may be derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus sequences of known human variable domains. The CDR-derived sequences of the invention, as defined in the preceding paragraph, may be introduced into a repertoire of variable domains lacking CDR regions, using recombinant DNA technology.

[000112] For example, Marks et al (Bio/Technology, 1992, 10:779-783) describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5' end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR/CDRs. Marks et al further describe how this repertoire may be combined with a CDR of a particular antibody. The repertoire may then be displayed in a suitable host system such as the phage display system of WO92/01047 so that suitable specific binding members may be selected. A repertoire may consist of from anything from 10⁴ individual members upwards, for example from 10⁶ to 10⁸ or 10¹⁰ members. Analogous shuffling or combinatorial techniques are also disclosed by Stemmer (Nature, 1994, 370:389- 391 ), who describes the technique in relation to a β-lactamase gene but observes that the approach may be used for the generation of antibodies.

[000113] A further alternative is to generate novel VH or VL regions carrying the CDR-derived sequences of the invention using random mutagenesis of, for example, the Ab VH or VL genes to generate mutations within the entire variable domain. Such a technique is described by Gram et al (1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-prone PCR. Another method which may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al, (1994, Proc. Natl. Acad. Sci., USA, 91 -.3809-3813) and Schier et al (1996, J. Mol. Biol. 263:551-567).

[000114] All the above described techniques are known as such in the art and in themselves do not form part of the present invention. The skilled person will be able to use such techniques to provide specific binding members of the invention using routine methodology in the art. [000115] A substantial portion of an immunoglobulin variable domain will comprise at least the three CDR regions, together with their intervening framework regions. Preferably, the portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C- terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of specific binding members of the present invention made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to join variable domains of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels as provided herein and/or known to those of skill in the art.

[000116] Although in a preferred aspect of the invention specific binding members comprising a pair of binding domains based on sequences substantially set out in Figures 1 and/or 2 are preferred, single binding domains based on either of these sequences form further aspects of the invention. In the case of the binding domains based on the sequence substantially set out in Figure 1 and/or 2, such binding domains may be used as targeting agents for FAP and/or TNFR on stromal cells, particularly tumor stroma, since it is known that immunoglobulin VH domains are capable of binding target antigens in a specific manner.

[000117] This may be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach as disclosed in U.S. Patent 5,969,108 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding member is selected in accordance with phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al, ibid. Phage library and phage display selection systems and techniques are also provided herein.

[000118] Specific binding members of the present invention may further comprise antibody constant regions or parts thereof. For example, specific binding members based on the sequences of Figures 2 and 10 may be attached at their C-terminal end to antibody light chain constant domains including human CK or Ck chains, preferably Ck chains. Similarly, specific binding members based on the sequences of Figures 10, 12 or 13 may be attached at their C-terminal end to all or part of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE, IgD and IgM and any of the isotype subclasses, particularly IgGl , IgG2b, and IgG4. IgGl is preferred.

[000119] The antibodies, or any fragments thereof, may be conjugated or recombinantly fused to any cellular toxin, bacterial or other, e.g. pseudomonas exotoxin, ricin, or diphtheria toxin. The part of the toxin used can be the whole toxin, or any particular domain of the toxin. Such antibody-toxin molecules have successfully been used for targeting and therapy of different kinds of cancers, see e.g. Pastan, Biochim Biophys Acta. 1997 Oct 24;1333(2):Cl-6; Kreitman et al., N Engl J Med. 2001 Jul 26;345(4):241-7; Schnell et al., Leukemia. 2000 Jan; 14(l): 129-35; Ghetie et al., Mol Biotechnol. 2001 Jul; 18(3):251-68.

[000120] Bi- and tri-specific multimers can be formed by association of different scFv molecules and have been designed as cross-linking reagents for T-cell recruitment into tumors (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics), see e.g. Todorovska et al., J Immunol Methods. 2001 Feb l ;248(l-2):47-66; Tomlinson et al., Methods Enzymol. 2000;326:461-79; McCall et al., J Immunol. 2001 May 15;166(10):61 12-7.

[000121] Fully human antibodies can be prepared by immunizing transgenic mice carrying large portions of the human immunoglobulin heavy and light chains. These mice, examples of such mice are the Xenomouse™ (Abgenix, Inc.) (US Patent Nos. 6,075,181 and 6,150,584), the HuMAb-Mouse™ (Medarex, Inc/GenPharm) (US patent 5545806 and 5569825), the TransChromo Mouse™ (Kirin) and the KM Mouse™ (Medarex/Kirin), are well known within the art. Antibodies can then be prepared by, e.g. standard hybridoma technique or by phage display. These antibodies will then contain only fully human amino acid sequences. Fully human antibodies can also be generated using phage display from human libraries. Phage display may be performed using methods well known to the skilled artisan, and as provided herein as in Hoogenboom et al and Marks et al (Hoogenboom HR and Winter G. (1992) J Mol Biol. 227(2):381-8; Marks JD et al (1991) J Mol Biol. 222(3):581-97; and also U.S. Patents 5885793 and 5969108).

[000122] Antibodies of the invention may be labelled with a detectable or functional label. Detectable labels include, but are not limited to, radiolabels such as the isotopes ³H, ¹⁴C, ³²P, ⁵S, ³⁶C1, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ^,2,I, ¹²⁴I, ¹²⁵I, ^13,I, ^{, n} In, ^{1 ,7}Lu, ^{2l ,}At, ¹⁹⁸Au, ⁶⁷Cu, ²²⁵Ac, ²¹³Bi, ⁹⁹Tc and ¹⁸⁶Re, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging. Labels also include fluorescent labels (for example fluorescein, rhodamine, Texas Red) and labels used conventionally in the art for MRJ-CT imaging. They also include enzyme labels such as horseradish peroxidase, β-glucoronidase, β-galactosidase, urease. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin. Functional labels include substances which are designed to be targeted to the site of a tumor to cause destruction of tumor tissue. Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which are capable of converting prodrugs into active drugs at the site of a tumor.

[000123] Also, antibodies including fragments thereof, and drugs that modulate the production or activity of the specific binding members, antibodies and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as cancer, precancerous lesions, conditions related to or resulting from hyperproliferative cell growth or the like. For example, the specific binding members, antibodies or their subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. Likewise, small molecules that mimic or antagonize the activity(ies) of the specific binding members of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.

[000124] The radiolabeled specific binding members, particularly antibodies and fragments thereof, are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques and in radioimmunotherapy. In the instance of in vivo imaging, the specific binding members of the present invention may be conjugated to an imaging agent rather than a radioisotope(s), including but not limited to a magnetic resonance image enhancing agent, wherein for instance an antibody molecule is loaded with a large number of paramagnetic ions through chelating groups. Examples of chelating groups include EDTA, porphyrins, polyamines crown ethers and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanium, holmium and ferbium. In a further aspect of the invention, radiolabeled specific binding members, particularly antibodies and fragments thereof, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabeled antibodies for cancer therapy. In a still further aspect, the radiolabeled specific binding members, particularly antibodies and fragments thereof, are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and/or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery to remove such cells. [000125J Immunoconjugates or antibody fusion proteins of the present invention, wherein the specific binding members, particularly antibodies and fragments thereof, of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.

[000126] Radioimmunotherapy (RAIT) has entered the clinic and demonstrated efficacy using various antibody immunoconjugates. ^13,I labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 has been evaluated in colorectal cancer (Behr TM et al (2002) Cancer 94(4Suppl):1373-81) and the same antibody with ⁹⁰Y label has been assessed in medullary thyroid carcinoma (Stein R et al (2002) Cancer 94(1):51-61). Radioimmunotherapy using monoclonal antibodies has also been assessed and reported for non-Hodgkin's lymphoma and pancreatic cancer (Goldenberg DM (2001) Crit Rev Oncol Hematol 39(1 -2): 195-201 ; Gold DV et al (2001) Crit Rev Oncol Hematol 39 (1-2) 147-54). Radioimmunotherapy methods with particular antibodies are also described in U.S. Patent 6,306,393 and 6,331,175. Radioimmunoguided surgery (RIGS) has also entered the clinic and demonstrated efficacy and usefulness, including using anti-CEA antibodies and antibodies directed against tumor-associated antigens (Kim JC et al (2002) Int J Cancer 97(4):542-7; Schneebaum S et al (2001) World J Surg 25(12): 1495-8; Avital S et al (2000) Cancer 89(8): 1692-8; Mcintosh DG et al (1997) Cancer Biother Radiopharm 12 (4):287-94).

[000127] Immunoconjugates of the invention may be generated by fusion or linkage of an immunoglobulin molecule and an active moiety using any of various standard methods or approaches known or used in the art. In particular, the immunoconjugate may be recombinantly constructed to be encoded via a single nucleic acid as recombinantly linked immunoglobulin and active moiety components. Immunoconjugates of antibody-molecules have previously been generated and are described. In particular, US2006/0045876 Al and WO 03/068924, incorporated herein by reference, describe methods and means for generating chimeric antibodies and fusion proteins, including fusion of biologically active portions of tumor necrosis factor or full length tumor necrosis factor. In this instance, a G250 specific antibody was fused to TNF whereby the cDNA for TNF was positioned right after the hinge region of the G250 heavy chain. Additional Ab-TNF immunoconjugates and their construction have been described (Bauer S et al (2004) J Immunol 172: 3930-3939; Bauer S et al (2006) J Immunol 177:2423-2430). In one of these constructs, the human TNF sequence started at amino acid 79 of the precursor protein and was directly linked to the IgGl hinge region of an anti-human FAP antibody, such that the CH2/CH3 region of the human IgG Fc portion was replaced by the human TNF sequence to prevent nonspecific binding to FcRs. This provides an antibody-linked TNF dimer, with reduced toxicity versus soluble TNF (sTNF) but a full TNF effector profile. IgGl -derived and scFv-derived immunocytokine immunoconjugates have been generated and compared in activity when fused to anti-huFAP antibody (Bauer S et al (2006) J Immunol 177:2423-2430). Particular immunoconjugate constructs of ECS11 and ESC14 FAP antibodies linked to TNF are provided herein and in Figures 7 and 8.

[000128] In vivo animal models of cancer or animal xenograft studies may be utilized by the skilled artisan to further or additionally screen, assess, and/or verify the specific binding members and antibodies or fragments thereof of the present invention, including further assessing FAP modulation and inhibiting stromal cell adhesion to ECM proteins in vivo and inhibiting tumor progression and/or infiltration. An exemplary cancer/tumor model is provided and utilized herein in the examples. Suitable animal models include, but are not limited to models of conditions associated with activated stroma, including wound healing, epithelial cancers, osteoarthritis, rheumatoid arthritis, cirrhosis and pulmonary fibrosis, particularly without the problems associated with normal tissue uptake. Any suitable cancer model may be utilized. Models of cancers whose progression, migration and/or invasion involves, is facilitated by, or is associated with stromal fibroblasts are particularly susceptible to and targeted by the immunoconjugates and antibodies of the present invention. Such cancers include epithelial cancers, including breast, lung, colorectal and ovarian cancer. For example but without limitation, the mouse sarcoma cell line lm8 expresses high levels of FAP and grows in syngeneic C3h mice. This may be utilized in xenograft experiments or in a sarcoma model for direct tumor targeting and/or to assess anti-tumor and anti-cancer effects of the immunoconjugates utilizing anti-FAP antibodies.

[000129] Immunoconjugates of the present invention may be administered to a patient in need of treatment via any suitable route, including by injection intramuscularly, into the bloodstream or CSF, or directly into the site of the tumor. The precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the tumor, the precise nature of the antibody (whether whole antibody, fragment, diabody, etc), and the nature of the detectable or functional label attached to the antibody. Where a radionuclide is used for therapy, a suitable maximum single dose may be about 45 mCi/m², to a maximum of about 250 mCi/m². Preferable dosage is in the range of 15 to 40 mCi, with a further preferred dosage range of 20 to 30 mCi, or 10 to 30 mCi. Such therapy may require bone marrow or stem cell replacement. A typical antibody dose for either tumor imaging or tumor treatment will be in the range of from 0.5 to 40 mg, preferably from 1 to 4 mg of antibody in F(ab')2 form. Naked antibodies are preferably administered in doses of 20 to 1000 mg protein per dose, or 20 to 500 mg protein per dose, or 20 to 100 mg protein per dose. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats, in proportion for example to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.

Pharmaceutical and Therapeutic Compositions

[000130] Immunoconjugates of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member. Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous, or by deposition at a tumor site.

[0001311 Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

[000132] For intravenous, injection, or injection at the site of affliction, the active ingredient may be in the form of a parenteral ly acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

[000133] A composition may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anticancer agents or therapeutics, hormones, anti-mitotic agents, anti-apoptotic agents, antibodies, or immune modulators. More generally these anti-cancer agents may be but are not limited to tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. The composition can be administered in combination (either sequentially (i.e. before or after) or simultaneously) with tyrosine kinase inhibitors (including, but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, lomustine, and/or other chemotherapeutic agents. Thus, these agents may be specific anti-cancer agents, or immune cell response modulators or may be more general anticancer and anti-neoplastic agents such as doxorubicin, cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine. In addition, the composition may be administered with hormones such as dexamethasone, immune modulators, such as interleukins, or other growth factors, colony stimulating factors, or cytokines which stimulate the immune response and reduction or elimination of cancer cells or tumors. The composition may also be administered with, or may include combinations along with other anti-tumor antigen antibodies.

[000134] In addition, the present invention contemplates and includes therapeutic compositions for the use of the binding member in combination with conventional radiotherapy.

[000135] The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a specific binding member, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient. In a preferred embodiment, the composition comprises an antigen capable of modulating the specific binding of the present binding member/antibody with a target cell.

[000136] The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions. However, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

[000137] A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[000138] The therapeutic antibody- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

[000139] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of peptide MHC or tumor antigen binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. Suitable regimes for initial administration and follow on administration are also variable, and may include an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain appropriate and sufficient concentrations in the blood or at the site of desired therapy are contemplated.

[000140] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. [000141] For intravenous, injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Diagnostic Assays

[000142] The present invention also relates to a variety of diagnostic applications, including methods for detecting the expression of or elevated presence of TNFR, FAP, FAP-mediated mediated cancer, epithelial cancer, or cancer more generally, wound healing, osteoarthritis, by reference to their ability to be recognized by the present specific binding member(s). Peptide complexes can be identified, targeted, labeled, and/or quantitated on stromal cells, fibroblast cells and/or tumor cells.

[000143] Diagnostic applications of the immunoconjugates of the present invention, include in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description. Diagnostic assays and kits for in vitro assessment and evaluation of tumor and cancer status, may be utilized to diagnose, evaluate and monitor patient samples including those known to have or suspected of having cancer, a precancerous condition, a condition related to hyperproliferative cell growth or from a tumor sample. The assessment and evaluation of cancer, tumor and metastatic disease status is also useful in determining the suitability of a patient for a clinical trial of a drug or for the administration of a particular chemotherapeutic agent or specific binding member, particularly an antibody, of the present invention, including combinations thereof, versus a different agent or binding member. This type of diagnostic monitoring and assessment is already in practice utilizing antibodies against the HER2 protein in breast cancer (Hercep Test, Dako Corporation), where the assay is also used to evaluate patients for antibody therapy using Herceptin. In vivo applications include imaging of tumors or assessing cancer status of individuals, including radioimaging.

(000144] Preferably, the antibody portion of the immunoconjugate used in the diagnostic methods of this invention is a monoclonal antibody or derivative thereof. Preferably, the antibody portion of the immunoconjugate used in the diagnostic methods of this invention is human antibody or a derivative or portion thereof. The antibody may be a single chain chain antibody or domain antibody. In addition, the antibody molecules used herein can be in the form of Fab, Fab', F(ab')2 or F(v) portions of whole antibody molecules, particularly Fab.

[000145] As described in detail above, antibody(ies) to FAP can be produced and isolated by standard methods including the phage display techniques and mutagenesis and recombinant techniques.

[000146] The presence of a target of an immunoconjugate of the present invention in cells can be ascertained by the usual in vitro or in vivo immunological procedures applicable to such determinations. A number of useful procedures are known. The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The "competitive" procedure is described in U.S. Patent Nos. 3,654,090 and 3,850,752. The "sandwich" procedure, is described in U.S. Patent Nos. RE 31,006 and 4,016,043. Still other procedures are known such as the "double antibody," or "DASP" procedure.

[000147] In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of aberrant expression of an immunoconjugate target including but not limited to amplified target and/or a target mutation, in suspected target containing or target expressing cells. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive," "sandwich," "DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

[000148] Accordingly, a test kit may be prepared for the demonstration of the presence of or elevated levels of FAP, comprising:

(a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the present specific binding member or a specific binding partner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

[000149] A test kit may be prepared for the demonstration of the presence of epithelial cancer, stromal cell mediated cancer, particularly selected from breast, lung, colorectal, ovarian cancer comprising: (a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the present specific binding member or a specific binding partner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

[000150] In accordance with the above, an assay system for screening potential drugs effective to modulate the presence or activity of FAP and/or the activity or binding of the antibody of the present invention may be prepared. The antigen peptide or the binding member or antibody may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the activity of the cells, binding of the antibody, or amount and extent of FAP due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known agent(s).

Nucleic Acids

[000151] The present invention further provides an isolated nucleic acid encoding a specific binding member of the present invention. Nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid which codes for a polypeptide of the invention as defined above, including a polypeptide as set out in Figures 1 , 2 , 7 or 8 or capable of encoding the CDR regions of the antibody protion of an immunoconjugate including as set out in Figure 2 or the tumor necrosis protion of an immunoconjugate as set out in Figure 7 or Figure 8.

[000152] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. The present invention also provides a recombinant host cell which comprises one or more constructs as above. A nucleic acid encoding any specific binding member as provided itself forms an aspect of the present invention, as does a method of production of the specific binding member which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.

[000153] Specific binding members and encoding nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes origin other than the sequence encoding a polypeptide with the required function. Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic.

[000154] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, cancer cells, ovarian cancer cells and many others. A common, preferred bacterial host is E.coli. The expression of antibodies and antibody fragments in prokaryotic cells such as E.coli is well established in the art.

[000155] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

[000156] Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a specific binding member or polypeptide as above. [000157] Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2u plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

[000158] Any of a wide variety of expression control sequences— sequences that control the expression of a DNA sequence operatively linked to it ~ may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[000159] A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSCl, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.

[000160] It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products. Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

[000161] As mentioned above, a DNA sequence encoding a specific binding member can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the specific binding member amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981 ); Nambair et al., Science, 223: 1299 (1984); Jay et al., J. Biol. Chem., 259:631 1 (1984). Synthetic DNA sequences allow convenient construction of genes which will express specific binding member analogs or "muteins". Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of native specific binding member genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.

[000162] The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1

[000163] Fibroblast Activation Protein (FAP) is a member of the dipeptidyl peptidase IV (DPPIV, CD26) protein family and capable of cleaving N-terminal dipeptides from polypeptides with proline or alanine in the penultimate position. FAP expression is up-regulated on activated fibroblasts such as carcinoma-associated fibroblasts and promotes malignant and invasive behaviour. Fully human Fab antibodies have been selected from a phage library that are cross-reactive to mouse and human FAP. Particular identified clones, ESC1 1 and ESC 14, show no binding to the highly homologous CD26 antigen. Both antibodies b to FAP at low nanomolar affinities when converted into human IgG antibodies and could be produced in mammalian cell lines at high levels. ESC1 1 and ESC 14 antibodies had no direct influence on the dipeptidyl peptidase IV activity of FAP. Their induced conversion of a FAP-positive into a FAP- negative phenotype was paralleled by a significantly inhibited adhesion of targeted cells to ECM proteins and enhanced target cell apoptosis. These FAP antibodies have application in areas where fibroblast dependent malignant and invasive behaviour is of relevance.

[000164] Fibroblast activation protein (FAP) was originally identified as a serine protease on reactive stromal fibroblasts [1, 2]. Subsequent molecular cloning revealed that FAP is identical to seprase, a 170 kDa membrane associated gelatinase that is expressed by melanoma cell lines [3, 4]. Full length cDNA encoded a type II transmembrane protease of 760 amino acids (aa) highly homologous to dipeptidyl peptidase IV (DPPIV) with a 52% aa identity over the entire sequence and almost 70% identity in the catalytic domain [3, 5]. FAP and DPPIV have similar gene sizes and are chromosomally adjacent to each other at 2q24, suggesting a gene duplication event (Genebank accession number U09278). Both proteins are members of the prolyl peptidase family [1, 6]. This class of enzymes is inducible, active on the cell surface or in extracellular fluids, and uniquely capable of cleaving N-terminal dipeptides from polypeptides with proline or alanine in the penultimate position [7], DPPIV, also termed CD26, is constitutively expressed by several cell types including fibroblasts, endothelial and epithelial cells, leukocyte subsets like N -cells, T-lymphocytes and macrophages. A small proportion of DPPrV circulates as soluble protein in the blood. In contrast to DPPIV, FAP is typically not expressed in normal adult tissue [1 ] and its proteolytically active soluble form is termed a2-Antiplasmin Cleaving Enzyme (APCE) [8]. Marked FAP expression occurs in conditions associated with activated stroma, including wound healing, epithelial cancers, osteoarthritis, rheumatoid arthritis, cirrhosis and pulmonary fibrosis [4, 9-1 1].

[000165] The FAP structure has been solved (PDB ID 1Z68) and is very similar to that of DPPIV [12]. FAP is anchored in the plasma membrane by an uncleaved signal sequence of approximately 20 aa and has a short, amino terminal, cytoplasmic domain of six aa [3-5]. The major part of the protein, including the catalytic domain, is exposed to the extracellular environment [13]. The FAP glycoprotein is a homodimer consisting of two identical 97-kDa subunits. Each FAP-monomer subunit consists of two domains, an αβ hydrolase domain (aa 27-53 and 493-760) and an eight-blade β propeller domain (aa 54- 492) that enclose a large cavity. A small pocket within this cavity at the interface of both domains contains the catalytic triad (Ser624, Asp702 and His734) [12]. FAP gains its enzymatic activity upon homodimerization of the subunits [14] and beside its dipeptidyl peptidase activity, FAP also has collagen type I specific gelatinase [15] and endopeptidase activity [16]. The β propeller acts as scaffolding for protein-protein interactions and determines substrate and extracellular matrix (ECM) binding [17]. Furthermore, the β propeller is involved in forming supra-molecular complexes of FAP with other prolyl peptidases or with other membrane-bound molecules [18, 19]. The formation of heteromeric or tetrameric complexes of FAP and DPPIV were found to be associated with invadopodia of migrating cells on a collagen substrate [20]. Type I collagen induces a close association of FAP with βΐ integrins, thereby playing major organizational roles in the formation and adhesion of invadopodia [21]. Although the involved mechanisms are not understood in detail, the formation of such proteinase-rich membrane domains at the cellular invasion front contributes to directed pericellular ECM degradation [22]. This indicates that FAP and ECM interactions may be closely related to invasive cell behaviour by influencing cell adhesion, migration, proliferation and apoptosis through integrin pathways [19, 21, 23] and supports o role of FAP in disease pathogenesis and progression [24]. In summary, FAP is recognized as a multifunctional protein that executes its biological functions in a cell dependent manner through a combination of its protease activity and its ability to form complexes with other cell-surface molecules. Over-expression of FAP in epithelial and fibroblastic cell lines promotes malignant behaviour [22], pointing to the clinical situation, where cellular expression levels of FAP are correlated with worse clinical outcome [25, 26].

[000166] The morphological and functional properties promote the investigation of FAP as a therapeutic target. The disease related and cell surface bound expression pattern especially qualifies FAP for antibody targeting. With regard to the pathophysiological involvement in ECM remodelling, targeting strategies should aim at the disruption of the signalling supra-molecular FAP complexes. Iincubation with the ESC 11 or ESC 14 anti-FAP antibodies significantly inhibited adhesion of targeted cells to ECM proteins and induced apoptotic signals.

[000167] MATERIALS AND METHODS

[000168] Recombinant expression of antigens. Human FAP and DPPIV/CD26 cDNA was obtained from pooled nasopharynx carcinoma and spleen tissue, respectively. Murine FAP cDNA was obtained from lung and skin tissue. The antigens were cloned into pEA 8 vector (Edge Biosystems) and stably expressed in HE 293 c-18 cells (ATCC CRL- 10852) using the FuGene6 transfection reagent (Roche) according to the manufacturers instructions. The stable cell lines were grown in DMEM supplemented with 10% FBS, penicillin-streptomycin, and puromycin (3μg ml). Monoclonal cultures were obtained by limited dilution. [000169] Immunoprecipitation of recombinant FAP. 5xl0⁷ HEK293 cells expressing human FAP or mock-transfected cells were solubilized in 5ml lysis buffer (50 mM Tris-HCl, 150 mM NaCl buffer, pH 7.4, 0.7% w/v β-octylglucopyranoside) by vortexing for 15 min at 4°C. Cell debris was removed by centrifugation and the supernatant was incubated for 4hr at 4°C with 50 μΐ protein A magnetic beads (Dynabeads, invitrogen) coated with monoclonal antibody F19. Beads were removed from suspension using a magnetic rack and washed five times with PBS 0.1% Tween 20 and stored at -80°C in ΙΟΟμΙ PBS. Accordingly, recombinant DPPIV/CD26 was immunoprecipitated with an anti-CD26 mAb. Immunoprecipitates were subjected to SDS-PAGE, western blot analysis and dipeptidyl-peptidase activity assay.

[000170] Selection of antibodies by phage display. For selection of huFAP-muFAP specific antibodies, a non-immunized phage library expressing antibody Fab fragments was used [27]. 10¹³ Phages were blocked in 2% milk powder in PBS and preabsorbed with 25μ1 of immunoprecipitate prepared from the mock-transfected cell line to remove phages that bind to protein A beads and F19 mAb. Preabsorbed phages were incubated with huFAP containing immunoprecipitates for 1 h at RT, washed with PBS 0.1% Tween 20 and with PBS, and subsequently eluted with lOOmM triethylamine. Neutralized phages were amplified in Escherichia coli TG-1 using M13K07 as helper phage. Four rounds of selection with decreasing antigen concentration were performed (50, 25, 12.5, and 6.5 μΐ of immunoprecipitate suspension). To enrich the phage pool for binders that recognize muFAP, a fifth round was appended by incubating the phages with HEK293 muFAP expressing cells. Prior to selection rounds four and five, an additional preabsorption step on 50μ1 DPPIV/CD26 immunoprecipitate suspension was included to remove FAP-binders that cross-react with DPPIV/CD26.

[000171] Screening of supernatants by ELISA and flow cytometry. Supernatants of the output libraries after each selection round, and supernatants of individual bacterial clones after rounds three, four, and five were screened for FAP-binding phage by ELISA. Ninety-six-well microtiter plates (MaxiSorp Nunc) were coated with cell extracts cell extracts from HE 293 huFAP, HEK293 huCD26, HEK293 muFAP, or mock-transfected HEK293, blocked with 5% milk powder in PBS, incubated with phage- containing supernatants for 1 hr at room temperature and developed using an anti-M13-HRP conjugated antibody. Positive clones were induced with ImM IPTG to produce soluble Fab and further screened for binding specificities using flow cytometry on antigen expressing HEK293 cell lines. Bound Fab antibody was detected with anti-myc tag antibody 9E10, followed by an anti-mouse immunoglobulin-PE conjugate. For competition assays, Fab antibodies were detected using biotinylated anti-myc tag 9E10, followed by strep-PE. [000172] Expression of Fab fragments. Fab fragments were produced in E. coli TG-1 by induction with I mM IPTG for 4 hr at 30°C. Soluble Fab was released from the periplasmic fraction by incubation in PBS, pH 8 at 4°C o/n, purified using His-tag purification with TALON beads and analyzed by SDS-PAGE.

[000173] Cloning, expression, and purification of IgGl. The variable sequences of heavy and light chain were cloned into a modified pEE12.4 vector (Lonza biologies) expressing human constant IgGl regions via Dralll and RsrII sites, respectively, with a PCR cloning kit (In-Fusion, BD clontech). The plasmid was linearized with Pvul, and transfected into GS-NSO cells (Lonza biologies) by electroporation. Positive clones were selected in glutamine-free medium with methionine sulphoximine. Stable cell lines were grown in glutamine-free medium with 5% FCS, which has been depleted from bovine IgG with protein G sepharose. IgG was purified from culture supernatant with protein A sepharose.

[000174] RESULTS

[000175] Selection and characterization of mouse-human FAP cross-reactive antibody Fab fragments

Monoclonal antibody Fab-fragments were selected from a large human Fab antibody library by phage display on recombinant human FAP obtained by immunoprecipitation from stably transfected cell lysatea. Briefly, lysates of HT1080 and FAP-transfected HT1080 (10) cells were separately incubated with magnetic protein A beads coated with a formerly described anti-huFAP antibody (11). FAP was immunoprecipitated from HT1080 FAP⁺ extracts at high purity (Fig 1A and B) and exhibited dipeptidyl- peptidase (DPP) activity (e.g. cleave the substrate Ala-Pro-AFC) when captured on magnetic beads (Fig 1C). Unspecifically immunoprecipitated proteins binding to anti-huFAP antibody loaded magnetic beads after incubation with HT1080 cells ('mock-immunocapture') were used in pre-absorption steps. After four selection rounds, 5% of the phages bound to human FAP and 1% recognized both human and murine FAP (Table 1 ). To further enrich for human-mouse cross-reactive binders, phages were panned on mouse FAP transfected HEK 293 cells for one additional selection round. Finally, 14% of the output phages bound to huFAP including 9% mouse-human cross-reactive phages. Subsequent screening of 300 clones by ELISA and sequencing of FAP-specific binders led to the identification of Fabs ESC1 1 and ESC 14 (Fig. 1 and 2). Both Fab fragments bound to human and murine FAP in a specific manner and did not cross-react with CD26 as pre-defined by our selection strategy (Fig. 3). Affinity measurements by surface plasmon resonance were performed on a low-density huFAP coated chip. K_D values of 10 ± 5 and 210 ± 35 nM were determined for Fabs ESC 1 1 and ESC 14, respectively. Affinities on muFAP were 51 ± 1 1 nM for ESC1 1 and 251 ± 42 nM for ESC 14, as determined by serial dilutions on 293 muFAP+ cells. [000176] Production and characterization of IgGl antibodies ESC11 and ESC14

[000177] The variable heavy (HC) and light (LC) chain domains of ESCl 1 and ESC 14 were cloned into a full human IgGl format. The original sequence obtained for the ESC1 1 HC could not been expressed in mammalian cells because of an anusual Histidine (H) in position 1 of the mature HC amino- acid sequence. The problem was solved when the Histidine was replaced by Glutamine (Q) (Figure 1). IgGs were produced in NS0 cells and purified by affinity chromatography on Protein A agarose from cell culture supernatant. Binding of purified IgG to FAP was confirmed by flow cytometry using FAP- transfected HT1080 cells. Affinities on antigen expressing cell lines were higher for the bivalent IgGs with apparent K_D values around 1 nM on human FAP. Competition assay demonstrated cross-inhibition for ESC1 1 IgG and ESC 14 IgG, while anti-huFAP antibody did not compete for FAP binding. These results suggest that antibodies ESCl 1 and ESC 14 recognise overlapping or closely linked epitopes that are clearly distinct from the epitope recognised by anti-hu FAP antibody.

[000178] ESC11 and ESC14 IgG down-regulate FAP expression, disrupt attachment of targeted cells to ECM proteins and induce apoptosis

[000179] Apart from its enzymatic activity, FAP has the ability to associate with CD26 and form heteromeric complexes that contribute to the invasive phenotype. FAP expression on transfected tumor cell lines could be abolished by ESC1 1 and ESC 14 antibody since both, after bivalent binding to the FAP antigen, induced rapid FAP down-regulation. To test the impact of ESCl 1 and ESC 14 on the attachment of targeted cells to ECM-proteins, a competition assay was performed. Both mAbs significantly inhibited the binding of FAP-expressing cells to matrigel and to type I collagen (data not shown). The F19 anti-FAP antibody did not interfere with the attachment to ECM proteins. As expected, binding of FAP-negative cells was not altered at all. Induction of apoptosis pathways was studied in a second step [29]. HEK293 cells stably transfected with mouse FAP were incubated with ESCl 1 and ESC 14 and underwent apoptosis, while mock-transfected cells were not altered at all. The extent of apoptosis induction was superior after the addition of ESC1 1 when compared to ESC 14. Apoptosis could not be induced at 0°C even in the presence of both antibodies, ESC1 1 and ESC 14. In addition, neither the mF19 nor a control antibody induced any significant level of apoptosis.

[000180] This provides a fully human, mouse/human cross-reactive, high affinity anti-FAP antibodies that induce relevant regulatory activities on target cells pointing out to their therapeutic impact. Since FAP has attracted increased interest as a target for antibody based immunotherapy, data of therapeutically active native FAP-specific antibodies are missing to date. The monoclonal antibody F19 was the first antibody investigated in a phase I clinical trial targeting metastatic colorectal cancer [30]. This trial served as a proof of principle for anti-FAP based tumor stroma targeting [1]. Although patients included in the trial had extensive scarring due to surgery, no specific enrichment of ¹³¹I-F19 could be detected at these sites. There were no toxic side effects associated with intravenous administration of iodine¹³¹ labelled F19 and carcinoma lesions were specifically detected by imaging down to a size of 1 cm in diameter. With regard to the immunogenicity of murine antibodies in humans, recent phase I and phase II clinical trials were conducted using the humanized version of F19, called Sibrotuzumab [31 , 32]. Results from these trials demonstrated the safe and well tolerated administration of Sibrotuzumab. Similar to the results obtained in the pivotal phase I trial [30], trace-labelling with ¹³¹I and imaging analysis revealed the specific accumulation of Sibrotuzumab at the tumor area. Unfortunately, unconjugated Sibrotuzumab demonstrated no anti-tumor or any therapeutic activity, respectively [32]. Albeit the biologic function of FAP is still not known in detail, its dipeptidyl peptidase activity was postulated to be involved in tumor progression and metastasis [15, 33]. The lack of Sibrotuzumab to affect FAP enzymatic function was suggested to be the reason for the lack of therapeutic efficacy [34]. In consequence, anti-FAP directed polyclonal antibodies have been raised in order to inhibit the catalytic activity in-vitro. Indeed, treatment of FAP-positive xenografts with anti-FAP anti-sera attenuated tumor growth [13]. However, since polyclonal sera were raised by immunization of rabbits with murine FAP, it is most likely that additional epitopes, different from the catalytic domain, have also been targeted. Therefore, it is difficult to conclude from this study that anti-tumor effects seen really depended on dipeptidyl-peptidase inhibition. These concerns are supported by work with catalytic mutants, reporting a functional impairment of FAP-related bioactivities independent from catalytic function that could also influence tumor growth and invasive cell behaviour [35, 36]. In addition, recent results demonstrated that FAP's proteolytic activity was not necessary for increased adhesion, migration and invasion of FAP over-expressing human hepatic stellate cells [22]. Focal cell adhesion to ECM proteins is mediated by the integrin family of transmembrane adhesion proteins and FAP is known to associate with βΐ integrin [19]. This complex is thought to participate in the formation of functional invadopodia [21]. In addition, FAP and DPP IV also form complexes that are localised at invadopodia of fibroblasts on collagenous fibres, thereby facilitating cell migration [37, 38]. As cellular adhesion to ECM proteins is the first step in the progression of invasive and metastatic diseases [39], its significant inhibition resulting from FAP-targeting by novel ESC1 1 and ESC14 antibodies provides therapeutic impact by disturbing the network architecture of the connective tissue. The observation that the antibody mediated interruption of the cell movement process does not depend on FAP's proteolytic activity is in accordance to above-mentioned data. Since we recently demonstrated that inhibition of dipeptidyl peptidase activity in vivo even significantly promoted the invasion of FAP-expressing fibroblasts (Ospelt et al., submitted), the proteolytically neutral activity of both antibodies should even be advantageous in view of the aimed therapeutic impact. The functional activity of both novel antibodies together with the fact that they do not compete with F 19 for FAP binding, suggest targeting of epitopes involved in the formation of supra-molecular complexes.

[000181] FAP targeting with ESC1 1/ESC14 IgG resulted in down-regulation of FAP expression and induced profound behavioural changes in signalling pathways as the disruption of cell adhesion capabilities. This supports a unique mode of action for ESC 1 1 and ESC 14 by interfering with complexes consisting of FAP and other components. Involvement of surface serine proteases in apoptosis was demonstrated for CD26, as interruption of survival signalling pathways was mediated through intrinsic and extrinsic apoptotic pathways upon restoration of neuroblastoma cells with DPPIV [29]. Furthermore, induction of late apoptosis in DPPIV -positive mesothelioma cells was reported upon cross-linking of a humanized anti-DPPIV antibody [40]. However, this trial was performed in T-cell deficient SCID-mice and antibody based targeting of DPPIV as an anticancer strategy in humans will be up against the ubiquitous expression of DPPIV that includes T-cells. This major obstacle even built the rationale for the pre-absorption step of selected FAP binders on immunoprecipitated DPPIV to exclude unwanted binding of DPPIV. Association with survival signalling has not yet been identified for FAP and only the novel rational displayed anti-FAP antibodies ESC 1 1 and ESC 14 are linked to apoptosis induction upon FAP- targeting. These antibodies provide antigen specific and highly effective, multifunctional anti-FAP antibodies with cross-reactivity for human and mouse FAP displaying a therapeutic profile.

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[000183] EXAMPLE

FAP-Mediated Tumor Stroma Targeted TNF-Immunokine Eradicates Established Colorectal

Cancer in a Syngeneic Mouse Model

[000184] Studies were undertaken to assess and determine whether FAP is a good target for delivery of biologically active compounds. Targeted delivery of dimeric TNF-immunoconjugates to solid tumors reduces the systemic toxicity and increases the efficacy of the targeted TNF molecules. The purpose of this study was to determine the feasibility of treating solid tumors by targeting tumor stroma associated Fibroblast activation protein (FAP) with a dimeric antibody-TNF conjugate and to evaluate its therapeutic efficacy in vivo.

[000185] A novel genetically engineered TNF construct was designed as CH2/CH3 truncated anti- FAP TNF fusion protein to target a shared epitope of human and mouse FAP enabling preclinical tolerability and preclinical efficacy assessments. The construct was generated using recombinant methods as previously described (Bauer S et al (2004) J Immunol 172:3930-3939; Bauer S et al (2006) J Immunol 177:2423-2430). The sequences of the ESC1 1-muTNF, ESC14-muTNF, ESC11-huTNF and ESC14- huTNF constructs are provided in FIGURES 7 and 8. In vitro characterization of the immunoconjugate basing on dimeric murine TNF comprised biochemical analysis and bioactivity assays. Biodistribution data on radiolabeled [^13,J] TNF-immunoconjugate and antitumor activity of dimeric anti-FAP TNF were measured on syngeneic tumors in immunocompetent mice.

[000186] Administration of dimeric anti-FAP TNF activates TNF-receptor type 1 and type 2 related pathways that represent key mechanisms displaying antitumor responses. Targeting FAP-expressing tumor stroma of syngeneic solid tumors in BALB/c mice with the murine TNF-based immunoconjugate induced long-lasting complete remission rates up to 80 percent without signs of intolerability. Six weeks after treatment termination, animals in complete remission were challenged with a second subcutaneous injection of tumor cells. There was no tumor growth detectable over the whole observation period of several weeks, suggesting crossactivation of the adaptive immune system following treatment with dimeric anti-FAP TNF.

[000187] HT1080 and HT1080 FAP are equivalently sensitive to killing by muTNF (FIGURE 4A). However, the ESC1 1-muTNF construct has a higher killing activity on HT1080FAP+ cells when compared to HT1080 wt cells (FIGURE 4B) due to rapid internalisation of the FAP-TNF. Therefore, FAP internalisation allows for a selective killing activity for ESC1 1-muTNF.

[000188] The same activity profile is true for ESC14muTNF. ESC14muTNF was tested on HT1080 and HT1080FAP. The cytotoxic activity of mu-TNF and the FAP-TNF construct can be amplified be preincubation of HT 1080 wt (FIGURE 5A) or HT1080FAP+ (B) cells with 20 μg/ml Cyclohexamide for 16h. Viability was measured with Cristal violet staining. Again the FAP positive HT1080FAP cell line led to internalisation of ESC 14-muTNF constructs which increased cytotoxic activity significantly as shown (FIGURE 5B)

[000189] Mouse colon cancer cell line CT 26 was established in syngeneic BALB/c mice (1 x 10⁶ cells were sc injected) and animals treated from day 8 with three consecutive daily injections of Escl l- muTNF, PBS or irrelevant antibody. Mice treated with the TNF construct had significantly prolonged survival (FIGURE 6A). In addition mice treated with the FAP-TNF construct were resistent against re- challenge with 1 x 10⁶ CT 26 tumor cells (FIGURE 6B). All naive mice developed tumors.

[000190] This study demonstrates excellent tolerability and efficacy of an anti-FAP TNF immunoconjugate providing a first assessment of the mode of action in an immunocompetent syngeneic model for tumor stroma targeting. Our results encourage further development of the novel dimeric TNF based drug candidate toward clinical testing. [000191] This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

[000192] Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

Claims

WHAT IS CLAIMED IS:

1. An isolated immunoconjugate comprising an active moiety molecule and an immunoglobulin molecule or fragment thereof wherein the immonglobulin molecule recognizes human and mouse FAP and does not react with CD26 (DPPIV).

2. The isolated immunoconjugate of claim 1 wherein the active moiety molecule is an anti-cancer molecule.

3. The isolated immunoconjugate of claim 2 wherein the anti-cancer molecule is tumor necrosis factor (TNF).

4. The isolated immunoconjugate of claim 3 wherein the TNF is mouse or human TNF.

5. The isolated immunoconjugate of claim 1 immunoglobulin molecule is a monoclonal antibody selected from ESC 1 1 and ESC 14.

6. The isolalated immunoconjugate of claim 1 wherein the immunoglobulin molecule comprises the CDR domain sequences as set out in Figure 2.

7. The isolated immunoconjugate of claim 1 wherein the immunoglobulin molecule is an antibody or antibody fragment comprising a heavy chain and a light chain variable region comprising an amino acid sequence selected from the amino acid sequence set out in Figure 1 or 2, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR region of Figure 2, wherein said variants retain human FAP reactivity and lack CD26 reactivity.

8. The isolated immunoconjugate of claim 1 wherein the immunoglobulin molecule is an antibody or fragment thereof in the form of an antibody F(ab')2, scFv fragment, diabody, triabody or tetrabody.

9. The isolated immunoconjugate of claim 1 comprising an amino acid sequence set out in Figure 7 or Figure 8.

10. The isolated immunoconjugate of claim 1 further comprising a detectable or functional label.

1 1. The isolated immunoconjugate of claim 10, wherein said label is a radiolabel.

12. An isolated nucleic acid which comprises a sequence encoding an immunoconjugate of claim 1.

13. The isolated nucleic acid of claim 12 comprising a nucleic acid sequence of Figure 7 or Figure 8 or capable of encoding an amino acid sequence set out in Figure 7 or Figure 8.

14. A method of preparing an immunoconjugate as defined in any one of claims 1 to 9 which comprises expressing the nucleic acid of claim 12 under conditions to bring about expression of said immunoconjugate.

15. An immunoconjugate according to any one of claims 1 to 1 1 for use in a method of treatment or diagnosis of the human or animal body.

16. A method of treatment of cancer in a human patient which comprises administering to said patient an effective amount of an immunoconjugate as defined in any one of claims 1 to 1 1.

17. A pharmaceutical composition comprising an immunoconjugate as defined in any one of claims 1 to 1 1 and a pharmaceutically acceptable vehicle, carrier or diluent.

18. A kit for the treatment of cancer in a human patient, comprising a pharmaceutical dosage form of the pharmaceutical composition of claim 17, and a separate pharmaceutical dosage form comprising an additional anti-cancer agent selected from the group consisting of chemotherapeutic agents,

radioimmunotherapeutic agents, and combinations thereof.

19. A method for targeting cancer in mammals, comprising administering to a mammal a

therapeutically effective amount of the pharmaceutical composition of claim 17 or the kit of claim 18.