HK1171032B - Anti-hepsin antibodies and methods using same - Google Patents

Description

anti-HEPSIN antibodies and methods of use thereof

Cross reference to related applications

This application claims the benefit of U.S. provisional application No.61/253,953 filed on day 22/10/2009, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates generally to the field of molecular biology. More specifically, the invention concerns anti-hepsin antibodies, and uses thereof.

Background

hepsin is a type II transmembrane serine protease (TTSP) expressed on the surface of epithelial cells. The 417 amino acid protein comprises a short N-terminal cytoplasmic domain, a transmembrane domain and a single scavenger receptor cysteine-rich domain that is tightly packed with a C-terminal protease domain (Somoza et al (2003) Structure 11(9), 1123-1131). The physiological function of hepsin is unclear. Although it is expressed very early in embryogenesis (Vu et al (1997) J Biol Chem 272(50),31315-31320), hepsin deficient mice are viable and develop normally (Yu et al (2000) Thromb Haemost 84(5),865-870; Wu et al (1998) JClin Invest 101(2), 321-326). Hepsin was found not to be critical for liver regeneration and for coagulation-related physiological functions (supra). A recent study demonstrated that hepsin knockout mice are hearing impaired (Guipponi et al (2007) Am J Pathol 171: 608-616). However, hepsin is implicated in the ovary [ (Tanimoto et al (1997) Cancer Res 57(14),2884-2887); WO2001/62271] and prostate Cancer. Several gene expression studies identified hepsin as one of the most highly induced genes in prostate Cancer (Dhanasekaran et al (2001) Nature 412,822-826; Luo et al (2001) Cancer Res61(12),4683-4688; Magee et al (2001) Cancer Res61 (15),5692-5696; Stamey et al (2001) J Urol 166(6),2171-2177; Stephan et al (2004) J Urol 171(1),187-191; Welsh et al (20010) Cancer Res61 (16), 5974-5978). Hepsin RNA levels were found to be lower in normal prostate and benign hyperplasia, but strongly elevated in prostate Cancer, particularly in advanced stages ((Dhanasekaran et al (2001) Nature 412,822-826; Luo et al (2001) Cancer Res61(12),4683-4688; Magee et al (2001) Cancer Res61 (15),5692-5696; Stamey et al (2001) J Urol 166(6), 2171-7; Stephan et al (2004) J Urol 171(1),187-191; Welsh et al (20010) Cancer Res61 (16), 5974-5978.) hepsin protein staining with monoclonal anti-hepsin antibodies showed highest hepsin expression (xulan (2006) Cancer Res 66(7),3611-3619) in primary tumors at sites of bone metastasis and in advanced stages consistent with increased tumor progression ((Luhaser et al) RNA levels 2001 and Cancer grade) (Luraso et 12),4683-4688, Magee et al (2001) Cancer Res61 (15),5692-5696, Stamey et al (2001) J Urol 166(6),2171-2177, Stephan et al (2004) JUrol 171(1),187-191, Chen et al (2003) J Urol 169(4), 1316-1319.

Experimental evidence for the role of hepsin in prostate Cancer comes from a study by Klezovitch et al (2004) Cancer Cell 6(2),185-195), which demonstrated that overexpression of hepsin leads to primary tumor progression and metastasis in a mouse model of non-metastatic prostate Cancer. Interestingly, hepsin overexpression was associated with basement membrane disruption (supra), suggesting the possibility that hepsin activity is somehow linked to degradation of basement membrane components. In vitro, hepsin is able to convert the latent growth factor pro-hepatocyte growth factor (pro-HGF) into its active double-stranded form (HGF), which induces Met receptor signaling (Herter et al (2005) Biochem J390 (Pt 1),125-136; Kirchhofer et al (2005) FEBS Lett 579(9),1945-1950; WO 2006/014928). hepsin is also capable of converting pro-uPA to its active form (Moran et al, (2006) J Biol chem.281(41):30439-46), and cleaving lamin in vitro (Tripathhi et al (2008) J Biol chem.283: 30576). Because the HGF/Met pathway is involved in invasive tumor growth and metastasis, it is likely that overexpression of hepsin activates the HGF/Met axis in prostate cancer (Herter et al (2005) Biochem J390 (Pt 1),125-136; Kirchhofer et al (2005) FEBS Lett 579(9),1945-1950; WO 2006/014928). hepsin has also been shown to cleave other substrates in vitro, mainly coagulation-related proteins (Herter et al, id; Kazama et al (1995) JBiol Chem 270(1), 66-72). However, their role in tumorigenesis is unknown.

It is clear that there is still a need for agents with clinical properties that are most suitable for development into therapeutic agents. The invention described herein satisfies this need and provides other benefits.

All references, including patent applications and publications, cited herein are hereby incorporated by reference in their entirety.

Summary of The Invention

The present invention is based, in part, on the identification of various hepsin binders, such as antibodies, and fragments thereof. hepsin represents an important and advantageous therapeutic target, and the present invention provides compositions and methods based on the binding of agents to hepsin. As described herein, hepsin conjugates of the invention provide important therapeutic and diagnostic agents for use in targeting pathological conditions associated with expression and/or activity of hepsin signaling pathways. Thus, the present invention provides methods, compositions, kits, and articles of manufacture relating to hepsin binding.

The active site of trypsin-like serine proteases, such as hepsin, is formed by several loops ("activation domains") that are intrinsically active (Huber and Bode, 1978). In particular, the 220 loop forms part of the S1 pocket and can adopt a variety of conformational states in some serine proteases (Johnson et al, 2005; Shia et al, 2005; Spraggon et al, 2009; Wilken et al, 2004). However, the co-crystal structure of serine proteases with active site inhibitors shows a correctly formed active site, most likely due to the stabilizing forces exerted by the inhibitors (Arni et al, 1994; Shia et al, 2005; Spraggon et al, 2009). It is theorized that the occupancy of the S1 pocket by the serpin exerts a stabilizing force on the flexibility of the serpin active site loop, facilitating antibody recognition of the serpin active site. Thus, to identify anti-hepsin antibodies that block hepsin enzymatic activity, antibodies were obtained that bound hepsin complexed with the serine protease inhibitor 3, 4-dichloro-isocoumarin (DCI), which occupied the S1 pocket.

The invention provides antibodies that bind hepsin. In one aspect, the invention features an isolated antibody that binds hepsin.

In one aspect, the invention provides an isolated anti-hepsin antibody, wherein a monovalent form of the antibody (such as the Fab form) specifically binds human hepsin with a binding affinity of about 10nM or better. In some embodiments, the antibody specifically binds human hepsin with a binding affinity of about 6nM or better. As is well established in the art, the binding affinity of a ligand to its receptor can be determined using a variety of assays and expressed in a variety of quantitative values. Thus, in one embodiment, the binding affinity is expressed as a Kd value and reflects the intrinsic binding affinity (e.g., with minimized affinity effects). Generally and preferably, binding affinity is measured in vitro, whether in a cell-free background or a cell-associated background. Binding affinity measurements can be obtained using any of a variety of assays known in the art, including the assays described herein, including, for example, Biacore, Radioimmunoassay (RIA), and ELISA.

In another aspect, the invention provides anti-hepsin antibodies that bind to the Kunitz (Kunitz) domain binding region in hepsin. In one aspect, the invention provides an isolated anti-hepsin antibody that competes with kunitz domain for binding to hepsin. In one embodiment, the Kunitz domain sequence is the Kunitz domain 1 of HAI-1 or HAI-1B (KD 1). In one embodiment, the kunitz domain sequence is a variant KD1 sequence having at least about 70%,75%,80%,85%,90%,95%,97%,98%,99% sequence identity to wild-type KD1 of human HAI-1, wherein the variant sequence has at least a comparable ability to wild-type KD1 to inhibit hepsin activity. In one embodiment, the kunitz domain sequence is one or both of the kunitz domains of HAI-2. In one embodiment, the variant HAI-2 kunitz domain sequence has about 70% to 99%, about 75% to 98%, about 80% to 97%, 85% to 95% sequence identity to a corresponding kunitz domain of wild-type human HAI-2, wherein the sequence has at least a comparable ability to inhibit hepsin activity as wild-type HAI-2.

In one aspect, the invention provides anti-hepsin antibodies that bind hepsin catalytic site.

In one aspect, the invention provides anti-hepsin antibodies that bind hepsin outside of the s1 subsite. In some embodiments, the antibody binds hepsin s2 and/or s3 subsite.

In one aspect, the invention provides anti-hepsin antibodies that bind catalytically inactivated hepsin.

In one aspect, the invention provides anti-hepsin antibodies that are resistant to hepsin proteolysis. In some embodiments, the antibody is exposed to hepsin for 24 hours under conditions that allow hepsin to cleave hepsin substrate.

In one aspect, the invention provides anti-hepsin antibodies that bind hepsin present in a complex comprising hepsin and a serine protease inhibitor that binds hepsin S1 subsite. In some embodiments, hepsin present in the complex is inactivated. In some embodiments, the serine protease inhibitor is 3,4-dichloro-isocoumarin (3, 4-dichoro-isocoumarin, DCI). In some embodiments, the serine protease inhibitor binds to hepsin catalytic amino acid residues Ser195 and His57, whereby hepsin is inactivated.

In one aspect, the invention provides anti-hepsin antibodies that specifically bind human hepsin and substantially inhibit hepsin enzymatic activity in vivo and/or in vitro. In one embodiment, the enzymatic activity comprises cleavage of a polypeptide substrate by hepsin. In one embodiment, the polypeptide substrate of hepsin is one or more of pro-macrophage stimulating protein (pro-MSP), pro-uPA, factor VII, and pro-HGF. pro-MSP activation by hepsin is described in co-pending, commonly owned U.S. provisional patent application No.61/253,990, filed 10/22/2009. In one embodiment, the enzymatic activity comprises cleavage of a synthetic substrate by hepsin. In some embodiments, the hepsin synthetic substrate is a substrate shown in table 1.

In one aspect, the invention provides anti-hepsin antibodies, wherein the antibodies substantially inhibit human and/or mouse hepsin catalytic activity. In some embodiments, a monovalent form of an anti-hepsin antibody inhibits human hepsin catalytic activity with a Ki of about (in some embodiments, less than or equal to) 4 nM. In some embodiments, a monovalent form of an anti-hepsin antibody inhibits mouse hepsin catalytic activity with a Ki of about (in some embodiments, less than or equal to) 330 nM.

In one aspect, the invention provides an anti-hepsin antibody, wherein the antibody substantially inhibits hepsin cleavage of pro-uPA. In some embodiments, the anti-hepsin antibody inhibits pro-uPA cleavage by hepsin with an IC50 of about 3nM or greater.

In one aspect, the invention provides anti-hepsin antibodies, wherein the antibodies substantially inhibit laminin-dependent cell migration.

In one aspect, the invention provides anti-hepsin antibodies, wherein the antibodies are produced by a method comprising selecting an antibody that binds to a complex comprising (a) hepsin and (b) a serine protease inhibitor that binds hepsin S1 subsite. In some embodiments, hepsin present in the complex is inactivated. In some embodiments, the serine protease inhibitor is 3, 4-dichloro-isocoumarin (DCI). In some embodiments, the serine protease inhibitor binds to hepsin catalytic amino acid residues Ser195 and His 57, whereby hepsin is inactivated. In some embodiments, the antibody is incubated with hepsin and a serine protease inhibitor prior to selecting the antibody. In some embodiments, the method further comprises the step of selecting an antibody that competes with the kunitz domain for hepsin binding. In some embodiments, the kunitz domain is KD 1.

In one aspect, the invention provides anti-hepsin antibodies, wherein the antibodies are generated by a method comprising selecting (identifying) antibodies that compete with kunitz domains for hepsin binding. In some embodiments, the kunitz domain is KD 1.

In one aspect, the invention provides anti-hepsin antibodies that are not substantially cleaved by hepsin. In some embodiments, the anti-hepsin antibody is substantially resistant to hepsin cleavage.

In general, the anti-hepsin antibodies of the invention are antagonist antibodies.

In one aspect, the invention provides an anti-hepsin antibody comprising at least one, two, three, four, five, and/or six hypervariable region (HVR) sequences selected from the group consisting of:

(a) HVR-L1, comprising sequence RASQSVSSAVA (SEQ ID NO:1),

(b) HVR-L2, comprising the sequence SASSLYS (SEQ ID NO:2),

(c) HVR-L3, comprising sequence QQYYSSYYLLT (SEQ ID NO:3),

(d) HVR-H1, comprising sequence GFNFSYSYMH (SEQ ID NO:4),

(e) HVR-H2 comprising sequence ASIYSYYGSTYYADSVKG (SEQ ID NO:5), and

(f) HVR-H3, comprising sequence ARSDSWSYKSGYTQKIYSKGLDY (SEQ ID NO: 6).

In one aspect, the invention provides an anti-hepsin antibody comprising (a) a light chain comprising (i) HVR-L1 comprising sequence RASQSVSSAVA (SEQ ID NO: 1); (ii) HVR-L2 comprising the sequence SASSLYS (SEQ ID NO: 2); and (iii) HVR-L3, comprising sequence QQYYSSYYLLT (SEQ ID NO: 3); and/or (b) a heavy chain comprising (i) HVR-H1, comprising sequence GFNFSYSYMH (SEQ ID NO: 4); (ii) HVR-H2, comprising sequence ASIYSYYGSTYYADSVKG (SEQ ID NO: 5); and (iii) HVR-H3, comprising sequence ARSDSWSYKSGYTQKIYSKGLDY (SEQ ID NO: 6).

In one aspect, the invention provides an anti-hepsin antibody comprising HVR-L1, HVR-L1 comprising sequence SEQ ID NO: 1. In one aspect, the invention provides an anti-hepsin antibody comprising HVR-L2, HVR-L2 comprising sequence SEQ ID NO. 2. In one aspect, the invention provides an anti-hepsin antibody comprising HVR-L3, HVR-L3 comprising sequence SEQ ID NO. 3. In one aspect, the invention provides an anti-hepsin antibody comprising a HVR-H1 region, said HVR-H1 region comprising the sequence SEQ ID NO. 4. In one aspect, the invention provides an anti-hepsin antibody comprising a HVR-H2 region, said HVR-H2 region comprising the sequence SEQ ID NO: 5. In one aspect, the invention provides an anti-hepsin antibody comprising a HVR-H3 region, said HVR-H3 region comprising the sequence SEQ ID NO 6.

In one aspect, an anti-hepsin antibody comprises a light chain variable region comprising HVR-L1, HVR-L2, HVR-L3, each comprising, in order, the sequences RASQDVN/STAVA (SEQ ID NO:7), SEQ ID NOS: 2, 3, and/or a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, each comprising, in order, SEQ ID NOS: 4, 5, 6.

In one aspect, an anti-hepsin antibody comprises a light chain variable region comprising HVR-L1, HVR-L2, HVR-L3, each comprising, in order, the sequence RASQDVN/STAVA (SEQ ID NO:7), SEQ ID NO:1, the sequence SASFLYS (SEQ ID NO:8), SEQ ID NO:3, and/or a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, each comprising, in order, SEQ ID NO:4, 5, 6.

In one aspect, an anti-hepsin antibody comprises a light chain variable region comprising HVR-L1, HVR-L2, HVR-L3, each comprising in sequence SEQ ID NOS 7, 8, 3, and/or a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, each comprising in sequence SEQ ID NOS 4, 5, 6.

Amino acid sequences SEQ ID NOS: 1-6 are numbered for each HVR (i.e., H1, H2, or H3) as shown in FIG. 1 in a manner consistent with the Kabat numbering system described below.

Antibodies of the invention may comprise any suitable variable domain framework sequence provided that binding affinity for hepsin is substantially retained. For example, in some embodiments, an antibody of the invention comprises a human subgroup III heavy chain framework consensus sequence. In one embodiment of these antibodies, the framework consensus sequence comprises a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is a, position 73 is T, and/or position 78 is a. In one embodiment, the antibodies comprise huMAb4D5-8 (h)Genentech，Inc.，South San Francisco, CA, USA) (in U.S. Pat. No.6,407,213&5,821,337, and Lee et al, J.mol.biol. (2004), 340(5): 1073-1093). In one embodiment, these antibodies further comprise a human kappa I light chain framework consensus sequence. In some embodiments, the framework consensus sequence comprises a substitution at position 66. In some embodiments, position 66 is G. In a specific embodiment, these antibodies comprise the light chain HVR sequence of huMAb4D5-8, e.g., U.S. Pat. No.6,407,213 &5,821,337. In one embodiment, the antibodies comprise huMAb4D5-8 (h)Genentech, Inc., South San Francisco, Calif., USA) (in U.S. Pat. No.6,407,213&5,821,337, and Lee et al, J.mol.biol. (2004), 340(5): 1073-1093).

In one embodiment, the antibody of the invention comprises a heavy chain variable domain, wherein the framework sequence comprises the sequences disclosed in figures 2A-B, and the HVR H1, H2, and H3 sequences are SEQ ID NOs 4, 5, and/or 6, respectively.

In one embodiment, the antibody of the invention comprises a heavy chain variable domain wherein the framework sequence comprises the sequence of SEQ ID NOs 14-15, 48, and/or 16 and the HVR H1, H2, and H3 sequences are the sequences of SEQ ID NOs 4, 5, and/or 6, respectively. In another embodiment, the framework sequences comprise the sequences of SEQ ID NOS 14-15, 43, and/or 16 and the HVR H1, H2, and H3 sequences are SEQ ID NOS 4, 5, and/or 6, respectively.

In a particular embodiment, the antibody of the invention comprises a light chain variable domain wherein the framework sequences comprise SEQ ID NOs 17-20; 49-51 and 20; 52-54 and 20; and/or 55-57 & 20, and the HVR L1, L2, and L3 sequences are SEQ ID NOs: 1, 2, and/or 3, respectively. In another embodiment, an antibody of the invention comprises a light chain variable domain wherein the framework sequence comprises the sequence of SEQ ID NOs 17-18, 58 and/or 20 and the HVR L1, L2, and L3 sequences are SEQ ID NOs 1, 2, and/or 3, respectively. In another embodiment, the antibody of the invention comprises a light chain variable domain, wherein the framework sequence comprises the sequence of SEQ ID NOs 17, 18, 19 and/or 20, and the HVR L1, L2, and L3 sequences are SEQ ID NOs 1, 2, and/or 3, respectively.

In one embodiment, the antibody of the invention comprises a heavy chain variable domain in which the framework sequences comprise the sequences of SEQ ID NOs 14-15, 43 and/or 16 and the HVRH1, H2 and H3 sequences are SEQ ID NOs 4, 5 and/or 6, respectively, and a light chain variable domain in which the framework sequences comprise the sequences of SEQ ID NOs 17-18, 58 and/or 20 and the HVR L1, L2 and L3 sequences are SEQ ID NOs 1, 2 and/or 3, respectively.

In another aspect, the antibody of the invention comprises a heavy chain variable domain comprising the sequence SEQ ID NO. 10 and/or a light chain variable domain comprising the sequence SEQ ID NO. 9.

In another aspect, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO. 10 and a light chain variable region.

In another aspect, the antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO 9 and a heavy chain variable domain.

Some embodiments of the antibodies of the invention comprise humanized 4D5 antibody (huMAb4D5-8) ((R))The light chain variable domain of Genentech, Inc., South San Francisco, Calif., USA) (also mentioned in U.S. Pat. Nos. 6,407,213 and Lee et al, J.mol.biol. (2004), 340(5): 1073-1093), as depicted in SEQ ID NO:11 below.

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser GlyVal Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107(SEQ ID NO:11)

(HVR residues underlined)

In one embodiment, the huMAb4D5-8 light chain variable domain sequence is modified at one or more of positions 30, 66, and 91 (Asn, Arg, and His, respectively, as indicated above in bold/italics). In one embodiment, the modified huMAb4D5-8 sequence comprises Ser at position 30, Gly at position 66, and/or Ser at position 91. Thus, in one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence shown in SEQ ID NO:45 below:

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser GlyVal Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107(SEQ ID NO:45)

(HVR residues are underlined).

In one aspect, the invention provides an anti-hepsin antibody that competes for binding to hepsin with any of the antibodies described above. In one aspect, the invention provides an anti-hepsin antibody that binds the same or a similar epitope on hepsin as any of the antibodies described above.

As known in the art, and as described in more detail below, the amino acid positions/boundaries that describe the hypervariable regions of an antibody can vary, depending on the context and various definitions known in the art (as described below). Some positions within a variable domain can be considered hybrid hypervariable positions in that those positions can be considered within a hypervariable region under one set of criteria and outside of the hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions (as further defined below).

In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is selected from the group consisting of: chimeric antibodies, affinity matured antibodies, humanized antibodies and human antibodies. In certain embodiments, the antibody is an antibody fragment. In some embodiments, the antibody is Fab, Fab '-SH, F (ab')₂Or scFv.

In one embodiment, the antibody is a chimeric antibody, e.g., an antibody comprising an antigen binding sequence from a non-human donor, and which sequence is grafted to a heterologous non-human, or humanized sequence (e.g., a framework and/or constant domain sequence). In one embodiment, the non-human donor is a mouse. In another embodiment, the antigen binding sequence is synthetic, such as by mutagenesis (e.g., phage display screen, etc.). In a specific embodiment, the chimeric antibody of the invention has a murine V region and a human C region. In one embodiment, the murine light chain V region is fused to a human kappa (κ) light chain. In another embodiment, the murine heavy chain V region is fused to a human IgG1C region.

Humanized antibodies of the invention include those affinity matured variants having amino acid substitutions in the Framework Regions (FR) and altered in the grafted CDRs. The substituted amino acids in the CDR or FR are not limited to those present in the donor or acceptor antibody. In other embodiments, the antibodies of the invention further comprise amino acid residue alterations in the Fc region that result in improved effector function (including enhanced CDC and/or ADCC function and B cell killing). Other antibodies of the invention include those with specific modifications that enhance stability. In other embodiments, the antibodies of the invention comprise an alteration of an amino acid residue in the Fc region that results in reduced effector function (e.g., reduced CDC and/or ADCC function and/or reduced B-cell killing). In some embodiments, the antibodies of the invention are characterized by reduced binding to human complement factor C1q and/or to human Fc receptors on Natural Killer (NK) cells. In some embodiments, the antibodies of the invention are characterized by reduced binding (such as loss of binding) to human Fc γ RI, Fc γ RIIA, and/or Fc γ RIIIA. In some embodiments, the antibody of the invention is of the IgG class (e.g., IgG1 or IgG 4) and comprises at least one mutation in E233, L234, G236, D265, D270, N297, E318, K320, K322, a327, a330, P331, and/or P329 (numbering according to the EU index). In some embodiments, the antibody comprises the mutations L234A/L235A or D265A/N297A.

In one aspect, the invention provides a hepsin binding polypeptide comprising any of the antigen binding sequences provided herein, wherein the hepsin binding polypeptide specifically binds hepsin, e.g., human and/or cynomolgus and/or mouse hepsin.

The antibodies of the invention bind (such as specifically bind) hepsin, and in some embodiments, can modulate (e.g., inhibit) one or more of the following: disruption of hepsin activity (such as hepsin enzymatic activity) and/or any biologically important hepsin and/or hepsin polypeptide substrate biological pathway, and/or treatment and/or prevention of a tumor, cell proliferative disorder or cancer; and/or treatment or prevention of a condition associated with hepsin expression and/or activity, such as increased hepsin expression and/or activity. In some embodiments, the hepsin antibody specifically binds a polypeptide consisting of or consisting essentially of hepsin (e.g., human and/or mouse hepsin). In one embodiment, hepsin enzymatic activity comprises cleavage of a polypeptide substrate by hepsin. In one embodiment, the polypeptide substrate of hepsin is one or more of pro-macrophage stimulating protein (pro-MSP), pro-uPA (pro-uPA), factor VII, and pro-HGF.

In one embodiment, the antibody of the invention is not an anti-hepsin antibody as described in Cancer Research volume 66, pages 3611-3619 published 2006 (e.g., antibodies 1a12, 85B11, 94a7, A6, a174, a21 and/or a24 as exemplified in figure 4), or an isolated hepsin antibody as disclosed in PCT publication WO2004/033630 (e.g., antibodies 47A5, 14C7, 46D12, 38E2, 37G10, 31C1, 11C1 and/or 72H6 as mentioned at page 93 and in figures 15A-D), or an Xuan et al (2006) Cancer Res 66(7), an isolated hepsin antibody as disclosed in 3611, or an isolated hepsin antibody as disclosed in WO 2007/149932.

In one embodiment, an antibody of the invention does not compete for binding to human hepsin with: an anti-hepsin antibody described in Cancer Research volume 66, pages 3611-3619, published 2006 (e.g., antibodies 1a12, 85B11, 94a7, a6, a174, a21 and/or a24, exemplified in figure 4), or an isolated hepsin antibody disclosed in PCT publication WO2004/033630 (e.g., antibodies 47a5, 14C7, 46D12, 38E2, 37G10, 31C1, 11C1 and/or 72H6, mentioned at page 93 and in figures 15A-D), or Xuan et al (2006) Cancer Res 66(7), an isolated hepsin antibody disclosed in 3611, or an isolated hepsin antibody disclosed in WO 2007/149932.

In one embodiment, the antibodies of the invention do not bind to the same epitope on human hepsin as the following antibodies: an anti-hepsin antibody described in Cancer Research volume 66, pages 3611-3619 published 2006 (e.g., antibodies 1a12, 85B11, 94a7, a6, a174, a21 and/or a24 illustrated in figure 4), or an isolated hepsin antibody disclosed in PCT publication WO2004/033630 (e.g., antibodies 47a5, 14C7, 46D12, 38E2, 37G10, 31C1, 11C1 and/or 72H6 mentioned at page 93 and in figures 15A-D), or Xuan et al (2006) Cancer Res 66(7), an isolated hepsin antibody disclosed in 3611, or a hepsin antibody disclosed in WO 2007/149932)

In one aspect, the invention provides a composition comprising one or more antibodies of the invention and a carrier. In one embodiment, the carrier is pharmaceutically acceptable.

In another aspect, the invention provides nucleic acids encoding the anti-hepsin antibodies of the invention.

In yet another aspect, the invention provides a vector comprising a nucleic acid of the invention.

In another aspect, the invention provides compositions comprising one or more of the nucleic acids of the invention and a vector. In one embodiment, the carrier is pharmaceutically acceptable.

In one aspect, the invention provides a host cell comprising a nucleic acid or vector of the invention. The vector may be of any type, e.g., a recombinant vector, such as an expression vector. Any of a variety of host cells may be used. In one embodiment, the host cell is a prokaryotic cell, such as E.coli. In another embodiment, the host cell is a eukaryotic cell, e.g., a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell.

In another aspect, the invention provides methods of making the antibodies of the invention. For example, the invention provides methods of making anti-hepsin antibodies (which as defined herein include full-length antibodies and fragments thereof) comprising culturing a host cell comprising a nucleic acid encoding a humanized antibody such that the nucleic acid is expressed. In some embodiments, the above methods further comprise recovering the antibody from the host cell culture. In some embodiments, the antibody is recovered from the host cell culture broth. In some embodiments, the method further comprises combining the recovered antibody with a pharmaceutically acceptable carrier, excipient, or vehicle to prepare a pharmaceutical formulation comprising the humanized antibody. In some embodiments, the invention provides methods of generating an anti-hepsin antibody, said method comprising selecting an antibody that binds to a complex comprising (a) hepsin and (b) a serine protease inhibitor that binds hepsin S1 subsite. In some embodiments, hepsin present in the complex is inactivated. In some embodiments, the serine protease inhibitor is 3, 4-dichloro-isocoumarin (DCI). In some embodiments, the serine protease inhibitor binds to hepsin catalytic amino acid residues Ser195 and His 57, whereby hepsin is inactivated. In some embodiments, the antibody is incubated with hepsin and a serine protease inhibitor prior to selecting the antibody. In some embodiments, the method further comprises the step of selecting an antibody that competes with the kunitz domain for hepsin binding. In some embodiments, the kunitz domain is KD 1.

In one aspect, the present disclosure provides an article comprising a container; and a composition contained within the container, wherein the composition comprises one or more hepsin antibodies of the invention. In one embodiment, the composition comprises a nucleic acid of the invention. In another embodiment, the composition comprising the antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, the article of manufacture of the invention further comprises instructions (such as instructions for any of the methods described herein) for administering a composition (e.g., an antibody) to an individual.

In another aspect, the invention provides a kit comprising a first container comprising a composition comprising one or more anti-hepsin antibodies of the invention; and a second container containing a buffer. In one embodiment, the buffer is pharmaceutically acceptable. In one embodiment, the composition comprising the antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In another embodiment, the kit further comprises instructions for administering a composition (e.g., an antibody) to the individual.

The hepsin pathway is involved in a variety of biological and physiological functions, including, for example, activation of the HGF/c-met pathway, activation of the MSP/Ron pathway, basement membrane disruption/degradation, matrix degradation, and the like. These functions, in turn, are often dysregulated in disorders such as cancer. Thus, in another aspect, the invention provides a method of inhibiting basement membrane destruction/degradation and/or matrix degradation, comprising contacting a cell or tissue with an antagonist of the invention, whereby basement membrane destruction/degradation and/or matrix degradation is inhibited. In yet another aspect, the invention provides methods of inhibiting basement membrane disruption/degradation and/or matrix degradation comprising administering an antagonist of the invention to a cell, tissue, and/or subject having a condition associated with abnormal basement membrane disruption/degradation and/or matrix degradation, whereby basement membrane disruption/degradation and/or matrix degradation is inhibited.

In one aspect, the present invention provides a method for treating or preventing a disorder associated with elevated hepsin activity, said method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the present invention, thereby effectively treating or preventing said disorder. In one embodiment, the disorder is cancer.

In a further aspect, the invention provides the use of an anti-hepsin antibody of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder. In some embodiments, the cancer, tumor, and/or cell proliferative disorder is prostate cancer. In some embodiments, the cancer, tumor, and/or cell proliferative disorder is ovarian or renal cancer.

In one aspect, the invention provides the use of a nucleic acid of the invention in the preparation of a medicament for the therapeutic and/or prophylactic treatment of a disorder (such as a cancer, a tumor, and/or a cell proliferative disorder). In some embodiments, the cancer, tumor, and/or cell proliferative disorder is prostate cancer. In some embodiments, the cancer, tumor, and/or cell proliferative disorder is ovarian or renal cancer.

In another aspect, the invention provides the use of an expression vector of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder (such as a cancer, a tumor, and/or a cell proliferative disorder). In some embodiments, the cancer, tumor, and/or cell proliferative disorder is prostate cancer. In some embodiments, the cancer, tumor, and/or cell proliferative disorder is ovarian or renal cancer.

In yet another aspect, the invention provides the use of a host cell of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder (such as a cancer, a tumor, and/or a cell proliferative disorder). In some embodiments, the cancer, tumor, and/or cell proliferative disorder is prostate cancer. In some embodiments, the cancer, tumor, and/or cell proliferative disorder is ovarian or renal cancer.

In a further aspect, the invention provides the use of an article of manufacture of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder. In some embodiments, the cancer, tumor, and/or cell proliferative disorder is prostate cancer. In some embodiments, the cancer, tumor, and/or cell proliferative disorder is ovarian or renal cancer.

In one aspect, the invention also provides the use of a kit of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder (such as a cancer, a tumor, and/or a cell proliferative disorder). In some embodiments, the cancer, tumor, and/or cell proliferative disorder is prostate cancer. In some embodiments, the cancer, tumor, and/or cell proliferative disorder is ovarian or renal cancer.

The present invention provides methods and compositions (.

The methods of the invention may be used to affect any suitable pathological condition. Exemplary conditions are described herein, and include cancers selected from the group consisting of: non-small cell lung cancer, ovarian cancer, thyroid cancer, testicular cancer, endometrial cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), brain cancer (e.g., neuroblastoma or meningioma), skin cancer (e.g., melanoma, basal cell carcinoma, or squamous cell carcinoma), bladder cancer (e.g., transitional cell carcinoma), breast cancer, gastric cancer, colorectal cancer (CRC), hepatocellular carcinoma, cervical cancer, lung cancer, pancreatic cancer, prostate cancer, and renal cancer, and endometrial cancer.

In one embodiment, the cells targeted by the methods of the invention are cancer cells. For example, the cancer cell may be a cancer cell selected from the group consisting of: breast cancer cells, colorectal cancer cells, lung cancer cells (e.g., non-small cell lung cancer cells), thyroid cancer cells, multiple myeloma cells, testicular cancer cells, papillary cancer cells, colon cancer cells, pancreatic cancer cells, ovarian cancer cells, cervical cancer cells, central nervous system cancer cells, osteogenic sarcoma cells, kidney cancer cells, hepatocellular carcinoma cells, bladder cancer cells (e.g., transitional cell carcinoma cells), gastric cancer cells, head and neck squamous cancer cells, melanoma cells, leukemia cells, endometrial cancer cells, and colon adenoma cells. In one embodiment, the cells targeted by the methods of the invention are highly proliferative and/or proliferative cells. In another embodiment, the cells targeted by the methods of the invention are dysplastic cells. In yet another embodiment, the cells targeted by the methods of the invention are metastatic cells.

The methods of the invention may further comprise additional therapeutic steps. For example, in one embodiment, the method further comprises the step of exposing the targeted cells and/or tissues (e.g., cancer cells) to radiation treatment (radiation treatment) or a chemotherapeutic agent.

In one aspect, the invention provides methods comprising administering an effective amount of an anti-hepsin antibody in combination with an effective amount of another therapeutic agent, such as an anti-angiogenic agent, another antibody, a chemotherapeutic agent, a cytotoxic agent, an immunosuppressive agent, a prodrug, a cytokine, cytotoxic radiotherapy, a steroid, an antiemetic, a cancer vaccine, an analgesic, or a growth inhibitory agent. For example, anti-hepsin antibodies are used in combination with anti-cancer or anti-angiogenic agents to treat various neoplastic or non-neoplastic conditions.

Depending on the particular cancer indication to be treated, the combination therapy of the present invention may be combined with another therapeutic agent such as a chemotherapeutic agent or another therapy such as radiation therapy or surgery. Many known chemotherapeutic agents may be used in the combination therapy of the present invention. Preferably, chemotherapeutic agents will be used that are standard treatments for the particular indication. The dose or frequency of each therapeutic agent to be used in combination is preferably the same as or less than the dose or frequency of the corresponding agent when used in the absence of the other agent.

In another aspect, the invention provides any of the anti-hepsin antibodies described herein, wherein the anti-hepsin antibody comprises a detectable label.

In another aspect, the invention provides a complex of any of the anti-hepsin antibodies described herein with hepsin. In some embodiments, the complex is in vivo or in vitro. In some embodiments, the complex comprises a cancer cell. In some embodiments, the anti-hepsin antibody is detectably labeled.

Brief Description of Drawings

FIG. 1: heavy and light chain HVR loop sequences of anti-hepsin antibodies. The figure shows the heavy chain HVR sequences, H1, H2 and H3, and the light chain HVR sequences, L1, L2 and L3. The sequence numbering is as follows: clone 25 (HVR-H1 is SEQ ID NO: 4; HVR-H2 is SEQ ID NO: 5; HVR-H3 is SEQ ID NO: 6; HVR-L1 is SEQ ID NO: 1; HVR-L2 is SEQ ID NO: 2; HVR-L3 is SEQ ID NO: 3); amino acid positions are numbered according to the Kabat numbering system as described below.

FIGS. 2A, 2B, and 3 depict exemplary recipient human consensus framework sequences for practicing the invention with the following sequence identifiers.

Heavy chain variable domain (VH) consensus framework (FIGS. 2A, 2B)

Human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NOS: 21-23 and 16, respectively)

Human VH subgroup I consensus framework minus extended hypervariable regions (SEQ ID NOS: 24-25, 23 and 16; 24-26 and 16; and 24-25, 27 and 16, respectively)

Human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID NOS: 28-30 and 16, respectively)

Human VH subgroup II consensus framework minus extended hypervariable regions (SEQ ID NOS: 31-32, 30 and 16; 31-33 and 16; and 31-32, 34 and 16, respectively)

Human VH subgroup II consensus framework minus extension

Human VH subgroup III consensus framework minus Kabat CDRs (SEQ ID NOS: 35-37 and 16, respectively)

Human VH subgroup III consensus framework minus extended hypervariable regions (SEQ ID NOS: 14-15, 37 and 16; 14-15, 38 and 16; and 14-15, 43 and 16, respectively)

Human VH acceptor framework minus Kabat CDRs (SEQ ID NOS: 39, 36, 40 and 16, respectively)

Human VH acceptor framework minus extended hypervariable regions (SEQ ID NOS: 14-15, 40 and 16; and 14-15, 41 and 16, respectively)

Human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NOS: 39, 36, 42 and 16, respectively)

Human VH acceptor 2 framework minus extended hypervariable regions (SEQ ID NOS: 14-15, 42 and 16; 14-15, 44 and 16; and 14-15, 48 and 16, respectively)

Light chain variable domain (VL) consensus boxHolder (Picture 3)

Human VL kappa subgroup I consensus framework (SEQ ID NOS: 17-20, respectively)

Human VL kappa subgroup II consensus framework (SEQ ID NOS: 49-51 and 20, respectively)

Human VL kappa subgroup III consensus framework (SEQ ID NOS: 52-54 and 20, respectively)

Human VL kappa subgroup IV consensus framework (SEQ ID NOS: 55-57 and 20, respectively)

FIG. 4 depicts the framework region sequences of the huMAb4D5-8 light chain (SEQ ID NOS: 17-18, 58, and 20, respectively, in order of appearance) and heavy chain (SEQ ID NOS: 14-15, 48, and 16, respectively, in order of appearance). The numbers indicated in superscript/bold indicate amino acid positions according to Kabat.

FIG. 5 depicts the modified/variant framework region sequences of the huMAb4D5-8 light chain (SEQ ID NOS: 17-20, respectively, in order of appearance) and heavy chain (SEQ ID NOS: 14-15, 43, and 16, respectively, in order of appearance). The numbers indicated in superscript/bold indicate amino acid positions according to Kabat.

FIG. 6 depicts the anti-hepsin antibody mAb 25 heavy chain variable region (SEQ ID NO:10) and light chain variable region (SEQ ID NO: 9).

FIG. 7: an embodiment of the amino acid sequence of native human hepsin.

Fig. 8A and B: another embodiment of the amino acid sequence of native human hepsin.

FIG. 9: inactivation of hepsin by 3, 4-dichloro-isocoumarin (DCI). A. Concentration-dependent inhibition of enzymatic activity against S2765 substrate after incubation of hepsin with increasing concentrations of DCI for 40 minutes. B. According to (Powers et al, 1989), the chemical structure of DCI and its potential adducts with hepsin.

FIG. 10: KD1 competed with anti-hepsin Fab phage for binding to hepsin. Binding of hepsin by Fab25 phage in the presence of increasing concentrations of KD 1.

FIG. 11: inhibition of human and murine hepsin enzyme activity by Fab 25. Human (hu) and murine (mu) hepsin were incubated with Fab25 or the control Fab (ctrl Fab) for 40 minutes before addition of the S2765 substrate. The initial linear velocity was measured on a kinetic microplate reader and the enzyme activity was expressed as fractional activity (vi/vo).

FIG. 12: specificity of Fab 25. Fab25 (1. mu.M) was incubated with hepsin and 9 trypsin-like serine proteases for 40 minutes, after which synthetic p-nitroaniline (pNA) substrate was added. The initial linear velocity was measured on a kinetic microplate reader and the enzyme activity was expressed as fractional activity (vi/vo).

FIG. 13: inhibition of macromolecular substrate processing of hepsin by Fab 25. Inhibition of pro-uPA activation by fab25. The effect of Fab25 on hepsin-mediated pro-uPA processing was determined in a two-stage enzyme assay. The initial linear velocity was measured on a kinetic microplate reader and the enzyme activity was expressed as fractional activity (vi/vo). By using the uPA standard curve, the rate of uPA formation without hepsin inhibition was determined to be 0.81 ± 0.22 μ Μ uPA/min (n = 3). Inhibition of hepsin substrate processing by Fab25 was performed on 3 known substrates: B. factor vii (fvii); pro-HGF; pro-msp. Cleavage of factor VII by hepsin was performed in the presence or absence of Fab25 and the control Fab (ctrl Fab) for 0.5 hours and 2.0 hours, while the pro-HGF and pro-MSP cleavage experiments were performed for 30 minutes. Reaction aliquots were analyzed by SDS-PAGE (reducing conditions) and the gels stained.

FIG. 14: prolonged exposure to hepsin, effects of Fab25 integrity. The CDR-H3 loop in Fab25 contained three lysine and one arginine residues that could potentially be cleaved by hepsin, but no proteolytic processing of Fab25 by hepsin was observed after 24 hours incubation at pH6.0 or pH 8.0. Proteolysis was monitored by gel mobility shift on 4-20% (w/v) polyacrylamide gradient gels and stained with Coomassie Brilliant blue.

FIG. 15: inhibition of laminin-dependent migration of DU145 cells by Fab 25. A. DU14 in serum-free Dulbecco's modified Eagle's Medium5 cells (2X 10)⁴) Added to the pretreated upper chamber of the fluoroblok insert and allowed to migrate for 5 hours at 37 ℃. After incubation, non-migrating cells and media were washed and those that migrated to the bottom of the filter were fixed, stained with YO-PRO-I, and imaged using an inverted microscope. Representative images were collected on pre-treated filters with cells immobilized (laminin, laminin co-incubated with hepsin: Fab25 complex, or PBS control). B. Measurement of relative fluorescence units (r.f.u) from cells stained with YO-PRO substrate, minus the reference PBS wells. DU145 cells treated with hepsin-processed laminin had significantly increased migration compared to cells treated with laminin alone. The absence of hepsin processing of laminin in the presence of Fab25 resulted in reduced migration of DU 145.

FIG. 16: pro-MSP activation by cell surface expressed hepsin in lncap-34 cells. Stably overexpressing hepsin LnCap-34 cells were serum-starved and used¹²⁵pro-I-MSP was treated for 3 hours either alone or in combination with different inhibitors. Recombinant hepsin (10nM) was used as positive control. A significant increase in pro-MSP processing compared to the experimental starting point was observed after 3 hours. Inhibitors KQLR (SEQ ID NO:12), KD1 and the anti-hepsin antibody Fab25 effectively blocked pro-MSP activation. pro-HGF activation by hepsin expressed on the cell surface in LnCap-34 cells. LnCap-34 cells were used in the presence of different concentrations of Fab25(20nM to 0.15nM)¹²⁵I-HGF pro-treatment and incubation for 3 hours. Recombinant hepsin (10nM) was used as positive control. A significant increase in HGF pro-processing compared to the starting point of the experiment was observed after 3 hours. The anti-hepsin antibody Fab25 effectively blocked activation of pro-HGF in a concentration-dependent manner.

FIG. 17: A. surface plasmon resonance was used to measure the binding affinity of Ab25 for active human hepsin. CM5 biosensor chip with captured Ab25 was injected with a serial dilution of hepsin (0.39nM to 200nM) for 3 min and monitored for dissociation for 15 min. Fitting of the experimental data gives an equilibrium dissociation constant rate (KD) of 10.6 nM. B. Since Ab25 exhibits rapid kinetics for binding of pro-hepsin, it was measured by steady-state affinity measurements Binding affinity. Serial dilutions of pro-hepsin (195nM to 80 μ M) were injected for 2 min into CM5 biosensor chips with Ab25 captured. Determination of equilibrium dissociation rate (K) from a plot of Req versus pro-hepsin concentration by steady state analysis_D=5.52μM)。

FIG. 18: isothermal titration calorimetry experiments for Fab25 binding to active hepsin. The binding reaction was exothermic and the stoichiometry of the binding was 1: 1, as predicted. Dissociation constant (K) from ITC_D) At 6.1nM, it correlates well with previous data from BIAcore. The enthalpy (Δ H) and entropy (T Δ S) of binding was-27.5 kcal/mol and-16.3 kcal/mol, indicating that the binding is enthalpy driven, with unfavorable entropy.

Detailed Description

The invention herein provides anti-hepsin antibodies that are useful, for example, for the treatment or prevention of disease states associated with expression and/or activity of hepsin (such as elevated expression and/or activity or unwanted expression and/or activity). In some embodiments, the antibodies of the invention are used to treat tumors, cancers, and/or cell proliferative disorders.

In another aspect, the anti-hepsin antibodies of the invention find utility as reagents for the detection and/or isolation of hepsin, such as the detection of hepsin in various tissues and cell types.

The invention further provides methods of making and using anti-hepsin antibodies, and polynucleotides encoding anti-hepsin antibodies.

General technique

The techniques and protocols described or referenced herein are generally well understood by those skilled in the art and are generally employed using conventional methods, such as, for example, the widely used methods described in the following documents: a Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y; CURRENT promoters IN MOLECULAR BIOLOGY (f.m. ausubel, et al. eds., (2003)); book of protocols IN enzymolygy (Academic Press, Inc.: PCR2: A PRACTICAL APPROACH (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)); harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL; and ANIMAL CELL CULTURE (r.i. freshney, ed. (1987)).

Definition of

An "isolated" antibody refers to an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified to (1) greater than 95% by weight, most preferably greater than 99% by weight, of the antibody as determined by the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and staining with Coomassie blue or preferably silver. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, an isolated antibody will typically be prepared by at least one purification step.

An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in its natural source. An isolated nucleic acid molecule is distinguished from the form or context in which it is found in nature. An isolated nucleic acid molecule is thus distinguished from a nucleic acid molecule when present in a natural cell. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that normally expresses the nucleic acid (e.g., an antibody-encoding nucleic acid), for example, when the chromosomal location of the nucleic acid molecule in the cell is different from its chromosomal location in a native cell.

The term "variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat" and variants thereof refers to the numbering system for heavy or light chain variable domains by antibody editing in Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may comprise fewer or additional amino acids, corresponding to a shortening or insertion of the variable domain FR or CDR. For example, the heavy chain variable domain may comprise a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after heavy chain FR residue 82. The Kabat residue numbering for a given antibody can be determined by aligning the antibody sequence to the region of homology with a "standard" Kabat numbered sequence.

The phrase "substantially similar" or "substantially the same" as used herein means a sufficiently high degree of similarity between two numerical values (typically one relating to an antibody of the invention and the other relating to a reference/comparison antibody) such that one of skill in the art would consider the difference between the two numerical values to be of little or no biological and/or statistical significance within the context of the biological property measured by the numerical values (e.g., Kd values). The difference between the two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, or preferably less than about 10%, as a function of the value for the reference/comparison antibody.

"binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). The desired Kd is 1x10^-7、1x10^-8、5x10^-8、1x10^-9、3x10^-9、5x10^-9Or even 1x10^-10Or stronger. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies typically bind antigen slowly and Tend to dissociate readily, while high affinity antibodies generally bind antigen more rapidly and tend to remain bound for a longer period of time. A variety of methods for measuring binding affinity are known in the art, any of which may be used for the purposes of the present invention. Specific exemplary embodiments are described below.

In one embodiment, "Kd" or "Kd value" according to the present invention is measured by a radiolabelled antigen binding assay (RIA) carried out using Fab-format antibodies of interest and their antigens as described in the following assays: by using the minimum concentration of the antigen in the presence of a titration series of unlabelled antigen¹²⁵I labelling of the antigen equilibrated Fab, and then capturing the bound antigen with anti-Fab antibody coated plates to measure the solution binding affinity of the Fab for the antigen (Chen et al, J Mol Biol 293:865-881 (1999)). To determine assay conditions, microtiter plates (Dynex) were coated with anti-Fab antibodies (Cappel Labs) overnight with 5. mu.g/ml capture in 50mM sodium carbonate (pH 9.6), followed by blocking with 2% (w/v) bovine serum albumin in PBS at room temperature (about 23 ℃) for 2-5 hours. In a non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]Mixing of antigen with serial dilutions of Fab of interest (e.g.in agreement with the evaluation of anti-VEGF antibodies, Fab-12, by Presta et al, Cancer Res.57: 4593-. The Fab of interest was then incubated overnight; however, the incubation may continue for a longer period of time (e.g., 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for room temperature incubation (e.g., 1 hour). The solution was then removed and the plate washed 8 times with PBS containing 0.1% Tween-20. After the plates were dried, 150. mu.l/well scintillation fluid (MicroScint-20; Packard) was added and the plates were counted for 10 minutes on a Topcount Gamma counter (Packard). The concentration at which each Fab gives less than or equal to 20% of the maximum binding is selected for use in competitive binding assays. According to another embodiment, the Kd or Kd value is determined by surface plasmon resonance assay using BIAcore ^TM-2000 or BIAcore^TM-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, N-ethyl-N '- (3-dimethylaminopropyl) hydrochloride was used according to the supplier's instructionsCarboxymethylated dextran biosensor chips (CM5, biacore inc.) were activated by yl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH 4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% Tween 20 were injected at 25 ℃ at a flow rate of about 25. mu.l/min. In some embodiments, the following modifications are used for the surface plasmon resonance assay: antibodies were immobilized to a CM5 biosensor chip to achieve approximately 400RU, and for kinetic measurements, two-fold serial dilutions (starting at 67 nM) of the target protein (e.g., hepsin-IIIb or-IIIc) in 25 ℃ PBST buffer were injected at a flow rate of approximately 30 ul/min. Binding rates (k) were calculated by simultaneous fitting of association and dissociation sensorgrams using a simple one-to-one Langmuir (Langmuir) binding model (BIAcore Evaluation Software version 3.2) _on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen, Y, et al, J Mol Biol 293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay⁶ M^-1 S^-1The binding rate can then be determined using fluorescence quenching techniques, i.e.measuring the increase or decrease in fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM band pass) at 25 ℃ of 20nM anti-antigen antibody (Fab form) in PBS in the presence of increasing concentrations of antigen, according to a measurement with a stirred cuvette in a spectrometer such as a spectrophotometer equipped with a flow-breaking device (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (Thermospectronic).

An "association rate" or "k" according to the present invention_on' also can be achieved by the same surface plasmon resonance technique as described above using BIAcore^TM-2000 or BIAcore^TM-3000(BIAcore,Inc.,Piscataway, NJ) was determined at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH 4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% Tween 20 were injected at 25 ℃ at a flow rate of about 25. mu.l/min. In some embodiments, the following modifications are used for the surface plasmon resonance assay: antibodies were immobilized to a CM5 biosensor chip to achieve approximately 400RU, and for kinetic measurements, two-fold serial dilutions (starting at 67 nM) of the target protein (e.g., hepsin-IIIb or-IIIc) in 25 ℃ PBST buffer were injected at a flow rate of approximately 30 ul/min. Binding rates (k) were calculated by simultaneous fitting of binding and dissociation sensorgrams using a simple one-to-one Langmuir (Langmuir) binding model (BIAcoreevaluation Software version 3.2) _on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen, Y, et al, J Mol Biol 293:865-881 (1999). However, if according to the above surface plasmon resonance determination method, the binding rate exceeds 10⁶ M^-1 S^-1The binding rate is then preferably determined using fluorescence quenching techniques, i.e.measuring the increase or decrease in fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM bandpass) at 25 ℃ of 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen, according to a measurement in a spectrometer such as a spectrophotometer equipped with a flow cut-off device (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (Thermospectronic) with a stirred cuvette.

The term "vector" as used herein means a nucleic acid molecule capable of transporting other nucleic acids to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used form of vector.

"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and/or analogs thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. Modifications to the nucleotide structure, if any, may be made before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties (e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with embedded pendant moieties, etc Modifications of agents (e.g., acridine, psoralen, and the like), modifications containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), modifications containing alkylating agents, modifications having modified linkages (e.g., alpha anomeric nucleic acids (anomeric nucleic acids), and the like), and unmodified forms of the polynucleotide. In addition, any hydroxyl groups typically present in sugars may be replaced with, for example, phosphonic acid (phosphonate) groups, phosphoric acid (phosphonate) groups, protected with standard protecting groups, or activated to make additional linkages to additional nucleotides, or may be coupled to a solid or semi-solid support. The 5 'and 3' terminal OH groups may be phosphorylated or substituted with amines or organic capping group moieties of 1-20 carbon atoms. Other hydroxyl groups can also be derivatized to standard protecting groups. Polynucleotides may also contain analog forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2 '-O-methyl, 2' -O-allyl, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methylribonucleosides. One or more phosphodiester linkages may be replaced with alternative linking groups. Such alternative linking groups include, but are not limited to, embodiments in which the phosphate ester is substituted with P (O) S ("thioester"), P (S) S ("dithioate"), (O) NR2 ("amidite"), P (O) R, P (O) OR', CO OR CH ₂(formacetal)), wherein R or R' are each independently H or a substituted or unsubstituted hydrocarbyl group (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or aryl hydrocarbyl (araldyl). Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

"oligonucleotide" as used herein generally refers to short polynucleotides, generally single-stranded, generally synthetic, generally but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides applies equally and fully to oligonucleotides.

"percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared. However, for purposes of the present invention,% amino acid sequence identity values are obtained using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table A below. The ALIGN-2 sequence comparison computer program was written by Genentech corporation and the source code has been submitted to the US Copyright Office (US Copyright Office, Washington d.c.,20559) along with the user document and registered with US Copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech corporation (South San Francisco, Calif.) or may be compiled from source code such as that provided in WO 2007/001851. The ALIGN2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a with respect to, with, or against a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not be equal to the% amino acid sequence identity of B relative to a.

In some embodiments, the two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

As used herein, unless otherwise clear or indicated by context, the term "hepsin" refers to any native or variant hepsin polypeptide. The term "native sequence" specifically encompasses naturally occurring truncated forms (e.g., an extracellular domain sequence or a transmembrane subunit sequence), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants. The term "wild-type hepsin" generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring hepsin protein. The term "wild-type hepsin sequence" generally refers to an amino acid sequence found in naturally occurring hepsin. In one embodiment, the native sequence hepsin polypeptide comprises the amino acid sequence of SEQ ID NO:46 (see FIG. 7). In some embodiments, the native sequence hepsin polypeptide comprises the amino acid sequence of SEQ ID NO:47 (see FIG. 8).

By "hepsin polypeptide variant" or variation thereof is meant a hepsin polypeptide having at least about 80% amino acid sequence identity to a native sequence hepsin polypeptide sequence disclosed herein, typically an active hepsin polypeptide as defined herein. Such hepsin polypeptide variants include, for example, hepsin polypeptides having one or more amino acid residues added or deleted at the N-or C-terminus of the native amino acid sequence. Typically, a hepsin polypeptide variant has at least about 80% amino acid sequence identity, or at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to a native sequence hepsin polypeptide sequence disclosed herein. Typically, hepsin variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acids in length, or more. Optionally, the hepsin variant polypeptide has no more than one conservative amino acid substitution as compared to the native hepsin polypeptide sequence, or no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to the native hepsin polypeptide sequence.

"tyrosine kinase inhibitor" refers to a molecule that inhibits to some extent the tyrosine kinase activity of tyrosine kinases such as hepsin receptors.

"inhibit" refers to a decrease or decrease in activity, function, and/or amount as compared to a reference.

Protein "expression" refers to the conversion of information encoded in a gene into messenger rna (mrna) and then into a protein.

As used herein, a sample or cell that "expresses" a protein of interest (such as hepsin) refers to a sample or cell in which the presence of mRNA or protein (including fragments thereof) encoding the protein is determined.

An "immunoconjugate" (interchangeably referred to as an "antibody-drug conjugate" or "ADC") refers to an antibody conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

A "blocking" antibody or antibody "antagonist" refers to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonistic antibodies completely inhibit the biological activity of the antigen.

"naked antibody" refers to an antibody that is not conjugated to a heterologous molecule such as a cytotoxic moiety or a radioactive label.

An antibody having the "biological characteristics" of a given antibody refers to an antibody that possesses one or more biological characteristics that distinguish the given antibody from other antibodies that bind the same antigen.

To screen for Antibodies that bind to an epitope on an antigen to which an antibody of interest binds, a conventional cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring harbor Laboratory, Ed Harlow and David Lane (1988).

To extend the half-life of an antibody or polypeptide comprising an amino acid sequence of the invention, a salvage receptor binding epitope can be attached to an antibody (particularly an antibody fragment) as described, for example, in U.S. Pat. No.5,739,277. For example, a nucleic acid molecule encoding a salvage receptor binding epitope can be linked in-frame to a nucleic acid encoding a polypeptide sequence of the invention such that the fusion protein encoded by the engineered nucleic acid molecule comprises the salvage receptor binding epitope and the polypeptide sequence of the invention. As used herein, the term "salvage receptor binding epitope" refers to an IgG molecule (e.g., IgG) ₁、IgG₂、IgG₃Or IgG₄) The Fc region of (A) is an epitope responsible for extending the serum half-life of the IgG molecule in vivo (e.g., Ghetie et al, Ann. Rev. Immunol.18:739-766(2000), Table 1). Antibodies with substitutions in their Fc region and increased serum half-life are also described in WO 00/42072; WO 02/060919; shields et al, J.biol.chem.276:6591-6604 (2001); hinton, J.biol.chem.279:6213-6216 (2004)). In another embodiment, serum half-life may also be extended, for example, by attaching other polypeptide sequences. For example, antibodies or other polypeptides useful in the methods of the invention can be attached to or bound in serum albuminThe FcRn receptor or that portion of the serum albumin binding peptide which allows serum albumin to bind to the antibody or polypeptide, for example such polypeptide sequences are disclosed in WO 01/45746. In a preferred embodiment, the serum albumin peptide to be attached comprises the amino acid sequence DICLPRWGCLW (SEQ ID NO: 13). In another embodiment, the half-life of the Fab is extended by these methods. Serum albumin binding peptide sequences can also be found in Dennis et al, J.biol.chem.277: 35035-.

"fragment" refers to polypeptides and portions of nucleic acid molecules that preferably contain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the full length of a reference nucleic acid molecule or polypeptide. The fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, or more nucleotides, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200 or more amino acids.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), and may also include certain antibody fragments (as described in more detail herein). The antibody may be human, humanized and/or affinity matured.

The term "variable" refers to the fact that certain portions of the variable domains differ widely between antibody sequences and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibodies. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light chain and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FRs, which mostly adopt a β -sheet conformation, connected by three CDRs which form loops connecting, and in some cases forming part of, the β -sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Proteins of immunological Interest, 5 th edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as participation of the antibody in antibody-dependent cellular (cellular) toxicity.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having an antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment to yield F (ab')₂A fragment which has two antigen binding sites and is still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. In the two-chain Fv species, this region consists of a dimer of one heavy and one light variable domain in tight, non-covalent association. In single-chain Fv species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure analogous to that of a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The six CDRs collectively confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, with only a lower affinity than the entire binding site.

The Fab fragment also comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domain carry a free thiol group. F (ab')₂Antibody fragments originally were paired F as hinge cysteines between Fab' fragmentsab' fragment. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), depending on the amino acid sequence of their constant domains.

Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which may be further divided into subclasses (isotypes), e.g. IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. The constant domains of the heavy chains corresponding to different classes of immunoglobulins are referred to as α, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. An "antibody fragment" comprises only a portion of an intact antibody, wherein said portion preferably retains at least one, preferably most or all of the functions normally associated with the portion when present in an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') ₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment comprises the antigen binding site of an intact antibody, such that the ability to bind antigen is retained. In another embodiment, an antibody fragment, e.g., an antibody fragment comprising an Fc region, retains at least one biological function normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may comprise one antigen-binding arm and is linked to an Fc sequence capable of conferring in vivo stability to the fragment.

The term "hypervariable region", "HVR" or "HV", when used herein, refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Typically, antibodies comprise six hypervariable regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). A description of many hypervariable regions is used and encompassed herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the position of the structural loops (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable region is based on an analysis of the available complex crystal structure. The residues for each of these hypervariable regions are noted below.

Hypervariable regions can comprise "extended hypervariable regions" as follows: 24-36 or 24-34(L1), 46-56 or 50-56(L2) and 89-97(L3) in VL and 26-35(H1), 50-65 or 49-65(H2) and 93-102, 94-102 or 95-102(H3) in VH. For each of these definitions, the variable region residues are numbered according to Kabat et al, supra.

"framework" or "FR" residues refer to those residues in the variable domain other than the hypervariable region residues defined herein.

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are those in which residues from a hypervariable region of a human immunoglobulin (recipient antibody) are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al, Nature 321:522-525(1986), Riechmann et al, Nature 332:323-329(1988), Presta, curr. Op. struct. biol.2:593-596 (1992). See also the following reviews and references cited therein: vaswani and Hamilton, Ann. allergy, Asthma & Immunol.1:105-115(1998); Harris, biochem. Soc. transactions 23:1035-1038(1995); Hurle and Gross, curr. Op. Biotech.5:428-433 (1994).

A portion of the heavy and/or light chain in a "chimeric" antibody (immunoglobulin) is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). As used herein, humanized antibodies are a subset of chimeric antibodies.

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein the domains are present on a single polypeptide chain. In general, scFv polypeptides are at V_HAnd V_LPolypeptide linkers are also included between the domains to enable the scFv to form the structure required for binding to the antigen. For a review of scFv see Pluckthun, edited by Rosenburg and Moore, The Pharmacology of monoclonal antibodies, vol 113, Springer-Verlag, New York, pp.269-315 (1994).

"antigen" refers to a predetermined antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide.

The term "diabodies" refers to small antibody fragments having two antigen-binding sites, which fragments are in the same polypeptide chain (V)_H-V_L) Comprising a linked heavy chain variable domain (V)_H) And a light chain variable domain (V)_L). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain, thereby creating two antigen binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93/11161, and Hollinger et al Proc. Natl. Acad. Sci. USA,90: 6444-.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more CDRs of the antibody that result in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody without the alterations. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies can be generated by procedures known in the art. Marks et al, Bio/Technology 10:779-783(1992) describe affinity maturation by VH and VL domain shuffling. The following documents describe random mutagenesis of CDR and/or framework residues: barbas et al, PNAS (USA)91: 3809-.

Antibody "effector functions" refer to those biological activities that can be attributed to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region) and vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted Ig bound to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The antibody "arms" (arm) cytotoxic cells and is absolutely required for such killing. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravatch and Kinet, annu.rev.immunol.9: 457-92(1991) 464 Page table 3 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. Pat. No.5,500,362 or 5,821,337 or Presta U.S. Pat. No.6,737,056. Useful effector cells that can be used in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of the molecule of interest may be assessed in vivo, for example in animal models such as Clynes et al, proc.natl.acad.sci.usa 95: 652-.

"human effector cells" refer to leukocytes which express one or more fcrs and which exert effector function. Preferably, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMC), Natural Killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, preferably PBMC and NK cells. Effector cells may be isolated from their natural source, e.g., blood.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody (gamma receptor), including receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see for review) Annu, rev, immunol.15: 203-234(1997)). For a review of fcrs see ravatch and Kinet, annu. 457-492 (1991); capel et al, immunolmethods 4: 25-34 (1994); deHaas et al, j.lab.clin.med.126: 330-41(1995). The term "FcR" encompasses other fcrs herein, including those that will be identified in the future. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, j.immunol.117: 587(1976) and Kim et al, j.immunol.24: 249(1994)) and for the regulation of immunoglobulin homeostasis. WO 00/42072(Presta) describes antibody variants with increased or decreased binding to FcR. The contents of this patent publication are expressly incorporated herein by reference. See also, shiplds et al, j.biol.chem.9(2):6591-6604 (2001).

Methods for measuring binding to FcRn are known (see e.g. Ghetie 1997, Hinton 2004). The in vivo binding and serum half-life of human FcRn high affinity binding polypeptides to human FcRn can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with the Fc variant polypeptides.

"complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to the antibody (of the appropriate subclass) to which its cognate antigen binds. To assess complement activation, CDC assays can be performed, for example as described in Gazzano-Santoro et al, J.Immunol.methods 202:163 (1996).

Polypeptide variants with altered Fc region amino acid sequence and increased or decreased C1q binding ability are described in U.S. patent No.6,194,551B1 and WO 99/51642. The contents of those patent publications are expressly incorporated herein by reference. See also Idusogene et al, J.Immunol.164: 4178-.

The term "Fc region-containing polypeptide" refers to a polypeptide comprising an Fc region, such as an antibody or immunoadhesin. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be eliminated, for example, during purification of the polypeptide or by recombinant engineering of the nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide having an Fc region according to the invention may comprise a polypeptide having K447, a polypeptide that eliminates all K447, or a mixture of polypeptides having and without the K447 residue.

For purposes herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence as it, or may comprise pre-existing amino acid sequence variations. When there are pre-existing amino acid changes, preferably there are no more than 5, preferably 4 or fewer, or 3 or fewer pre-existing amino acid changes. When pre-existing amino acid changes are present in the VH, preferably those changes are located only at three, two, or one of positions 71H, 73H, and 78H; for example, the amino acid residues at those positions may be 71A, 73T, and/or 78A. In one embodiment, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"human consensus framework" refers to a framework representing the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subgroups are subgroups as in Kabat et al. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al.

A "VH subgroup III consensus framework" comprises a consensus sequence obtained from amino acid sequences in variable heavy chain subgroup III, Kabat et al. In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion of, or all of:

EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:14) -H1-

WVRQAPGKGLEWV(SEQ ID NO:15) -H2-

RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:43) -H3-

WGQGTLVTVSS(SEQ ID NO:16)。

a "VL subgroup I consensus framework" comprises a consensus sequence obtained from amino acid sequences in variable light chain kappa subgroup I of Kabat et al. In one embodiment, the VL subgroup I consensus framework amino acid sequence comprises at least a portion of, or the entire of, each of the following sequences:

DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:17) -L1-

WYQQKPGKAPKLLIY(SEQ ID NO:18) -L2-

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:19) -L3-

FGQGTKVEIK(SEQ ID NO:20)。

as used herein, "antibody mutant" or "antibody variant" refers to an amino acid sequence variant of an antibody in which one or more amino acid residues of a species-dependent antibody are modified. Such mutants necessarily have less than 100% sequence identity or similarity to species-dependent antibodies. In one embodiment, the antibody mutant has an amino acid sequence that has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% amino acid sequence identity or similarity to the amino acid sequence of the heavy or light chain variable domain of the species-dependent antibody. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same residues) or similar (i.e., amino acid residues from the same group according to common side chain characteristics, see below) to species-dependent antibody residues after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Neither N-terminal, C-terminal, nor extension, deletion, or insertion into the interior of antibody sequences outside of the variable domains should be considered to affect sequence identity or similarity.

"disorder" or "disease" refers to any condition that would benefit from treatment with the agents/molecules or methods of the invention. This includes chronic and acute conditions or diseases, including those pathological conditions that predispose a mammal to the condition in question. Non-limiting examples of conditions to be treated herein include malignant and benign tumors; carcinoma, blastoma, and sarcoma.

"treatment" and "treatment" (treatment) refer to both therapeutic treatment and prophylactic measures. Subjects in need of treatment include those already suffering from benign, precancerous, or non-metastatic tumors and who are to be prevented from developing or relapsing.

The term "therapeutically effective amount" refers to a therapeutic dose for treating or preventing a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a therapeutic agent can reduce the number of cancer cells; reducing the size of the primary tumor; inhibit (i.e., slow to some extent, preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent, preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or to alleviate one or more symptoms associated with the condition to some extent. Depending on the extent to which the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can be measured by, for example, assessing survival duration, time to disease progression (TTP), Response Rate (RR), response duration, and/or quality of life.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. "early cancer" or "early tumor" refers to a cancer that is non-invasive or metastatic, or is classified as a stage 0, stage I, or stage II cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumor (including carcinoid tumor, gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric cancer (gastric or stomach cancer) including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer or hepatoma), bladder cancer, hepatoma (hepatoma), breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (kidney cancer), prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, cholangiocarcinoma, and head and neck cancer and multiple myeloma.

The term "precancerous" refers to a condition or growth that typically precedes or develops cancer. "precancerous" growth will have cells characterized by abnormal cell cycle regulation, proliferation, or differentiation, as measured by markers of cell cycle regulation, cell proliferation, or differentiation.

"dysplasia" refers to any abnormal growth or development of a tissue, organ, or cell. Preferably, the dysplasia is advanced or precancerous.

"metastasis" refers to the spread of cancer from its primary site to other locations in the body. Cancer cells can detach from the primary tumor, penetrate into the lymph and blood vessels, circulate through the bloodstream and grow in distant foci (metastases) in normal tissues elsewhere in the body. The transfer may be local or remote. Metastasis is a continuous process, depending on the shedding of tumor cells from the primary tumor, spreading through the bloodstream, and stopping at a distal site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass.

Stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in distal sites are also important.

"non-metastatic" refers to cancers that are benign or remain at the primary site and have not penetrated into the lymphatic or vascular system or penetrated to tissues outside the primary site. In general, a non-metastatic cancer refers to any cancer that is a stage 0, stage I, or stage II cancer and occasionally a stage III cancer.

"Primary tumor" or "primary cancer" refers to the initial cancer, and not a metastatic lesion located in another tissue, organ, or location in the body of the subject.

"benign tumor" or "benign carcinoma" refers to a tumor that remains localized to the site of origin and has no ability to penetrate, invade, or metastasize to distant sites.

"tumor burden" refers to the number of cancer cells, the size of the tumor, or the amount of cancer in the body. Tumor burden is also referred to as tumor burden.

"tumor number" refers to the number of tumors.

"subject" refers to a mammal, including but not limited to a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. Preferably, the subject is a human.

The term "anti-cancer therapy" refers to a therapy useful in the treatment of cancer. Examples of anti-cancer therapeutics include, but are not limited to, for example, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, anti-CD 20 antibodies, platelet-derived growth factor inhibitors (e.g., Gleevec) ^TM(Imatinib Mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets (ErbB 2, ErbB3, ErbB4, PDGFR- β, BlyS, APRIL, BCMA or VEGF receptor, TRAIL/Apo 2), and other biologically active and organic chemicals, and the like. The present invention also includes combinations thereof.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. The term is intended to include radioisotopes (e.g., I)¹³¹、I¹²⁵、Y⁹⁰And Re¹⁸⁶) Chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof.

"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include chemical compounds useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and thiotepaCyclophosphamide (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa), and uretepa (uredepa); ethyleneimines and methylmelamines including altretamine, triethylenemelamine Tetramethylene melamine (triethyleneamine), triethylene phosphoramide (triethylenephosphoramide), triethylene thiophosphoramide (triethylenephosphoramide), and trimethylolmelamine (trimetylomelamine); annonaceous acetogenins (especially bullatacin and bullatacin); camptothecin (camptothecin) (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycins (especially cryptophycins 1 and 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards (nitrosgen mustards), such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics such as enediynes antibiotics (enediynes) (e.g., calicheamicins, especially calicheamicin γ 1I and calicheamicin ω I1 (see, e.g., Agnew (1994) chem. intl. Ed. Engl.33: 183-186)), anthracyclines (dynemicins), including dynemicin A, bisphosphonates (e.g., clodronate), esperamicins (esperamicin), and neocarzinostatin (neocarzinostatin) and related chromogenes of tryptophans enediynes, aclacinomycin (aclacinomycin), actinomycins (actinomycin), anthranomycin (antromycin), azaserines (azaserines), bleomycin (bleomycin), actinomycins (calcimycin C) n), carabicin, carminomycin (carminomycin), carzinophilin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorexin (detorbicin), 6-diazepin-5-oxo-L-norleucine, norvaline, norleucine, norvaline,doxorubicin (doxorubicin) (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcelomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (polyplomycin), pofiromycin (potfiromycin), puromycin (puromycin), triiron (quelamycin), rodobicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptazocin), tubercidin (tubercidin), ubulin (ubulin), ubulin (streptonigrosine), zocin (zorubicin), zorubicin (stazostatin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); aldehyde phosphoramidates Glycosides (aldophosphamide glycosides); aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine);polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sizofuran (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2',2 "-trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin A, rorodin A and snake-fish (anguidin); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); cyclophosphamide (cyclophosphamide); thiotepa (thiotepa); taxoids, e.g. taxol Paclitaxel (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE^TMCremophor-free, albumin-engineered nanoparticle dosage form of paclitaxel (American P)Pharmaceutical Partners, Schaumberg, Illinois) anddocetaxel (doxetaxel) ((doxetaxel))Rorer, antonyy, France); chlorambucil (chlorambucil);gemcitabine (gemcitabine); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum (platinum); etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); vincristine (vincristine);vinorelbine (vinorelbine); oncostatin (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); (xiloda); ibandronate (ibandronate); irinotecan (irinotecan) (Camptosar, CPT-11) (treatment regimens that include irinotecan with 5-FU and folinic acid); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoic acid); capecitabine (capecitabine); combretastatin (combretastatin); VELCADE bortezomib; revalidatenalidomide; leucovorin (LV); oxaliplatin (oxaliplatin), including oxaliplatin treatment regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva (TM)) and VEGF-A that reduce cell proliferation; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

This definition also includes anti-hormonal agents such as anti-estrogens and Selective Estrogen Receptor Modulators (SERMs) that act to modulate or inhibit the effects of hormones on tumors, including, for example, tamoxifen (tamoxifen) ((R))Comprises thatTamoxifen), raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxytamoxifene, trioxifene (trioxifene), naloxifene (keoxifene), LY117018, onapristone (onapristone), andtoremifene (toremifene); aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland, such as, for example, 4(5) -imidazole, aminoglutethimide,Megestrol acetate (megestrolacetate),Exemestane (exemestane), formestane (formestane), fadrozole (fadrozole),Vorozole (vorozole),Letrozole (letrozole) andanastrozole (anastrozole); anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide (leuprolide), and goserelin (goserelin); and troxacitabine (a 1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly antisense oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal (atherant) cell proliferation, such as, for example, PKC- α, Raf, and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g. Nucleic acids) and HEAn inhibitor of R2 expression; vaccines, such as gene therapy vaccines, e.g.A vaccine,A vaccine anda vaccine;rIL-2；a topoisomerase 1 inhibitor;rmRH; vinorelbine (Vinorelbine) and Esperamicins (Esperamicins) (see U.S. patent No.4,675,187); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

The term "prodrug" as used herein refers to precursor and derivative forms of a pharmaceutically active substance that are less cytotoxic to tumor cells than the parent drug and are capable of enzymatic activation or conversion to a more active parent drug form. See, for example, Wilman, "Prodrugs in cancer Chemotherapy", Biochemical Society Transactions,14, pp.375-382,615 through Belfast (1986) and Stella et al, "Prodrugs: A Chemical Approach to targeted Drug Delivery", direct Drug Delivery, Borchardt et al, (ed.), pp.247-267, Humana Press (1985). Prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β -lactam-containing prodrugs, prodrugs containing optionally substituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide, 5-fluorocytosine and other 5-fluorouridine prodrugs convertible to more active, non-cytotoxic drugs. Examples of cytotoxic drugs that may be derivatized into prodrug forms for use in the present invention include, but are not limited to, those chemotherapeutic agents described above.

"radiotherapy" or "radiotherapy" refers to the use of directed gamma rays or beta rays to induce sufficient damage to cells to limit their ability to function normally or to destroy them altogether. It will be appreciated that many ways are known in the art to determine the dosage and duration of treatment. Typical treatments are given as one administration, while typical doses range from 10-200 units per day (Gray).

A "biological sample" (interchangeably referred to as a "sample" or "tissue or cell sample") encompasses a variety of sample types obtained from an individual and that may be used in diagnostic or monitoring assays. This definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and the progeny thereof. This definition also covers samples that have been manipulated after they have been obtained, such as by treatment with reagents, solubilization, or enrichment for certain components such as proteins or polynucleotides, or buried in a semi-solid or solid matrix for slicing purposes. The term "biological sample" encompasses clinical samples, but also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The source of the biological sample may be a solid tissue, like from a fresh, frozen and/or preserved organ or tissue sample or biopsy sample or punch sample; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; from cells at any time during pregnancy or ontogeny. In some embodiments, the biological sample is obtained from a primary or metastatic tumor. Biological samples may contain compounds that do not naturally mix with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

For the purposes of the present invention, a "section" of a tissue sample refers to a piece or sheet of the tissue sample, such as a thin slice of tissue or cells cut from the tissue sample. It should be understood that multiple slices of tissue sample may be made and analyzed in accordance with the present invention. In some embodiments, the same section of tissue sample is used for both morphological and molecular level analysis or methods for analyzing both proteins and nucleic acids.

The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a nucleic acid probe or antibody, to facilitate detection of the agent to which it is conjugated or fused. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

Compositions of the invention and methods of making and using the same

The present invention encompasses compositions (including pharmaceutical compositions) comprising an anti-hepsin antibody; and polynucleotides comprising sequences encoding anti-hepsin antibodies. As used herein, a composition comprises one or more antibodies that bind hepsin, and/or one or more polynucleotides comprising sequences encoding one or more antibodies that bind hepsin. These compositions may further comprise suitable carriers such as pharmaceutically acceptable excipients, including buffers, which are well known in the art.

The invention also encompasses embodiments of the isolated antibodies and polynucleotides. The invention also encompasses embodiments of substantially pure antibodies and polynucleotides.

The invention also encompasses methods of treating disorders, such as prostate cancer, using anti-hepsin antibodies (as described herein or as known in the art).

Composition comprising a metal oxide and a metal oxide

The anti-hepsin antibodies of the invention are preferably monoclonal. The scope of the invention also encompasses fabs of the anti-hepsin antibodies provided hereinFab ', Fab ' -SH and F (ab ')₂And (3) fragment. These antibody fragments may be prepared by conventional means, such as enzymatic digestion, or may be generated by recombinant techniques. Such antibody fragments may be chimeric or humanized. These fragments are useful for diagnostic and therapeutic purposes as set forth below.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of different antibodies.

The anti-hepsin monoclonal antibodies of the invention may be prepared using the hybridoma method originally described by Kohler et al, Nature 256:495(1975), or may be prepared by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies to hepsin can be generated by multiple subcutaneous (sc) or intraperitoneal (ip) injections of hepsin and adjuvant in animals. hepsin may be prepared using methods well known in the art, some of which are further described herein. For example, recombinant production of human and mouse hepsin is described below. In one embodiment, the animal is immunized with hepsin fused to the Fc portion of an immunoglobulin heavy chain. In a preferred embodiment, the animal is immunized with hepsin-IgG1 fusion protein. Animals are typically immunized against an immunogenic conjugate or derivative of hepsin and monophosphoryl lipid a (mpl)/trehalose mycolic acid bis (trehalose dicrynynomycolate, TDM) Ribi immunochem. After 2 weeks, animals were boosted. After 7-14 days, animals were bled and sera were assayed for anti-hepsin titers. Animals were boosted until the titer reached a plateau (plateaus).

Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, monoclonal antibodies: Principles and Practice, pp.59-103, Academic Press, 1986).

The hybridoma cells so prepared are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent HGPRT-deficient cells from growing.

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these cells, preferred myeloma Cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center (San Diego, California, USA) and SP-2 or X63-Ag8-653 cells available from the American Type culture Collection (American Type culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human Monoclonal antibodies (Kozbor, J.Immunol.133:3001(1984); Brodeur et al, Monoclonal antibody production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., NewYork, 1987).

The culture medium in which the hybridoma cells are growing can be assayed for production of monoclonal antibodies directed against hepsin. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. biochem.107:220 (1980).

After identification of hybridoma cells producing Antibodies with the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, academic Press, 1986). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors.

Monoclonal antibodies secreted by the subclones can be suitably separated from the culture fluid, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Anti-hepsin antibodies of the invention can be prepared by screening synthetic antibody clones with the desired activity using combinatorial libraries. In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying various antibody variable region fragments (Fv) fused to phage coat proteins. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and are thus separated from the non-binding clones in the library. The bound clones are then eluted from the antigen and can be further enriched by additional antigen adsorption/elution cycles. Any anti-hepsin antibody of the invention may be obtained by designing an appropriate antigen screening protocol to select phage clones of Interest, followed by construction of full-length anti-hepsin antibody clones using Fv Sequences from phage clones of Interest and appropriate constant region (Fc) Sequences as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, NIHPublication 91-3242, Bethesda MD (1991), volumes 1-3. An exemplary method for generating anti-hepsin antibodies is disclosed in the examples.

The antigen-binding domain of an antibody is formed by two variable (V) regions of about 110 amino acids, from the heavy (VL) and light (VH) chains, respectively, that both exhibit three hypervariable loops or Complementarity Determining Regions (CDRs). The variable domains can be functionally displayed on phage, either as single chain fv (scFv) fragments, in which VH and VL are covalently linked by a short, flexible peptide, or as Fab fragments, in which each is fused to a constant domain and interacts non-covalently, as described by Winter et al, Ann.Rev.Immunol.,12:433-455 (1994). As used herein, scFv-encoding phage clones and Fab-encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones".

The repertoire of VH and VL genes can be separately cloned by Polymerase Chain Reaction (PCR) and recombined randomly in a phage library, and then can be searched for antigen-binding clones, as described by Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Libraries from immunized sources can provide antibodies with high affinity for the immunogen without the need to construct hybridomas. Alternatively, the non-immunized repertoire can be cloned to provide a single human antibody source for a broad range of non-self and self antigens without any immunization as described by Griffiths et al, EMBO J,12:725 (1993). Finally, unimmunized libraries can also be constructed synthetically, i.e., by cloning unrearranged V gene fragments from stem cells, using PCR primers comprising random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992).

Filamentous phage was used to display antibody fragments by fusion with the minor coat protein pIII. Antibody fragments may be displayed as single chain Fv fragments in which the VH and VL domains are linked by a flexible polypeptide spacer on the same polypeptide chain, for example as described by Marks et al, J.mol.biol.,222: 581-.

Generally, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If a bias of the library towards anti-hepsin clones is desired, the individual may be immunized with hepsin to generate an antibody response, and splenocytes and/or circulating B cells or other Peripheral Blood Lymphocytes (PBLs) recovered for library construction. In a preferred embodiment, a library of human antibody gene fragments biased for anti-hepsin cloning is obtained by generating anti-hepsin antibody responses in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that hepsin immunization generates B cells that produce human antibodies against hepsin. The production of human antibody-producing transgenic mice is described below.

Further enrichment of the anti-hepsin-reactive cell population may be obtained by isolating B-cells expressing hepsin-specific membrane-bound antibodies using a suitable screening procedure, e.g. by cell separation using hepsin affinity chromatography or adsorption of fluorescent dye-labeled hepsin by the cells followed by Flow Activated Cell Sorting (FACS).

Alternatively, the use of splenocytes and/or B cells or other PBLs from non-immunized donors provides a better representation of the possible repertoire of antibodies, and also allows the construction of antibody libraries using any animal (human or non-human) species in which hepsin is not antigenic. For library construction of in vitro incorporated antibody genes, stem cells are harvested from an individual to provide nucleic acids encoding unrearranged antibody gene segments. Immune cells of interest can be obtained from a variety of animal species (such as human, mouse, rat, rabbit, luprine, dog, cat, pig, cow, horse, and avian species, among others).

Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. For the rearranged VH and VL gene libraries, the desired DNA may be obtained by isolating genomic DNA or mRNA from lymphocytes followed by Polymerase Chain Reaction (PCR) using primers matched to the 5 'and 3' ends of the rearranged VH and VL genes, as described by Orlandi et al, Proc.Natl.Acad.Sci.USA,86: 3833-. The V gene can be amplified from cDNA and genomic DNA, with a reverse primer located at the 5' end of the exon encoding the mature V domain, and a forward primer based within the J segment, as described by Orlandi et al (1989) and Ward et al, Nature,341:544, 546 (1989). However, for amplification from cDNA, the reverse primer may also be based within the leader exon (leader exon), as described by Jones et al, Biotechnol.,9:88-89(1991), and the forward primer is based within the constant region, as described by Sastry et al, Proc. Natl. Acad. Sci. (USA),86: 5728-. To maximize complementarity, degeneracy may be incorporated into the primers, as described by Orlandi et al (1989) or by Sasty et al (1989). Preferably, library diversity is maximized by amplifying all available VH and VL permutations present in immune cell Nucleic acid samples using PCR primers targeting each V gene family, for example as described in the methods of Marks et al, J.mol.biol.,222: 581-4497 (1991) or Orum et al, Nucleic Acids Res.,21:4491-4498 (1993). For cloning the amplified DNA into an expression vector, rare restriction sites may be introduced as tags at one end of the PCR primers, as described by Orlandi et al (1989), or further PCR amplification may be performed using tagged primers, as described by Clackson et al, Nature,352: 624-.

The artificially synthesized rearranged complete set of V genes can be derived from V gene segments in vitro. Most human VH gene segments have been cloned and sequenced (Tomlinson et al, J.mol.biol.,227:776-798(1992)) and located (Matsuda et al, Nature Genet.,3:88-94 (1993)); segments of these clones (including all major configurations of the H1 and H2 loops) can be used to generate a diverse repertoire of VH genes using PCR primers for the H3 loop with coding sequence and length diversity, as described in Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). VH repertoires can also be generated by concentrating all sequence diversity in a single length long H3 loop, as described by Barbas et al, Proc. Natl. Acad. Sci. USA,89:4457-4461 (1992). The human V.kappa.and V.lambda.segments have been cloned and sequenced (Williams and Winter, Eur. J.Immunol.,23: 1456-. Synthetic V gene repertoires based on a series of VH and VL folds and lengths of L3 and H3 will encode antibodies with considerable structural diversity. After amplification of the DNA encoding the V gene, the germline V gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992).

The repertoire of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be created in different vectors and the vectors recombined in vitro, e.g.as described by Hogrefe et al, Gene,128:119-126(1993), or in vivo by combinatorial infection, e.g.the loxP system described by Waterhouse et al, Nucl. acids Res.,21:2265-2266 (1993). The in vivo recombination method exploits the double-stranded nature of the Fab fragment to overcome the library volume limitation imposed by e. The non-immunized VH and VL repertoires were cloned separately, one into the phagemid and the other into the phage vector. The two libraries were then combined by infecting phage-containing bacteria with phage so that each cell contained a different combination, the library capacity being limited only by the number of cells present (about 10) ¹²Individual clones). Both vectors contain in vivo recombination signals, such that the VH and VL genes are recombined onto a single replicon and co-packaged into phage virions. These giant libraries provide large numbers of libraries with good affinity (Kd)^-1Is about 10^-8M) of a diverse antibody.

Alternatively, the repertoires can be cloned sequentially into the same vector, e.g., as described by Barbas et al, Proc. Natl. Acad. Sci. USA,88: 7978-. PCR assembly can also be used to link VH and VL DNA with DNA encoding flexible peptide spacers to form single chain fv (scfv) repertoires. In another technique, "intracellular PCR assembly" is used to combine VH and VL genes in lymphocytes by PCR, and then to clone a repertoire of linked genes, as described by Embleton et al, Nucl. acids Res.,20:3831-3837 (1992).

Antibodies generated by non-immune libraries (natural or synthetic) may have intermediate affinity (Kd)^-1Is about 10⁶-10⁷M^-1) However, it is also possible to simulate affinity maturation in vitro by constructing and reselecting secondary genesLibraries, such as Winter et al (1994), supra. For example, in the method of Hawkins et al, J.mol.biol.,226:889-896(1992) or the method of Gram et al, Proc.Natl.Acad.SciUSA,89:3576-3580(1992), mutations were randomly introduced in vitro using an error-prone polymerase (Leung et al, Technique,1:11-15 (1989)). In addition, affinity maturation can be performed by randomly mutating one or more CDRs, e.g., PCR using primers that carry random sequences across the CDRs of interest in selected individual Fv clones and screening for higher affinity clones. WO 96/07754 (published on 3/14 1996) describes a method for inducing mutagenesis in the complementarity determining regions of immunoglobulin light chains to create a light chain gene library. Another highly efficient method is to combine VH or VL domains selected by phage display with naturally occurring V domain variants from non-immune donors and screen for higher affinity in rounds of strand shuffling, as described in Marks et al, Biotechnol.,10:779-783 (1992). This technique allows the generation of affinities at 10 ^-9Antibodies and antibody fragments in the M range.

hepsin nucleic acid and amino acid sequences are known in the art. The amino acid sequence of the desired hepsin region can be used to design a nucleic acid sequence encoding hepsin. As is well known in the art, hepsin has two major splicing isoforms (isosorm), hepsin IIIb and hepsin IIIc. hepsin sequences are well known in the art and may include the sequences shown in FIGS. 7 and 8.

Nucleic acids encoding hepsin may be prepared by a variety of methods known in the art. These methods include, but are not limited to, chemical synthesis by any of the methods described in Engels et al, Agnew. chem. int. Ed. Engl.,28:716-734(1989), such as the triester, phosphite, phosphoramidate, and H-phosphonate methods. In one embodiment, the DNA encoding hepsin is designed using codons preferred by the expression host cell. Alternatively, hepsin-encoding DNA may be isolated from genomic or cDNA libraries.

After construction of a DNA molecule encoding hepsin, the DNA molecule is operably linked to expression control sequences in an expression vector, such as a plasmid, wherein the control sequences are recognized by a host cell transformed with the vector. Generally, plasmid vectors contain replication and control sequences that are derived from a species compatible with the host cell. Vectors typically carry a site of replication, as well as sequences encoding proteins capable of providing phenotypic selection in transformed cells. Vectors suitable for expression in prokaryotic and eukaryotic host cells are known in the art, and some are described further herein. Eukaryotic organisms such as yeast or cells derived from multicellular organisms such as mammals can be used.

Optionally, the hepsin-encoding DNA is operably linked to a secretion leader sequence, resulting in secretion of the expression product into the culture medium by the host cell. Examples of secretory leaders include stII, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and α -factor. Also suitable for use herein is the 36 amino acid leader sequence of protein A (Abrahmsen et al, EMBO J.,4:3901 (1985)).

Host cells are transfected, preferably transformed, with the expression or cloning vectors of the invention described above and cultured in conventional nutrient media, which may be modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transfection refers to the uptake of an expression vector by a host cell, whether or not any coding sequence is actually expressed. One of ordinary skill in the art will know of a number of transfection methods, e.g., CaPO₄Precipitation and electroporation. Transfection is considered successful if there is any indication of the operation of this vector within the host cell. Methods for transfection are well known in the art, and some are described further herein.

Transformation refers to the introduction of DNA into an organism such that the DNA may be replicated, either as an extrachromosomal element, or by chromosomal integration. Depending on the host cell used, transformation is carried out using standard techniques appropriate for the cell. Methods for transformation are well known in the art, and some are described further herein.

Prokaryotic host cells for production of hepsin may be cultured as described generally above in Sambrook et al.

Mammalian host cells used to produce hepsin can be cultured in a variety of media, which are well known in the art and some of which are described herein.

The host cells disclosed herein encompass cells in culture in vitro as well as cells in vivo in a host animal.

Purification of hepsin may be achieved using art-recognized methods, some of which are described herein.

Purified hepsin can be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic acid copolymers, hydroxy methacrylate gels, polyacrylic and polymethacrylic acid copolymers, nylon, neutral and ionic carriers, and the like, for affinity chromatography separation of phage display clones. Attachment of hepsin proteins to substrates can be achieved by Methods described in Methods in enzymology, volume 44 (1976). A common technique for attaching protein ligands to polysaccharide matrices, such as agarose, dextran or cellulose, involves activation of the support with cyanogen halides followed by coupling of the primary aliphatic or aromatic amines of the peptide ligands to the activated matrix.

Alternatively, hepsin can be used to coat the wells of the adsorption plate, expressed on host cells attached to the adsorption plate, or used for cell sorting, or coupled to biotin for capture with streptavidin-coated beads, or used in any other method known in the art for panning phage display libraries.

Contacting a phage library sample with the immobilized hepsin under conditions suitable for binding of at least a portion of the phage particles to the adsorbent. Normally, conditions including pH, ionic strength, temperature, and the like are selected to mimic physiological conditions. Phage bound to the solid phase are washed and then eluted with an acid, for example as described by Barbas et al, Proc. Natl.Acad. Sci USA,88: 7978-. Phages can be enriched 20-1,000-fold in a single round of selection. In addition, the enriched phage can be cultured in bacterial culture and subjected to more rounds of selection.

The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage are capable of binding antigen simultaneously. Antibodies with faster dissociation kinetics (and weak binding affinity) can be retained by using short washes, multivalent phage display, and high antigen coating density in the solid phase. The high density not only stabilizes the phage through multivalent interactions, but also facilitates the recombination of dissociated phage. Selection of antibodies with slower dissociation kinetics (and strong binding affinity) can be facilitated by the use of prolonged washing and monovalent phage display (as described by Bass et al, Proteins,8:309-314(1990) and WO 92/09690) and low antigen coating density (as described by Marks et al, Biotechnol.,10:779-783 (1992)).

It is possible to select between phage antibodies with different affinities for hepsin, even with slightly different affinities. However, random mutagenesis of selected antibodies (e.g., as performed by affinity maturation techniques, some of which are described above) is likely to produce many mutants, most of which bind antigen, and a few of which have higher affinity. By limiting hepsin, rare high affinity phage can compete out. To retain all higher affinity mutants, phages may be incubated with an excess of biotinylated hepsin, but at a molar concentration of biotinylated hepsin that is below the target molar affinity constant for hepsin. High affinity binding phage were then captured with streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows selection of antibodies according to binding affinity, with sensitivity that allows isolation of mutant clones with only 2-fold the original affinity from a large excess of low affinity phage. Conditions for washing phage bound to the solid phase can also be manipulated to perform dissociation kinetic-based differentiation.

hepsin clones can be actively selected. Fv clones corresponding to such hepsin antibodies can be selected as follows: (1) isolating hepsin clones from the phage library as described above, and optionally amplifying the population of isolated phage clones by culturing the population in a suitable bacterial host; (2) selecting hepsin for which blocking of activity is desired and a second protein for which blocking of activity is not desired; (3) adsorbing an anti-hepsin phage clone to the immobilized hepsin; (4) eluting any unwanted clones that recognize hepsin binding determinants that overlap or share with the binding determinants of the second protein using an excess of the second protein; and (5) eluting the clones that remain adsorbed after step (4). Optionally, clones with the desired blocking/non-blocking properties can be further enriched by repeating the selection procedure described herein one or more times.

The DNA encoding the hybridoma-derived monoclonal antibodies or phage display Fv clones of the invention are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from a hybridoma or phage DNA template). Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulin protein, such as an escherichia coli cell, a simian COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell, to obtain synthesis of the desired monoclonal antibody in the recombinant host cell. A review of the recombinant expression of DNA encoding antibodies in bacteria includes Skerra et al, curr. opinion in Immunol.,5:256(1993) and Pluckthun, Immunol. Rev.,130:151 (1992).

The DNA encoding Fv clones of the invention may be combined with known DNA sequences encoding the heavy and/or light chain constant regions (e.g., suitable DNA sequences are available from Kabat et al, supra) to form clones encoding full-length or partial heavy and/or light chains. It will be appreciated that constant regions of any isotype may be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and that such constant regions may be derived from any human or animal species. Fv clones derived from variable domain DNA of one animal species (such as human) and then fused with constant region DNA of another animal species to form coding sequences for "hybrid" full-length heavy and/or light chains are included in the definition of "chimeric" and "hybrid" antibodies as used herein. In a preferred embodiment, Fv clones derived from human variable DNA are fused to human constant region DNA to form fully human, full-length or partial heavy and/or light chain coding sequences.

The DNA of the invention encoding an anti-hepsin antibody derived from a hybridoma may also be modified, for example by replacing, with the coding sequences for the human heavy and light chain constant domains, homologous murine sequences derived from hybridoma clones (e.g.as in Morrison et al, Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). The DNA encoding the hybridoma or Fv clone-derived antibody or fragment may be further modified by covalently joining the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide. "chimeric" or "hybrid" antibodies having the binding specificity of the Fv clone or hybridoma clone-derived antibodies of the invention can be prepared in this manner.

Antibody fragments

The invention encompasses antibody fragments. In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and may result in easier access to solid tumors.

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and biological Methods 24:107-117(1992); Brennan et al, Science 229:81 (1985)). However, these fragments can now be produced directly from recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted by E.coli, thus allowing easy production of large quantities of these fragments. Antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab' -SH fragments can be recovered directly from E.coli and chemically coupled to Form F (ab')₂Fragments (Carter et al, Bio/Technology10:163-167 (1992)). According to another method, F (ab') can be isolated directly from recombinant host cell cultures₂And (3) fragment. Fab and F (ab') with extended in vivo half-life comprising salvage receptor binding epitope residues₂Fragments are described in U.S. Pat. No.5,869,046. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (see, e.g., WO 93/16185; U.S. Pat. No.5,571,894; and 5,587,458). Fv and sFv are the only types with intact binding sites, lacking constant regions; as such, they are suitable for reducing non-specific binding when used in vivo. sFv fusion proteins can be constructed to generate fusions of effector proteins at the amino or carboxy terminus of an sFv. See, for example, Antibody Engineering, eds. Borebaeck, supra. Antibody fragments may also be "linear antibodies," for example as described in U.S. Pat. No.5,641,870. Such linear antibody fragments may be monospecific or bispecific.

Humanized antibodies

The present invention encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed essentially following the method of Winter and co-workers (Jones et al, Nature 321:522-525(1986); Riechmann et al, Nature 332:323-327(1988); Verhoeyen et al, Science 239:1534-1536 (1988)), using the (non-human) hypervariable region sequences in place of the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which significantly less than an intact human variable domain is replaced with the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

The choice of human light and heavy chain variable domains for making humanized antibodies is very important for reducing antigenicity. The entire library of known human variable domain sequences is screened with the variable domain sequences of rodent antibodies according to the so-called "best-fit" method. The closest human sequence to rodents is then selected as the human framework for the humanized antibody (Sims et al, J.Immunol.151:2296(1993); Chothia et al, J.mol.biol.196:901 (1987)). Another approach uses a specific framework derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains (subgroups). The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA 89:4285(1992); Presta et al, J. Immunol.151:2623 (1993)).

More importantly, the antibodies retain high affinity for the antigen and other favorable biological properties after humanization. To achieve this, according to one method, humanized antibodies are prepared by a process of analyzing the parent sequence and various conceptual humanized products using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional models of immunoglobulins are generally available, as will be familiar to those skilled in the art. Computer programs are also available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these display images allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the acceptor and import sequences and combined to obtain a desired antibody characteristic, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in the effect on antigen binding.

Human antibodies

Human anti-hepsin antibodies of the invention may be constructed by combining Fv clone variable domain sequences selected from human-derived phage display libraries with known human constant domain sequences as described above. Alternatively, the human monoclonal anti-hepsin antibodies of the invention may be produced by hybridoma methods. Human myeloma and mouse-human heteromyeloma cell lines for generating human Monoclonal antibodies have been described, for example, in Kozbor J.Immunol.,133:3001(1984), Brodeur et al, Monoclonal Antibody Production Techniques and applications, pp.51-63(Marcel Dekker, Inc., New York,1987), and Borner et al, J.Immunol.,147:86 (1991).

It is now possible to generate transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of large numbers of human germline immunoglobulin genes in such germline mutant mice results in the production of human antibodies following antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA 90:2551(1993), Jakobovits et al, Nature 362:255-258(1993), Bruggemann et al, Yeast in Immunol.7:33 (1993).

Gene shuffling can also be used to derive human antibodies from non-human (e.g., rodent) antibodies, where the human antibodies have similar affinity and specificity as the starting non-human antibody. According to this method, which is also known as "epitope imprinting", the variable regions of the heavy or light chains of the non-human antibody fragments obtained by phage display techniques as described above are replaced with a repertoire of human V domain genes, resulting in a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in the isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site that was destroyed upon elimination of the corresponding non-human chain in the primary phage display clone, i.e., the epitope determines (imprints) the selection of the human chain partner. When this process is repeated to replace the remaining non-human chains, human antibodies are obtained (see PCT WO 93/06213, published at 1/4 of 1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides fully human antibodies that do not contain FR or CDR residues of non-human origin.

Bispecific antibodies

Bispecific antibodiesRefers to a monoclonal antibody, preferably a human or humanized antibody, having binding specificity for at least two different antigens. In the present case, one of the binding specificities is for hepsin and the other binding specificity is for any other antigen. Exemplary bispecific antibodies can bind two different epitopes of hepsin. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing hepsin. These antibodies possess a hepsin binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-alpha, vinca alkaloids, ricin a chain, methotrexate, or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab') ₂Bispecific antibodies).

Methods for constructing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two pairs of immunoglobulin heavy-light chains, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305:537 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. A similar procedure is disclosed in WO 93/08829, published in 1993 at 13.5.13 and Trunecker et al, EMBO J.10:3655 (1991).

According to a different and more preferred method, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. Preferably, at least part of the hinge, C_H2 and C_HRegion 3 immunoglobulin heavy chain constant domain fusion. Preferably, a first heavy chain constant region (C) comprising the site necessary for light chain binding is present in at least one of the fusions _H1). The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. In the case of unequal ratios of the three polypeptide chains used for constructionThis provides great flexibility in adjusting the mutual ratio of the three polypeptide fragments in embodiments that provide optimal yields. However, where expression of at least two polypeptide chains in equal ratios results in high yields or where the ratio is of no particular significance, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector.

In a preferred embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient separation route, it was found that this asymmetric structure facilitates the separation of the desired bispecific complex from the unwanted immunoglobulin chain combinations. This method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, methods Enzymology 121:210 (1986).

According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise at least part of an antibody constant domain C_H3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size to the large side chains are created at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody of the heterologous conjugate may be conjugated to avidin and the other antibody to biotin. For example, such antibodies have been suggested for use in targeting immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for use in the treatment of HIV infection (WO 91/00360; WO 92/00373; and EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No.4,676,980, along with a number of crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science 229:81(1985) describe proteolytic cleavage of intact antibodies to F (ab')₂Protocol for fragmentation. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reverted back to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization reagents for enzymes.

Recent advances have facilitated the direct recovery of Fab' -SH fragments from E.coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, J.Exp.Med.175:217-225(1992) describe a fully humanized bispecific antibody F (ab')₂And (4) generation of molecules. Each Fab' fragment was secreted separately from E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibody so formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for the direct production and isolation of bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been generated using leucine zippers. Kostelny et al, J.Immunol.148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. Such asThe methods can also be used to generate antibody homodimers. The "diabody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. The fragment comprises heavy chain variable domains (V) connected by a linker_H) And a light chain variable domain (V)_L) The linker is too short to allow pairing between the two domains on the same strand. Thus, V on a segment is forced_HAnd V_LDomain and complementary V on another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for constructing bispecific antibody fragments by using single chain fv (sfv) dimers has also been reported. See Gruber et al, J.Immunol.152:5368 (1994).

Antibodies with more than two titers are contemplated. For example, trispecific antibodies can be prepared. Tutt et al, J.Immunol.147:60 (1991).

Multivalent antibodies

Multivalent antibodies can be internalized (and/or catabolized) by a cell expressing an antigen to which the antibody binds more rapidly than bivalent antibodies. The antibodies of the invention can be multivalent antibodies (other than the IgM class) having three or more antigen binding sites (e.g., tetravalent antibodies) that can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. A multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites at the amino terminus of the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight, but preferably four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chains comprise two or more variable domains. For example, a polypeptide chain can comprise VD1- (X1)_n-VD2-(X2)_n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. Example (b) For example, the polypeptide chain can comprise: VH-CH 1-flexible linker-VH-CH 1-Fc region chain; or VH-CH1-VH-CH1-Fc domain chain. Preferably, the multivalent antibody herein further comprises at least two (and preferably four) light chain variable domain polypeptides. A multivalent antibody herein may comprise, for example, about two to about eight light chain variable domain polypeptides. Light chain variable domain polypeptides encompassed herein comprise a light chain variable domain, and optionally further comprise a CL domain.

Antibody variants

In some embodiments, amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics. Amino acid changes can be introduced into the subject antibody amino acid sequence at the time the sequence is prepared.

One method that can be used to identify certain residues or regions of an antibody that are preferred mutagenesis positions is referred to as "alanine scanning mutagenesis" as described in Cunningham and Wells, Science 244:1081-1085 (1989). Here, a residue or set of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions that exhibit functional sensitivity to substitution are then refined by introducing more or other variants at or to the substitution site. Thus, while the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the consequences of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region and the expressed immunoglobulin is screened for the desired activity.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion of the N-or C-terminus of the antibody with an enzyme (e.g. for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate module to an asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding or replacing one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

If the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, U.S. patent application No. US 2003/0157108(Presta, L.) describes antibodies with a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody. See also US 2004/0093621(Kyowa Hakko kogyo co., Ltd.). Antibodies having an aliquot of N-acetylglucosamine (GlcNAc) in a carbohydrate attached to the Fc region of the antibody are described in WO 2003/011878(Jean-Mairet et al) and U.S. Pat. No.6,602,684(Umana et al). Antibodies having at least one galactose residue in an oligosaccharide attached to the Fc region of an antibody are reported in WO 1997/30087(Patel et al). For antibodies with altered carbohydrates attached to their Fc regions see also WO 1998/58964(Raju, S.) and WO 99/22764(Raju, S.). For antigen binding molecules with improved glycosylation see also US 2005/0123546(Umana et al).

Preferred glycosylation variants herein comprise an Fc region, wherein the carbohydrate structure attached to the Fc region lacks fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions that further improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (Eu residue numbering). Examples of publications relating to "defucose" or "fucose-deficient" antibodies include: US 2003/0157108, WO 2000/61739, WO 2001/29246, US2003/0115614, US 2002/0164328, US 2004/0093621, US 2004/0132140, US2004/0110704, US 2004/0110282, US 2004/0109865, WO 2003/085119, WO2003/084570, WO 2005/035586, WO 2005/035778, WO2005/053742, Okazaki et al J.mol.biol.336:1239-1249(2004), Yamane-Ohnuki et al Biotech.Bioeng.87:614 (2004). Examples of cell lines producing defucosylated antibodies include protein fucosylated deficient Lec13 CHO cells (Ricka et al Arch. biochem. Biophys.249:533-545(1986); U.S. patent application No. US 2003/0157108A1, Presta, L; and WO 2004/056312A1, Adams et al, especially example 11) and knock-out cell lines such as the alpha-1, 6-fucosyltransferase gene, FUT8, knock-out CHO cells (Yamane-Ohnuki et al Biotech. Bioeng.87:614 (2004)).

Another class of variants are amino acid substitution variants. These variants have at least one amino acid residue (at least two, at least three, at least four or more) in the antibody molecule replaced with a different residue. Sites of greatest interest for substitutional mutagenesis include hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table A under the heading "preferred substitutions". If such substitutions result in a change in biological activity, more substantial changes, referred to in Table A as "exemplary substitutions," or as described further below with reference to amino acid classifications, can be introduced and the products screened.

TABLE A

Original residues	Example alternatives	Preferred alternatives
			Ala(A)	Val;Leu;Ile	Val
Arg(R)	Lys;Gln;Asn	Lys
			Asn(N)	Gln;His;Asp;Lys;Arg	Gln
Asp(D)	Glu;Asn	Glu
			Cys(C)	Ser;Ala	Ser
Gln(Q)	Asn;Glu	Asn
			Glu(E)	Asp;Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn;Gln;Lys;Arg	Arg
Ile(I)	Leu, Val, Met, Ala, Phe, norleucine	Leu
			Leu(L)	Norleucine, Ile, Val, Met, Ala, Phe	Ile
Lys(K)	Arg;Gln;Asn	Arg
			Met(M)	Leu;Phe;Ile	Leu
Phe(F)	Trp;Leu;Val;Ile;Ala;Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val;Ser	Ser
Trp(W)	Tyr;Phe	Tyr
			Tyr(Y)	Trp;Phe;Thr;Ser	Phe
Val(V)	Ile, Leu, Met, Phe, Ala, norleucine	Leu

Substantial modification of antibody biological properties can be achieved by selecting substitutions that differ significantly in their effectiveness in maintaining: (a) the structure of the polypeptide backbone in the replacement region, e.g., (folded) sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Naturally occurring residues may be grouped as follows, according to common side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp and Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro; and

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions may entail replacing one of these classes with a member of the other class.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were generated. One convenient method for generating such surrogate variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) were mutated to generate all possible amino acid substitutions at each site. The antibodies so generated are displayed on filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. The contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

Nucleic acid molecules encoding antibody amino acid sequence variants can be prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants), or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications in the Fc region of the immunoglobulin polypeptides of the invention, thereby generating Fc region variants. Fc region variants may include human Fc region sequences (e.g., human IgG) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions, including hinge cysteines₁、IgG₂、IgG₃Or IgG₄An Fc region).

It is contemplated that in some embodiments, the antibodies used in the methods of the invention may comprise one or more alterations in, for example, the Fc region as compared to a wild-type counterpart antibody, in accordance with this description and the teachings of the art. These antibodies will still retain essentially the same properties required for therapeutic efficacy compared to their wild-type counterparts. For example, it is believed that certain changes in the Fc region may be made which will result in altered (i.e. either enhanced or attenuated) C1q binding and/or Complement Dependent Cytotoxicity (CDC), for example as described in WO 99/51642. See also Duncan and Winter, Nature 322:738-40(1988), U.S. Pat. No.5,648,260, U.S. Pat. No.5,624,821, and WO94/29351, which are directed to other examples of Fc region variants. Variants of antibodies with increased or decreased binding to FcR are described in WO00/42072(Presta) and WO 2004/056312 (Lowman). The contents of these patent publications are expressly incorporated herein by reference. See also Shields et al J.biol.chem.9(2):6591-6604 (2001). Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249(1994)) are described in US2005/0014934A1(Hinton et al). These antibodies comprise an Fc region having one or more substitutions that improve binding of the Fc region to FcRn. Polypeptide variants having altered amino acid sequences of the Fc region and increased or decreased binding of C1q are described in U.S. Pat. No.6,194,551B1, WO 99/51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogene et al J.Immunol.164: 4178-.

Antibody derivatives

The antibodies of the invention can be further modified to include additional non-proteinaceous moieties known in the art and readily available. Preferably, the moiety suitable for derivatization of the antibody is a water-soluble polymer. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or function of the antibody to be improved, whether the antibody derivative will be used for therapy under specified conditions, and the like.

Screening for antibodies with desired Properties

The antibodies of the invention can be characterized for their physical/chemical properties and biological functions using various assays known in the art, some of which are disclosed herein. In some embodiments, the antibody is characterized by any one or more of: decreasing or blocking hepsin binding, decreasing or blocking hepsin activity, decreasing or blocking hepsin and/or hepsin substrate (e.g. pro-MSP, pro-uPA, factor VII, pro-HGF) downstream molecular signaling, and/or treating and/or preventing a tumor, a cell proliferative disorder or cancer, and/or treating or preventing a disorder associated with hepsin expression and/or activity, such as increased hepsin expression and/or activity. In some embodiments, the hepsin activity is hepsin enzymatic activity. In one embodiment, the enzymatic activity comprises a polypeptide substrate that cleaves hepsin. In one embodiment, the polypeptide substrate of hepsin is one or more of pro-macrophage stimulating protein (pro-MSP), pro-uPA, factor VII, and pro-HGF. pro-MSP activation by hepsin is described in co-pending, commonly owned U.S. provisional patent application No.61/253,990, filed 10/22/2009. In one embodiment, the enzymatic activity comprises a synthetic substrate that cleaves hepsin. In some embodiments, the hepsin synthetic substrate is a substrate shown in table 1.

The purified antibody may also be characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion High Pressure Liquid Chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

In certain embodiments of the invention, the antibodies produced herein are assayed for their biological activity. In some embodiments, antibodies of the invention are tested for their antigen binding activity. Antigen binding assays known in the art and that may be used herein include, but are not limited to, any direct or competitive binding assay that utilizes techniques such as: western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich/sandwich" immunoassay, immunoprecipitation assay, fluorescent immunoassay, and protein A immunoassay. Exemplary antigen binding and/or other assays are provided in the examples section below.

If an anti-hepsin antibody that inhibits cell growth is desired, the candidate antibody can be tested in an in vitro and/or in vivo assay that measures inhibition of cell growth. Methods for examining the growth and/or proliferation of cancer cells are well known in the art. Exemplary methods for determining cell growth and/or proliferation and/or apoptosis include, for example, BrdU incorporation assays, MTT, [3H ] -thymidine incorporation (e.g., TopCount assay (PerkinElmer)), cell viability assays (e.g., CellTiter-glo (promega)), and the like.

In one embodiment, the invention encompasses antibodies with effector function. In certain embodiments, the Fc activity of the antibody is measured. In vitro and/or in vivo cytotoxicity assays may be performed to demonstrate the reduction/elimination of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and thus may lack ADCC activity), but retains FcRn binding ability. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in table 3 on page 464 of ravatch and Kinet, annu.rev.immunol 9:457-92 (1991). One example of an in vitro assay for assessing ADCC activity of a molecule of interest is described in U.S. Pat. No.5,500,362 or 5,821,337. An assay for detecting ADCC activity is also exemplified herein. Useful effector cells that can be used in these assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al PNAS (USA)95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and thereby lacks CDC activity. To assess complement activation, CDC assays can be performed, for example, as described by Gazzano-Santoro et al, J.Immunol.methods 202:163 (1996). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art.

Vectors, host cells, and recombinant methods

For recombinant production of the antibody of the invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector will depend in part on the host cell to be used. Generally, preferred host cells are of prokaryotic or eukaryotic (typically mammalian) origin. It will be appreciated that constant regions of any isotype may be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and that such constant regions may be obtained from any human or animal species.

a. Production of antibodies using prokaryotic host cells:

i. vector construction

The polynucleotide sequences encoding the polypeptide building blocks of the antibodies of the invention can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated from antibody producing cells such as hybridoma cells and sequenced. Alternatively, polynucleotides may be synthesized using nucleotide synthesizers or PCR techniques. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replication and expression of the heterologous polynucleotide in a prokaryotic host. For the purposes of the present invention, a wide variety of vectors available and known in the art may be used. The choice of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains a variety of components, depending on its function (either to amplify or express the heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. Carrier members generally include, but are not limited to: an origin of replication, a selectable marker gene, a promoter, a Ribosome Binding Site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

In general, plasmid vectors for use with host cells contain replicon and control sequences that are derived from species compatible with these hosts. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells. For example, E.coli is typically transformed with plasmid pBR322 derived from the E.coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thus providing easy means for identifying transformed cells. pBR322, derivatives thereof, or other microbial plasmids or phages may also contain or be modified to contain promoters that can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives useful for the expression of particular antibodies are described in detail in Carter et al, U.S. Pat. No.5,648,237.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transformation vectors for these hosts. For example, bacteriophages such as λ GEM can be used^TM11 to construct a recombinant vector which can be used to transform susceptible host cells such as E.coli LE 392.

The expression vectors of the invention may comprise two or more promoter-cistron pairs which encode each polypeptide building block. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that regulates its expression. Prokaryotic promoters are generally divided into two classes, inducible and constitutive. An inducible promoter is a promoter that initiates an elevated level of transcription of a cistron under its control in response to a change in culture conditions (e.g., the presence or absence of a nutrient or a change in temperature).

Numerous promoters recognized by a variety of potential host cells are well known. The selected promoter may be operably linked to cistron DNA encoding the light or heavy chain by excising the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Both native promoter sequences and many heterologous promoters can be used to direct amplification and/or expression of a target gene. In some embodiments, heterologous promoters are used because they generally allow for higher transcription and higher yields of expressed target genes as compared to the native target polypeptide promoter.

Promoters suitable for use in prokaryotic hosts include the PhoA promoter, the β -galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoters. However, other promoters functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, whereby the skilled worker is able to operably link them to cistrons encoding the target light and heavy chains using linkers or adaptors providing any desired restriction sites (Siebenlist et al, Cell 20:269 (1980)).

In one aspect of the invention, each cistron within the recombinant vector contains a secretory signal sequence component that directs the transmembrane transport of the expressed polypeptide. In general, the signal sequence may be a component of the vector, or it may be part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purposes of the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected, for example, from the group consisting of: alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin ii (stii) leader sequences, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the invention, the signal sequence used in both cistrons of the expression system is a STII signal sequence or a variant thereof.

In another aspect, the production of immunoglobulins according to the present invention may occur in the cytoplasm of the host cell, and thus does not require the presence of a secretion signal sequence within each cistron. At that point, the immunoglobulin light and heavy chains are expressed, folded and assembled within the cytoplasm to form functional immunoglobulins. Certain host strains (e.g., E.coli trxB-strains) provide cytoplasmic conditions favorable for disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun, Gene 159:203 (1995)).

Prokaryotic host cells suitable for expression of the antibodies of the invention include Archaebacteria (Archaebaceria) and Eubacteria (Eubacterium), such as gram-negative or gram-positive organisms. Examples of useful bacteria include Escherichia (e.g. Escherichia coli), Bacillus (e.g. Bacillus subtilis), enterobacter (enterobacter), Pseudomonas (e.g. Pseudomonas aeruginosa), Salmonella typhimurium (Salmonella typhimurium), Serratia marcescens (Serratia marcans), Klebsiella (Klebsiella), Proteus (Proteus), Shigella (Shigella), Rhizobium (rhizium), Vitreoscilla (vitroscilla), or Paracoccus (Paracoccus). In one embodiment, gram-negative cells are used. In one embodiment, E.coli cells are used as hosts in the present invention. Examples of E.coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol.2, Washington, D.C., society of microbiology, 1987, pp. 1190-1219; ATCC accession No. 27,325) and derivatives thereof, including strain 33D3 (U.S. Pat. No.5,639,635) having the genotype W3110. delta. fhuA (. totnA) ptr3 lac Iq lacL 8. delta. ompT. delta. mpc-fepE) degP41 kanR. Other strains and derivatives thereof, such as E.coli 294 (ATCC 31,446), E.coli B, E.coli lambda 1776 (ATCC 31,537) and E.coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative only and not limiting. Methods for constructing any of the above bacterial derivatives having a defined genotype are known in the art, see, e.g., Bass et al, Proteins 8: 309-. It is generally necessary to select an appropriate bacterium in consideration of replicability of the replicon in bacterial cells. For example, E.coli, Serratia, or Salmonella species may be suitable for use as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to provide the replicon. In general, the host cell should secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors in the cell culture.

Antibody production

Host cells are transformed with the above expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transformation is the introduction of DNA into a prokaryotic host so that the DNA can be replicated, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is carried out using standard techniques appropriate for these cells. Calcium treatment with calcium chloride is commonly used for bacterial cells with a strong cell wall barrier. Another transformation method used polyethylene glycol/DMSO. Yet another technique used is electroporation.

Prokaryotic cells for producing the polypeptides of the invention are cultured in media known in the art and suitable for culturing the selected host cells. Examples of suitable media include LB media (Luria broth) supplemented with essential nutrient supplements. In some embodiments, the medium further contains a selection agent selected based on the construction of the expression vector to selectively permit growth of the prokaryotic cell comprising the expression vector. For example, ampicillin is added to a medium for culturing cells expressing an ampicillin resistance gene.

In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may be present at appropriate concentrations, either alone or as a mixture with another supplement or medium, such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of: glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol.

Prokaryotic host cells are cultured at a suitable temperature. For example, for culturing E.coli, preferred temperatures range from about 20 ℃ to about 39 ℃, more preferably from about 25 ℃ to about 37 ℃, and even more preferably about 30 ℃. The pH of the medium may be any pH ranging from about 5 to about 9, depending primarily on the host organism. For E.coli, the pH is preferably from about 6.8 to about 7.4, more preferably about 7.0.

If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for activating the promoter. In one aspect of the invention, the PhoA promoter is used to control transcription of the polypeptide. Thus, for induction, the transformed host cells are cultured in phosphate-limited medium. Preferably, the phosphate-limited medium is C.R.A.P medium (see, e.g., Simmons et al, J.Immunol. methods 263:133-147 (2002)). Depending on the vector construct employed, a variety of other inducers may be employed, as is known in the art.

In one embodiment, the expressed polypeptide of the invention is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery typically involves the destruction of microorganisms, typically by means such as osmotic shock (osmoticshock), sonication, or lysis. Once the cells are disrupted, the cell debris or whole cells can be removed by centrifugation or filtration. The protein may be further purified by, for example, affinity resin chromatography. Alternatively, the protein may be transported into the culture broth and isolated therefrom. The cells can be removed from the culture broth and the culture supernatant filtered and concentrated for further purification of the produced protein. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

In one aspect of the invention, antibody production is carried out in large quantities by a fermentation process. A variety of large-scale fed-batch fermentation protocols are available for the production of recombinant proteins. Large scale fermentations have a capacity of at least 1000 liters, preferably about 1,000 to 100,000 liters. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source). Small-scale fermentation generally refers to fermentation in a fermentor that is no more than about 100 liters in volumetric capacity, and may range from about 1 liter to about 100 liters.

During fermentation, induction of protein expression is typically initiated after culturing the cells under appropriate conditions to a desired density (e.g., OD550 of about 180-. Depending on the vector construct employed, a variety of inducers may be used, as is known in the art and described above. Cells can be cultured for a shorter time before induction. Cells are typically induced for about 12-50 hours, although longer or shorter induction times may be used.

In order to improve the production and quality of the polypeptides of the invention, a variety of fermentation conditions may be modified. For example, to improve proper assembly and folding of the secreted antibody polypeptide, additional vectors that overexpress chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and/or DsbG) or FkpA (a peptidylprolyl-cis, trans-isomerase with chaperone activity) may be used to co-transform the host prokaryotic cell. Chaperonins have been shown to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al J.biol.chem.274: 19601-.

In order to minimize proteolysis of the expressed heterologous protein, particularly one that is sensitive to proteolysis, certain host strains deficient in proteolytic enzymes may be used in the present invention. For example, a host cell strain can be modified to make genetic mutations in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Some E.coli protease deficient strains are available, see for example Joly et al, (1998) supra, Georgiou et al, U.S. Pat. No.5,264,365, Georgiou et al, U.S. Pat. No.5,508,192, Hara et al, Microbial Drug Resistance 2:63-72 (1996).

In one embodiment, an E.coli strain deficient in proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins is used as host cell in the expression system of the invention.

Antibody purification

Standard protein purification methods known in the art can be used. The following protocol is illustrative of a suitable purification protocol: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

In one aspect, protein a immobilized on a solid phase is used for immunoaffinity purification of a full-length antibody product of the invention. Protein a is a 41kD cell wall protein from Staphylococcus aureus (Staphylococcus aureus), which binds with high affinity to the antibody Fc region. Lindmark et al, J.Immunol.Meth.62:1-13 (1983)). The solid phase on which protein A is immobilized is preferably a column having a glass or quartz surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column is coated with a reagent, such as glycerol, in an attempt to prevent non-specific adhesion of contaminants.

As a first step of purification, a preparation derived from the cell culture as described above is applied to a protein a immobilized solid phase such that the antibody of interest specifically binds to protein a. The solid phase is then washed to remove contaminants that are non-specifically bound to the solid phase. Finally the antibody of interest is recovered from the solid phase by elution.

b. Production of antibodies using eukaryotic host cells:

carrier members typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(i) Signal sequence component

Vectors used in eukaryotic host cells may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. Preferably a heterologous signal sequence that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretion leaders, such as the herpes simplex virus gD signal, can be used.

The DNA of these precursor regions is ligated in-frame to the DNA encoding the antibody.

(ii) Origin of replication

Typically, mammalian expression vectors do not require an origin of replication component. For example, the SV40 origin may typically only be used because it contains the early promoter.

(iii) Selection gene components

Expression and cloning vectors may comprise a selection gene, also referred to as a selectable marker. Typical selection genes encode the following proteins: (a) conferring resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) complementing the corresponding nutritional deficiency; or (c) provide key nutrients not available from complex media.

One example of a selection scheme utilizes drugs to retard the growth of host cells. Those cells successfully transformed with the heterologous gene produce a protein conferring drug resistance, thus surviving the selection protocol. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of a suitable selectable marker for mammalian cells is one that is capable of identifying cells competent to take up antibody nucleic acids, such as DHFR, thymidine kinase, metallothionein I and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is employed, a suitable host cell is a Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts comprising endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, a wild-type DHFR protein, and another selectable marker such as aminoglycoside 3' -phosphotransferase (APH) can be selected by culturing the cells in a medium containing a selection agent for the selectable marker such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No.4,965,199.

(iv) Promoter component

Expression and cloning vectors typically comprise a promoter recognized by the host organism and are operably linked to the antibody polypeptide nucleic acid. Promoter sequences for eukaryotic cells are known. Virtually all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is the CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence, which may be a signal to add a poly A tail to the 3' end of the coding sequence. All these sequences are suitably inserted into eukaryotic expression vectors.

Transcription of antibody polypeptides by vectors in mammalian host cells is under the control of promoters from heat shock promoters, e.g., obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis b virus, and simian virus 40(SV40), from heterologous mammalian promoters (e.g., actin promoter or immunoglobulin promoter), provided these promoters are compatible with the host cell system.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. U.S. Pat. No.4,419,446 discloses a system for expressing DNA in a mammalian host using bovine papilloma virus as a vector. A modification of this system is described in U.S. patent No.4,601,978. Alternatively, the rous sarcoma virus long terminal repeat can be used as a promoter.

(v) Enhancer element component

Transcription of DNA encoding an antibody polypeptide of the invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. For enhanced elements for activation of eukaryotic promoters see also Yaniv, Nature 297:17-18 (1982). Enhancers may be spliced into the vector at positions 5' or 3' to the coding sequence of the antibody polypeptide, but are preferably located at sites 5' to the promoter.

(vi) Transcription termination component

Expression vectors used in eukaryotic host cells also typically contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are typically available from the 5 'and occasionally 3' ends of untranslated regions of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated region of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and expression vectors disclosed therein.

(vii) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (293 cell or 293 cell subcloned for suspension culture, Graham et al, J.Gen.Virol.36:59 (1977)), baby hamster kidney cell (BHK, ATCC CCL 10), Chinese hamster ovary cell/-DHFR (CHO, Urlaub et al, Proc.Natl.Acad.Sci.USA 77:4216 (1980)), mouse Sertoli (Sertoli) cell (TM 4, Mather, biol.Reprod.23:243-251 (1980)), monkey kidney cell (CV 1, ATCC CCL 70), African green monkey kidney cell (VERO-76, ATCC CRL 1587), human cervical cancer cell (HELA, ATCC CCL 2), canine kidney cell (CCL, CCL 34), bovine mouse kidney (ATCC 1443) cell (BRfallo) and human lung cell (ATCC 144138), human lung liver cell (CCL) 75, ATCC 14465), human lung H75, ATCC 14465, and mouse lung H cell (CCL) transformed by SV40, Mouse breast tumor (MMT 060562, ATCC CCL 51), TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982)), MRC 5 cells, FS4 cells, and human hepatosarcoma (Hep G2) line.

For antibody production, host cells are transformed with the expression or cloning vectors described above and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

(viii) Culture of host cells

Host cells for producing the antibodies of the invention can be cultured in a variety of media. Commercial media such as Ham's F10 (Sigma), minimal essential medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in the following documents can be used as the medium for the host cells: ham et al, meth.Enz.58:44(1979), Barnes et al, anal. biochem.102:255(1980), U.S. Pat. No.4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469, WO90/03430, WO 87/00195, or U.S. Pat. No. Re.30,985. Any of these media may be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN) ^TMDrugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included at suitable concentrations. Culture conditions such as temperature, pH, etc. are previously used for the host cell selected for expression, as will be apparent to the ordinarily skilled artisan.

(ix) Purification of antibodies

When recombinant techniques are used, the antibodies can be produced intracellularly or secreted directly into the culture medium. If the antibody is produced intracellularly, the particulate debris, either the host cells or the lysed fragments, are first removed, for example, by centrifugation or ultrafiltration. If the antibody is secreted into the culture medium, the supernatants from these expression systems are typically first concentrated using a commercial protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (the preferred purification technique is affinity chromatography). The suitability of protein a as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify human gamma 1, gamma 2, or gamma 4 heavy chain-based antibodies (Lindmark et al, J.Immunol. meth.62:1-13 (1983)). Protein G is recommended for all mouse isoforms and human gamma 3 (Guss et al, EMBO J.5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices may be used. Physically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow faster flow rates and shorter processing times than agarose. If the antibody comprises a CH3 domain, Bakerbond ABX may be used ^TMPurification was performed on resin (j.t. baker, phillips burg, NJ). Depending on the antibody to be recovered, other protein purification techniques such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, heparin Sepharose may also be used^TMChromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification step, the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.25M salt).

Immunoconjugates

The invention also provides immunoconjugates (interchangeably referred to as "antibody-drug conjugates" or "ADCs") comprising any of the anti-hepsin antibodies described herein conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

Use Of Antibody-drug conjugates for the local delivery Of cytotoxic or cytostatic agents (i.e., drugs that kill or inhibit tumor cells) In the treatment Of Cancer (Syrigos And Epenetos, anticancer research 19:605-614(1999); Niculescu-Duvaz And Springer, adv. drug Del. Rev.26:151-172(1997); U.S. Pat. No.4,975,278) allows targeted delivery Of drug moieties to tumors And intracellular accumulation there, whereas systemic administration Of these unconjugated drug agents may result In unacceptable levels Of toxicity to normal cells beyond the tumor cells sought to be eliminated (Baldwin et al, Lancet 603-05(1986, 15.; Thoror, "Antibodies Of Cytoxigen In Cancer: A Review", In Monoclonal' 84: Biological, Pictures, 1985). Thereby attempting to achieve maximum efficacy and minimal toxicity. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al, Cancer Immunol. Immunother.21:183-87 (1986)). Drugs used in these methods include daunomycin (daunomycin), doxorubicin (doxorubicin), methotrexate (methotrexate) and vindesine (vindesine) (Rowland et al, 1986, supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al, journal.of the Nat. Cancer Inst.92(19):1573-1581(2000); Mandler et al, Bioorganic & Med. chem. letters 10:1025-1028(2000); Mandler et al, Bioconjugate chem.13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al, Proc. Natl.AcSci.USA 93:8618-8623 (1996)), and calicheamicin (Lode et al, Cancer Res.58:2928(1998); Hinman et al, Cancer Res.53:3336-3342 (1993)). Toxins may exert their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when coupled to large antibody or protein receptor ligands.

(ibritumomab tiuxetan, Biogen/Idec) is a monoclonal antibody against murine IgG1 kappa to the CD20 antigen found on the surface of normal and malignant B lymphocytes bound by a thiourea linker-chelator¹¹¹In or⁹⁰Antibody-radioisotope conjugates of Y-radioisotope (Wiseman et al, Eur. Jour. Nucl. Med.27(7):766-77 (2000); Wiseman et al, Blood99(12):4336-42(2002); Witzig et al, J. Clin. Oncol.20(10):2453-63(2002); Witzig et al, J. Clin. Oncol.20(15):3262-69 (2002)). Although zevanin has activity against B-cell non-Hodgkin's (Hodgkin) lymphoma (NHL), administration results in severe and prolonged cytopenia in most patients. MYLOTARG^TM(gemtuzumab ozogamicin, Wyeth pharmaceuticals), an antibody-drug conjugate composed of human CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myelogenous leukemia by injection (Drugs of the Future25(7):686(2000); U.S. Pat. No.4970198;5079233;5585089;5606040;5693762;5739116;5767285; 5773001). Phase II trials for the treatment of CanAg expressing cancers such as colon, pancreatic, gastric and other cancers are ongoing with Cantuzumab mertansine (immunogen inc.), an antibody-drug conjugate consisting of the huC242 antibody linked via a disulfide linker SPP to maytansinoid drug module DM 1. MLN-2704 (Millennium pharm., BZLBIOGICS, Immunogen In c.) antibody-drug conjugates consisting of monoclonal antibodies against Prostate Specific Membrane Antigen (PSMA) linked to maytansinoid drug module DM1, are being developed for potential treatment of prostate tumors. Synthetic analogs of dolastatin (dolastatin), Auristatin E (AE) and monomethyl auristatin (MMAE), were coupled to chimeric monoclonal antibodies cBR96 (specific for Lewis Y on carcinomas) and cAC10 (specific for CD30 on hematologic malignancies) (Doronina et al, Nature Biotechnology 21(7):778-784 (2003)) and are under therapeutic development.

Chemotherapeutic agents useful for generating immunoconjugates are described herein (e.g., above). Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α -sarcin (sarcocin), Aleurites fordii (Aleuties fordii) toxic protein, dianthus toxic protein, Phytolacca americana (Phytolaccariella americana) toxic protein (PAPI, PAPII and PAP-S), Momordica charantia (Momoracia chacotiana) inhibitor, Jatropha curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin alba (gelonin), mitomycin (gelonin), trichostatin (strictin), tricin (triphenomycin), trichothecin (triomycin), and enomycin (enomycin). See, for example, WO 93/21232 published on month 10 and 28, 1993. A variety of radionuclides are available for use in the production of radioconjugated antibodies. Examples include ²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y and¹⁸⁶re. Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-azidobenzoyl) hexanediamine, and the likeBifunctional derivatives of bis (p-diazobenzoyl) ethylenediamine), diisocyanates such as toluene 2, 6-diisocyanate, and bis-active fluorine compounds such as 1, 5-difluoro-2, 4-dinitrobenzene. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026.

Also contemplated herein are conjugates of the antibody with one or more small molecule toxins such as calicheamicin (calicheamicin), maytansinoids (maytansinoids), dolastatins (dolastatins), aurostatins, trichothecenes (trichothecene), and CC1065, and derivatives of these toxins that have toxin activity.

i. Maytansine and maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (full length or fragment) of the invention conjugated to one or more maytansinoid molecules.

Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was originally isolated from the east African shrub Maytenus serrata (Maytenus serrata) (U.S. Pat. No.3,896,111). It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No.4,151,042). The synthesis of maytansinol and its derivatives and analogues is disclosed, for example, in the following U.S. patents: 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drug conjugates because they: (i) relatively easy to prepare by fermentation or chemical modification, derivatization of the fermentation product; (ii) are readily derivatized with functional groups suitable for conjugation of antibodies via non-disulfide linkers; (iii) is stable in plasma; and (iv) is effective against a variety of tumor cell lines.

Immunoconjugates comprising maytansinoids, methods of making and therapeutic uses thereof are disclosed, for example, in the following patents: U.S. Pat. Nos. 5,208,020, 5,416,064, and European patent EP 0425235B 1, the disclosures of which are expressly incorporated herein by reference. Liu et al, Proc.Natl.Acad.Sci.USA93:8618-8623(1996) describe immunoconjugates comprising a maytansinoid called DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic against cultured colon cancer cells and showed antitumor activity in an in vivo tumor growth assay. Chari et al, Cancer Research 52:127-131(1992) describe immunoconjugates in which a maytansinoid is conjugated via a disulfide linker to the murine antibody A7 that binds to an antigen on human colon Cancer cell lines or to another murine monoclonal antibody TA.1 that binds to the HER-2/neu oncogene. Cytotoxicity of TA.1-maytansinoid conjugates was tested in vitro on human breast cancer cell line SK-BR-3, which expresses 3x10 per cell⁵A HER-2 surface antigen. The drug conjugates achieved cytotoxicity to a similar extent as the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules conjugated per antibody molecule. The a 7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates can be prepared by chemically linking an antibody to a maytansinoid molecule without significantly impairing the biological activity of the antibody or the maytansinoid molecule. See, for example, U.S. Pat. No.5,208,020, the disclosure of which is expressly incorporated herein by reference. An average of 3-4 maytansinoid molecules per antibody molecule coupled showed efficacy in enhancing cytotoxicity against target cells without negatively affecting the function or solubility of the antibody, although it is expected that even one molecule of toxin/antibody will enhance cytotoxicity compared to the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.5,208,020 and in the other patents and non-patent publications referred to above. Preferred maytansinoids are maytansinol and maytansinol analogs modified at aromatic rings or other positions of the maytansinol molecule, such as various maytansinol esters.

A number of linking groups are known in the art for use in preparing antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No.5,208,020 or European patent 0425235B 1, Chari et al, cancer research 52:127-131(1992), U.S. patent application Ser. No.10/960,602, filed on 8/10/2004, the disclosure of which is expressly incorporated herein by reference. Antibody-maytansinoid conjugates comprising linker component SMCC can be prepared as disclosed in U.S. patent application Ser. No.10/960,602, filed on 8/10/2004. The linking group includes a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group, or an esterase labile group, as disclosed in the patents mentioned above, disulfide and thioether groups being preferred. Additional linking groups are described and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. Particularly preferred coupling agents include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al, biochem. J.173:723-737 (1978)) and N-succinimidyl-4- (2-pyridylthio) valerate (SPP), thereby providing disulfide linkages.

Depending on the type of linkage, linkers can be attached to various positions of the maytansinoid molecule. For example, ester linkages can be formed by reaction with hydroxyl groups using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.

Auristatin and dolastatin

In some embodiments, immunoconjugates comprise an antibody of the invention conjugated to dolastatins (dolastatins) or dolastatin peptide analogs and derivatives, auristatins (U.S. Pat. Nos. 5,635,483;5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemothers.45 (12):3580-3584) and to have anti-cancer (U.S. Pat. No.5,663,149) and anti-fungal activity (Pettit et al (1998) Antimicrob. Agents Chemothers.42: 2961-2965). Dolastatin or auristatin drug moieties can be attached to an antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02/088172).

Exemplary auristatin embodiments include N-terminally attached monomethyl auristatin drug moieties DE and DF, disclosed in "monomer compositions Cable of Conjugation to Ligands", U.S. Ser. No. 10/983,340 filed on 5.11.2004, the disclosure of which is expressly incorporated herein by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the art of peptide chemistry (see E. And K.L u bke, The Peptides, Vol.1, pp 76-136,1965, Academic Press). The auristatin/dolastatin drug module can be prepared according to the methods in the following references: U.S. Pat. Nos. 5,635,483 and 5,780,588, Pettit et al (1989) J.Am.chem.Soc.111:5463-5465, Pettit et al (1998) Anti-Cancer Drug Design 13:243-277, Pettit et al Synthesis,1996,719-725, Pettit et al (1996) J.chem.Soc.perkin Trans.15:859-863, and Doronina (2003) nat. Biotechnol.21(7):778-784; "monomer Synthesis Package of ligation to Ligands", U.S. Ser. No. 10/983,340, filed on 5.2004, 11.S.A., incorporated herein in their entirety (disclosing, for example, linkers and methods for making Monomethylvaline Compounds such as MMAE and MMAF coupled to linkers).

iii. calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of the invention conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of generating double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (both to the company Canamid). Useful calicheamicin structural analogs include, but are not limited to, γ 1I, α 2I, α 3I, N-acetyl- γ 1I, PSAG and θ I1 (Hinman et al, Cancer Research 53:3336-3342(1993); Lode et al, Cancer Research 58:2925-2928(1998); and the aforementioned U.S. patents to American Cyanamid corporation). Another anti-tumor drug that can be conjugated to an antibody is QFA, which is an antifolate drug. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, cellular uptake of these agents via antibody-mediated internalization greatly enhances their cytotoxic effects.

Other cytotoxic agents

Other anti-tumor agents that can be conjugated to the antibodies of the invention include BCNU, streptavidin, vincristine (vincristine), 5-fluorouracil, the family of agents collectively referred to as the LL-E33288 complex described in U.S. Pat. Nos. 5,053,394 and 5,770,710, and esperamicins (U.S. Pat. No.5,877,296).

Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α -sarcin (sarcocin), Aleutites fordii (Aleuties fordii) toxic protein, dianthus dianilin toxic protein, Phytolacca americana (Phytolaca americana) toxic protein (PAPI, PAPII and PAP-S), Momordica charantia (Mordicacharantia) inhibitor, Jatropha curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin alba toxic protein (gelonin), mitomycin (gelonin), tricin (restrictocin), trichothecin (enomycin), and enomycin (enomycin). See, for example, WO 93/21232 published on month 10 and 28, 1993.

The invention also encompasses immunoconjugates formed between an antibody and a compound having nucleic acid degrading activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of tumors, the antibody may comprise a highly radioactive atom. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. Where the conjugate is to be used for detection, it may contain a radioactive atom for scintigraphic studies, e.g. tc^99mOr I¹²³Or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, or mixtures thereof,Gadolinium, manganese or iron.

Radiolabels or other labels may be incorporated into the conjugates in known manner. For example, the peptides may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels can be attached via cysteine residues in the peptide, such as tc99m or I¹²³、Re¹⁸⁶、Re¹⁸⁸And In¹¹¹. Yttrium-90 can be attached via lysine residues. The IODOGEN method (Fraker et al, biochem. Biophys. Res. Commun.80:49-57 (1978)) can be used to incorporate iodine-123. Other methods are described in detail in Monoclonal Antibodies in Immunoscintigraphy (Chatal, CRC Press, 1989).

Conjugates of the antibody and cytotoxic agent may be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Research 52:127-131(1992); U.S. Pat. No.5,208,020).

The compounds of the present invention specifically encompass, but are not limited to, ADCs prepared with the following crosslinkers: commercially available (e.g., BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate) from Pierce Biotechnology Inc., Rockford, IL, U.S.A.). See 2003-2004 Applications handbook and catalogs (Applications handbook and Catalog) pp.467-498.

v. preparation of antibody-drug conjugates

In the antibody-drug conjugates (ADCs) of the invention, the antibody (Ab) is conjugated via a linker (L) to one or more drug moieties (D), for example from about 1 to about 20 drug moieties per antibody. The ADCs of formula I can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) the nucleophilic group of the antibody reacts with the bivalent linker reagent via a covalent bond to form Ab-L, which then reacts with the drug moiety D; and (2) reacting the nucleophilic group of the drug moiety with a bivalent linker reagent via a covalent bond to form D-L, followed by reaction with the nucleophilic group of the antibody. Additional methods for making ADCs are described herein.

Ab-(L-D)p I

The joint may be made up of one or more joint members. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4- (2-pyridylthio) pentanoate ("SPP"), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"). Other linker components are known in the art, some of which are also described herein. See also "monomer ingredients Cable of coupling to Ligands", U.S. Ser. No. 10/983,340 filed on 5.11.2004, the entire contents of which are incorporated herein by reference.

In some embodiments, the linker may comprise amino acid residues. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine (gly-gly-gly). Amino acid residues that make up the amino acid linker moiety include those naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid linker components can be designed and optimized for their selectivity in enzymatic cleavage by specific enzymes (e.g., tumor associated proteases, cathepsin B, C and D, or plasmin proteases).

Nucleophilic groups of antibodies include, but are not limited to: (i) an N-terminal amino group; (ii) side chain amino groups, such as lysine; (iii) side chain thiol groups, such as cysteine; and (iv) glycosylating the hydroxyl or amino groups of the sugar in the antibody. The amino, thiol, and hydroxyl groups are nucleophilic and capable of reacting with electrophilic groups on a linker moiety to form covalent bonds, and linker reagents include: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) hydrocarbyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl groups and maleimide groups. Some antibodies have reducible interchain disulfide bonds, i.e., cysteine bridges. The antibody can be rendered reactive for conjugation to a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge would theoretically so form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody via reaction of lysine with 2-iminothiolane (Traut's reagent), resulting in conversion of the amine to a thiol. Reactive thiol groups can be introduced into an antibody by introducing one, two, three, four, or more cysteine residues (e.g., preparing a mutant antibody comprising one or more non-native cysteine amino acid residues).

Antibody-drug conjugates of the invention can also be produced by modifying the antibody, i.e., introducing electrophilic moieties that can react with nucleophilic substituents on the linker reagent or drug. The sugar of the glycosylated antibody can be oxidized with, for example, a periodate oxidizing agent to form an aldehyde or ketone group that can react with the amine group of the linker reagent or drug moiety. The resulting imine Schiff base groups may form stable linkages, or may be reduced with, for example, a borohydride reagent to form stable amine linkages. In one embodiment, reaction of the carbohydrate moiety of a glycosylated antibody with galactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate technologies). In another embodiment, a protein containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate, resulting in the formation of an aldehyde at the first amino acid (Geoghegan and Stroh, bioconjugateChem.3:138-146(1992); U.S. Pat. No.5,362,852). Such aldehydes may react with the drug moiety or linker nucleophile.

Likewise, nucleophilic groups on the drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting with electrophilic groups on a linker moiety to form covalent bonds, and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) hydrocarbyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl groups, and maleimide groups.

Alternatively, fusion proteins comprising an antibody and a cytotoxic agent may be prepared, for example, by recombinant techniques or peptide synthesis. The length of the DNA may comprise regions encoding the two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

In yet another embodiment, an antibody can be conjugated to a "receptor" (such as streptavidin) for tumor pre-targeting, wherein the individual is administered an antibody-receptor conjugate, followed by clearing of unbound conjugate from the circulation using a clearing agent, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

Methods of using anti-hepsin antibodies

The invention features the use of hepsin antibodies as part of a specific therapeutic regimen intended to provide a beneficial effect from the activity of such therapeutic agents. The invention is particularly useful in the treatment of various stages and types of cancer.

The term cancer encompasses a collection of proliferative disorders including, but not limited to, pre-cancerous growths, benign tumors, and malignant tumors. Benign tumors remain localized to the site of origin and have no ability to penetrate, invade, or metastasize to distant sites. Malignant tumors can invade and damage other tissues around them. They also gain the ability to break free from the site of origin and spread to other parts of the body (metastasis), usually via the bloodstream or via the lymphatic system where lymph nodes are located. Primary tumors are classified by the type of tissue in which they occur; metastatic tumors are classified by the tissue type from which the cancer cells are derived. Over time, malignant cells become more and more abnormal and appear less like normal cells. This change in the appearance of cancer cells is called tumor grade (tumor grade), and cancer cells are described as fully-differentiated (low grade), moderately-differentiated (modular-differentiated), poorly-differentiated (pore-differentiated), or undifferentiated (high grade). Fully differentiated cells are fairly normal, appearing as normal cells of similar origin. Undifferentiated cells refer to cells that have become so abnormal that it is no longer possible to determine their origin.

Cancer staging system describes how far the cancer has spread anatomically and attempts are made to place patients with similar prognosis and treatment in the same staging group. Several tests may be performed to aid in staging cancer, including biopsies and certain image detections such as chest x-rays, mammograms, bone scans, CT scans, and MRI scans. Blood tests and clinical assessments are also used to assess the overall health of a patient and to detect whether cancer has spread to certain organs.

To stage cancer, the American Joint Committee on cancer first places the cancer (particularly solid tumors) into a letter classification using the TNM classification system. Cancers are assigned the letters T (tumor size), N (palpable nodules), and/or M (metastasis). T1, T2, T3, and T4 describe increasing primary lesion sizes; n0, N1, N2, N3 indicate node involvement in progressive progression; and M0 and M1 reflect the presence or absence of distant metastasis.

In a second Staging approach, also known as Overall Stage Grouping or Roman numerical Staging, the cancer is divided into stages 0 to IV, incorporating the size of the primary disorder and the presence of node spread and distant metastases. In this system, cancers are grouped into four stages, represented by roman numerals I to IV, or classified as "relapsed". For some cancers, stage 0 is referred to as "in situ" or "Tis," an in situ ductal carcinoma such as breast cancer or in situ lobular carcinoma. High-grade adenomas may also be classified as stage 0. Generally, stage I cancer is a small localized cancer that is usually curable, while stage IV usually represents an inoperable or metastatic cancer. Stage II and III cancers are often locally advanced and/or exhibit involvement of regional lymph nodes. In general, a higher number of stages indicates more severe (extensive) disease, including larger tumor size and/or spread of the cancer to nearby lymph nodes and/or organs adjacent to the primary tumor. These stages are precisely defined, but the definition is different for each cancer and is known to the skilled artisan.

Many cancer registers such as the NCI "watch Epidemiology and End Results Program" (SEER) use generalized staging. Such systems are used for all types of cancer. It divides cancer medical records into five major categories:

"in situ" refers to an early stage cancer that is present only in the cell layer from which it originated.

By "localized" is meant that the cancer is confined to the organ in which it begins, with no evidence of spread.

By "regional" is meant that the cancer has spread beyond the site of origin (primary) to nearby lymph nodes or organs and tissues.

"distal" refers to the spread of the cancer from the primary site to a distant organ or a distant lymph node.

"unknown" is used to describe the situation where there is insufficient information to indicate a stage.

In addition, it is common for cancer to recur months or years after the primary tumor has cleared. Cancers that recur after all visible tumors have been eradicated are referred to as recurrent disease. Recurrent disease in the area of the primary tumor is locally recurrent, while recurrent disease as a metastasis is referred to as distant recurrence.

The tumor may be a solid tumor or a non-solid tumor or a soft tissue tumor. Examples of soft tissue tumors include leukemias (e.g., chronic myelogenous leukemia (chronic myelogenous leukemia), acute myelogenous leukemia (acute myelogenous leukemia), adult acute lymphoblastic leukemia (adult acute lymphoblastic leukemia), acute myelogenous leukemia (acute myelogenous leukemia), mature B-cell acute lymphoblastic leukemia (mass B-cell acute lymphoblastic leukemia), chronic lymphocytic leukemia (chronic lymphocytic leukemia), polymorphonuclear leukemia (polymorphonuclear leukemia), or hairy cell leukemia (hairy cell leukemia)) or lymphomas (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors include any cancer of body tissues other than the blood, bone marrow, or lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Examples of solid epithelial tumors include tumors of the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, lip (labium), nasopharynx, skin, uterus, male reproductive organs, urinary organs, bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors. Other examples of tumors are described in the definitions section.

In some embodiments, the patient herein is subjected to a diagnostic test, e.g., before and/or during and/or after treatment. Generally, if a diagnostic test is performed, a sample may be obtained from a patient in need of treatment. If the subject has cancer, the sample may be a tumor sample or other biological sample, such as a biological fluid, including but not limited to blood, urine, saliva, ascites fluid, or derivatives such as serum and plasma, and the like.

The biological sample herein may be a fixed sample, such as a formalin fixed, paraffin embedded (FFPE) sample, or a frozen sample.

There are a variety of methods for determining mRNA or protein expression, including but not limited to gene expression profiling, Polymerase Chain Reaction (PCR) including quantitative real-time PCR (qRT-PCR), microarray analysis, Serial Analysis of Gene Expression (SAGE), MassARRAY, gene expression analysis by Massively Parallel Signature Sequencing (MPSS), proteomics, Immunohistochemistry (IHC), and the like. Preferably, mRNA is quantified. The mRNA analysis is preferably performed using Polymerase Chain Reaction (PCR) techniques or by microarray analysis. If PCR is used, the preferred form of PCR is quantitative real-time PCR (qRT-PCR). In one embodiment, a positive expression is considered if the expression of one or more of the genes mentioned above is at or above the median value, e.g., compared to other samples of the same tumor type. The median expression level may be determined at substantially the same time as the measurement of gene expression, or may be determined in advance.

The steps of a representative gene expression profiling scheme using fixed, paraffin-embedded tissues as a source of RNA are given in various published journal papers (e.g., Godfrey et al, J.Molec.diagnostics 2:84-91 (2000); Specht et al, am.J.Pathol.158:419-29 (2001)): including mRNA isolation, purification, primer extension and amplification. Briefly, one representative method begins by cutting a paraffin-embedded tumor tissue sample into sections approximately 10 microns thick. RNA is then extracted and proteins and DNA are removed. After analysis of the RNA concentration, RNA repair and/or amplification steps may be included, if desired, and the RNA is reverse transcribed using a gene-specific promoter, followed by PCR. Finally, the data is analyzed to determine the best treatment options available to the patient based on the characteristic gene expression patterns identified in the tumor samples examined.

Detection of gene or protein expression may be determined directly or indirectly.

Hepsin expression or amplification in cancer can be determined (directly or indirectly). A variety of diagnostic/prognostic assays can be used for this purpose. In one embodiment, hepsin overexpression can be analyzed by IHC. Paraffin-embedded tissue sections from tumor biopsies can be subjected to IHC assays and controlled against the hepsin protein staining intensity criteria as follows:

Score 0: no staining was observed or membrane staining was observed in less than 10% of the tumor cells.

Score 1 +: faint/barely detectable membrane staining was detected in more than 10% of tumor cells. The cells have staining in only a portion of their membranes.

And the score is 2 +: weak to moderate complete membrane staining was observed in more than 10% of tumor cells.

Score 3 +: moderate to intense complete membrane staining was observed in more than 10% of tumor cells.

In some embodiments, those tumors that assess hepsin overexpression that score 0 or 1+ may be characterized as not overexpressing hepsin, while those tumors that score 2+ or 3+ may be characterized as overexpressing hepsin.

Alternatively, or in addition, FISH assays can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the presence and/or extent (if any) of hepsin amplification or translation in the tumor.

Hepsin activation can be measured directly (e.g., by a phosphate ELISA assay, or other means of detecting phosphorylated receptors) indirectly (e.g., by detecting activated downstream signaling pathway components, detecting receptor dimers (e.g., homodimers, heterodimers), detecting gene expression profiles, etc.).

Methods for detecting nucleic acid mutations are well known in the art. Typically, but not necessarily, the target nucleic acid in the sample is amplified to provide a desired amount of material to determine whether a mutation is present. Amplification techniques are well known in the art. For example, an amplification product may or may not encompass all nucleic acid sequences encoding a protein of interest, so long as the amplification product comprises the particular amino acid/nucleic acid sequence position at which a mutation is suspected.

Samples containing the target nucleic acid can be obtained by methods well known in the art, and they are suitable for a particular type and location of tumor. Tissue biopsies are commonly used to obtain representative pieces of tumor tissue. Alternatively, tumor cells may be obtained indirectly in the form of a tissue/fluid known or believed to contain tumor cells of interest. For example, samples of lung cancer lesions may be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushings, or from sputum, pleural fluid, or blood. Mutated genes or gene products can be detected from tumors or from other body samples such as urine, sputum or serum. The same techniques described above for detecting mutated target genes or gene products in tumor samples can be used for other body samples. Cancer cells shed from the tumor and appear in these body samples. By screening these body samples, a simple early diagnosis of diseases such as cancer can be achieved. In addition, by testing these body samples for mutated target genes or gene products, the progress of the treatment can be monitored.

Means for enriching a tissue preparation for tumor cells are known in the art. For example, tissue may be isolated from paraffin or cryogenically preserved sections. Cancer cells can also be separated from normal cells by flow cytometry or laser capture microdissection. These and other techniques for isolating tumors from normal cells are well known in the art. Detection of mutations can be difficult if the tumor tissue is highly contaminated with normal cells, however techniques are known to minimize contamination and/or false positive/negative results, some of which are described below. For example, the sample may also be assessed for the presence of biomarkers (including mutations) that are known to be associated with the tumor cells of interest but not with the corresponding normal cells, and vice versa.

In some cases, the cancer overexpresses or does not overexpress hepsin. Hepsin overexpression can be determined in a diagnostic or prognostic assay by assessing an increase in hepsin levels present on a cell (e.g., by immunohistochemistry assay; IHC). Alternatively or additionally, the level of hepsin-encoding nucleic acid in the cell may be measured, for example, by fluorescence in situ hybridization (FISH; see WO 98/45479 published 10.1998), Southern blotting or Polymerase Chain Reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR). In addition to the above assays, a variety of in vivo assays may be utilized by the skilled practitioner. For example, cells within the body of a patient may be exposed to an antibody that is optionally labeled with a detectable label, such as a radioisotope, and binding of the antibody to cells within the body of the patient may be assessed, such as by external scanning for radioactivity or by analyzing a biopsy taken from a patient that has been previously exposed to the antibody.

Chemotherapeutic agents

The combination therapy of the present invention may further comprise one or more chemotherapeutic agents. Combined administration includes co-administration or simultaneous administration and sequential administration in either order using separate formulations or a single pharmaceutical formulation, wherein preferably all active agents exert their biological activity simultaneously for a period of time.

Chemotherapeutic agents, if administered, are generally administered at doses known or optionally reduced with respect to them due to the combined effects of the drugs or negative side effects attributable to the administration of the antimetabolite chemotherapeutic agent. The preparation and dosing schedule for such chemotherapeutic agents can be used according to the manufacturer's instructions or as determined empirically by the practitioner.

Disclosed herein are a plurality of chemotherapeutic agents that can be combined.

Formulation, dosage and administration

The therapeutic agents used in the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular subject being treated, the clinical condition of the individual patient, the cause of the condition, the site at which the agent is delivered, the method of administration, the schedule of administration, the drug-drug interactions of the agents to be combined, and other factors known to medical practitioners.

Therapeutic formulations are prepared by mixing the active ingredient of the desired purity with optional physiologically acceptable carriers, excipients or stabilizers using standard methods known in the art (Remington's pharmaceutical Sciences (20 th edition), a.gennaro eds., 2000, Lippincott, Williams&Wilkins, philiadelphia, PA). Acceptable carriers include saline, or buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN^TM、PLURONICS^TMOr PEG.

Optionally but preferably, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride, and preferably at about physiological concentrations. Optionally, the formulations of the present invention may contain a pharmaceutically acceptable preservative. In some embodiments, the concentration of the preservative ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methyl paraben, and propyl paraben are preferred preservatives. Optionally, the formulation of the present invention may include a pharmaceutically acceptable surfactant at a concentration of 0.005-0.02%.

The formulations herein may also contain more than one active compound necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Suitably, such molecules are combined in amounts effective for the intended purpose.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's pharmaceutical sciences, supra.

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and L-glutamic acid gamma-ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of sustained release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulating antibodiesWhen maintained in vivo for extended periods of time, they may denature or aggregate by exposure to a humid environment at 37 ℃, resulting in a loss of biological activity and possible changes in immunogenicity. Stabilization rational strategies can be designed according to relevant mechanisms. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation via thiol-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, employing appropriate additives, and developing specific polymer matrix compositions.

The therapeutic agents of the invention are administered to a human patient according to known methods, such as intravenous administration (like bolus injection or by continuous infusion over a period of time), by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Ex vivo strategy may also be used for therapeutic applications. Ex vivo strategies involve transfecting or transducing cells obtained from a subject with a polynucleotide encoding a hepsin antagonist. The transfected or transduced cells are then returned to the subject. The cells can be any of a wide variety of types, including but not limited to hematopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.

For example, if the hepsin antagonist is an antibody, the antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if local immunosuppressive therapy is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, it is suitable to administer the antibody by pulse infusion, in particular with decreasing doses of the antibody. Preferably, the dosage is administered by injection, most preferably, the dosage is administered by intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

In another example, the hepsin antagonist compound is administered locally, e.g., by direct injection, as allowed at the site of the disorder or tumor, and the injection may be repeated periodically. Hepsin antagonists can also be delivered systemically to the subject or directly to tumor cells, such as a tumor or tumor bed, following surgical resection of the tumor to prevent or reduce local recurrence or metastasis.

Administration of the therapeutic agents in the combination is typically carried out over a defined period of time (typically minutes, hours, days or weeks, depending on the combination selected). Combination therapy is intended to encompass administration of these therapeutic agents in a sequential manner (that is, where each therapeutic agent is administered at a different time), as well as administration of at least two of these therapeutic agents or therapeutic agents in a substantially simultaneous manner.

The therapeutic agents may be administered by the same route or by different routes. For example, the anti-hepsin antibodies in the combination may be administered by intravenous injection, while the chemotherapeutic agents in the combination may be administered orally. Alternatively, for example, the two therapeutic agents may be administered orally, or the two therapeutic agents may be administered by intravenous injection, depending on the particular therapeutic agent. The order of administration of the therapeutic agents also varies with the particular agent.

Depending on the type and severity of the disease, the initial candidate dose for administration to the patient is about 1 μ g/kg to 100mg/kg of each therapeutic agent, e.g., either by one or more separate administrations or by continuous infusion. Typical daily dosages may range from about 1 μ g/kg to 100mg/kg or more, depending on the factors discussed above. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the cancer is treated as measured according to the methods described above. However, other dosage regimens may also be useful.

The application encompasses administration of hepsin antibodies by gene therapy. See, e.g., WO96/07321 published 3/14/1996, which focuses on the use of gene therapy to generate intrabodies.

Article of manufacture

In another aspect of the invention, there is provided an article of manufacture comprising a substance useful in the treatment, prevention and/or diagnosis of the disorders described above. The article of manufacture comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of various materials, such as glass or plastic. The container contains the composition, by itself or in combination with other compositions, effective for the treatment, prevention and/or diagnosis of the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is for use in treating a selected condition, such as cancer. In addition, an article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition therein, wherein the composition comprises an additional cytotoxic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the first and second antibody compositions are useful for treating a particular condition, such as cancer. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in accordance with the general description provided above.

Examples

Materials and methods

Reagents and proteins: synthesis of p-nitroaniline substrate S2765(= DiaPharma FXa substrate), S2266, S2288, S2366, S2444 from DiaPharma (Westchester, OH), ChromozymTH from Roche Diagnostics (Indianapolis, IN), Spectrozyme(#299) andFVIIa is from American diagnostic (Greenwich, CT). 3, 4-dichloro-isocoumarin, BSA and Tween-20 were from Sigma-Aldrich.

Plasmin and factor XIa are from Haematologic Technologies Inc (Essex Junction, VT), plasma kallikrein from Calbiochem (La Jolla, CA), factor VII and factor XIIa from Enzyme Research (South find, IN), urokinase-type plasminogen activator (uPA) from American Diagnostica, uPA originally from Fitzgerald Industries Int. Rat laminin is from Millipore (Temecula, CA). hepsin (human), matriptase, piliferin (marapsin), Hepatocyte Growth Factor Activator (HGFA), pro-Hepatocyte Growth Factor (HGF), Kunitz domain inhibitor-1 (HAI-1) derived from HGFA inhibitor-1 and HAI-2 (KD1) were recombinantly expressed and purified as previously described (Kirchhofer et al, 2003; Kirchhofer et al, 2005; Li et al, 2009; Moran et al, 2006; Peek et al, 2002; Shia et al, 2005). As control antibodies we used anti-HGFA Fab or IgG (Fab40, Fab58, Fab75, IgG75) generated by antibody phage display (Ganesan et al, 2009; Wu et al, 2007).

M13-KO7 helper phage was from New England Biolabs. Maxisorp immunoplates were from Nalgen NUNC International (Naperville, Ill.).MyOne streptavidin was from Invitrogen (Carlsbad, Calif.). 3,3 ', 5, 5' -tetramethyl-benzidine/H₂O₂(TMB) peroxidase substrate was from Kirkegaard and Perry Laboratories, Inc. Neutravidin and streptavidin are available from Pierce Biotechnology, Inc.

Cloning, expression, and purification of prostasin: ni cells used a baculovirus expression system to express the extracellular domain of human prostasin containing a C-terminal flag tag (Ala33-Leu 321). After 72 hours incubation, the supernatant was harvested and the clarified culture broth was loaded on an anti-Flag M2 antibody-sepharose column (Sigma). Bound proteins were eluted with 100mM glycine, pH 3.0 and immediately neutralized with 1M Tris pH 8.0. The obtained prostasin in zymogen form was activated with recombinant matriptase at room temperature for 16 hours. Thereafter, the activated double-stranded prostasin was purified by clearing matriptase with a (His)8 tag ("His8" disclosed as SEQ ID NO:59) on a Ni-NTA column. Prostasin was further purified by size exclusion chromatography using an S-200 column.

Cloning, expression, and purification of mouse hepsin: mouse hepsin-containing C-terminal His-tagged extracellular domain (Ser45-Pro416) was expressed using a baculovirus expression system with t.ni cells under conditions similar to human hepsin (Moran et al, 2006). After 72 hours incubation, the supernatant was harvested and the clarified culture broth loaded on a Ni-NTA column (Qiagen) pre-equilibrated with buffer containing 50mM Tris-HCl, pH 8.0,300mM NaCl. Bound proteins were eluted with a buffer containing 50mM Tris-HCl, pH 8.0,300mM NaCl and 250mM imidazole.

Cloning, expression and purification of recombinant MSP and KD 1: recombinant MSP was expressed in chinese hamster ovary cells as described (Wahl et al, 1997). KD1 was expressed and purified as described (Shia et al, 2005).

Library construction for Fab phage display: a library called YSGX library was constructed as previously described using phagemids (pF1359) for Fab phage display (library D in Fellouse et al, 2007). The diversity of the library was 2X10¹⁰Left and right.

Selection and characterization of phage-displayed anti-hepsin fabs: for phage display experiments, we used biotinylated hepsin (= biotin-hepsin) and biotinylated hepsin complexed with 3, 4-dichloro-isocoumarin (DCI) (= biotin-hepsin: DCI). Hepsin was biotinylated using the Sulfo-NHS-LC-biotin kit (Pierce, Cat #21327) according to the manufacturer's instructions. Some biotinylated hepsin was used to form biotin-hepsin DCI complexes, i.e., incubated with 100 μ M DCI and maintained in subsequent panning experimentsThis DCI concentration. Previous experiments with DCI showed that hepsin activity was completely inhibited by exposing hepsin to 25-50 μ M DCI for 40 minutes in an enzymatic assay with S2765 substrate. For the first round of panning, 10 μ g of biotin-hepsin or biotin-hepsin in the absence or presence of 100 μ M DCI, respectively, was mixed with 1ml of 1 × 10 ¹³The YSGX library of pfu/ml was incubated together at 4 ℃ for 1.5 hours. Using 100. mu.lMyOne streptavidin was captured for 15 min for phage bound to the target. Bound phage were eluted with 0.1M HCl, immediately neutralized, and then amplified following standard protocols (Sidhu et al, 2000). From the second round, 2. mu.g of biotin-hepsin or biotin-hepsin, respectively, were incubated with 400. mu.l of amplified phage for 1.5 hours at 4 ℃ in the absence or presence of 100. mu.M DCI. Phages bound to biotin-hepsin or biotin-hepsin: DCI were captured for 15 minutes using Maxisorp immunoplates (NUNC) coated with neutravidin or streptavidin (or between rounds) and blocked with blocking buffer (PBS,0.5% (w/v) BSA). After five rounds of selection, phage were generated from each clone cultured in 96-well format and culture supernatants were diluted three-fold in PBS,0.5% (w/v) BSA,0.1% (v/v) Tween 20(PBT buffer). The diluted phage supernatants were incubated for 1 hour with biotin-hepsin or biotin-hepsin DCI immobilized on 384-well Maxisorp immunoplates (NUNC) coated with neutravidin. The plate was washed six times with PBS,0.05% (v/v) Tween 20(PT buffer) and incubated with horseradish peroxidase/anti-M13 antibody conjugate (1: 5000 dilution in PBT buffer) (Pharmacia) for 30 minutes. The plate was washed six times with PT buffer, twice with PBS, and 3,3 ', 5, 5' -tetramethyl-benzidine/H ₂O₂Peroxidase substrate (Kirkegaard)&Perry Laboratories) for 15 minutes with 1.0M H₃PO₄Quenched and the absorbance was measured spectrophotometrically at 450 nm.

Single-site phage competition ELISA was used to identify phage-displayed fabs that bound the same hepsin epitope as KD 1. Each clone was cultured in 96-well format, and the culture supernatant was diluted five-fold in PBT buffer with or without 200nM KD1 and incubated for 1 hour with biotin-Hepsin immobilized on 384-well Maxisorp immunoplates (NUNC) coated with neutravidin. Plates were washed and bound phage detected by anti-M13-HRP as described above. For each clone, the ratio of the 200nM KD1 deletion to A450 in the presence of 200nM KD1 was calculated. Any clone with a ratio above 2 was considered to target a similar epitope as KD 1. Such clones were subjected to a detailed phage competition ELISA with KD1, using a series of concentrations of KD1 (1: 3 serial dilutions starting at 10 μ M) to compete with phage display fabs that bind to hepsin.

Anti-hepsin Fab25 and IgG25 expression and purification: generally, throughout this application, antibody 25 in the IgG form is designated with the prefix Ab, while antibody 25 in the Fab form is designated with the prefix Fab. A stop codon was introduced between the heavy chain and gene 3 on the phagemid encoding Fab 25. The resulting phagemid was transformed into E.coli strain 34B 8. A single colony was cultured overnight at 37 ℃ in 30ml LB medium supplemented with 50. mu.g/ml carbenicillin. 5ml of the culture was inoculated into 500ml of complete C.R.A.P.medium (for preparation of 1 liter: 3.57g (NH) ₄)₂SO₄0.71g of sodium citrate-2H₂O,1.07g KCl,5.36g yeast extract, 5.36g Hycase SF. 872ml of deionized water were added. The pH was adjusted to 7.3 with KOH. And (5) autoclaving. Cooled to 55 ℃ and 110ml of 1M MOPS pH 7.3,11ml of 50% glucose and 7ml of 1M MgSO supplemented with carbenicillin (50. mu.g/ml) were added₄) And cultured at 30 ℃ for 24 hours. The Fab25 protein was purified using protein a agarose resin.

The light and heavy chain variable domains of selected fabs were cloned into pRK 5-based plasmids with constant domains of human light or heavy chains (human IgG1) for transient expression in Chinese Hamster Ovary (CHO) cells. The IgG25 protein was purified by using protein a sepharose chromatography.

Enzyme assay with synthetic substrate: fab25, IgG25, control Fab or control IgG were combined with hepsin (1nM for human, 2nM for mouse hepsin) in Hepes buffer (20mM Hepes, pH 7.5,150mM NaCl,5mM CaCl₂,0.01% Triton X-100) Incubated at room temperature for 40 minutes. For experiments with 3, 4-dichloro-isocoumarin (DCI), hepsin (1nM) was incubated with DCI in Hepes buffer containing 0.5% DMSO for 40 minutes. S2765 (final concentration 0.2mM) was added and the linear rate of absorbance rise at 405nm was measured on a dynamic microplate reader (Versamax, Molecular Devices, Sunnyvale, Calif.). Data were expressed as fractional enzyme activity (vi/vo) and fitted to a four parameter regression curve fitting program (Kaleidagraph Version 3.6, Synergy Software) to determine IC50 values. Values are mean ± SD of three independent experiments. Kiapp values for Fab25 and IgG25 were calculated by fitting the data to the equation for the tight binding inhibition system (Morrison,1969; Olivero et al, 2005).

To check Fab25 specificity, a panel of 9 trypsin-like serine proteases was incubated with 1 μ MFab25 in Hepes buffer for 40 minutes. The appropriate chromogenic substrate is added and the linear rate of the increase in absorbance at 405nm is measured on a dynamic microplate reader. The concentrations of each enzyme and chromogenic substrate used were as follows: 1nM Hepsin-0.5mM S2765,0.5nM matriptase-0.5mM MS2765,5nM prostasin-0.5mM S2765,2nM plasmin-0.5 mM S2366,2nM plasma kallikrein-0.5 mM S2366,0.5nM factor XIa-0.5 mM S2366,5 nFXIIa-0.5 mM S2288,5nM uPA-0.5 mM S2444,50nM parnaphrin-0.2 mM S2366FVIIa,10nM HGFA–0.2mMFVIIa. Data are expressed as fractional enzyme activity (vi/vo) and are the mean. + -. SD of 3 independent experiments.

A panel of commercial pNA substrates (all having P1Arg residues) was used to examine hepsin inhibition by Fab 25. The 8 substrates are S2765, S2266, S2288, S2366, S2444, Chromozym TH, Spectrozyme fIXa and Spectrozyme FVIIa. The assay was performed as described above except that the concentration of Fab25 and control Fab was fixed at 100 nM. The concentration of the substrate was 0.5 mM. Data are expressed as percent inhibition of no inhibitory activity (no Fab) and are the mean ± SD of four independent experiments.

Enzyme assay with macromolecular substrate: to measure hepsin-mediated pro-uPA activation, Fab25 was placed in HBSA buffer (20mM Hepes, pH 7.5,150nM NaCl,5mM CaCl)₂0.5mg/ml BSA) and incubated with hepsin for 35 minutes at room temperature. The enzymatic reaction was initiated by addition of uPA. The concentrations of hepsin and pro-uPA in the reaction mixture were 0.5nM and 100nM, respectively. At 45 second intervals, 50. mu.l aliquots were transferred to 96-well plates containing 150. mu.l/well of the stop reagent HAI-2(667 nM). After addition of S2444(0.5mM), the linear rate of absorbance rise at 405nm was measured on a dynamic microplate reader. Data were expressed as fractional enzyme activity (vi/vo) and a four parameter regression curve fitting program (Kaleidagraph) was fitted to determine IC50 values.

For the factor VII activation assay, hepsin was incubated with Fab25 or control Fab in Hepes buffer for 5 minutes before addition of factor VII. The concentrations in the reaction mixture were: 50nM hepsin,500nM Fab and 8. mu.M factor VII. After incubation at 37 ℃ for 0.5 and 2 hours, aliquots were removed and analyzed by SDS-PAGE (4-20% gradient gel, InVitrogen) under reducing conditions. The gel was washed with Simplyblue^TM(InVitrogen) staining.

In vitro proteolytic processing of Fab25 by hepsin: fab25 (3. mu.M) was incubated with 10nM hepsin for 24 hours at room temperature either in low pH buffer (100mM Mes pH 6.0,150mM NaCl) or in high pH buffer (50mM Tris-HCl pH 8.0,150mM NaCl). The reaction was stopped by adding 20uL of 2X sample buffer (with/without β -mercaptoethanol) and boiling for 5 min at 95 ℃. Proteolysis was monitored by gel mobility shift on 4-20% (w/v) polyacrylamide gradient gels stained with Coomassie Brilliant blue.

Cell migration assay: FluoroBlok with 24-pore and 8.0 μm pore diameter is used^TMCell migration assays can be performed on supports (BDBiosciences, Bedford, MA) as previously described (Tripathi et al, 2008). The bottom surface of the filter was coated with untreated or treated rat laminin (1 μ g/ml). Contacting laminin with hepsin orhepsin-Fab25 complex or Phosphate Buffered Saline (PBS) were co-incubated overnight at 4 ℃. The inserts were then blocked with superblock buffer for 1 hour. DU145 human prostate cancer cells were trypsinized, resuspended in serum-free media, washed twice with serum-free media, and seeded in the upper chamber of the insert (20,000). At 5% CO ₂After 5 hours incubation at 37 ℃, the remaining cells on the upper filter were gently wiped with a cotton swab and the insert gently washed with PBS. Those cells that migrated to the lower chamber were fixed with 500. mu.l of methanol for 30 minutes, air-dried for 20 minutes, and stained with 500. mu.l of 10. mu.M YO-PRO-I (Invitrogen, Carlsbad, Calif.) for 10 minutes. Plates were washed with PBS and fluorescence was measured in a plate reader (Spectramax M5, Molecular Devices, Sunnyvale, Calif.) using an excitation wavelength of 485nm and an emission wavelength of 555 nm. Subsequently, the plate was imaged with an inverted microscope (IX81, Olympus) with a 10x objective lens attached to a CCD camera.

Binding of Fab25 to hepsin: the Octet-RED system equipped with streptavidin SBC biosensor tips was purchased from ForteBio (Menlo Park, CA). Prior to starting the experiment, the sensor tips were prewetted for 15 minutes at 25 ℃ in a buffer containing 50mM Hepes pH 7.5,150mM NaCl and 0.05% tween-20. Biotinylated hepsin (7.5. mu.g/ml) was captured on streptavidin sensor for 5 min with shaking at 1000 rpm. The sensors were washed briefly in buffer before they were immersed in sample wells containing Fab25 (2-fold serial dilution, starting at 200 nM) and buffer controls. Binding was monitored for 10 minutes and dissociation was followed for 30 minutes. Data were fitted to a 1: 1 binding model using Octet-RED analysis software.

Activation of pro-MSP by cell surface-expressed hepsin in LnCap cells: LnCaP-34 cells were generated as described (Moran et al, 2006) to stably overexpress hepsin, resulting in a 5-fold increase in hepsin cell surface expression and a 3-fold increase in hepsin enzymatic activity compared to LnCaP-17 cells expressing endogenous hepsin only at relatively low levels comparable to the parental LnCaP cells. Confluent LnCaP-17 and LnCap-34 cells cultured in 24-well plates were washed with serum-free RPMI-1640 medium and either alone or with different anti-hepsin inhibitors (1. mu.M anti-hepsin antibody Fab 25/1. mu.M KD11 μ MAc-KQLR-cmk ("KQLR" disclosed as SEQ ID NO:12)) were incubated together in serum-free RPMI-1640 medium at 37 ℃ for 15 minutes, after which addition¹²⁵I-labeled pro-MSP (25. mu.g/ml). After 3 hours incubation at 37 ℃, aliquots were removed and analyzed by SDS-PAGE (4-20% gradient gel) (Invitrogen, Carlsbad, CA), followed by exposure to X-ray film.

Activation of pro-HGF by cell surface-expressed hepsin in LnCap cells: confluent LnCap-34 cells cultured in 24-well plates were washed with serum-free RPMI-1640 medium and incubated at 37 ℃ for 15 minutes in serum-free RPMI-1640 medium either alone or with recombinant hepsin (10nM) or with different concentrations of Fab25(20nM-0.15nM), after which addition ¹²⁵pro-HGF labeled I (25. mu.g/ml). After 3 hours incubation at 37 ℃, aliquots were removed and analyzed by SDS-PAGE (4-20% gradient gel) (Invitrogen, Carlsbad, CA), followed by exposure to X-ray film.

Binding of Ab25 to hepsin as determined by surface plasmon resonance: in BIAcore^TMSurface plasmon resonance measurements were performed on a-3000 instrument (GE Health Care, NJ) to determine the binding affinity of Ab 25. Rabbit anti-human IgG was chemically immobilized (amine coupled) on CM5 biosensor chip and Ab25 was captured to give approximately 350 Response Units (RU). For kinetic measurements, two-fold serial dilutions (1nM to 500nM) of active hepsin in HBS-P buffer were injected at 25 ℃ at a flow rate of 30. mu.l/min. Binding rates (k) were obtained using a simple one-to-one Langmuir binding model (BIA-Evaluation)_a) And dissociation rate (k)_d) And calculate (k)_d/k_a) Equilibrium dissociation constant (K)_D). Because of the rapid kinetics, a steady state measurement was used to determine the binding affinity of pro-hepsin to Ab 25. Two-fold serial dilutions of pro-hepsin (195nM to 100mM) were injected on the captured antibody (Ab25) sensor chip to achieve maximal binding (Rmax) and reach steady state equilibrium. Req values were calculated using BIA-Evaluation and plotted individually against C (pro-hepsin concentration) to determine K _D。

Isothermal titration calorimetry: the TC experiments were performed on an ITC200 instrument (GE healthcare). Fab25 and hepsin were purified separately on a size exclusion chromatography column (Superdex S200, GE healthcare) using a buffer containing 50mM HEPES pH 7.5 and 150mM NaCl. The sample chamber (204. mu.l) was loaded with hepsin (14. mu.M) and the concentration of Fab25 in the syringe was 138. mu.M. Titration experiments typically consisted of 20 injections, each 2 μ Ι _ volume and 180 seconds duration, with 3 minutes between each addition. The agitation rate was 1000 rpm. Raw data were integrated, non-specific thermal correction, concentration normalized and analyzed according to the 1:1 binding model, assuming a set of identical binding sites. (isothermal titration curves were registered and analyzed using ORIGIN software provided with the ITC instrument the data were integrated to provide titration curves and the binding constant KA, heat of binding (Δ H), and binding stoichiometry were extracted from the curves by fitting using a non-linear least squares method.

Results/discussion

To identify anti-hepsin antibodies that block hepsin enzymatic activity, we used solution sorting against biotinylated hepsin (no inhibitor) (biotin-hepsin) and against biotinylated hepsin complexed with DCI (biotin-hepsin: DCI). DCI is a mechanism-based serine protease inhibitor that occupies the S1 pocket (fig. 9A) by forming a covalent mono-or diacyl adduct with catalytic Ser195 and His57 (chymotrypsinogen numbering) (Powers et al, 1989). hepsin DCI Complex molecular modeling based on factor D DCI Complex crystal structure (PDB code 1DIC) (Cole et al, 1998) indicates that the aromatic ring of the DCI inhibitor will occupy the S1 pocket. Our previous findings (Wu et al, 2007) indicate that the functional blocking anti-HGFA antibodies derived from our Fab phage display library do not occupy the S1 pocket. Thus, hepsin-bound DCI may not interfere with antibody binding, but may exert a beneficial effect on the active site conformation, facilitating interaction with the antibody. The active site of trypsin-like serine proteases is formed by several loops ("activation domains") that are intrinsically mobile (Huber and Bode, 1978). In particular, the 220 loop forms part of the S1 pocket and can adopt a variety of conformational states in some serine proteases (Johnson et al, 2005; Shia et al, 2005; Spraggon et al, 2009; Wilken et al, 2004). Only the apo (apo) structure reveals such a loop conformation, while the co-crystal structure with the active site inhibitor shows a correctly formed active site, most likely due to the stabilizing forces exerted by the inhibitor (Arni et al, 1994; Shiaet al, 2005; Spraggon et al, 2009). The Hepsin apoptopic structure is unknown, and all published Hepsin structures are co-crystal structures with active site inhibitors (Herter et al, 2005; Somoza et al, 2003). Therefore, we reasoned that DCI occupancy of the S1 pocket could exert stabilizing forces on potential loop flexibility, favoring antibody recognition. Enzyme assays showed that DCI concentrations above 20 μ M resulted in complete hepsin inhibition after a 40 min incubation period (fig. 9). Thus, phage sorting experiments against biotinylated hepsin DCI complexes were performed in the presence of 100 μ M DCI.

Inhibitory antibodies against hepsin were searched by solution sorting using a minimal synthetic antibody library of limited chemical diversity at the Complementarity Determining Regions (CDRs) named YSGX library (Fellhouse et al,2007), in which either biotin-hepsin or biotin-hepsin: DCI complexes were incubated with the phage display Fab library. Panning against biotin-hepsin DCI yielded one hepsin binding clone, designated hpfab 25 (also referred to as "Fab 25"), which became dominant after 5 rounds of selection. The HVR sequence of Fab25 is shown in figure 1. The CDR-H3 of HpsFab25 is very long (21 residues), and HpsFab25 contains three Lys residues.

ELISA experiments were performed to test whether Fab25 binding was inhibited by KD1, an inhibitor of hepsin that binds to the active site of hepsin. As shown in fig. 10, KD1 inhibited hepsin binding by Fab25 phage in a concentration-dependent manner, suggesting that Fab25 binds at or near the hepsin active site region and thus may interfere with enzymatic activity.

The ability of Fab25 to inhibit human and mouse activity was tested using the synthetic hepsin substrate S2765. As shown in fig. 11, Fab25 inhibited human and mouse hepsin activity in a concentration-dependent manner, reaching complete inhibition at the highest concentrations tested. Kiapp was calculated to be 4.1 + -1.0 nM (n =3) for human hepsin and 329 + -51 nM (n =3) for mouse hepsin. Additional experiments with IgG25 showed that it inhibited human hepsin with a Kiapp of 11.3 ± 1.7nM (n =3), whereas control IgG was not effective (data not shown). The results show that Fab25 inhibited human hepsin with about 80-fold higher potency than murine hepsin. This suggests that the antibody binding site is not completely conserved in mouse hepsin. Because the protease domain of mouse hepsin has neither insertions nor deletions compared to human hepsin, the reduced potency of Fab25 is presumably due to amino acid changes important for interaction with Fab 25.

The binding affinity of Fab25 to human hepsin was determined using Octet RED analysis. Kd was 2.55+/-0.45 nM.

The specificity of Fab25 was tested by asking if Fab25 inhibits the enzymatic activity of a panel of trypsin-like serine proteases. The group of trypsin-like serine proteases includes some of the closest protease domain homologs of hepsin, such as plasma kallikrein, prostasin, marapsin and plasmin. The results (fig. 12) demonstrate that Fab25 has exclusive specificity for hepsin, as it is aligning the Ki determined for hepsin^appA concentration 250 times higher does not affect the enzymatic activity of the serine protease examined.

The effect of Fab25 on hepsin-mediated pro-uPA and factor VIII processing was determined. As shown in fig. 13A, Fab25 inhibited hepsin activity against pro-uPA with an IC50 value of 3.3 ± 0.4nM (n =3), comparable to its potency in the pNA assay (table 1). At Fab concentrations above 100nM, the inhibition was complete and comparable to the strong inhibition in the factor VII activation assay (fig. 13B). Factor VII is a relatively poor hepsin substrate, requiring higher hepsin concentrations (50nM) in the assay. Thus, although Fab25 was used at 500nM, this resulted in a relatively low Fab25: hepsin ratio (10:1) compared to the uPA pro-activation assay (up to a ratio of 600: 1), which might indicate that inhibition was incomplete over an extended 2 hour period of the assay.

Hepsin preferentially cleaves the substrate behind P1 Arg (her et al, 2005), but also recognizes substrates with P1Lys (Moran et al, 2006). Therefore, we considered the possibility that hepsin might cleave the Fab CDR-H3 loop, which contains three Lys residues and is abnormally long. Priority comes from a recent study on anti-matriptase antibody E2, which undergoes matriptase cleavage after the P1-Arg residue in the long CDR-H3 loop (faray et al, 2008). Thus, Fab25 was exposed to hepsin for extended periods of time at different pH conditions. Fab25 migrated as a 50kDa band under non-reducing conditions and a 25kDa band under reducing conditions, and no low molecular weight degradation products were identified (fig. 14). Thus, Fab25 was resistant to hepsin proteolysis when exposed to hepsin for extended periods of time. This conclusion is consistent with our hypothesis that the bio-hepsin-DCI complex facilitates the selection of phage display fabs that bind outside the S1 pocket.

It was recently established that laminin is a substrate for hepsin (Tripathi et al). This study also suggested that cleavage of laminin by hepsin could physiologically enhance migration of the prostate cancer cell line DU 145. We examined whether Fab25 inhibited laminin-dependent DU145 cell migration. The results of this experiment (fig. 15) demonstrate that Fab25 effectively neutralizes the proteolytic activity of hepsin and thus blocks processing of its substrate laminin.

We also tested the ability of Fab25 to inhibit hepsin activity against a panel of synthetic substrates. The results shown in table 1 demonstrate that Fab25 inhibited hepsin-mediated cleavage of all pNA substrates by > 90%. Since the pNA substrate occupies the S1-S3 subsites on hepsin, it can be concluded that the antibody strongly interferes with substrate interactions at these subsites. Despite the chemical diversity of the P2 and P3 positions of the pNA substrates, the finding that inhibition of all substrates is >90% supports strong antibody effects at the S2 and/or S3 sites rather than minor effects at these sites. Whether these effects are allosteric in nature or direct steric hindrance remains to be elucidated. In addition, because Fab25 was selected for DCI-inhibited hepsin, it is unlikely that Fab would inhibit hepsin by directly occupying the S1 pocket.

Table 1: inhibition of hepsin activity against a panel of synthetic substrates by Fab 25. Hepsin was incubated with 100 nFab 25 or control Fab for 40 minutes before addition of 8 different pNA substrates. Initial linear velocities were measured on a dynamic microplate reader and enzyme activity was expressed as a percentage of hepsin activity in the absence of Fab.

TABLE 1

^a100nM Fab in the reaction mixture

^bControl hepsin enzymatic Activity without Fab

Fab25 was shown to consistently inhibit hepsin activity by >90% in all functional assays using a variety of synthetic and macromolecular substrates.

Proteolytic processing of pro-MSP by hepsin naturally expressed on the cell surface was monitored on LnCap-34 cell line (Moran et al, 2006). LnCap-34 cells expressing hepsin are capable of processing¹²⁵I-pro-MSP (FIG. 16). The proteolytic activity of pro-MSP processing is due primarily to hepsin, as all three hepsin inhibitors (Ac-KQLR-chloromethyl ketone ("KQLR" disclosed as SEQ ID NO:12), KD1 and Fab25) effectively block proteolytic cleavage.

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Although the foregoing invention has been described in some detail by way of illustration for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention.

Claims (14)

1. An isolated anti-hepsin antibody, wherein the antibody binds hepsin present in a complex comprising hepsin and a serine protease inhibitor that binds human hepsin S1 subsite, and wherein the antibody comprises:

(a) HVR-H1, consisting of sequence GFNFSYSYMH (SEQ ID NO: 4);

(b) HVR-H2, consisting of sequence ASIYSYYGSTYYADSVKG (SEQ ID NO: 5);

(c) HVR-H3, consisting of sequence ARSDSWSYKSGYTQKIYSKGLDY (SEQ ID NO: 6);

(d) HVR-L1, consisting of sequence RASQSVSSAVA (SEQ ID NO: 1);

(e) HVR-L2, consisting of the sequence SASSLYS (SEQ ID NO: 2); and

(f) HVR-L3, consisting of sequence QQYYSSYYLLT (SEQ ID NO: 3).

2. The antibody of claim 1, wherein the serine protease inhibitor is 3, 4-dichloro-isocoumarin (DCI).

3. The antibody of claim 1, wherein the antibody inhibits hepsin-mediated macrophage-stimulating protein (MSP) activation.

4. The antibody of claim 1, wherein the antibody inhibits laminin-dependent cell migration.

5. The antibody of claim 1, further comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14-15, 48 and 16; and SEQ ID NO: 14-15, 43 and 16 or a light chain variable domain framework sequence selected from SEQ ID NOs: 17-20; SEQ ID NO: 49-51 and 20; SEQ ID NO: 52-54 and 20; SEQ ID NO: 55-57 and 20; and SEQ ID NO: 17-18, 58 and 20.

6. The antibody of claim 1, comprising (a) a VH sequence set forth in amino acid sequence SEQ ID No. 10; or (b) a VL sequence shown by an amino acid sequence SEQ ID NO. 9.

7. The antibody of claim 6, comprising a VH sequence of SEQ ID NO 10 and a VL sequence of SEQ ID NO 9.

8. The antibody of any one of the preceding claims, which is a full length IgG1 antibody.

9. The antibody of any one of claims 1-7, wherein the antibody comprises human k subgroup consensus framework sequence and/or the antibody comprises heavy chain human subgroup III consensus framework sequence.

10. The antibody of claim 8, wherein the antibody comprises human k subgroup consensus framework sequence and/or the antibody comprises heavy chain human subgroup III consensus framework sequence.

11. An isolated nucleic acid encoding the antibody of any one of the preceding claims.

12. An immunoconjugate comprising the antibody of claim 1 and a cytotoxic agent.

13. A pharmaceutical formulation comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.

14. A method of generating an anti-serine protease antibody comprising selecting an antibody that binds to a serine protease present in a complex comprising (a) a serine protease and (b) a serine protease inhibitor that binds to the S1 subsite, wherein the serine protease inhibitor is 3, 4-dichloro-isocoumarin, and wherein the serine protease is hepsin.