Anti-c-met antibody formulations   

Abstract: Provided herein are pharmaceutical formulations comprising a one-armed, anti-c-met antibody and uses of the same.

This application claims benefit of priority to U.S. Patent Application 61/503,513, filed Jun. 30, 2011, the entire contents of which is incorporated herein by reference in its entirety.

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 16, 2012, is named P4690R1WO.txt and is 22,553 bytes in size.

TECHNICAL FIELD

Provided herein are pharmaceutical formulations comprising an anti-c-met antibody and uses of the same.

Excipients are added to pharmaceutical formulations to aid in the stabilization of the active compound. The compatibility of the excipients with the active compound is crucial for the quality and stability of the pharmaceutical formulation. While excipients are important in the stabilization of the active compound, excipients can cause problems: the excipient may degrade and thus lose its mechanism of stabilization or it may produce degradants that interact with the active compound.

Pharmaceutical formulations in which the active compound is a polypeptide, e.g. an antibody, can pose special problems as polypeptides generally are larger and more complex than traditional organic and inorganic molecules (for example, polypeptides possess multiple functional groups, in addition to complex three-dimensional structures). In addition, for a polypeptide to remain biologically active, the pharmaceutical polypeptide formulation must preserve intact the conformational integrity of at least a core sequence of the polypeptide\'s amino acids, while at the same time maintaining physical and chemical stability of the pharmaceutical polypeptide formulation. Excipients are generally stable in aqueous solution; however, excipients in a pharmaceutical polypeptide formulation can interact with the polypeptide to undergo degradation in a formulation and can prevent the stabilization of the protein or the degradants could interact with the polypeptide to pose challenges (such as a loss in activity). Therefore, the evaluation of the interaction of the non-active components of the pharmaceutical formulation and polypeptide active agent is crucial for ensuring chemical and physical stability.

Polysorbates are non-ionic surfactants used to stabilize an active compound against interface induced aggregation and surface adsorption. Polysorbates can be effective against various stresses such as agitation (for example, shaking or stirring), freeze/thawing, and lyophilization. In pharmaceutical polypeptide formulations, polysorbates minimize adsorption to surfaces and reduce the air-liquid interfacial surface tension and thus the rate of protein denaturation. Loss of polysorbate in the pharmaceutical formulation can result in instability of the formulation. Further, polysorbates can be degraded by oxidation and hydrolysis which can lead to a decrease in the apparent concentration of polysorbate in the pharmaceutical formulation over long shelf life. Polysorbates (e.g., polysorbate 20) can be cleaved to produce degradants (e.g., free lauric acid and sorbitan polyoxyethylene side chain). These polysorbate degradants are less surface-active than nondegraded polysorbate and hence the chemical and physical stability of the pharmaceutical formulation may be compromised. Further, some polysorbate degradants are insoluble and may form particles if they are not solubilized by intact polysorbate, i.e., if the ratio of degraded polysorbate 20: intact polysorbate 20 is too high.

The rate and extent of degradation of polysorbate is influenced by the chemical and physical properties of the active compound, and the stabilizing ability of polysorbate can vary between different pharmaceutical formulations comprising different active compounds. Particularly since polysorbates are included in protein formulations to stabilize the protein, the decrease in the concentration of polysorbate and the accumulation of degradant molecules in a pharmaceutical polypeptide formulation is of potential concern for protein stability.

Numerous molecules targeted at the HGF/c-met pathway have been reported. These molecules include a portion of the extracellular domain of c-met and anti-c-met antibodies such as those described in U.S. Pat. No. 5,686,292, Martens, T. et al., Clin. Cancer Res. 12 (20 Pt. 1):6144 (2006); U.S. Pat. No. 6,468,529; WO2006/015371; WO2007/063816, and WO2010/045345. Bivalent forms of anti-c-met antibodies have been shown to promote dimerization and lead to activation of c-met (agonistic function), while conversely monovalent antibodies have been shown to inhibit c-met activity (antagonistic function). For treatment of pathological conditions requiring an antagonistic function, bivalency of an anti-c-met antibody could result in an undesirable agonistic effect, and therefore, the monovalent trait is required to ensure an antagonistic activity upon binding of the anti-c-met antibody to the target for treatment of the pathological condition. Fab fragments and one-armed antibodies are examples of monovalent antibodies. One-armed antibodies generally have a longer half-life than Fabs. However, as a one-armed antibody comprises a single light chain and a single heavy chain (as well as an additional Fc region), if the one-armed antibody structure is not stabilized, the polypeptides could potentially form a bivalent antibody with two heavy chain and two light chains. Aggregation of monovalent antibodies (formation of multimer and oligomers) and/or failure to maintain monovalent structure in a pharmaceutical formulation comprising anti-c-met antibodies could lead to an undesirable agonistic effect. Minimization of anti-c-met antibody aggregation in the pharmaceutical formulation is thus particularly important. Therefore, despite the significant advancement in the molecules which target the HGF/c-met pathway, stable pharmaceutical formulations, which minimize aggregation of c-met antibodies, are still needed.

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

DISCLOSURE OF THE INVENTION

Provided herein are pharmaceutical formulations comprising an anti-c-met antibody. In some embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a stable pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a stable liquid pharmaceutical formulation. In some embodiments, the anti-c-met antibody is an antagonist anti-c-met antibody.

For example, provided herein are pharmaceutical formulations comprising: (a) an anti-c-met antibody; (b) a histidine buffer at pH 5.0-5.4; (c) a saccharide; and (d) a polysorbate, wherein the polysorbate is present at greater than 0.02% w/v.

In some embodiments of any of the formulations, the anti-c-met antibody comprising a HVR-L1 comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO:1), a HVR-L2 comprising sequence WASTRES (SEQ ID NO:2), a HVR-L3 comprising sequence QQYYAYPWT (SEQ ID NO:3), a HVR-H1 comprising sequence GYTFTSYWLH (SEQ ID NO:4), a HVR-H2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO:5), and a HVR-H3 comprising sequence ATYRSYVTPLDY (SEQ ID NO:6). In some embodiments, the anti-c-met antibody comprises (a) a heavy chain variable domain comprising the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRF NPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO:19) and (b) a light chain variable domain comprising the sequence: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTR ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO:20). In some embodiments, the anti-c-met antibody comprises a single antigen binding arm and comprises a Fc region, wherein the Fc region comprises a first and a second Fc polypeptide, and wherein the first and second Fc polypeptides are present in a complex. In some embodiments, the first and second Fc polypeptides form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. In some embodiments, the anti-c-met antibody comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:19, a CH1 sequence, and a first Fc polypeptide and (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:20 and CL1 sequence. In some embodiments, the anti-c-met antibody further comprises (c) a third polypeptide comprising a second Fc polypeptide. In some embodiments, the first Fc polypeptide comprises the Fc sequence depicted in FIG. 2 (SEQ ID NO: 17) and the second Fc polypeptide comprises the Fc sequence depicted in FIG. 3 (SEQ ID NO: 18). In some embodiments, the anti-c-met antibody is onartuzumab. In some embodiments, the anti-c-met antibody binds the same epitope as onartuzumab.

In some embodiments of any of the formulations, the anti-c-met antibody is present at a concentration between about 10 mg/mL and about 100 mg/mL (e.g. about 15 mg/mL and about 75 mg/mL). In some embodiments, the anti-c-met antibody is present at a concentration of about 60 mg/mL.

In some embodiments of any of the formulations, the saccharide is present at a concentration of about 75 mM to about 200 mM (e.g., about 100 mM to about 150 mM). In some embodiments, the saccharide is present at a concentration of about 120 mM. In some embodiments, the saccharide is a disaccharide. In some embodiments, the disaccharide is trehalose. In some embodiments, the disaccharide is sucrose.

In some embodiments of any of the formulations, the histidine buffer is at a concentration of about 1 mM to about 50 mM (e.g. about 1 mM to about 25 mM). In some embodiments, the histidine buffer is at a concentration of about 10 mM. In some embodiments, the histidine buffer is histidine acetate.

In some embodiments of any of the formulations, the polysorbate is present at a concentration greater than 0.02% and less than about 0.1%. In some embodiments, the polysorbate is present at a concentration of about 0.04%. In some embodiments, the polysorbate is polysorbate 20.

In some embodiments of any of the formulations, the formulation is diluted with a diluent (e.g., 0.9% NaCl). In some embodiments, the anti-c-met antibody is present at a concentration of about 1 mg/mL.

Provided herein are methods of inhibiting c-met activated cell proliferation, said method comprising contacting a cell or tissue with an effective amount of the pharmaceutical formulation described herein (e.g., upon dilution).

Also provided herein are methods of modulating a disease associated with dysregulation of the HGF/c-met signaling axis, said method comprising administering to a subject an effective amount of the pharmaceutical formulation described herein (e.g., upon dilution).

Further provided are methods of treating a subject having a proliferative disorder, said method comprising administering to the subject an effective amount of the pharmaceutical formulation described herein (e.g., upon dilution). In some embodiments, the proliferative disorder is cancer. In some embodiments, the cancer is lung cancer (non-small cell lung cancer (NSCLC)), glioblastoma, pancreatic cancer, sarcoma, renal cell carcinoma, hepatocellular carcinoma, gastric cancer, colorectal cancer, and/or breast cancer. In some embodiments, the methods further comprise a second therapeutic agent.

Provided are methods of making the pharmaceutical formulation described herein.

In addition, provided herein are articles of manufacture comprising a container with a pharmaceutical formulation described herein (e.g., upon dilution) contained therein. Provided herein are also methods of making the articles of manufacture comprising a pharmaceutical formulation described herein (e.g., upon dilution).

FIG. 1 depicts the general structures of short half-life and long half-life agonists and antagonists of c-Met.

FIG. 2 depicts amino acid sequences of the framework (FR), hypervariable region (HVR), first constant domain (CL or CH1) and Fc region (Fc) of MetMAb (onartuzumab or OA5D5.v2). The Fc sequence depicted comprises “hole” (cavity) mutations T366S, L368A and Y407V, as described in WO 2005/063816.

FIG. 3 depicts sequence of an Fc polypeptide comprising “knob” (protuberance) mutation T366W, as described in WO 2005/063816. In some embodiments, an Fc polypeptide comprising this sequence forms a complex with an Fc polypeptide comprising the Fc sequence of FIG. 1 to generate an Fc region. The sequence disclosed in FIG. 3 represents residues 6-227 of SEQ ID NO: 18.

FIG. 4 depicts the rate of aggregate formation as indicated by the percentage of high molecular weight species (HMWS) over time (days) at 40° C. for formulations of 20 mg/mL onartuzumab, 10 mM histidine acetate, 120 mM trehalose, and 0.02% polysorbate 20 at pH 5.2, 5.7, and 6.2.

FIG. 5 depicts the rate of aggregate formation as indicated by the percentage of high molecular weight species (HMWS) over time (days) at 25° C. for formulations of 40 mg/mL onartuzumab, 10 mM histidine acetate, 120 mM trehalose, and 0.02% polysorbate 20 at pH 5.1, 5.4, and 5.7.

FIG. 6 depicts the rate of aggregate formation as indicated by the percentage of high molecular weight species (HMWS) over time (days) at 40° C. for formulations of 40 mg/mL onartuzumab, 10 mM histidine acetate, 120 mM trehalose, and 0.02% polysorbate 20 at pH 5.1, 5.4, and 5.7.

FIG. 7 depicts the chemical stability as measured by ion-exchange chromatography (IEC) as indicated by the percent main peak over time (days) at 25° C. and 40° C. for formulations of 40 mg/mL onartuzumab, 10 mM histidine acetate, 120 mM trehalose, and 0.02% polysorbate 20 at pH 5.1, 5.4, and 5.7.

FIG. 8 depicts the percentage of intact polysorbate over time (weeks) at 40° C. for formulations of 60 mg/mL onartuzumab, 10 mM histidine acetate, pH 5.4, and 120 mM sucrose with 0.02% polysorbate 20 or 0.04% polysorbate 20.

FIG. 9 depicts the rate of aggregate formation of onartuzumab diluted to 1 mg/mL in IV bags containing 0.9% NaCl. The rate of aggregation is indicated by the percentage of high molecular weight species (HMWS) over time (hours) of agitation for diluted formulations of (a) 60 mg/mL onartuzumab, 10 mM histidine acetate, 120 mM trehalose, and 0.02% polysorbate 20 at pH 5.4 kept at room temperature as shown by squares and (b) 60 mg/mL onartuzumab, 10 mM histidine acetate, 120 mM sucrose, and 0.04% polysorbate 20 at pH 5.4 kept at 30° C. as shown by circles.

DETAILED DESCRIPTION

Provided herein are stable pharmaceutical formulations comprising an anti-c-met antibody. In some embodiments, the anti-c-met antibody is an antagonist anti-c-met antibody. In some embodiments, the anti-c-met antibody is a monovalent anti-c-met antibody. In addition, kits and articles of manufacture comprising the anti-c-met antibody pharmaceutical formulations and uses of the anti-c-met antibody pharmaceutical formulations are provided.

The term “pharmaceutical formulation” refers to preparations which are in such form as to permit the biological activity of the active compound(s) to be effective, and which contain no additional components which are toxic to the subjects to which the formulation is administered. “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject to provide an effective dose of the active compound.

A “stable” formulation is one in which the polypeptide therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage and/or during administration (e.g., after dilution in an IV bag). Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. Stability can be measured at a selected temperature for a selected time period.

A polypeptide “retains its physical stability” in a pharmaceutical formulation if the chemical stability at a given time is such that the protein is considered to still retain its biological activity and an acceptable safe profile, for example, as determined by International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines (e.g., commercially acceptable levels of aggregation, precipitation and/or denaturation, for example, upon visual examination of color and/or clarity or as measured by UV light scattering or by size exclusion chromatography).

A polypeptide “retains its chemical stability” in a pharmaceutical formulation, if the chemical stability at a given time is such that the protein is considered to still retain its biological activity. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical alteration may involve size modification (e.g., clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example. Other types of chemical alteration include charge alteration which can be evaluated by ion-exchange chromatography, for example.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

A “histidine buffer” is a buffer comprising histidine ions.

A “saccharide” herein comprises the general composition (CH2O)n and derivatives thereof.

An “anti-c-met antibody” and “an antibody that binds to c-met” refer to an antibody that is capable of binding c-met with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting c-met. In some embodiments, the extent of binding of an anti-c-met antibody to an unrelated, non-c-met protein is less than about 10% of the binding of the antibody to c-met as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antibody that binds to c-met has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10−8 M or less, e.g. from 10−8 M to 10−13 M, e.g., from 10−9 M to 10−13 M). In some embodiments, an anti-c-met antibody binds to an epitope of c-met that is conserved among c-met from different species.

The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, and antibody fragments so long as they exhibit the desired biological activity (e.g., Fab and/or single-armed antibodies).

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

A “blocking” antibody or an “antagonist” antibody is one which significantly inhibits (either partially or completely) a biological activity of the antigen it binds.

An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “hypervariable region” or “HVR,” as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).) With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

“Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The phrase “N-terminally truncated heavy chain”, as used herein, refers to a polypeptide comprising parts but not all of a full length immunoglobulin heavy chain, wherein the missing parts are those normally located on the N terminal region of the heavy chain. Missing parts may include, but are not limited to, the variable domain, CH1, and part or all of a hinge sequence. Generally, if the wild type hinge sequence is not present, the remaining constant domain(s) in the N-terminally truncated heavy chain would comprise a component that is capable of linkage to another Fc sequence (i.e., the “first” Fc polypeptide as described herein). For example, said component can be a modified residue or an added cysteine residue capable of forming a disulfide linkage.

The term “Fc region”, as used herein, generally refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc sequence is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl-terminus of the Fc sequence. However, the C-terminal lysine (Lys447) of the Fc sequence may or may not be present. The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. By “Fc polypeptide” herein is meant one of the polypeptides that make up an Fc region. An Fc polypeptide may be obtained from any suitable immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM. In some embodiments, an Fc polypeptide comprises part or all of a wild type hinge sequence (generally at its N terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild type hinge sequence.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see, e.g., Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

Binding to human FcRn in vivo and serum half life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered. WO 2000/42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

The “hinge region,” “hinge sequence”, and variations thereof, as used herein, includes the meaning known in the art, which is illustrated in, for example, Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209:193-202.

Unless indicated otherwise, the expression “multivalent antibody” is used throughout this specification to denote an antibody comprising three or more antigen binding sites. The multivalent antibody is preferably engineered to have the three or more antigen binding sites and is generally not a native sequence IgM or IgA antibody.

An “Fv” fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six HVRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

The “Fab” fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F(ab′)2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

The phrase “antigen binding arm”, as used herein, refers to a component part of an antibody fragment that has an ability to specifically bind a target molecule of interest. Generally and preferably, the antigen binding arm is a complex of immunoglobulin polypeptide sequences, e.g., HVR and/or variable domain sequences of an immunoglobulin light and heavy chain.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create 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, 90:6444-6448 (1993).

The expression “linear antibodies” refers to the antibodies described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which 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 represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

An “affinity matured” antibody refers to an antibody with one or more alterations in one or more HVRs, compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

An antibody having a “biological characteristic” of a designated antibody is one which possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies that bind to the same antigen.

A “functional antigen binding site” of an antibody is one which is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen. Moreover, the antigen binding affinity of each of the antigen binding sites of a multivalent antibody herein need not be quantitatively the same. For the multimeric antibodies herein, the number of functional antigen binding sites can be evaluated using ultracentrifugation analysis as described in Example 2 of U.S. Patent Application Publication No. 20050186208. According to this method of analysis, different ratios of target antigen to multimeric antibody are combined and the average molecular weight of the complexes is calculated assuming differing numbers of functional binding sites. These theoretical values are compared to the actual experimental values obtained in order to evaluate the number of functional binding sites.

A “species-dependent antibody” is one which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody “binds specifically” to a human antigen (i.e. has a binding affinity (Kd) value of no more than about 1×10−7 M, preferably no more than about 1×10−8 M and most preferably no more than about 1×10−9 M) but has a binding affinity for a homologue of the antigen from a second nonhuman mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be any of the various types of antibodies as defined above. In some embodiments, the species-dependent antibody is a humanized or human antibody.

An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The term “substantially similar” or “substantially the same,” as used herein, refers to a sufficiently high degree of similarity between two numeric values (for example, one associated with an antibody and the other associated with a reference/comparator antibody), such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values).

The phrase “substantially reduced” or “substantially different,” as used herein, refers to a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values).

“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

A “disorder” is any condition that would benefit from treatment with a substance/molecule or method described herein. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include malignant and benign tumors; non-leukemias and lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, immunologic and other angiogenesis-related disorders.

The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

“Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer”, “cancerous,” “cell proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin\'s and non-Hodgkin\'s lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer. In some embodiments, the cancer is triple-negative (ER-, PR-, HER2-) cancer. In some embodiments, the cancer is triple-negative metastatic breast cancer, including any histologically confirmed triple-negative (ER-, PR-, HER2-) adenocarcinoma of the breast with locally recurrent or metastatic disease, e.g., where the locally recurrent disease is not amenable to resection with curative intent.

By “metastasis” is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies are used to delay development of a disease or to slow the progression of a disease.

An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

A “therapeutically effective amount” refers to an amount of a therapeutic agent to treat or prevent a disease or disorder in a mammal. In the case of cancers, the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.

An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

The term “anti-cancer therapy” refers to a therapy useful in treating cancer. Examples of anti-cancer therapeutic agents include, but are limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, anti-CD20 antibodies, platelet derived growth factor inhibitors (e.g., Gleevec™ (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR-beta, BlyS, APRIL, BCMA receptor(s), TRAIL/Apo2, and other bioactive and organic chemical agents, etc. Combinations thereof are also included.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

A “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide; onapristone; anti-progesterones; estrogen receptor down-regulators (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

The term “prodrug” as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs 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, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use include, but are not limited to, those chemotherapeutic agents described above.

A “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell (e.g., a cell whose growth is dependent upon HGF/c-met activation either in vitro or in vivo). Thus, the growth inhibitory agent may be one which significantly reduces the percentage of HGF/c-met-dependent cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

By “radiation therapy” is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one time administration and typical dosages range from 10 to 200 units (Grays) per day.

The term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).

By “reduce or inhibit” is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

It is understood that aspect and embodiments of the invention described herein include “consisting” and/or “consisting essentially of” aspects and embodiments.

As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise.

As is understood by one skilled in the art, reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

Provided herein are pharmaceutical formulations comprising an anti-c-met antibody. In some embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a stable pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a stable liquid pharmaceutical formulation. In some embodiments, the anti-c-met antibody is an antagonist anti-c-met antibody. In some embodiments, the anti-c-met antibody is a monovalent anti-c-met antibody. Minimization in the pharmaceutical formulation of anti-c-met antibody aggregation is particularly important. Bivalent forms of anti-c-met antibodies have been shown to promote dimerization and lead to activation of c-met (agonistic function), while conversely monovalent antibodies inhibit c-met activity (antagonistic function). Aggregation of monovalent antibodies (formation of multimer and oligomers) and/or failure to maintain monovalent structure in a pharmaceutical formulation comprising anti-c-met antibodies could lead to an undesirable agonistic effect.

In particular, provided herein are pharmaceutical formulations comprising (a) an anti-c-met antibody and (b) a polysorbate, wherein the polysorbate concentration is greater than 0.02% w/v. In some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody; (b) a polysorbate, wherein the polysorbate concentration is greater than 0.02% w/v; and (c) a histidine buffer (e.g., a histidine buffer at a pH between 5.0 and 5.4). In some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody; (b) a histidine buffer at pH 5.0-5.4; (c) a saccharide; and (d) a polysorbate, wherein the polysorbate concentration is greater than 0.02% w/v. In some embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a stable pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a stable liquid pharmaceutical formulation.

Anti-c-met antibodies useful in the pharmaceutical formulations are described in Section III. In some embodiments, the anti-c-met antibody is an antagonist anti-c-met antibody. In some embodiments, the anti-c-met antibody is a monovalent anti-c-met antibody. For example, in some embodiments, the anti-c-met antibody comprises a single antigen binding arm. In some embodiments, the anti-c-met antibody comprises a single antigen binding arm and comprises a Fc region, wherein the Fc region comprises a first and a second Fc polypeptide, wherein the first and second Fc polypeptides are present in a complex. In some embodiments, the first and second Fc polypeptides form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. In some embodiments, the anti-c-met antibody comprises a HVR-L1 comprising the amino acid sequence of SEQ ID NO:1, a HVR-L2 comprising the amino acid sequence of SEQ ID NO:2, a HVR-L3 comprising the amino acid sequence of SEQ ID NO:3, a HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, a HVR-H2 comprising the amino acid sequence of SEQ ID NO:5, and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the anti-c-met antibody comprises (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:19 and (b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the anti-c-met antibody comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:19, a CH1 sequence, and a first Fc polypeptide, (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:20 and CL1 sequence, and (c) a third polypeptide comprising a second Fc polypeptide. In some embodiments, the first Fc polypeptide comprises the Fc sequence depicted in FIG. 2 (SEQ ID NO: 17) and the second Fc polypeptide comprises the Fc sequence depicted in FIG. 3 (SEQ ID NO: 18). In some embodiments, the anti-c-met antibody is onartuzumab.

In some embodiments of any of the pharmaceutical formulations described herein, the anti-c-met antibody of the pharmaceutical formulation is present at a concentration between about 10 mg/mL and about 100 mg/mL. In some embodiments, the concentration of the anti-c-met antibody (e.g., onartuzumab) is between about any of 10 mg/mL to 50 mg/mL, 10 mg/mL to 75 mg/mL, 25 mg/mL to 75 mg/mL, 50 mg/mL to 100 mg/mL, 50 mg/mL to 75 mg/mL, and/or 75 mg/mL to 100 mg/mL. In some embodiments, the concentration of the anti-c-met antibody (e.g., onartuzumab) is greater than about any of 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, or 100 mg/mL. For example, in some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody (e.g., onartuzumab), wherein the anti-c-met antibody is present at a concentration between about 50 mg/mL and about 75 mg/mL; (b) a histidine buffer at pH 5.0-5.4; (c) a saccharide; and (d) a polysorbate, wherein the polysorbate concentration is greater than 0.02% w/v. In some embodiments, the concentration of the anti-c-met antibody (e.g., onartuzumab) is less than about any of 150 mg/mL, 125 mg/mL, 100 mg/mL, or 75 mg/mL. In some embodiments, the concentration of the anti-c-met antibody (e.g., onartuzumab) is about any of 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, or 80 mg/mL. In some embodiments, the concentration of the anti-c-met antibody (e.g., onartuzumab) is about 60 mg/mL.

The pharmaceutical formulation preferably comprises a polysorbate. The polysorbate is generally included in an amount which reduces aggregate formation (such as that which occurs upon shaking or shipping). Examples of polysorbate include, but are not limited to, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and/or polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In some embodiments, the polysorbate is polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate). In some embodiments of any of the pharmaceutical formulations described herein, the polysorbate concentration is sufficient to minimize aggregation and/or maintain stability upon long term storage and/or during administration (e.g., after dilution in an IV bag). In some embodiments, the polysorbate concentration is greater than 0.02% w/v, greater than or equal to about 0.03% w/v, or greater than or equal to about 0.04% w/v. In some embodiments, the polysorbate concentration is greater than 0.02% w/v and less than about 0.1% w/v. In some embodiments, the polysorbate concentration is greater than 0.03% w/v and less than about 0.1% w/v. In some embodiments, the polysorbate concentration is about any of 0.03% w/v, 0.04% w/v, or 0.05% w/v. In some embodiments, the polysorbate is present at a concentration of about 0.04% w/v. For example, in some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody (e.g., onartuzumab); (b) a histidine buffer at pH 5.0-5.4; (c) saccharide; and (d) polysorbate 20, wherein the polysorbate 20 concentration is about 0.04% w/v.

The pharmaceutical formulation preferably comprises a saccharide. Saccharides include monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, nonreducing sugars, etc. Further examples of saccharides include, but are not limited to, glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc. In some embodiments, the saccharide is a disaccharide. In some embodiments, the saccharide is a nonreducing disaccharide. In some embodiments, the saccharide is trehalose. In some embodiments, the saccharide is sucrose. For example, in some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody (e.g., onartuzumab); (b) a histidine buffer at pH 5.0-5.4; (c) sucrose; and (d) a polysorbate, wherein the polysorbate concentration is greater than 0.02% w/v.

The saccharide is generally included in an amount which reduces aggregate formation. In some embodiments of any of the pharmaceutical formulations described herein, the saccharide is present at a concentration of between about any of 50 mM to 250 mM, 75 mM to 200 mM, 75 mM to 150 mM, 100 mM to 150 mM, or 110 mM to 130 mM. In some embodiments, the saccharide is present at a concentration greater than about any of 50 mM, 75 mM, 100 mM, 110 mM, or 115 mM. In some embodiments, the saccharide is present at a concentration of about any of 100 mM, 110 mM, 120 mM, 130 mM, or 140 mM. In some embodiments, the saccharide is present at a concentration of about 120 mM. For example, in some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody (e.g., onartuzumab); (b) a histidine buffer at pH 5.0-5.4; (c) sucrose, wherein the sucrose is present at a concentration of about 120 mM; and (d) a polysorbate, wherein the polysorbate concentration is greater than 0.02% w/v.

The pharmaceutical formulation preferably comprises a histidine buffer. Examples of histidine buffers include, but are not limited to, histidine chloride, histidine succinate, histidine acetate, histidine phosphate, histidine sulfate. In some embodiments, the histidine buffer is histidine acetate. In some embodiments of any of the pharmaceutical formulations described herein, the histidine buffer concentration is between about any of 1 mM to 50 mM, 1 mM to 35 mM, 1 mM to 25 mM, 1 mM to 20 mM, 7.5 mM to 12.5 mM, or 5 mM to 15 mM. In some embodiments, the histidine buffer concentration is greater than or equal to about any of 5 mM, 7.5 mM, or 10 mM. In some embodiments, the histidine buffer concentration is about any of 5 mM, 7.5 mM, 10 mM, 12.5 mM, or 15 mM. In some embodiments, the histidine buffer concentration is about 10 mM. In some embodiments of any of the pharmaceutical formulations described herein, the histidine buffer is at a pH of between pH 5.0 and 5.4, for example, about any of pH 5.0, pH 5.1, pH 5.2, pH 5.3, or pH 5.4. In some embodiments, the pH is between pH 5.1 and 5.4. For example, in some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody (e.g., onartuzumab); (b) a histidine acetate buffer at pH 5.4, wherein the histidine acetate buffer is at a concentration of about 10 mM; (c) saccharide; and (d) a polysorbate, wherein the polysorbate concentration is greater than 0.02% w/v.

In some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody (e.g., onartuzumab), wherein the anti-c-met antibody is present at a concentration between about 50 mg/mL and about 75 mg/mL; (b) a histidine acetate buffer at pH 5.0-5.4, wherein the histidine acetate buffer is at a concentration between about 1 mM and about 20 mM; (c) sucrose, wherein the sucrose is at a concentration between about 100 mM to about 150 mM; and (d) polysorbate 20, wherein the polysorbate 20 concentration is greater than 0.02% w/v. In some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody (e.g., onartuzumab), wherein the anti-c-met antibody is present at a concentration of about 60 mg/mL; (b) a histidine acetate buffer at pH 5.4, wherein the histidine acetate buffer is at a concentration of about 10 mM; (c) sucrose, wherein the sucrose is at a concentration of about 120 mM; and (d) polysorbate 20, wherein the polysorbate 20 concentration is about 0.04% w/v.

The pharmaceutical formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

Further, provided herein are vials and methods of filing a vial comprising a pharmaceutical formulation described herein. In some embodiments, the pharmaceutical formulation is provided inside a vial with a stopper pierceable by a syringe, preferably in aqueous form. The vial is desirably stored at about 2-8° C. as well as up to 30° C. for 24 hours until it is administered to a subject in need thereof. The vial may for example be a 15 cc vial (for example for a 600 mg dose) or 20 cc vial (for example for a 900 mg dose).

The pharmaceutical formulation for administration is preferably a liquid formulation (not lyophilized) and has not been subjected to prior lyophilization. While the pharmaceutical formulation may be lyophilized, preferably it is not. In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation, the pharmaceutical formulation is a lyophilized pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a liquid formulation. In some embodiments, the pharmaceutical formulation does not contain a tonicifying amount of a salt such as sodium chloride. In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation is diluted.

In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation comprising the anti-c-met antibody is stable. In some embodiments, the pharmaceutical formulation comprising the anti-c-met antibody is physically stable. In some embodiments, the pharmaceutical formulation comprising the anti-c-met antibody is chemically stable. In some embodiments, the pharmaceutical formulation comprising the anti-c-met antibody is physically stable and chemically stable. In some embodiments, the pharmaceutical formulation comprises an antagonistic anti-c-met antibody and agonistic activity of the pharmaceutical formulation is substantially undetectable. Methods of detecting agonistic and/or agonistic activity are known in the art, for example, U.S. Pat. No. 6,207,152, which is incorporated by reference in its entirety. In some embodiments, the pharmaceutical formulation is substantially nonimmunogenic.

In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation does not significantly result in increased aggregate formation after storage at about 40° C. for about two weeks or about four weeks, at about 25° C. for about one month or about three months; at about 5° C. for about six months, about one year, or about two years, and/or about −20° C. for about three months, about six months, or about a year. In some embodiments, the pharmaceutical formulation has reduced or lower levels of aggregate formation after storage at about 40° C. for about two weeks or about four weeks, at about 25° C. for about one month or about three months; at about 5° C. for about six months, about one year, or about two years, and/or about −20° C. for about three months, about six months, or about a year (e.g., compared to a similar formulation at pH 5.7).

High Molecular Weight Species (HMWS) are generally larger than the reference molecule. For example, onartuzumab is about 100 kDa (99,161 Daltons), therefore, a HMWS is greater than about 100 kDa. The size of a bivalent antibody is approximately 150 kDa and a dimer of onartuzumab is about 200 kDa. In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation comprises less than about any of 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25%, 0.20% or 0.15% HMWS (e.g., upon storage). In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation does not significantly increase the percentage of HMWS after storage at about 40° C. for about two weeks or about four weeks, at about 25° C. for about one month or about three months; at about 5° C. for about six months, about one year, or about two years, and/or about −20° C. for about three months, about six months, or about a year. In some embodiments, the pharmaceutical formulation has reduced or lower levels of HMWS after storage at about 40° C. for about two weeks or about four weeks, at about 25° C. for about one month or about three months; at about 5° C. for about six months, about one year, or about two years, and/or about −20° C. for about three months, about six months, or about a year (e.g., compared to a similar formulation at pH 5.7). In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation does not significantly increase the percentage of HMWS after storage at about 40° C. for about any of 15 days, 30 days, 45 days, or 60 days or at about 25° C. for about 30 days or about 60 days. In some embodiments, the pharmaceutical formulation has reduced or lower levels of HMWS after storage at about 40° C. for about any of 15 days, 30 days, 45 days, or 60 days or at about 25° C. for about 30 days or about 60 days (e.g., compared to a similar formulation at pH 5.7).

Low Molecular Weight Species (LMWS) are generally smaller than the reference molecule. For example, onartuzumab is about 100 kDa (99,161 Daltons), therefore, a LMWS is less than about 100 kDa. In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation does not significantly increase degradation and/or percentage of LMWS after storage at about 40° C. for about two weeks or about four weeks, at about 25° C. for about one month or about three months; at about 5° C. for about six months, about one year, or about two years, and/or about −20° C. for about three months, about six months, or about a year (e.g., compared to a similar formulation at pH 5.7). In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation does not significantly increase degradation and/or percentage of LMWS after storage at about 40° C. for about any of 15 days, 30 days, 45 days, or 60 days, at about 25° C. for a for about 30 days or about 60 days (e.g., compared to a similar formulation at pH 5.7).

In some embodiments of any of the pharmaceutical formulations, the percentage of intact polysorbate in the pharmaceutical formulation is greater than about any of 75%, 80%, 85%, or 90% after storage at about 40° C. for about two weeks or about four weeks, at about 25° C. for about one month or about three months; at about 5° C. for about six months, about one year, or about two years, and/or about −20° C. for about three months, about six months, or about a year. In some embodiments of, the percentage of intact polysorbate in the pharmaceutical formulation is greater than about any of 75%, 80%, 85%, or 90% after storage at about 40° C. for about any of one, two, three, four, five, six, seven, or eight weeks.

In some embodiments of any of the pharmaceutical formulations, the percentage of degraded polysorbate in the pharmaceutical formulation is less than about any of 25%, 20%, 15%, or 10% after storage at about 40° C. for about two weeks or about four weeks, at about 25° C. for about one month or about three months; at about 5° C. for about six months, about one year, or about two years, and/or about −20° C. for about three months, about six months, or about a year. In some embodiments, the percentage of degraded polysorbate in the pharmaceutical formulation is less than about any of 25%, 20%, 15%, or 10% after storage at about 40° C. for one, two, three, four, five, six, seven, or eight weeks.

In some embodiments, the ratio of degraded polysorbate to intact polysorbate in the pharmaceutical formulation is less than about any of 0.25, 0.20, 0.15 or 0.10 after storage at about 40° C. for about two weeks or about four weeks, at about 25° C. for about one month or about three months; at about 5° C. for about six months, about one year, or about two years, and/or about −20° C. for about three months, about six months, or about a year. In some embodiments, the ratio of degraded polysorbate to intact polysorbate in the pharmaceutical formulation is less than about any of 0.25, 0.20, 0.15 or 0.10 after storage at about 40° C. for about any of one, two, three, four, five, six, seven, or eight weeks. In some embodiments, the pharmaceutical formulation comprising the anti-c-met antibody is more stable than a similar formulation at pH 5.7 and/or with a polysorbate concentration of 0.02% or less.

Moreover, the pharmaceutical formulation is desirably one which has been demonstrated to be stable upon storage and/or during administration (e.g., after dilution in an IV bag). Various stability assays are available to the skilled practitioner for confirming the stability of the formulation. For example, the formulation may be one which is found to be stable upon storage: at about 25° C. for at least about one month or at least about three months, about 5° C. for at least about six months or at least about one year; and/or about −20° C. for at least about six months or at least about one year. Furthermore, the pharmaceutical formulation is preferably stable following freezing (to, e.g., −70° C.) and thawing of the pharmaceutical formulation. Freezing of the aqueous pharmaceutical formulation, without simultaneous drying that occurs during freeze-drying, is specifically contemplated herein, facilitating longer term storage thereof, for instance in a stainless steel tank. Freezing of the pharmaceutical formulation is specifically contemplated herein. Hence, the pharmaceutical formulation can be tested for stability upon freezing and thawing. In another embodiment, the formulation is provided inside a stainless steel tank. The formulation in the stainless steel tank is optionally frozen and not freeze-dried.

The pharmaceutical formulation to be used for in vivo administration should be sterile. This can be achieved according to the procedures known to the skilled person for generating sterile pharmaceutical formulations suitable for administration to human subjects, including filtration through sterile filtration membranes, prior to, or following, preparation of the formulation.

In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation comprising the anti-c-met antibody is stable upon dilution with a diluent (e.g., saline). For example, provided herein are IV bags comprising a diluted pharmaceutical formulation described herein. In some embodiments, the diluent is saline (e.g., 0.9% sodium chloride). In some embodiments, the concentration of the anti-c-met antibody is diluted to about any of 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL or 5 mg/mL. In some embodiments, the concentration of the anti-c-met antibody is diluted to between about any of 0.5-5 mg/mL, 0.5-2.5 mg/mL, or 0.5-1.5 mg/mL. Therefore, for example, provided herein are pharmaceutical formulations comprising a) an anti-c-met antibody, wherein the antibody concentration is about 1 mg/mL, and (b) a polysorbate, wherein the polysorbate concentration is greater than 0.00033% w/v. In some embodiments, the pharmaceutical formulation comprises (a) an anti-c-met antibody, wherein the antibody concentration is about 1 mg/mL; (b) a polysorbate, wherein the polysorbate concentration is greater than 0.00033% w/v; (c) a histidine buffer (e.g., a histidine buffer at a pH between 5.0 and 5.4). In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation is stable (e.g., physically stable) in an IV bag and/or IV administration set.

In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation (for example, after dilution) is stable upon agitation for any of about thirty minutes, one hour, 1.5 hours, or two hours at about any of 75, 100, 125, or 150 rpm. In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation (for example, after dilution) comprises less than about any of 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25%, 0.20% or 0.15% HMWS (for example, upon agitation).

Provided herein are also methods of making a pharmaceutical formulation comprising preparing the formulation as described herein. In some embodiments, the methods further comprise evaluating physical stability, chemical stability, or biological activity of the anti-c-met antibody in the formulation.

Stability can be tested by evaluating physical stability, chemical stability, and/or biological activity of the antibody in the formulation around the time of formulation as well as following storage, during administration, and/or upon agitation at the noted temperatures. Physical and/or stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may result in aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc. Biological activity or antigen binding function can be evaluated using various techniques available to the skilled practitioner.

One or more other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington\'s Pharmaceutical Sciences 18th edition, Gennaro, A. Ed. (1990) may be included in the formulation provided that they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include; additional buffering agents; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers such as polyesters; preservatives; and/or salt-forming counterions such as sodium.

Provided herein are anti-c-met antibodies for use in the pharmaceutical formulations described herein. Useful anti-c-met antibodies include antibodies that bind with sufficient affinity and specificity to c-met and can reduce or inhibit one or more c-met activities. Anti-c-met antibodies in the pharmaceutical formulations can be used to modulate one or more aspects of HGF/c-met-associated effects, including but not limited to c-met activation, downstream molecular signaling (e.g., mitogen activated protein kinase (MAPK) phosphorylation), cell proliferation, cell migration, cell survival, cell morphogenesis and angiogenesis. These effects can be modulated by any biologically relevant mechanism, including disruption of ligand (e.g., HGF) binding to c-met, c-met phosphorylation and/or c-met multimerization. In some embodiments, the anti-c-met antibody is an antagonist anti-c-met antibody. In some embodiments, the anti-c-met antibody interferes with diseases or conditions wherein c-met/HGF activity is involved.

In some embodiments of any of the anti-c-met antibody formulations described herein, the anti-c-met antibody is an anti-c-met antibody fragment. In some embodiments, the anti-c-met antibody is an antagonist anti-c-met antibody. In some embodiments, the anti-c-met antibody is monovalent. In some embodiments, the anti-c-met antibody fragment may comprise a single antigen binding arm and an Fc region. Anti-c-met antibody fragments are described herein and are known in the art, in the one-armed format. Accordingly, in some embodiments, the anti-c-met antibody fragment is a one-armed antibody (i.e., the heavy chain variable domain and the light chain variable domain form a single antigen binding arm) comprising an Fc region, wherein the Fc region comprises a first and a second Fc polypeptide, wherein the first and second Fc polypeptides are present in a complex. In some embodiments, the first and second Fc polypeptides form a Fc region that increases stability of the anti-c-met antibody compared to a Fab molecule comprising said antigen binding arm. In some embodiments, the anti-c-met antibody comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:19, a CH1 sequence, and a first Fc polypeptide and (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:20 and CL1 sequence. In some embodiments, the anti-c-met antibody further comprises (c) a third polypeptide comprising a second Fc polypeptide.

In some embodiments, the anti-c-met antibody fragment of the pharmaceutical formulation described herein comprises an antigen binding site of the bivalent antibody and thus retains the ability to bind antigen. In some embodiments, the anti-c-met antibody fragment comprises the Fc region and retains at least one of the biological functions normally associated with the Fc region when present in an bivalent antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding. In some embodiments, the anti-c-met antibody fragment does not have ADCC function and/or complement binding activity. In some embodiments, the anti-c-met antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to a bivalent antibody. For example, such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment. In some embodiments, an Fc polypeptide comprises part or all of a wild type hinge sequence (generally at its N terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild type hinge sequence. In some embodiments, the anti-c-met antibody fragment is a one-armed antibody as described in WO2005/063816. In some embodiments, the one-armed antibody comprises Fc mutations constituting “knobs” and “holes” as described in WO2005/063816; Ridgeway, J et al, Prot Eng (1996) 9:617-21; and Zhu Z et al. Prot Sci (1997) 6:781-8. In some embodiments, the Fc region comprises at least one protuberance (knob) and at least one cavity (hole), wherein presence of the protuberance and cavity enhances formation of a complex between an Fc polypeptide comprising the protuberance and an Fc polypeptide comprising the cavity, for example as described in WO 2005/063816. In some embodiments, the Fc region of the anti-c-met antibodies comprises a first and a second Fc polypeptide, wherein the first and second polypeptide each comprises one or more mutations with respect to wild type human Fc. In some embodiments, a cavity mutation is T366S, L368A and/or Y407V. In some embodiments, a protuberance mutation is T366W. In some embodiments, the first polypeptide comprises the Fc sequence depicted in FIG. 2 and the second polypeptide comprises the Fc sequence depicted in FIG. 3. In some embodiments, the anti-c-met antibody may comprise at least one characteristic that promotes heterodimerization, while minimizing homodimerization, of the Fc sequences within the antibody fragment.

In some embodiments of any of the anti-c-met antibodies described herein, the anti-c-met antibody is an antagonist anti-c-met antibody. In some embodiments, blocking anti-c-met antibodies or antagonist anti-c-met antibodies completely inhibit the biological activity of the antigen. For treatment of pathological conditions requiring an antagonistic function and where bivalency of an anti-c-met antibody results in an undesirable agonistic effect upon binding to a target antigen (even though it is an antagonistic anti-c-met antibody as a Fab fragment), the monovalent trait of a one-armed antibody (i.e., an antibody comprising a single antigen binding arm) results in and/or ensures an antagonistic function upon binding of the anti-c-met antibody to a target molecule. Furthermore, the one-armed antibody comprising a Fc region is characterized by superior pharmacokinetic attributes (such as an enhanced half life and/or reduced clearance rate in vivo) compared to Fab forms having similar/substantially identical antigen binding characteristics, thus overcoming a major drawback in the use of conventional monovalent Fab antibodies.

Anti-c-met antibodies (which may be provided as one-armed antibodies) useful in the pharmaceutical formulation described herein include those known in the art (see, e.g., Martens, T. et al., Clin. Cancer Res. 12 (20 Pt. 1):6144 (2006); U.S. Pat. No. 6,468,529; WO2006/015371; WO2007/063816, and WO2010/045345, which are incorporated by reference in their entirety). In some embodiments, the anti-c-met antibody for use in the pharmaceutical formulations described herein comprises one or more of the HVR sequences of the monoclonal antibody produced by the hybridoma cell line deposited under American Type Culture Collection (ATCC) Accession Number ATCC HB-11894 (hybridoma 1A3.3.13) or HB-11895 (hybridoma 5D5.11.6). In some embodiments, the anti-c-met antibody is a one-armed antibody comprising one or more of the HVRs of the light chain variable domain and/or one or more of the HVRs of the heavy chain variable domain of ATCC Accession Number ATCC HB-11894 (hybridoma 1A3.3.13) or HB-11895 (hybridoma 5D5.11.6) and an Fc polypeptide.

In some embodiments of the anti-c-met antibody pharmaceutical formulation, the anti-c-met antibody comprises a light chain variable domain comprising one or more of HVR1-LC, HVR2-LC and HVR3-LC sequence depicted in FIG. 2 (SEQ ID NOs:1-3). In some embodiments, the anti-c-met antibody comprises a heavy chain variable domain comprising one or more of HVR1-HC, HVR2-HC and HVR3-HC sequence depicted in FIG. 2 (SEQ ID NOs:4-6). In some embodiments, the anti-c-met antibody comprises a light chain variable domain comprising one or more of HVR1-LC, HVR2-LC and HVR3-LC sequence depicted in FIG. 2 (SEQ ID NOs:1-3) and one or more of HVR1-HC, HVR2-HC and HVR3-HC sequence depicted in FIG. 2 (SEQ ID NOs:4-6). In some embodiments, the heavy chain variable domain comprises one or more of HVR1-HC, HVR2-HC and HVR3-HC sequence depicted in FIG. 2 (SEQ ID NOs:4-6) and one or more of FR1-HC, FR2-HC, FR3-HC and FR4-HC sequence depicted in FIG. 2 (SEQ ID NOs:11-14). In some embodiments, the light chain variable domain comprises one or more of HVR1-LC, HVR2-LC and HVR3-LC sequence depicted in FIG. 2 (SEQ ID NOs:1-3) and one or more of FR1-LC, FR2-LC, FR3-LC and FR4-LC sequence depicted in FIG. 2 (SEQ ID NOs:7-10). In some embodiments, the anti-c-met antibody is a one-armed antibody comprising one or more of the HVRs of the light chain variable domain (SEQ ID NOs:1-3) and/or one or more of the HVRs of the heavy chain variable domain (SEQ ID NOs:4-6) and an Fc polypeptide.

In some embodiments of the anti-c-met antibody pharmaceutical formulation, the anti-c-met antibody comprises: (a) at least one, two, three, four, or five HVR sequences selected from the group consisting of: (i) HVR-L1 comprising sequence A1-A17, wherein A1-A17 is KSSQSLLYTSSQKNYLA (SEQ ID NO:23) (ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is WASTRES (SEQ ID NO:24); (iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is QQYYAYPWT (SEQ ID NO:25); (iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GYTFTSYWLH (SEQ ID NO:26); (v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is GMIDPSNSDTRFNPNFKD (SEQ ID NO:27); and (vi) HVR-H3 comprising sequence F1-F11, wherein F1-F11 is XYGSYVSPLDY (SEQ ID NO:28) and X is not R; and (b) at least one variant HVR, wherein the variant HVR sequence comprises modification of at least one residue of the sequence depicted in SEQ ID NOs:23, 24, 25, 26, 27, or 28. In some embodiments, HVR-L1 of the anti-c-met antibody comprises the sequence of SEQ ID NO:23. In some embodiments, HVR-L2 comprises the sequence of SEQ ID NO:24. In some embodiments, HVR-L3 comprises the sequence of SEQ ID NO:25. In some embodiments, HVR-H1 comprises the sequence of SEQ ID NO:26. In some embodiments, HVR-H2 comprises the sequence of SEQ ID NO:27. In some embodiments, HVR-H3 the sequence of SEQ ID NO:28. In some embodiments, HVR-H3 comprises TYGSYVSPLDY (SEQ ID NO: 29). In some embodiments, HVR-H3 comprises SYGSYVSPLDY (SEQ ID NO:30). In some embodiments, the anti-c-met antibody comprising these sequences (in combination as described herein) is humanized or human. In some embodiments, the anti-c-met antibody is a one-armed antibody comprising one or more of the HVRs of the light chain variable domain (SEQ ID NOs:23-25) and/or one or more of the HVRs of the heavy chain variable domain (SEQ ID NOs:26-30) and an Fc polypeptide.

Provided herein are also anti-c-met antibodies for use in the pharmaceutical formulation comprising one, two, three, four, five or six HVRs, wherein each HVR comprises, consists or consists essentially of a sequence selected from the group consisting of SEQ ID NOs:23, 24, 25, 26, 27, 28, and 29, and wherein SEQ ID NO:23 corresponds to an HVR-L1, SEQ ID NO:24 corresponds to an HVR-L2, SEQ ID NO:25 corresponds to an HVR-L3, SEQ ID NO:26 corresponds to an HVR-H1, SEQ ID NO:27 corresponds to an HVR-H2, and SEQ ID NOs:26, 27, or 28 corresponds to an HVR-H3. In some embodiments, the anti-c-met antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NOs:23, 24, 25, 26, 27 and 29. In some embodiments, the anti-c-met antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NOs:23, 24, 25, 26, 27 and 30.

Variant HVRs can have modifications of one or more residues within the HVR. In some embodiments, a HVR-L2 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combination of the following positions: B1 (M or L), B2 (P, T, G or S), B3 (N, G, R or T), B4 (I, N or F), B5 (P, I, L or G), B6 (A, D, T or V) and B7 (R, I, M or G). In some embodiments, a HVR-H1 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combination of the following positions: D3 (N, P, L, S, A, I), D5 (I, S or Y), D6 (G, D, T, K, R), D7 (F, H, R, S, T or V) and D9 (M or V). In some embodiments, a HVR-H2 variant comprises 1-4 (1, 2, 3 or 4) substitutions in any combination of the following positions: E7 (Y), E9 (I), E 10 (I), E14 (T or Q), E15 (D, K, S, T or V), E16 (L), E17 (E, H, N or D) and E18 (Y, E or H). In some embodiments, a HVR-H3 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combination of the following positions: F1 (T, S), F3 (R, S, H, T, A, K), F4 (G), F6 (R, F, M, T, E, K, A, L, W), F7 (L, I, T, R, K, V), F8 (S, A), F10 (Y, N) and F11 (Q, S, H, F). Letter(s) in parenthesis following each position indicates an illustrative substitution (i.e., replacement) amino acid; as would be evident to one skilled in the art, suitability of other amino acids as substitution amino acids in the context described herein can be routinely assessed using techniques known in the art and/or described herein. In some embodiments, a HVR-L1 comprises the sequence of SEQ ID NO:23. In some embodiments, F1 in a variant HVR-H3 is T. In some embodiments, F1 in a variant HVR-H3 is S. In some embodiments, F3 in a variant HVR-H3 is R. In some embodiments, F3 in a variant HVR-H3 is S. In some embodiments, F7 in a variant HVR-H3 is T. In some embodiments, the anti-c-met antibody comprises a variant HVR-H3 wherein F1 is T or S, F3 is R or S, and F7 is T.

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation comprises a variant HVR-H3 wherein F1 is T, F3 is R and F7 is T. In some embodiments, the anti-c-met antibody comprises a variant HVR-H3 wherein F1 is S. In some embodiments, the anti-c-met antibody comprises a variant HVR-H3 wherein F1 is T, and F3 is R. In some embodiments, the anti-c-met antibody comprises a variant HVR-H3 wherein F1 is S, F3 is R and F7 is T. In some embodiments, the anti-c-met antibody comprises a variant HVR-H3 wherein F1 is T, F3 is S, F7 is T, and F8 is S. In some embodiments, the anti-c-met antibody comprises a variant HVR-H3 wherein F1 is T, F3 is S, F7 is T, and F8 is A. In some embodiments, said variant HVR-H3 antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H2 wherein each comprises, in order, the sequence depicted in SEQ ID NOs:1, 2, 3, 4 and 5. In some embodiments, these antibodies further comprise a human subgroup III heavy chain framework consensus sequence. In some embodiments of these antibodies, the framework consensus sequence comprises substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or 78 is A. In some embodiments of these antibodies, these antibodies further comprise a human κI light chain framework consensus sequence.

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation comprises a variant HVR-L2 wherein B6 is V. In some embodiments, said variant HVR-L2 anti-c-met antibody further comprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each comprises, in order, the sequence depicted in SEQ ID NOs:23, 25, 26, 27 and 28. In some embodiments, said variant HVR-L2 anti-c-met antibody further comprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each comprises, in order, the sequence depicted in SEQ ID NOs:23, 25, 26, 27 and 29. In some embodiments, said variant HVR-L2 anti-c-met antibody further comprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each comprises, in order, the sequence depicted in SEQ ID NOs:23, 25, 26, 27 and 30. In some embodiments, these anti-c-met antibodies further comprise a human subgroup III heavy chain framework consensus sequence. In some embodiments of these anti-c-met antibodies, the framework consensus sequence comprises substitution at position 71, 73 and/or 78. In some embodiments of these anti-c-met antibodies, position 71 is A, 73 is T and/or 78 is A. In some embodiments of these anti-c-met antibodies, these antibodies further comprise a human κI light chain framework consensus sequence.

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation comprises a variant HVR-H2 wherein E14 is T, E15 is K and E17 is E. In some embodiments, the anti-c-met antibody comprises a variant HVR-H2 wherein E17 is E. In some embodiments, said variant HVR-H3 anti-c-met antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, and HVR-H3 wherein each comprises, in order, the sequence depicted in SEQ ID NOs:23, 24, 25, 26, and 28. In some embodiments, said variant HVR-H2 anti-c-met antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, and HVR-H3, wherein each comprises, in order, the sequence depicted in SEQ ID NOs:23, 24, 25, 26, and 29. In some embodiments, said variant HVR-H2 anti-c-met antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, and HVR-H3, wherein each comprises, in order, the sequence depicted in SEQ ID NOs:23, 24, 25, 26 and 30. In some embodiments, these anti-c-met antibodies further comprise a human subgroup III heavy chain framework consensus sequence. In some embodiments of these anti-c-met antibodies, the framework consensus sequence comprises substitution at position 71, 73 and/or 78. In some embodiments of these anti-c-met antibodies, position 71 is A, 73 is T and/or 78 is A. In some embodiments of these antibodies, these anti-c-met antibodies further comprise a human κI light chain framework consensus sequence.

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation comprises (a) a heavy chain variable domain comprising the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTR FNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVS S (SEQ ID NO:19) and/or (b) a light chain variable domain comprising the sequence: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTR ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO:20). In some embodiments, the anti-c-met antibody is a one-armed antibody comprising (a) the light chain variable domain (SEQ ID NO:20) and/or (b) the heavy chain variable domain (SEQ ID NO:19) and (c) a Fc polypeptide.

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation comprises (a) HVR-H1, HVR-H2, and HVR-H3 of a heavy chain variable domain comprising the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTR FNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVS S (SEQ ID NO:19) and/or (b) HVR-L1, HVR-L2, and HVR-L3 of a light chain variable domain comprising the sequence: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTR ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO:20). In some embodiments, the anti-c-met antibody is a one-armed antibody comprising (a) the light chain variable domain (SEQ ID NO:20) and/or (b) the heavy chain variable domain (SEQ ID NO:19) and (c) a Fc polypeptide.

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation is an anti-c-met antibody fragment, wherein the antibody fragment comprises (a) a first polypeptide comprising a heavy chain variable domain comprising SEQ ID NO:19, CH1 sequence (e.g., SEQ ID NO:16), and a first Fc polypeptide; and (b) a second polypeptide comprising a light chain variable domain comprising SEQ ID NO:20, and CL1 sequence (e.g., SEQ ID NO:15).

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation is an anti-c-met antibody fragment, wherein the antibody fragment comprises (a) a first polypeptide comprising a heavy chain variable domain comprising SEQ ID NO:19, CH1 sequence (e.g., SEQ ID NO:16), and a first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain comprising SEQ ID NO:20, and CL1 sequence (e.g., SEQ ID NO:15); and (c) a third polypeptide comprising a second Fc polypeptide, wherein the heavy chain variable domain and the light chain variable domain are present as a complex and form a single antigen binding arm and wherein the first and second Fc polypeptides are present in a complex. In some embodiments, the first and second Fc polypeptides form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. In some embodiments, the Fc region is that of a human IgG (e.g., IgG1, 2, 3 or 4). In some embodiments, the first Fc polypeptide comprises the Fc sequence depicted in FIG. 2 (SEQ ID NO:17) and the second Fc polypeptide comprises the Fc sequence depicted in FIG. 3 (SEQ ID NO:18). In some embodiments, the first Fc polypeptide comprises the Fc sequence depicted in FIG. 3 (SEQ ID NO:18) and the second Fc polypeptide comprises the Fc sequence depicted in FIG. 2 (SEQ ID NO:17).

In some embodiments, the anti-c-met antibody is an anti-c-met antibody or antibody fragment thereof, wherein the antibody comprises (a) a first polypeptide comprising a heavy chain variable domain comprising SEQ ID NO:19, CH1 sequence, and a first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain comprising SEQ ID NO:20, and CL1 sequence; and (c) a third polypeptide comprising a second Fc polypeptide, wherein the heavy chain variable domain and the light chain variable domain are present as a complex and form a single antigen binding arm, wherein the first and second Fc polypeptides are present in a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm.

In some embodiments, the anti-c-met antibody comprises (a) a first polypeptide comprising a heavy chain variable domain, said polypeptide comprising the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFT SYWLHWVRQAPGKGLEWVGMIDPSNS FNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:21); (b) a second polypeptide comprising a light chain variable domain, the polypeptide comprising the sequence DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRE SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:22); and a third polypeptide comprising a Fc sequence, the polypeptide comprising the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:18), wherein the heavy chain variable domain and the light chain variable domain are present as a complex and form a single antigen binding arm and wherein the first and second Fc polypeptides are present in a complex. In some embodiments, the first and second Fc polypeptides form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm.

In some embodiments, polynucleotides encoding any of the anti-c-met antibodies described herein are expressed such that the anti-c-met antibody is produced. In some embodiments, polynucleotides encoding any of the anti-c-met antibody are expressed in vitro or in vivo (for example, in CHO cells or E. coli cells).

In some embodiments, the anti-c-met antibody for use in the pharmaceutical formulation described herein is onartuzumab (interchangeably termed MetMAb), a one-armed antibody comprising a Fc region. A sequence of MetMAb is shown in FIGS. 2 and 3. MetMAb (also termed OA5D5v2 and onartuzumab) is also described in, e.g., WO2006/015371; WO2010/04345; and Jin et al, Cancer Res (2008) 68:4360. Biosimilar version of MetMAb are also contemplated and encompassed herein for use in the formulation.

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation specifically binds at least a portion of c-met Sema domain or variant thereof. In some embodiments, the anti-c-met antibody is an antagonist. In some embodiments, the anti-c-met antagonist antibody specifically binds at least one of the sequences selected from the group consisting of LDAQT (SEQ ID NO:31) (e.g., residues 269-273 of c-met), LTEKRKKRS (SEQ ID NO:32) (e.g., residues 300-308 of c-met), KPDSAEPM (SEQ ID NO: 33) (e.g., residues 350-357 of c-met) and NVRCLQHF (SEQ ID NO:34) (e.g., residues 381-388 of c-met). In some embodiments, the anti-c-met antagonist antibody specifically binds a conformational epitope formed by part or all of at least one of the sequences selected from the group consisting of LDAQT (SEQ ID NO:31) (e.g., residues 269-273 of c-met), LTEKRKKRS (SEQ ID NO:32) (e.g., residues 300-308 of c-met), KPDSAEPM (SEQ ID NO: 33) (e.g., residues 350-357 of c-met) and NVRCLQHF (SEQ ID NO:34) (e.g., residues 381-388 of c-met). In some embodiments, an antagonist antibody specifically binds an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98% sequence identity or similarity with the sequence LDAQT (SEQ ID NO:31), LTEKRKKRS (SEQ ID NO:32), KPDSAEPM (SEQ ID NO:33) and/or NVRCLQHF (SEQ ID NO:34). In some embodiments, the anti-c-met antibody is an antagonist anti-c-met antibody. In some embodiments, the anti-c-met antibody is a one-armed antibody. In order to screen for antibodies which bind to an epitope on an antigen bound by an antibody of interest, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed.

In some embodiments of any of the anti-c-met antibodies described herein, the anti-c-met antibody may interfere with HGF/c-met activation, including but not limited to interfering with HGF binding to the extracellular portion of c-met and receptor multimerization. In some embodiments, the anti-c-met antibody are useful in treating or diagnosing pathological conditions associated with abnormal or unwanted signaling of the HGF/c-met pathway. In some embodiments, the anti-c-met antibody may modulate the HGF/c-met pathway, including modulation of c-met ligand binding, c-met dimerization, activation, and other biological/physiological activities associated with HGF/c-met signaling. In some embodiments, the anti-c-met antibody may disrupt HGF/c-met signaling pathway. In some embodiments of any of the anti-c-met antibodies described herein, binding of the anti-c-met antibody to c-met inhibits c-met activation by HGF. In some embodiments of any of the anti-c-met antibodies described herein, binding of the anti-c-met antibody to c-met in a cell inhibits proliferation, survival, scattering, morphogenesis and/or motility of the cell.

In some instances, it may be advantageous to have an anti-c-met antibody that does not interfere with binding of a ligand (such as HGF) to c-met. Accordingly, in some embodiments, the anti-c-met antibody does not bind an HGF binding site on c-met. In some embodiment, the anti-c-met antibody does not substantially inhibit HGF binding to c-met. In some embodiments, the anti-c-met antibody does not substantially compete with HGF for binding to c-met. In one example, the anti-c-met antibody can be used in conjunction with one or more other antagonists, wherein the antagonists are targeted at different processes and/or functions within the HGF/c-met axis. Thus, in some embodiments, the anti-c-met antibody binds to an epitope on c-met distinct from an epitope bound by another c-met antagonist (such as the Fab fragment of the monoclonal antibody produced by the hybridoma cell line deposited under American Type Culture Collection Accession Number ATCC HB-11894 (hybridoma 1A3.3.13)). In another embodiment, the anti-c-met antibody is distinct from (i.e., it is not) a Fab fragment of the monoclonal antibody produced by the hybridoma cell line deposited under American Type Culture Collection Accession Number ATCC HB-11894 (hybridoma 1A3.3.13).

In some embodiments, the anti-c-met antibody binds to c-met of a first animal species, and does not specifically bind to c-met of a second animal species. In some embodiments, the first animal species is human and/or primate (e.g., cynomolgus monkey), and the second animal species is murine (e.g., mouse) and/or canine. In some embodiments, the first animal species is human. In some embodiments, the first animal species is primate, for example cynomolgus monkey. In some embodiments, the second animal species is murine, for example mouse. In some embodiments, the second animal species is canine.

In some embodiments, the anti-c-met antibody elicits little to no immunogenic response in said subject. In some embodiments, the anti-c-met antibody elicits an immunogenic response at or less than a clinically-acceptable level.

In some embodiments of any of the anti-c-met antibodies described herein, an altered antibody that possesses some but not all effector functions. In some embodiments, the anti-c-met antibody does not possess complement depletion and/or ADCC activity. In some embodiments, the Fc activities of the produced immunoglobulin are measured to ensure that only the desired properties are maintained (e.g., half-life but not complement depletion and/or ADCC activity). In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). An example of an in vitro assay to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art. In some embodiments, the anti-c-met antibody is glycosylated. In some embodiments, the anti-c-met antibody is substantially aglycosylated.

The anti-c-met antibodies of the formulations described herein can be characterized for their physical/chemical properties and biological functions by various assays known in the art. The purified anti-c-met antibodies can be further 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 some embodiments of any of the anti-c-met antibodies described herein, the anti-c-met antibody may be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue, or silver stain.

Further, in some embodiments, the anti-c-met antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-8 below:

1. Antibody Affinity

In some embodiments, the anti-c-met antibody provided herein has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g. 10−8M or less, e.g. from 10−8M to 10−13 M, e.g., from 10−9M to 10−13 M).

Binding affinity of a ligand to its receptor can be determined using any of a variety of assays, and expressed in terms of a variety of quantitative values. Antigen binding assays are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, enzyme-linked immunoabsorbent assay (ELISA), “sandwich” immunoassays, surface plasmon resonance based assay (such as the BIAcore assay as described in PCT Application Publication No. WO2005/012359), immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays.

Accordingly, in some embodiments, the binding affinity is expressed as Kd values and reflects intrinsic binding affinity (e.g., with minimized avidity effects). The anti-c-met antibody selected will normally have a sufficiently strong binding affinity for c-met, for example, the antibody may bind human c-met with a Kd value of between 100 nM−1 pM.

2. Antibody Fragments

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation described herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH, F(ab′)2, Fv, one-armed antibodies, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Other monovalent antibody forms are described in, e.g., WO2007048037, WO2008145137, WO2008145138, and WO2007059782. One-armed antibodies are described, e.g., in WO2005/063816. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

3. Chimeric and Humanized Antibodies

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation described herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat\'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-HVR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall\'Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

4. Human Antibodies

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation described herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal\'s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELocIMousE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., 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 Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-Derived Antibodies

The anti-c-met antibody of the pharmaceutical formulation described herein may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O\'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In some phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In some embodiments, the anti-c-met antibody of the pharmaceutical formulation described herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In some embodiments, one of the binding specificities is for an antigen and the other is for any other antigen. In some embodiments, bispecific antibodies may bind to two different epitopes of an antigen. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express an antigen. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to c-met as well as another, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In some embodiments, amino acid sequence variants of the anti-c-met antibody for use in the pharmaceutical formulation described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

a. Substitution, Insertion, and Deletion Variants

In some embodiments, anti-c-met antibody variants having one or more amino acid substitutions for use in the pharmaceutical formulation described herein are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of “conservative substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N)

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