WO2013173745A1 - Monoclonal antibodies to macrophage stimulating protein - Google Patents

Abstract

The present invention is directed toward a neutralizing monoclonal antibody to Macrophage Stimulating Protein (MSP), a pharmaceutical composition comprising same, and methods of treatment comprising administering such a pharmaceutical composition to a patient.

Description

MONOCLONAL ANTIBODIES TO MACROPHAGE STIMULATING PROTEIN

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of the provisional application U.S. Patent Application No. 61 /849,192 filed May 18, 2012, which is incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the combination of monoclonal antibody (mAb) and recombinant DNA technologies for developing novel biologies, and more particularly, for example, to the production of monoclonal antibodies that bind to and neutralize Macrophage Stimulating Protein.

BACKGROUND OF THE INVENTION

[0002] Macrophage Stimulating Protein (MSP) is a multifunctional heterodimeric glycosylated polypeptide, belonging to the plasminogen/prothrombin family of proteins, and more specifically having about 45% sequence identity (in humans) to Hepatocyte Growth Factor (HGF); MSP has therefore also been called Hepatocyte Growth Factor- like (HGFL) protein (E. Leonard et aL, Advances in Cancer Research, Academic Press, 2000, p. 139-187). The precursor of human MSP is secreted as an inactive single-chain 71 1 -amino acid protein, pro-MSP, which is cleaved between Arg483 and Val484 by trypsin-iike serine proteases to generate the mature disuifide-linked heterodimer. The a-chain (43 kDa) consists of an N-terminal hairpin loop followed by four kringie domains. The β-chain (25 - 30 kDa) is a serine proteinase-like subunit that is enzymaticaliy inactive because the catalytic triad has been replaced by other amino acids. Hepatocytes of the liver constitutive!y produce and release pro-MSP into the blood. Certain proteases of the coagulation system in serum can cleave pro-MSP, but to act on target cells in extravascular sites, pro-MSP diffuses into tissues where it can be cleaved by one or more pro-MSP convertases, which have been found in wound fluid exudates and on the surface of macrophages and other cells (Leonard et ai., supra). Such convertases include matriptase (MT-SP1 ) (Bhatt et a!., Proc Natl Acad Sci USA 104:5771 -5776, 2007), hepatocyfe growth factor activator (HGFA; Kawaguchi et a!., FEBS J: 276:3481 -3490, 2009), and hepsin (Ganesan et al., Mol Cancer Res 9:1 175- 1 186, 201 1 ).

[0003] The cellular receptor for MSP is RON (also designated as STK in mice), which is in the same family as the HGF receptor MET (Camp et aL, Ann Surg Oncol 12:273-281 , 2005). RON is a 180 kDa disulfide-linked heterodimer of an a-chain and β-chain. The a-chain (40 kDa) is extracellular, whereas the larger β-chain (150 kDa) comprises an extracellular domain, a short transmembrane segment, and a cytoplasmic tyrosine kinase domain (Lu et ai., Cancer Let 257:157-164, 2007). The a-chain and the first part of the β-chain together constitute the SEMA domain of RON, which contains the ligand (MSP) binding site (Angeion et al., J Biol Chem 279: 3726-3732, 2004). MSP binds to RON primarily through a high affinity binding site on the MSP β-chain (Wang et al., J Biol Chem 272:16999-17004, 1997), although there is a lower affinity binding site for RON on the MSP a-chain, and the two sites together mediate receptor dimerization and subsequent activation through aufophosphorylation (Danilkovich et al., J Biol Chem 274:29937-29943, 1999). RON activation initiates multiple downstream signaling pathways including the Ras/MAPK and PI3-K/Akt pathways; Src and Focal adhesion kinase (FAK) also participate in MSP-induced mitogenic signaling (Leonard et al., supra).

[0004] MSP has a variety of effects on tissue-resident macrophages, but does not act on peripheral blood monocytes, as they lack the RON receptor (Leonard et al., supra). MSP generally promotes motility, acting as a chemoattractant for macrophages and inducing shape change. It also alters mediator production, for example inhibiting induction of NO-synthase by macrophages in response to endotoxin. MSP also induces morphologic changes including cytoskeietai reorganization in osteoclast-like ceils, thereby facilitating bone resorption by these cells (Kurihara et ai., Blood 87:3704-3710). In various experimental systems, MSP increased ceil adhesion, stimulated ceil motility and invasion, inhibited apoptosis, and mediated the epithelial to mesenchymal transition (EMT) characteristic of embryonic development, tissue repair and tumorigenesis (Camp et aL, supra). However, MSP knock-out mice are viable and generally normal, except with regard to certain inflammatory responses (Bezerra et aL, J Clin Invest 01 :1 175- 1 183, 1998).

[0005] MSP and especially the RON receptor, for which MSP is the only known ligand, have been associated with various tumors, Overexpression of RON has been detected by immunohistochemistry or reverse transcriptase-PCR in more than half of examined breast, colorectal, non-small cell lung, ovarian, and head and neck squamous ceil carcinomas (Camp et aL, supra) and over 90% of pancreatic cancers (Camp et aL, Cancer 109:1030-1039, 2007). While normal cells generally express a single form of RON rrsRNA, a number of variant isoforms of RON, usually generated by alternative splicing, have been detected in various cancers and tumor ceil lines (Lu et aL, supra). Many of these RON variants stimulate migration, invasion, and proliferation, which contribute to the invasive phenotype and promote malignant progression (Lu et aL, supra). In an experimental breast cancer model, MSP promoted tumor growth and metastasis; while coordinate overexpression of MSP, RON and the convertase MT-SP1 in breast cancer patients is a strong predictor of poor prognosis (Welm et aL, Proc Natl Acad Sci USA 104:7570-7575, 2007). In addition, there is evidence for the involvement of MSP in endometriosis (Matsuzaki et aL, Moi Hum Reprod 1 1 :345-349, 2005), glomerulonephritis (Rampino et aL, J Am Soc Nephrol 18:1486-1496, 2007) and inflammatory bowel disease (Goyette et aL, Immunol 1 :131 -138, 2007).

[0006] A number of monoclonal antibodies (mAbs) have been developed against RON. For example, the mAbs ID-1 and ID-2 bind two distinct epitopes on RON and block binding of MSP to RON (Montero-Julian et aL, Hybridoma 17:541 -551 ,1998). Similarly, human mAbs have been developed that bind with high affinity to RON, block interaction with MSP, inhibit downstream signaling, and inhibit growth of colon, lung, and pancreatic tumor xenografts (OToole et aL, Cancer Res 2008: 66:9162-9170, 2008; US Patent Nos. 7,947,81 1 and 8,133,489). MAbs to MSP that inhibit its biological activity have also been reported (Wang et aL, J Leukocyte Biol 54: 289-295, 1993). A mouse mAb available from R&D Systems (Clone #88801 , Cat. No. MAB735, of the lgG2b isotype) binds to the MSP β-chain and inhibits ligarid/receptor interaction, and a rabbit mAb from Epitomics binds to MSP for use in western blotting and IHC (Clone EPR8207, Cat. # 5350-1 ). On the other hand, soluble forms of the RON SEMA domain inhibit binding of MSP to RON, presumably by competitively binding MSP, and are antagonists of MSP-induced signaling and cell growth (Ange!on et a!., supra).

SUMMARY OF THE CLAIMED INVENTION

[0007] In one embodiment, the invention provides a genetically engineered neutralizing monoclonal antibody (mAb) to human Macrophage Stimulating Protein (MSP). In a preferred embodiment, the mAb fully competes with the C15.2 mAb for binding to MSP. In another preferred embodiment, the mAb binds an epitope that comprises amino acids from both the a-chain and β-chain of MSP. In yet another preferred embodiment, the mAb does not bind to pro-MSP. The mAb inhibits at least one, and preferably several or all biological activities of MSP including binding to its cellular receptor RON. Advantageously, the anti-MSP mAb inhibits growth of a human tumor xenograft in a mouse. Preferably, the mAb of the invention is chimeric, humanized or human. Exemplary antibodies are C15.2 and mAbs that comprise a light chain variable region having three CDRs from the light chain variable region sequence of C15.2 and a heavy chain variable region having three CDRs from the heavy chain variable region sequence of C15.2, for example chimeric and humanized forms of C15.2. Cell lines producing any of these anti-MSP mAbs are also provided. In another aspect, a pharmaceutical composition comprising one of these anti-MSP mAbs, e.g., a chimeric or humanized C15.2 mAb, is provided. In a third aspect, a pharmaceutical composition comprising a neutralizing anti-MSP mAb, for example a mAb that competes with C15.2 for binding to MSP, is administered to a patient to treat cancer or other disease. In yet another aspect, a mAb that binds and neutralizes mouse MSP is provided for research purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 . Binding ELISA of C15.2 mAb to human MSP. [0009] Figure 2. Blocking ELISA showing that C15.2 but not control mouse mAb mlgG inhibits binding of Flag-MSP to RON-Fc.

[0010] Figure 3. Binding ELISA of C15.2 and other anti-MSP mAbs to Flag-scMSP.

[0011] Figure 4. Competitive binding assay showing that AB735 partially competes with C15.2 for binding to MSP.

[0012] Figure 5. Binding ELISA of control mAb m!gG and anti-MSP mAbs A7.7.2, C15.2 and MAB735 to mouse MSP-kF (mMSP), human MSP-kF (hMSP) and chimeric proteins HaMpMSP-kF and MaHBMSP-kF.

[0013] Figure 6. Biological assay showing that chimeric C15.2 (chC15.2) but not control mAb hlgG inhibits shape change of murine peritoneal macrophages induced by MSP.

[0014] Figure 7. Biological assay showing that C15.2 and RON-Fc but not control mouse mAb mlgG inhibits proliferation of T47D breast tumor cells induced by MSP, measured by metabolism of WST-1 . The dashed line is the level of proliferation in the absence of MSP and mAb.

[0015] Figure 8. Inhibition of growth of DU145 human prostate tumor xenografts by C15.2.

[0016] Figure 9. Inhibition of growth of HCC-2998 colon tumor xenografts by C15.2.

[0017] Figures 10A, B. Sequences of the mature light chain variable region (SEQ ID NO:1 ) (A) and mature heavy chain variable region (SEQ ID NO:2) (B) of C15.2. The CDRs as defined by Kabat are underlined.

[0018] Figure 1 1 . Photomicroscopy (100x) of macrophages adhering to a membrane after migration, performed as described in Examples 7 and 10 under the indicated conditions. The small, empty circles are pores in the membrane.

[0019] Figures 12A, B. (A) Binding ELISA of E2.3 mAb to mouse MSP (mMSP). (B)

Blocking ELISA showing that E2.3 but not control rat IgG inhibits binding of mMSP-kF to mRon-Fc.

[0020] Figure 13. Binding ELISA of E2.3 to human MSP-kF (hMSP-kF), mMSP-kF, single chain mMSP-kF, and chimeric proteins HaMpMSP-kF and MaH^MSP-kF. [0021] Figures 14A, B. Sequences of the mature light chain variable region (SEQ ID NO:3) (A) and mature heavy chain variable region (SEQ ID NO:4) (B) of E2.3. The CDRs as defined by Kaba are underlined.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1 . Antibodies

[0022] As used herein, "antibody" means a protein containing one or more domains capable of binding an antigen, where such domain(s) are derived from or homologous to the variable domain of a natural antibody. A monoclonal antibody ("mAb") is simply a unique species of antibody, in contrast to a mixture of different antibodies. The antibodies described herein are generally monoclonal, unless otherwise indicated by the context. An "antigen" of an antibody means a compound to which the antibody specifically binds and is typically a polypeptide, but may also be a small peptide or small-molecule hapten or carbohydrate or other moiety. Examples of antibodies include natural, full-length tetrameric antibodies; antibody fragments such as Fv, Fab, Fab' and (Fab')2; single-chain (scFv) antibodies (Huston et al., Proc. Natl. Acad. Sci. USA 85:5879, 1988; Bird et al ., Science 242:423, 1988); single-arm antibodies (Nguyen et al., Cancer Gene Ther 10:840, 2003); and bispecific, chimeric and humanized antibodies, as these terms are further explained below. Antibodies may be derived from any vertebrate species, including chickens, rodents (e.g., mice, rats and hamsters), rabbits, primates and humans. An antibody comprising a constant domain may be of any of the known isotypes IgG, IgA, IgM, IgD and IgE and their subtypes, i.e., human lgG1 , lgG2, lgG3, lgG4 and mouse lgG1 , lgG2a, lgG2b, and lgG3, and their allotypes and isoallofypes, including permutations of residues occupying polymorphic positions in allotypes and isoallotypes. An antibody can also be of chimeric isotype, that is, one or more of its constant (C) regions can contain regions from different isotypes, e.g., a gamma-1 C_H1 region together with hinge, C_H2 and/or C_H3 domains from the gamma-2, gamma-3 and/or gamma-4 genes. The antibody may also contain replacements in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,824,821 ; Tso et al., US Patent No. 5,834,597; and Lazar et aL, Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et a!., J. Biol. Chem. 279:6213, 2004).

[0023] A natural antibody molecule is generally a etramer consisting of two identical heterodimers, each of which comprises one light chain paired with one heavy chain. Each light chain and heavy chain consists of a variable (V_L or V_H, or simply V) region followed by a constant (CL or CH, or simply C) region. The CH region itself comprises C_H1 , hinge (H), C_H2, and C_H3 regions. In 3-dimensional (3D) space, the V_L and V_H regions fold up together to form a V domain, which is also known as a binding domain since it binds to the antigen. The CL region folds up together with the CH1 region, so that the light chain V_L-CL and the V_H-CH1 region of the heavy chain together form a part of the antibody known as a Fab: a naturally Ύ-shaped" antibody thus contains two Fabs, one from each heterodimer, forming the arms of the Y. The C_H2 region of one heterodimer is positioned opposite the CH2 region of the other heterodimer, and the respective CH3 regions fold up with each other, forming together the single Fc domain of the antibody (the base of the Y), which interacts with other components of the immune system.

[0024] Within each light or heavy chain variable region, there are three short segments (averaging about 10 amino acids in length) called the complementarity determining regions ("CDRs"). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3D space to form the actual antibody binding site which locks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et aL, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework, which forms the environment for the CDRs. Chothia et a!., J. Mo!. Biol. 198:901 , 1987, have defined the related concept of hypervariable regions or loops determined by structure.

[0025] As used herein, a "genetically engineered" mAb is one for which the genes have been constructed or put in an unnatural environment (e.g., human genes in a mouse or on a bacteriophage) with the help of recombinant DNA techniques, and therefore includes chimeric antibodies and humanized antibodies, as described below, but would not encompass a mouse or other rodent mAb made with conventional hybridoma technology. A chimeric antibody (or respectively chimeric antibody light or heavy chain) is an antibody (or respectively antibody light or heavy chain) in which the variable region of a mouse (or other non-human species) antibody (or respectively antibody light or heavy chain) is combined with the constant region of a human antibody; their construction by means of genetic engineering is well-known. Such antibodies retain the binding specificity of the mouse antibody, while being about two- thirds human. Genetically engineered antibodies also include veneered or resurfaced antibodies, which, like humanized antibodies, have CDRs entirely or substantially from a non-human donor antibody. Veneered antibodies are made more human-like by replacing specific amino acids in the variable region frameworks of the non-human donor antibody that may contribute to B- or T-cel! epitopes, for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991 ). Other types of genetically engineered antibodies include human antibodies made using phage display methods (Dower et al., W091/17271 ; McCafferty et al., WO92/001047; Winter, WO92./20791 ; and Winter, FEBS Lett. 23:92, 1998, each of which is incorporated herein by reference) or by using transgenic animals (Lonberg et al., WO93/12227; Kucherlapati WO91/10741 , each of which is incorporated herein by reference).

[0026] A humanized antibody is a genetically engineered antibody in which CDRs from a non-human "donor" antibody (e.g., chicken, mouse, rat, rabbit or hamster) are grafted into human "acceptor" antibody sequences, so that the humanized antibody retains the binding specificity of the donor antibody (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat, No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. Nos. 5,859,205 6,881 ,557; Foote, U.S. Pat. No. 6,881 ,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a consensus sequence of human antibody sequences, a germiine human antibody sequence, or a composite of two or more such sequences. Thus, a humanized antibody is an antibody having some or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly, a humanized light chain (respectively heavy chain) has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light (resp. heavy) chain, and a light (resp, heavy) chain variable region framework and light (resp. heavy) chain constant region, if present, substantially from a human light (resp. heavy) acceptor chain. A humanized antibody generally comprises a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding amino acids (as defined by Kabat) are identical between the respective CDRs. The variable region framework or constant region of an antibody chain are substantially from a human variable region or human constant region respectively when at least 85, 90, 95 or 100% of corresponding amino acids (as defined by Kabat) are identical.

[0027] Here, as elsewhere in this application, percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention (Eu index for the CH region). After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.

[0028] In order to retain high binding affinity in a humanized antibody, at least one of two additional structural elements can be employed. See, US Patent No. 5,530,101 and 5,585,089, incorporated herein by reference, which provide detailed instructions for construction of humanized antibodies, !n the first structural element, the framework of the heavy chain variable region of the acceptor or humanized antibody is chosen to have high sequence identity (between 85% and 95%) with the framework of the heavy chain variable region of the donor antibody, by suitably selecting the acceptor antibody heavy chain from among the many known human antibodies. In the second structural element, in constructing the humanized antibody, selected amino acids in the framework of the human acceptor antibody (outside the CDRs) are replaced with corresponding amino acids from the donor antibody, in accordance with specified rules. Specifically, the amino acids to be replaced in the framework are chosen on the basis of their ability to interact with the CDRs. For example, the replaced amino acids can be adjacent to a CDR in the donor antibody sequence or within 4-6 angstroms of a CDR in the humanized antibody as measured in 3-dimensionai space.

[0029] The term "antibody" also encompasses bispecific antibodies. A "bispecific antibody" is an antibody that contains a first domain binding to a first antigen and a second (different) domain binding to a second antigen, where the first and second domains are derived from or homologous to variable domains of natural antibodies. The first antigen and second antigen may be the same antigen, in which case the first and second domains can bind to different epitopes on the antigen. The term bispecific antibody encompasses muitispecific antibodies, which in addition to the first and second domains contain one or more other domains binding to antigens and derived from or homologous to variable domains of natural antibodies. The term bispecific antibody also encompasses an antibody containing a first binding domain derived from or homologous to a variable domain of a natural antibody, and a second binding domain derived from another type of protein, e.g., the extracellular domain of a receptor, (a "bispecific antibody-immunoadhesin").

[0030] Bispecific antibodies have been produced in a variety of forms (see, e.g., R.E. Kontermann, mAbs 4:182-197, 2012 and references cited therein), for example !gG-single chain variable fragment (scFv), Fab-scFv, and scFv-scFv fusion proteins (Coloma et al., Nat Biotechnol 15:125-8, 1997; Lu et ai., J Immunol Methods 267:213- 26, 2002; Mailender, J Biol Chem 269:199-206, 1994), dual variable domain antibodies (DVD-lg; Wu et al., Nat Biotechnol 25:1290-7, 2007), and diabodies (Hoiiiger et ai., Proc Natl Acad Sci USA 90:6444-8, 1993). Bispecific F(ab')₂ antibody fragments have been produced by chemical coupling (Brennan et al., Science 229:81 , 1985) or by using leucine zippers (Kostelny et ai., J Immunol 148:1547-53, 1992). A more naturally shaped bispecific antibody, with each heavy chain - light chain pair having a different V region, can be made by chemically cross-linking the two heavy chain - light chain pairs produced separately (Karpovsky et al., J Exp Med 160:1686-701 , 1984), Naturally shaped bispecific antibodies can also be produced by expressing both required heavy chains and light chains in a single cell, made by fusing two hybridoma ceil lines (a "quadroma"; Milstein et a!., Nature 305: 537-40) or by transfection. Association of the correct light and heavy chains expressed in a eel! to form the desired bispecific antibody can be promoted by using "knobs-into-hoies" technology (Ridgway et a!., Protein Eng 9:817-21 , 1996; Atwel! et aL, J Mo! Biol 270:26-35, 1997; and US Patent No, 7,895,938); optionally with exchange or "crossing over" of heavy chain and light chain domains within the antigen binding fragment (Fab) of one light chain - heavy chain pair, thus creating bispecific antibodies called "Cross abs" (Schaefer et aL, Proc Natl Acad Sci USA 108:1 1 187-92, 201 1 ; WO 2009/080251 ; WO 2009/080252; WO 2009/080253).

[0031] An antibody is said to bind "specifically" to an antigen if it binds to a significantly greater extent than irrelevant antibodies not binding the antigen, and thus typically has binding affinity (K_a) of at least about 10⁶ but preferably 10⁷, 10⁸, 10⁹ or 10^1ϋ M^"1 for the antigen. Generally, when an antibody is said to bind to an antigen, specific binding is meant. If an antibody is said not to bind an antigen, it is meant that any signal indicative of binding is not distinguishable within experimental error from the signal of irrelevant control antibodies. The epitope of a mAb is the region of its antigen to which the mAb binds. Two antibodies are judged to bind to the same or overlapping epitopes if each competitively inhibits (blocks) binding of the other to the antigen. Competitively inhibits binding means that a 1 x or 5x excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, or that a 10x, 20x or 100x excess of one antibody inhibits binding of the other by at least 75% but preferably 90% or even 95% or 99% as measured in a competitive binding assay (see, e.g., Junghans et aL, Cancer Res. 50:1495, 1990). One mAb (the second mAb) is said to "fully" compete for binding an antigen with another mAb (the first mAb) if the inhibitory concentration 50 (IC50) of the second mAb to inhibit binding (of the first mAb) is comparable to, that is, within 2-fold or 3-fold, of the IC50 of the first mAb to inhibit binding of itself, in competitive binding assays. A second mAb is said to "partially" compete for binding an antigen with a first mAb if the IC50 of the second mAb to inhibit binding (of the first mAb) is substantially greater than, e.g., greater than 3-fold or 5-fold or 10-fold, the IC50 of the first mAb to inhibit binding. In general, two mAbs have the same epitope on an antigen if each fully competes for binding to the antigen with the other, and have overlapping epitopes if at least one mAb partially competes for binding with the other mAb. Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other, while two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

2. Anti-MSP Antibodies

[0032] A monoclonal antibody that binds MSP (i.e., an anti-MSP mAb) is said to neutralize MSP, or be neutralizing, if the binding partially or completely inhibits one or more biological activities of MSP (i.e., when the mAb is used as a single agent). Among the biological properties of MSP that a neutralizing antibody may inhibit are the ability of MSP to bind to its RON receptor, to cause the scattering of certain cells, to inhibit apoptosis, and to stimulate motility and invasion by certain cells including macrophages. A neutralizing mAb of the invention at a concentration of, e.g., 0.01 , 0.1 , 0.5, 1 , 2, 5, 10, 20 or 50 μο/ml inhibits a biological function of MSP by about at least 50% but preferably 75%, more preferably by 90% or 95% or even 99%, and most preferably approximately 100% (essentially completely) as assayed by methods described under Examples or known in the art. Typically, the extent of inhibition is measured when the amount of MSP used is just sufficient to fully stimulate the biological activity, or is 0.05, 0.1 , 0.5, 1 , 3 or 10 ^g/ml. Preferably, the mAb neutralizes not just one but two, three or several of the biological activities listed above; for purposes herein, an anti-MSP mAb that used as a single agent neutralizes ail the biological activities of MSP is called "fully neutralizing", and such mAbs are most preferable. MAbs of the invention are preferably specific for MSP, that is they do not (specifically) bind, or only bind to a much lesser extent (e.g., less than ten-fold), proteins that are related to MSP such as hepatocyte growth factor (HGF) and other members of the plasminogen/prothrombin family, and fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF). MAbs of the invention typically have a binding affinity (K_a) for MSP of at least 10⁷ M^"1 but preferably 10⁸ M^"1 or higher, and most preferably 10⁹ M^" or higher or even 10¹⁰ M^"1 or higher. The mAb binds human MSP, but advantageously also MSP from other species, e.g., mice or non- human primates such as cynomolgus monkeys, ideally with binding affinity similar to (e.g., within 10-fold) the binding affinity to human MSP. Alternatively, the mAb may only bind MSP from a non-human species, for example mice; such mAbs are usefui to conduct research using animal models from that species. MAbs of the invention include all the various forms of antibodies described above, including bispecific antibodies having a binding domain that binds MSP. The precursor form of human MSP (pro- MSP) is assigned UniProtKB No. P26927. Residues 1 -18 are a signal sequence, residues 19-483 are the alpha chain and residues 484-71 1 are the beta chain. After cleavage, the alpha and beta chains together constitute human MSP (i.e., the mature form).

[0033] The anfi-MSP mAb C15.2 described below is an example of the invention. Neutralizing mAbs with the same or overlapping epitope as C15.2 provide other examples. Preferred antibodies bind to an epitope comprising amino acids in both the a-chain and β-chain of MSP. These or other preferred antibodies do not bind pro-MSP or do so only with low affinity (e.g., with K_a less than 10⁶ or 10⁷ or 10⁸) or bind pro-MSP significantly less well than MSP (e.g., bind pro-MSP with less than 2-, 5-, 10- or 100-fold the affinity for mature MSP). Neutralizing anti-MSP mAbs that are chimeric, humanized or human, e.g., a chimeric or humanized form of C15.2, are especially preferred embodiments. In another preferred embodiment, the mAb is a bispecific antibody comprising one binding domain from an anti-MSP mAb (e.g., C15.2 or a humanized form of C15.2) that has one or more of the properties mentioned above (e.g., neutralizing MSP), and a second binding domain from a mAb that optionally binds and neutralizes HGF (e.g., the L2G7 mAb or a humanized form of it such as HuL2G7, as described in U.S. Patent No. 7,220,410 and 7,632,928). Most preferably, the anti-MSP mAb inhibits growth of a human tumor xenograft in a mouse as assessed by any of the assays in the Examples or otherwise known in the art. MAbs that have CDRs that individually or collectively are at least 90%, 95% or 98% or completely identical to the CDRs of C15.2 in amino acid sequence and that maintain its functional properties, or which differ from C15.2 by a small number of functionally inconsequential amino acid substitutions (e.g., conservative substitutions), deletions, or insertions are also included in the invention. [0034] The E2.3 mAb described below, which binds to mouse MSP (mMSP), is another example of the invention, as are mAbs that partially or fully compete for binding to mMSP with E2.3, or which comprise the CDRs of E2.3, or which neutralize mMSP. The invention also encompasses a method of predicting or testing the efficacy of treatment with an anti-MSP mAb in a human disease, comprising administering E2.3 or other anti-mMSP mAb of the invention in a mouse model of that disease, such as cancer or other disease associated with macrophages, e.g., rheumatoid arthritis or inflammatory bowel disease, and determining whether such treatment has an ameliorative or curative effect in the disease model.

[0035] Once a single, archetypal anti-human-MSP mAb, for example C15.2, has been isolated that has the desired properties described herein, it is straightforward to generate other mAbs with similar properties by using art-known methods, including mAbs that compete with C15.2 for binding to MSP and/or have the same epitope. For example, mice may be immunized with MSP, hybridomas produced, and the resulting mAbs screened for the ability to compete with C15.2 for binding to MSP. Mice can also be immunized with a smaller fragment of MSP containing the epitope to which C15.2 binds. The epitope can be localized by, e.g., screening for binding to a series of overlapping peptides spanning MSP. Mouse mAbs generated in these ways can then be humanized. Alternatively, the method of Jespers et al., Biotechnology 12:899, 1994, which is incorporated herein by reference, may be used to guide the selection of mAbs having the same epitope and therefore similar properties to C15.2. Using phage display, first the heavy chain of C15.2 is paired with a repertoire of (preferably human) light chains to select a MSP-binding mAb, and then the new light chain is paired with a repertoire of (preferably human) heavy chains to select a (preferably human) MSP- binding mAb having the same epitope as C15.2. Alternatively variants of C15.2 can be obtained by mutagenesis of cDNA encoding the heavy and light chains of C15.

[0036] Genetically engineered mAbs, e.g., chimeric or humanized mAbs, may be expressed by a variety of art-known methods. For example, genes encoding their light and heavy chain V regions may be synthesized from overlapping oligonucleotides and inserted together with available C regions into expression vectors (e.g., commercially available from !nvitrogen) that provide the necessary regulatory regions, e.g., promoters, enhancers, poly A sites, etc. Use of the CMV promoter-enhancer is preferred. The expression vectors may then be transfected using various well-known methods such as iipofection or eiectroporation into a variety of mammalian cell lines such as CHO or non-producing myelomas including Sp2/0 and NSO, and cells expressing the antibodies selected by appropriate antibiotic selection. See, e.g., US Patent No. 5,530,101 . Larger amounts of antibody may be produced by growing the ceils in commercially available bioreactors.

[0037] Once expressed, the mAbs of the invention may be purified according to standard procedures of the art such as microfiitration, ultrafiltration, protein A or G affinity chromatography, size exclusion chromatography, anion exchange chromatography, cation exchange chromatography and/or other forms of affinity chromatography based on organic dyes or the like. Substantially pure antibodies of at least about 90 or 95% homogeneity are preferred, and 98% or 99% or more homogeneity most preferred, for pharmaceutical uses. It is also understood that when the mAb is manufactured by conventional procedures, one to several amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules, and such a composition is still considered to be the same mAb.

3. Therapeutic Methods

[0038] In a preferred embodiment, the present invention provides a pharmaceutical formulation comprising an antibody described herein. Pharmaceutical formulations contain the mAb in a physiologically acceptable carrier, optionally with excipients or stabilizers, in the form of lyophilized or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or acetate at a pH typically of 5.0 to 8.0, most often 8.0 to 7.0; salts such as sodium chloride, potassium chloride, etc. to make isotonic; antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophiiic polymers such as poiysorbate 80, amino acids, carbohydrates, chelating agents, sugars, and other standard ingredients known to those skilled in the art (Remington's Pharmaceutical Science 18 edition, Osol, A. Ed. 1980). The mAb is typically present at a concentration of 1 - 100 mg/nil, but most often 10 - 50 mg/mi, e.g., 10, 20, 30, 40 or 50 mg/ml.

[0039] In another preferred embodiment, the invention provides a method of treating a patient with a disease by administering an anti-MSP mAb in a pharmaceutical formulation. The mAb prepared in a pharmaceutical formulation can be administered to a patient by any suitable route, especially parentally by intravenous infusion or bolus injection, intramuscularly or subcutaneously. Intravenous infusion can be given over as little as 15 minutes, but more often for 30 minutes, or over 1 , 2 or even 3 hours. The mAb can also be injected directly into the site of disease (e.g., a tumor), or encapsulated into carrying agents such as liposomes. The dose given is sufficient to alleviate the condition being treated ("therapeutically effective dose") and is likely to be 0.1 to 5 mg/kg body weight, for example 1 , 2, 3, 4 or 5 mg/kg, but may be as high as 10 mg/kg or even 15 or 20 or 30 mg/kg, e.g., in the ranges 1 - 10 mg/kg or 1 - 20 mg/kg. A fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 1000 mg/m². Usually between 1 and 8 doses, (e.g., 1 , 2, 3, 4, 5, 6, 7 or 8) are administered to treat cancer, but 10, 20 or more doses may be given. The mAb can be administered daily, biweekly, weekly, every other week, monthly or at some other interval, depending, e.g. on the half-life of the mAb, for 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3-8 months or longer. Repeated courses of treatment are also possible, as is chronic administration.

[0040] Diseases especially susceptible to therapy with the anti-MSP mAbs of this invention include solid tumors, especially those associated with elevated levels of MSP or RON expression, for example ovarian cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, pancreatic cancer, gastric cancer, liver cancer (hepatocellular carcinoma), kidney cancer (renal cell carcinoma), head-and-neck tumors, melanoma, sarcomas, and brain tumors (e.g., glioblastomas). Hematologic malignancies such as leukemias and lymphomas may also be susceptible. In a preferred embodiment, the anti-MSP mAb is administered in combination with (i.e., together with, that is, before, during or after) other therapy. For example, to treat cancer, the anti-MSP mAb may be administered together with any one or more of the known chemotherapeutic drugs, for example alkylating agents such as carmustine, chlorambucil, cisplatin, carbopiatin, oxa!iplatin, procarbazine, and cyclophosphamide; antimetabolites such as fluorouracil, floxuridine, fludarabine, gemcitabine, methotrexate and hydroxyurea; natural products including plant alkaloids and antibiotics such as bleomycin, doxorubicin, daunorubicin, idarubicin, etoposide, mitomycin, mitoxantrone, vinblastine, vincristine, and Taxol (paclitaxel) or related compounds such as Taxotere©; the topoisomerase 1 inhibitor irinotecan; and inhibitors of tyrosine kinases such as Gleevec® (imatinib), Sutent® (sunitinib), Nexavar® (sorafenib), Tarceva® (eriotinib), Tykerb® (lapatinib) and Iressa® (gefitinib); Rapamycin® (siroiimus) and other mTOR inhibitors; and inhibitors of angiogenesis; and ail approved and experimental anti-cancer agents listed in WO 2005/017107 A2 (which is herein incorporated by reference). The anti-MSP mAb may be used in combination with 1 , 2, 3 or more of these other agents, preferably in a standard chemotherapeutic regimen. Normally, the other agents are those already believed or known to be effective for the particular type of cancer being treated.

[0041] Other agents with which the anti-MSP mAb can be administered to treat cancer include biologies such as monoclonal antibodies, including Herceptin® against the HER2 antigen; Avastin® against VEGF; or antibodies to the Epidermal Growth Factor (EGF) receptor such as Erbitux® (cetuximab) and Vectibix® (panifumumab), as well as antibody-drug conjugates such as Kadcyla™ (ado-trastuzumab emtansine). MAbs against HGF are especially preferred for use with the anti-MSP mAb, including mAb L2G7 (Kim et a!., Clin Cancer Res 12:1292, 2006 and US Patent No. 7,220,410) and particularly its chimeric and humanized forms such as HuL2G7 (US patent No. 7,832,928); the human anti-HGF mAbs described in WO 2005/017107 A2, particularly 2.12.1 ; and the HGF binding proteins described in WO 07143090 A2 or WO 07143098 A2; and other neutralizing anti-HGF mAbs that compete for binding with any of the aforementioned mAbs. A mAb that binds to RON or to the Met receptor of HGF is also preferred, for example the anti-cMet mAb OA-5D5 (Martens et al., Clin. Cancer Res. 12:6144, 2008) that has been genetically engineered to have only one "arm", i.e. binding domain. Moreover, the anti- MSP mAb can be used together with any form of surgery and/or radiation therapy. [0042] Treatment (e.g., standard chemotherapy) including the anti-MSP mAb antibody may increase the median progression-free survival or overall survival time of patients with a particular type of cancer such as those listed above by at least 20% or 30% or 40% but preferably 50%, 80% to 70% or even 100% or longer, compared to the same treatment (e.g., chemotherapy) but without anti-MSP mAb; or by (at least) 2, 3, 4, 8 or 12 months. In addition or alternatively, treatment (e.g., standard chemotherapy) including the anti-MSP mAb may increase the complete response rate, partial response rate, or objective response rate (complete + partial) of patients (especially when relapsed or refractory) by at least 30% or 40% but preferably 50%, 80% to 70% or even 100% compared to the same treatment (e.g., chemotherapy) but without the anti-MSP mAb.

[0043] Typically, in a clinical trial (e.g., a phase II, phase Π/ΙΠ or phase III trial), the aforementioned increases in median progression-free survival and/or response rate of the patients treated with chemotherapy plus the anti-MSP mAb, relative to the control group of patients receiving chemotherapy alone (or plus placebo), is statistically significant, for example at the p = 0.05 or 0.01 or even 0.001 level. It is also understood that response rates are determined by objective criteria commonly used in clinical trials for cancer, e.g., as accepted by the National Cancer Institute and/or Food and Drug Administration, for example the RECIST criteria (Response Evaluation Criteria In Solid Tumors).

[0044] The anti-MSP mAb may also be used to treat endometriosis and inflammatory and autoimmune diseases, especially those involving macrophages and/or associated with MSP, including inflammatory bowel disease (Crohn's disease and ulcerative colitis) in which a role for MSP has been shown (see Gorlatova et a!., PLoS One 8:e27269, 201 1 and Hauser et al., Genes Immun 13:321 -7, 2012), rheumatoid arthritis, and kidney disease such as glomerulonephritis, and osteoporosis.

4. Other Methods

[0045] The anti-MSP mAbs of the invention also find use in diagnostic, prognostic and laboratory methods. They may be used to measure the level of MSP in a tumor or in the circulation of a patient with a tumor, and therefore to follow and guide treatment of the tumor. For example, a tumor associated with high levels of MSP would be especially susceptible to treatment with an anti-MSP mAb. In particular embodiments, the mAbs can be used in an ELISA or radioimmunoassay to measure the level of MSP, e.g., in a tumor biopsy specimen or in serum or in media supernatant of MSP-secreting ceils in ceil culture. The use of two anti-MSP mAbs binding to different epitopes (i.e., not competing for binding) is especially useful in developing a sensitive "sandwich" ELISA to detect MSP. For various assays, the mAb may be labeled with fluorescent molecules, spin-labeled molecules, enzymes or radioisotopes, and may be provided in the form of kit with all the necessary reagents to perform the assay for MSP. In other uses, the anti-MSP mAbs are used to purify MSP, e.g., by affinity chromatography.

5. Examples

[0046] Example 1 : Generation of anti-MSP mAbs

[0047] To generate and assay mAbs that bind to and block the activities of human MSP, recombinant human MSP was first produced in a mammalian expression system. cDNAs encoding full length human MSP or Flag-MSP (8 amino acid residues of Flag peptide attached to the N-terminus of amino acids 19-71 1 of MSP) were constructed and inserted into a derivative of the pCR3 expression vector (Invitrogen), and transfected and expressed in 293F human kidney fibroblast cells (Invitrogen), using standard methods of molecular biology. MSP-Fc, in which the human immunoglobulin (Ig) gamma-1 constant region (C_H1 , H, C_H2 and C_H3 domains) is linked to the carboxy- terminus of MSP, was similarly produced. Single chain Flag-scMSP, a form of MSP which cannot be cleaved into separate a and β chains, was generated by using in vitro mutagenesis to change amino acid Arg483 to Ala. Human MSP-kF and mouse MSP-kF, as well as the chimeric proteins ΗαΜβΜ5Ρ- Ρ and MaHpMSP-kF in which the human MSP a-chain was linked to the mouse MSP β-chain or respectively vice versa, in all cases followed by the human Ig kappa constant region and Flag peptide for detection, were similarly produced. For functional assays, most of the extracellular domain of the MSP receptor RON (amino acids 25 to 884) was linked to the human !g gamma-1 constant region and similarly produced to generate RON-Fc. Human and mouse MSP were also purchased from R&D systems. MSP-Fc and Flag-MSP were purified from culture medium of stable transfectant cell lines respectively using a protein A column or an anti-Flag M2 mAb affinity column (Sigma-Aldrich). The anti-MSP mAb MAB735 (clone #68801 ) was purchased from R&D Systems for comparative purposes.

[0048] Baib/c mice were immunized in each hind footpad weekly 12 times with 5 pg of purified MSP-Fc in Ribi adjuvant (10 g for the first injection), and boosted later with 5 pg of purified Flag-MSP. Three days after this final boost, popliteal lymph node cells were fused with murine myeloma ceils, P3X83AgU.1 (ATCC CRL1597), using 35% polyethylene glycol. Hybridomas were selected in HAT medium as described (Chuntharapai and Kim, J Immunol 183:768, 1997). Ten days after the fusion, hybridoma culture supernatants were screened in a Flag-MSP binding ELISA, and as a secondary screen in a blocking ELISA; these assays are described below. Selected hybridomas were cloned, assayed again as above and then assayed for ability to bind Fiag-scMSP. Because of its desirable binding and blocking properties, the mAb C15.2, which is of the lgG2b, kappa isotype was chosen for further study. Two other anti-MSP mAbs designated A7.7.2 and C5.2 (both of the lgG2a isotype) were also selected.

[0049] Example 2: Binding and blocking properties of C15.2 mAb

[0050] Each step of each ELISA assay described herein was performed by room temperature incubation with the appropriate reagent for 1 hour, except the initial plate coating step was done overnight at 4 , followed by blocking with 2% BSA. Between each step, plates were washed 3 times in PBS containing 0.05% Tween 20. Data points were generally in triplicate; there was generally little variability between triplicate data points. To measure binding of mAbs to MSP, plates coated with 2 pg/ml goat anti- mlgG-Fc were incubated with hybridoma supernatant for screening or with increasing concentrations of purified C15.2 or other anti-MSP mAb to be tested, and then with 1 pg/rn! Flag-MSP plus 30 pg/rn! mouse IgG to block binding of the anti-mlgG on the plate to the HRP-anti-Flag mouse M2 mAb used in the next step for detection. The bound Flag-MSP was detected by addition of HRP-anti-Flag M2 antibody (Sigma-Aldrich; 1 :5000 dilution) plus 10 pg/ml mouse IgG for blocking and then TMB substrate. As seen in Figure 1 , C15.2 strongly bound to MSP. The isotype of C15.2 was determined to be !gG2b using an isotyping kit.

[0051] For the blocking ELISA, plates coated with 2 pg/ml goat anti-hlgG-Fc were first incubated with culture media containing RON-Fc (at a concentration of approximately 1 pg/ml). The plates were then incubated with 1 pg/ml Flag-MSP plus hybridoma supernatant for screening or increasing concentrations of purified C15.2 or other anti-MSP mAb to be tested, followed by HRP-anti-Flag M2 antibody and then TMB substrate for detection. As seen in Figure 2, C15.2 potently blocked binding of MSP (in the form of Flag-MSP) to its receptor RON (in the form of RON-Fc), with an IC5Q less than or equal to about 0.1 pg/ml. MAB735 also blocked in this assay, comparably to C15.2, as did the C5.2 mAb but not A7.7.2. To determine whether C15.2 binds to pro- MSP (unprocessed single chain), the same ELISA assay described above to measure binding to MSP was used, but with Fiag-scMSP (0.7 pg/ml in culture media) in place of Flag-MSP. As seen in Figure 3, neither C15.2 nor MAB735 bound significantly to Flag- scMSP, but mAbs A7.7.2 and C5.2 did. !t was also determined in a similar ELISA that none of these mAbs bound to mouse MSP.

[0052] Example 3: Epitope of C15.2

[0053] To determine whether MAB735 competes with C15.2 for binding to MSP, a plate coated with 2 pg/ml C15.2 was incubated with 0.2 pg/ml Flag-MSP and increasing concentrations of competitor mAbs C15.2 or MAB735. As seen in Figure 4, C15.2 of course competes with itself for binding, but MAB735 also competes, although less well. In fact, the IC50 for inhibition of binding by MAB735 is between about 5 and 10-fold higher than the IC50 for C15.2, so using the terminology introduced above, MAB735 partially competes for binding with C15.2 but does not fully compete for binding. This indicates that the epitopes of C15.2 and MAB375 on MSP are overlapping but not the same.

[0054] To confirm this, the binding of these mAbs as well as A7.7.2 to the chimeric proteins HaM MSP-kF and Mah^MSP-kF was measured in an ELISA. For this purpose, wells of a plate coated with 2 pg/ml goat anti-mlgG-Fc were incubated with 1 pg/ml of each of the mAbs, followed by human MSP-kF, mouse MSP-kF, ΗαΜβΜ8Ρ- kF or MaH MSP-kF (about 0.15 pg/ml in culture media), which were detected with HRP-goat-an i-kappa. Figure 5 shows that none of the mAbs bind mouse MSP. A7.7.2 binds well to ΗαΜβΜΘΡ but not to ΜαΗβΜ8Ρ, indicating that it binds to the (human) a-chain of MSP (since replacement with the mouse a-chain prevents binding). Conversely, MAB735 binds well to ΜαΗβΜ8Ρ but not ΗαΜβΜ8Ρ, indicating it binds to the β-chain, but C15.2 does not bind well to either ΗαΜβΜδΡ or ΜαΗβΜ8Ρ, indicating that both the a and β-chains participate in binding. Hence, C15.2 and MAB735 have different epitopes, but which overlap in the β-chain. These results are consistent with the ability of C15.2 and MAB735 but not A7.7.2 to block binding of MSP to its receptor RON, because this interaction is known to be primarily mediated through the β-chain of MSP.

[0055] Example 4: Neutralization of biological activities of MSP by C15.2

[0056] One known biological activity of MSP is the ability to induce shape change in macrophages. To show that the C15.2 mAb can inhibit this activity, murine peritoneal resident macrophages were obtained by injecting a Balb/c mouse with 1 .5 ml of 4% thioglycollate (Sigma), 4 days later washing the peritoneal cavity with 10 ml of serum- free DMEM medium, washing the resulting ceils and resuspending them in DM EM/

0.1 % BSA. These cells, primarily macrophages, were seeded at 2x10 ceils/well in a 24-well plate and cultured overnight, and non-adherent cells were removed. Wells were incubated with 100 ng/m! MSP and 5 pg/m! of control hlgG or chimeric C15.2 mAb in DMEM or with media alone. After 1 hour, the cells were washed and stained with Diff- Quick Stain solution (Dade Behring), then examined by microscopy. As seen by comparing panels A and B in Figure 8, the MSP (in the presence of control hlgG) induces a major shape change in many of the macrophage, but the C15.2 mAb completely inhibits this shape change (panel C).

[0057] Another biological activity of MSP is the ability to stimulate the proliferation of certain cells, including human T47D breast tumor ceils (ATCC HTB-133). To show that C15.2 mAb can inhibit this activity, T47D ceils at 2000/weil in DMEM/ 0.1 %FCS were incubated with 100 ng/ml of MSP plus various concentrations of negative control mlgG mAb or C15.2, or as a positive control RON-Fc (which binds MSP and thus inhibits it from binding to the ceils). After 3 days, proliferation was measured by addition of W8T-1 (Roche Bioscience) for 7 hours. As seen in Figure 7, MSP moderately stimulated proliferation (increase of OD 450 from 0.3 to 0.4). The C15.2 mAb, but not the control mAb, inhibited this stimulation, at least as well as RON-Fc, with strong inhibition at 1 pg/ml and essentially complete inhibition at 4 or 16 pg/ml.

[0058] Example 5: Ability of C15.2 to inhibit growth of tumor xenografts

[0059] Xenograft experiments are carried out as described previously (Kim et al., Nature 382:841 , 1993). Human tumor cells typically grown in complete DMEM medium are harvested in HBSS. Female athymic nude mice or ΝΙΗ-ΠΙ Xid/Beige/nude mice (4-6 wks old) are injected subcutaneousiy with 2-10 x 10° ceils in 0.1 mi of HBSS in the dorsal areas. When the tumor size reaches 50-100 mm³, the mice are grouped randomly and 5 mg/kg (100 pg total) of mAbs are administered i.p. twice per week in a volume of 0.1 ml. Tumor sizes are determined twice a week by measuring in two dimensions [length (a) and width (b)]. Tumor volume is calculated according to V = ab^/2 and expressed as mean tumor volume ± SEM. The number of mice in each treatment group is typically 5-7 mice. Statistical analysis can be performed, e.g., using Student's t test on the final data point.

[0060] Figure 8 shows that treatment with C15.2 inhibited the growth of xenografts of DU145 human prostate tumor cells (ATCC HTB-81 ) to a statistically significant extent (p = 0.01 ). Similarly, Figure 9 shows that treatment with C15.2 inhibited the growth of xenografts of HCC-2998 colon tumor cells (National Cancer Institute) to a statistically significant extent (p = 0.05). These tumor cell lines were chosen for xenograft experiments because they secrete MSP at relatively high levels and express RON. Similar tumor inhibition experiments are performed with an anti-MSP mAb such as C15.2 administered in combination with one or more chemotherapeutic agents such as 5-FU (5-fiuorouracil) or cisplatin to which the tumor type is expected to be responsive, as described by Ashkenize et al., J. Clin. Invest. 104:155, 1999, or with other biologic agents including mAbs that bind to other growth factors or angiogenic factors or their receptors, such as HGF, FGF2, EGFR, Met, RON and VEGF. The combination of the anti-MSP antibody and other agent may produce a greater inhibition of tumor growth than either agent alone. The effect may be additive or synergistic, and strongly inhibit growth, e.g. by 80% or 90% or more, or even cause tumor regression or disappearance. [0061] Example 6: Cloning of C15.2 genes

[0062] Cloning of the light and heavy chain variable regions of the C15.2 mAb, and construction and expression of a chimeric C15.2 mAb (chC15.2) were performed using standard methods of molecular biology, e.g. as described in US Patent No. 7,832,928 for the L2G7 mAb, which is herein incorporated by reference for ail purposes. The amino acid sequences of the (mature) light and heavy chain variable (V) regions of Ch15.2 are shown respectively in Figs. 10A (SEQ ID NO:1 ) and 10B (SEQ ID NO:2).

[0063] Example 7: Ability of C15.2 to inhibit migration of macrophages and tumor cells

[0064] MSP is a chemoattractant for macrophages (Leonard et al., Exp Cell Res 1 14:1 17-26, 1978) and promotes migration and invasion by tumor ceils (Thangasamy et al., J Biol Chem 283:5335-5343, 2008). To verify that C15.2 also neutralizes these activities of MSP, mouse peritoneal macrophages were isolated as described in Example 4 and resuspended in DMEM-0.1 % BSA. Experiments were carried out using 24-well BD BioCoat Matrigei Invasion Chambers (BD Biosciences). The lower chamber of a well was filled with 0.75 ml/well of DM EM containing 0.1 % BSA with or without 200 ng/ml hMSP +/- 10 pg/mi C15.2. Macrophages (5 x 10⁵ cells in 0.5 ml) or T-47D (ATCC HTB-133) human breast tumor cells (10⁵ cells in 0.5 ml) were placed in the upper chamber of a cell culture insert having an 8 μ,ηη pore-size membrane. The chamber was incubated at 37°C for 20 hours to allow the macrophage to migrate through the membrane from the upper chamber to the reverse surface of the membrane. The cells retained on the reverse surface of the membrane were then stained with Diff-Quik stain, and visualized by photomicroscopy. As seen in Figure 1 1 , compared to medium alone, human MSP (hMSP, 200 ng/ml) greatly increased the number of macrophage that migrated through the membrane, but this effect was strongly inhibited by addition of C 5.2 but not by negative control mouse mlgG. Similar results were observed with T47-D tumor cells.

[0065] Infiltration of macrophages into a tumor stimulates tumor progression and metastasis (Pollard, Nat Rev Cancer, 4:71 -78, 2004; Grugan et al., J Immunol 189:5457-66, 2012) so the ability of C15.2 and other antibodies of the invention to inhibit this activity contributes to their therapeutic efficacy in patients. [0066] Example 8: Generation of a rat rnAb that binds mouse MSP

For research purposes, a mAb that binds mouse MSP (mMSP) was generated as follows. A gene encoding a fusion protein of mMSP linked to human Ig Fc (mMSP-hFc) was constructed and expressed in 293F human kidney fibroblast cell line similarly to MSP-Fc described in Example 1 , using standard molecular biological methods; the mMSP-hFc protein was purified using a protein A column. Fisher rats (8-week old female) were immunized in each hind footpad once or twice per week with 10 pg of mMSP-Fc in Ribi adjuvant, a total of 17 to 19 times. The rats were then sacrificed and hybridomas generated as in Example 1 , For assays, niMSP-Kappa-Fiag (mMSP-kF), and murine Ron - human Fc (mRON-Fc) fusion proteins were made as in Example 1 . Hybridoma culture supernatants were screened for their ability to capture mMSP-kF, followed by their ability to block binding of mMSP-kF to mRON-Fc, using ELISA assays described in Example 9 below. Selected hybridomas were cloned by limiting dilution, and antibodies were purified from culture media by protein A affinity chromatography. Because of its desirable binding and blocking activities, a mAb designated as mMSP E2.3 or simply E2.3 was chosen for further study; it is of the rat lgG2b isotype.

[0067] Example 9: Binding and blocking properties of mMSP E2.3 mAb

[0068] To measure binding of mAbs to mMSP, EL!SA plates were coated with 2 pg/ml goat anti-hlgG-kappa overnight, blocked with 2% BSA, and then incubated with 0.2 pg/ml mMSP-kF followed by various concentrations of the anti-mMSP mAbs. The bound mAbs were detected by addition of HRP-mouse-anti-rat Ig (BD Pharmingen). Figure 12A shows that E2.3 (but not negative control rat IgG) strongly binds to mMSP in this ELISA assay.

[0069] To measure the ability of the anti-mMSP mAbs to block binding of mMSP to mRON, ELISA plates were coated with 2 pg/mi goat anti-hlgG-Fc and then incubated with mRon-Fc followed by various concentrations of the mAbs together with 0.1 pg/mi mMSP-kF. The amount of mMSP-kF was determined using HRP-anti-Fiag M2 antibody. Figure 12B shows that E2.3 (but not negative control rat IgG) inhibits (blocks) binding of mouse MSP to its receptor RON, with an IC50 of about 2 pg/mi.

[0070] To define the epitope of E2.3 on mMSP, binding of E2.3 to human MSP-Fc (hMSP-Fc), mMSP-Fc, the chimeric proteins HaM|3MSP-kF and MaH|3MSP-kF (described in Example 3), and the single chain form of mMSP (SCmMSP-kF; mMSP(R694C)-kF) was measured by an ELJSA, in which the reagents were respectively bound to a plate coated with goat-anti-hlgG-kappa, and then incubated with E2.3, which was then detected with HRP-mouse anti-rat !g. As seen in Figure 13, E2.3 bound to mMSP but not hMSP, and to HaMpMSP-kF but not MaHf»M8P-kF, indicating that E2.3 binds to the β chain but not a chain of mouse MSP. Interesting, E2.3 binds to the single chain (unprocessed) form of MSP, in contrast to C15.2.

[0071] Example 10: Neutralization of biological activities of mMSP by mMSP E2.3

[0072] The ability of E2.3 to block binding of mMSP to RON implies that it neutralizes the biological activities of mMSP, for example the ability to induce migration of macrophages. To verify this experimentally, mouse peritoneal macrophages were isolated and attracted through a membrane by mMSP, as in Example 7. Compared to medium alone, mMSP (200 ng/ml) greatly increased the number of macrophage that migrated through the membrane, but this effect was strongly inhibited by addition of 10 pg/ml E2.3, but not by negative control rat IgG (Figure 1 1 ). In another assay, E2.3 (10 pg/ml) inhibited the shape change in mouse macrophages induced by mMSP (200 ng/ml), in the same manner that C15.2 inhibits the shape changed induced by human MSP (Example 4 and Figure 8).

[0073] Example 1 1 : Cloning of mMSP E2.3 mAb genes

[0074] The light and heavy chain variable regions of the E2.3 mAb were cloned using standard methods of molecular biology, as the C15.2 genes were cloned in Example 6, but using primers suitable for cloning of mouse mAbs. The amino acid sequences of the (mature) light and heavy chain variable (V) regions of E2.3 are shown respectively in Figures 14A (SEQ ID NO:3) and 14B (SEQ !D NO:4). To produce E2.3 antibody, these V regions (after synthesis from their sequence if necessary) can be opera ively linked to rat lgG2b constant region genes in expression vectors and expressed in suitable mammalian cells such as 293 cells. Alternatively, the V regions can be linked to mouse or human constant regions to produce rat-mouse or rat-human chimeric antibodies respectively, using methods well-known in the art. In fact, these V regions were linked to human lgG1 constant regions in suitable expression vectors, which were then transfected into human 293 cells; the chimeric E2.3 mAb that was thus expressed bound to mMSP comparably to the E2.3 mAb itself, establishing that the correct V regions of E2.3 were cloned. Any of these forms can be used for experiments in immunodeficient mice such as nude mice.

[0075] Example 12: Ability of C15.2 plus E2.3 to inhibit growth of tumor xenografts

[0076] C15.2 inhibits the growth of human tumor xenografts in mice, e.g., DU145 human prostate tumor xenografts (Example 5 and Figure 8), but this inhibition is not complete. A potential reason for this is that while C15.2 can neutralize the activity of human MSP produced by the tumor cells, it cannot neutralize endogenous mouse MSP processed at the tumor site from pro-MSP produced by the mouse itself (as described under Background of the Invention). Therefore, to obtain stronger or even complete inhibition of the growth of xenografts, the xenograft models are performed as described in Example 5, but the mice are treated with both C15.2 and E2.3 so as to neutralize MSP of both human and mouse origin in the tumor. For this purpose and to achieve the best effect, it may be necessary to use higher dose levels of E2.3 than C15.2, for example 10 mg/kg, 25 mg/kg, 50 mg/kg or even 100 mg/kg of E2.3 per dose, because of the relatively large amount of unprocessed mouse MSP (pro-MSP) in the blood circulation of the mouse, which serves as a sink for the E2.3 mAb.

[0077] The ability of C15.2 plus E2.3 to strongly inhibit the growth of human xenografts in mice provides further evidence of the therapeutic efficacy of C15.2 (and other mAbs of the invention such as humanized forms of C15.2) in human patients, because in humans such a mAb is by itself capable of neutralizing MSP from all sources.

[0078] Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the invention. Unless otherwise apparent from the context any step, element, embodiment, feature or aspect of the invention can be used with any other. All publications, patents and patent applications including accession numbers and the like cited are herein incorporated by reference in their entirety for ail purposes to the same extent as if each individual publication, patent and patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. If more than one sequence is associated with an accession number at different times, the sequence associated with the accession number as of the effective filing date of this application is intended, the effective filing date meaning the actual filing date or earlier date of a filing of a priority application disclosing the accession number in question.

Claims

We claim:

1 . A genetically engineered monoclonal antibody (mAb) that binds and neutralizes human Macrophage Stimulating Protein (MSP).

2. The mAb of claim 1 which fully competes for binding to human MSP with a C15.2 mAb, where the C15.2 mAb has the light chain variable region sequence of Figure 10A (SEQ ID NO:1 ) and the heavy chain variable region sequence of Figure 10B (SEQ ID NO:2), wherein fully competes means that the IC50 of the mAb to inhibit binding of C15.2 is within 3-fold of the IC50 of C15.2 to inhibit binding of itself in a competitive binding assay.

3. The mAb of claim 1 that binds to an epitope on MSP comprising amino acids in the a-chain and the β-chain of MSP.

4. The mAb of claim 1 which does not bind human pro-MSP.

5. The mAb of claim 1 which is humanized.

6. The mAb of claim 1 which is human.

7. The mAb of claim 1 which inhibits binding of MSP to its cellular receptor

RON.

8. The mAb of claim 1 which neutralizes ail biological activities of MSP.

9. The mAb of claim 1 which inhibits growth of a human tumor xenograft in a mouse.

10. The mAb of claim 9 wherein the tumor xenograft is a xenograft of the DU145 human prostate tumor ceil line or the HCC-2998 colon tumor ceil line.

1 1 . The mAb of claim 1 which is a bispecific antibody.

12. The mAb of claim 1 which is a Fab or F(ab')₂ fragment or single-chain antibody.

13. An anti-MSP monoclonal antibody (mAb) comprising a light chain variable region having three CDRs from the light chain variable region sequence of C15.2 in Figure 10A (SEQ ID NO:1 ) and a heavy chain variable region having three CDRs from the heavy chain variable region sequence of C15.2 in Figure 10B (SEQ ID NO:2).

14. The mAb of claim 13 which is chimeric or humanized.

15. A cell line producing a mAb of claim 1 .

16. A cell line producing a mAb of claim 13.

17. A pharmaceutical composition comprising a mAb of claim 1 .

18. A pharmaceutical composition comprising a mAb of claim 13.

19. A method of treating cancer in a patient comprising administering to the patient a pharmaceutical composition comprising a neutralizing anti-MSP mAb.

20. The method of claim 19, wherein the mAb is a mAb of claim 2.

21 . A monoclonal antibody that binds and neutralizes mouse Macrophage Stimulating Protein (mMSP).