GPC3-specific antibodies and methods of using same

Abstract

The present disclosure provides antibodies specific to glypican-3 (GPC3). Nucleic acids encoding one or both variable chain polypeptides of the antibodies of the present disclosure are also provided, as are cells containing such nucleic acids. Compositions, including in some instances pharmaceutical compositions, containing the antibodies of the present disclosure are also provided. Methods of making and using the antibodies of the present disclosure are also provided. In certain aspects, methods are provided that include administering a therapeutically effective amount of an antibody of the present disclosure to an individual with a cell proliferative disorder, where the antibody is administered to the individual to enhance an immune response (e.g., a T cell response) against abnormally proliferating cells of the cell proliferative disorder. The antibodies of the present invention are useful in a variety of diagnostic and monitoring applications, as also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 881,547, filed August 1, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporating a sequence listing The sequence listing (file name ''RDWD-024WO Seq Listing_ST25'', created on July 29, 2020, size: 33KB) is incorporated herein by reference in its entirety. [Background technology]

[0003] Introduction Glypican-3 (GPC3) is a heparan sulfate proteoglycan expressed on the surface of many types of malignant cells, such as hepatocellular carcinoma (HCC) cells. Glypican-3 is linked to the cell surface via a glycosyl-phosphatidylinositol (GPI) anchor. GPC3 has been shown to be highly expressed in over 70% of HCC biopsies, but not in adjacent non-tumor tissues. Patients with GPC3-positive HCC have significantly lower disease-free survival rates than those with GPC3-negative HCC.

[0004] There is a need in the art for safe and effective agents that target GPC3 for the diagnosis and treatment of various GPC3-associated conditions, such as cancer. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure provides an antibody specific to GPC3. Nucleic acids encoding one or both variable chain polypeptides of the antibody of the present disclosure are also provided, as are cells containing such nucleic acids. Compositions, including pharmaceutical compositions in some instances, containing the antibody of the present disclosure are also provided. Methods of making and using the antibody of the present disclosure are also provided. In certain aspects, a method is provided comprising administering a therapeutically effective amount of an antibody of the present disclosure to an individual with a cell proliferative disorder, wherein the antibody is administered to the individual to enhance an immune response (e.g., a T cell response) against abnormally proliferating cells of the cell proliferative disorder. The antibodies of the present invention are useful in a variety of diagnostic and monitoring applications, which are also provided. [Brief explanation of the drawings]

[0006] [Figure 1] The CAT-07 monoclonal antibody is greater than 99% monomeric as determined by size exclusion chromatography (SEC). [Figure 2] The CAT-07 monoclonal antibody is shown to bind to recombinant human glypican-3 protein as assessed by ELISA. [Figure 3] The CAT-07 monoclonal antibody is shown to bind to glypican-3, but not to other human glypican proteins, as assessed by ELISA. [Figure 4] 1 provides flow cytometry data showing that the CAT-07 monoclonal antibody binds to glypican-3 protein from cynomolgus monkey, rat, and mouse. DETAILED DESCRIPTION OF THE INVENTION

[0007] definition The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), intact antibodies (e.g., antibodies composed of a tetramer composed of two dimers of a heavy chain polypeptide and a light chain polypeptide), single-chain antibodies (e.g., scFv), antibody fragments (e.g., fragments of intact antibodies or single-chain antibodies) that retain specific binding to an antigen (including, but not limited to, Fab, Fv, scFv, and Fd fragments), chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. Antibodies may be detectably labeled, for example, with radioisotopes, enzymes that generate a detectable product, fluorescent proteins, and the like. Antibodies may further be conjugated to other moieties, such as members of specific binding pairs, such as biotin (a member of the biotin-avidin specific binding pair) and similar members. Antibodies may also be bound to solid supports, including, but not limited to, polystyrene plates or beads and similar solid supports. Also encompassed by this term are Fab', Fv, F(ab')2 and / or other antibody fragments that retain specific binding to the antigen, as well as monoclonal antibodies. Antibodies may be monovalent or bivalent.

[0008] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062(1995)), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment (a name reflecting its ease of crystallization). Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and still has the ability to cross-link antigen.

[0009] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. The three CDRS of each variable domain interact to form the antigen-binding site. H -V L It is in this configuration that the six CDRs define the surface of the dimer. Together, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0010] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0011] The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of its constant domain. Immunoglobulins can be assigned to a variety of different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes): e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0012] "Single-chain Fv" or "sFv" antibody fragments are fragments of the V chain of an antibody. H Domains and V L In some aspects, the Fv polypeptide further comprises a polypeptide linker which enables the sFv to form the desired structure for antigen binding. H Domains and V LFor a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0013] The term "diabody" refers to a group of molecules that share the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) to a heavy chain variable domain (V H (Figure 1 illustrates a small antibody fragment having two antigen-binding sites, each containing a linker that is too short to allow pairing between these two domains on the same chain, forcing these domains to pair with complementary domains on another chain, resulting in two antigen-binding sites. Various diabodies are described in more detail, for example, in EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0014] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, expressed as the dissociation constant (Kd). The affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of the antibody for an unrelated amino acid sequence. The affinity of an antibody for a target protein can be, for example, between about 100 nanomolar (nM) and about 0.1 nM, between about 100 nM and about 1 picomolar (pM), or between about 100 nM and about 1 femtomolar (fM), or greater. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0015] The term "binding" refers to a direct association between two molecules due to ionic and / or hydrogen-bond interactions, including various interactions such as covalent interactions, electrostatic interactions, hydrophobic interactions, and salt and water bridges. The subject anti-GPC3 antibodies specifically bind to an epitope within a GPC3 polypeptide. Non-specific binding occurs at approximately 10 -7 Binding with an affinity less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 It will exhibit binding with an affinity such as M.

[0016] The term "specifically binds" in the context of antibodies and antigens means that the antibody specifically binds to the antigen, e.g., at a concentration of about 10 5 M-1 or greater affinity or K a (i.e., the equilibrium association constant of a particular binding interaction in units of 1 / M) or associates with the antigen.

[0017] "High affinity" binding is defined as K a is at least 10 7 M -1 is at least 10 8 M -1 is at least 10 9 M -1 is at least 10 10 M -1 is at least 10 11 M -1 is at least 10 12 M -1 is at least 10 13 M -1 Alternatively, affinity can be expressed as the equilibrium dissociation constant (K) of a particular binding interaction in units of M. D ) (e.g., 10 -5 M~10 -13 In some embodiments, specific binding occurs when the antibody binds to a target molecule at a concentration of about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, or about 10 -9 M or less, 10 -10 M, 10 -11 M or 10 -12 K below M D , or a smaller K D The binding affinity of an antibody for a given antigen can be readily determined using conventional techniques, such as by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., a BIAcore2000 instrument using the general procedures outlined by the manufacturer), or by radioimmunoassay, etc.

[0018] As used herein, the term "CDR" or "complementarity-determining region" is intended to mean the noncontiguous antigen-binding sites found in the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where these definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDRs of a given antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues that encompass the CDRs as defined by each of the above-cited references are set forth below in Table 1 for comparison. [Table 1]

[0019] Throughout this disclosure, the numbering of residues in immunoglobulin heavy and light chains is that in Kabat et al., Sequences of Proteins of Immunological Interest (5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is specifically incorporated herein by reference).

[0020] As used herein, the term "framework," when used in reference to an antibody variable region, is intended to refer to all amino acid residues outside the CDR regions within the variable region of the antibody. Variable region frameworks are generally discontinuous amino acid sequences between about 100 and 120 amino acids in length, but are intended to refer only to those amino acids outside the CDRs. As used herein, the term "framework region" is intended to refer to each domain of the framework separated by the CDRs.

[0021] An "original Ig polypeptide" is a polypeptide comprising an amino acid sequence lacking an aldehyde-tagged constant region as described herein. The original polypeptide may comprise a native-sequence constant region or may comprise a constant region with pre-existing amino acid sequence modifications (e.g., additions, deletions, and / or substitutions).

[0022] In the context of Ig polypeptides, the term "constant region" is well understood in the art and refers to the C-terminal region of an Ig heavy chain or an Ig light chain. The Ig heavy chain constant region comprises CH1, CH2, and CH3 domains (and a CH4 domain if the heavy chain is a μ or ε heavy chain). In native Ig heavy chains, the CH1, CH2, CH3 (and CH4, if present) domains begin immediately (C-terminally) after the heavy chain variable (VH) region, and each domain is about 100 to 130 amino acids in length. In native Ig light chains, the constant region begins immediately (C-terminally) after the light chain variable (VL) region and is about 100 to 120 amino acids in length.

[0023] An "epitope" is a site on an antigen surface (e.g., a site on the surface of GPC3) to which an antibody binds. Epitopes can be formed from both contiguous or noncontiguous amino acids juxtaposed by protein folding (e.g., tertiary folding). Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three amino acids, more usually at least five or eight to ten amino acids, in a linear or spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996). Several commercial laboratories offer epitope mapping services. Epitopes to which antibodies immunoreactive with membrane-associated antigens bind may be present on the surface of a cell (e.g., in the extracellular region of a transmembrane protein); consequently, such epitopes are considered to be cell surface accessible, solvent accessible, and / or cell surface exposed.

[0024] By "genetically encodable" as used in reference to an amino acid sequence of a polypeptide, peptide, or protein, it is meant that the amino acid sequence is composed of amino acid residues that can be produced by transcription and translation of a nucleic acid encoding the amino acid sequence, where transcription and / or translation can occur in a cell or in a cell-free in vitro transcription / translation system.

[0025] The term "control sequence" refers to DNA sequences that promote the expression of an operably linked coding sequence in a particular expression system, such as mammalian cells, bacterial cells, cell-free synthesis, etc. Control sequences that are suitable for prokaryotic systems include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. In eukaryotic systems, promoters, polyadenylation signals, and enhancers may be utilized.

[0026] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. Alternatively, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Alternatively, a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation initiation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Linking is accomplished by ligation or via an amplification reaction. Synthetic oligonucleotide adapters or linkers may be used to link sequences in accordance with conventional practice.

[0027] The term "expression cassette," as used herein, refers to a segment of nucleic acid (usually DNA) that can be inserted into a nucleic acid (e.g., by use of restriction sites compatible with nucleic acid ligation into a target construct, or by homologous recombination into a target construct or into a host cell genome). Generally, such a nucleic acid segment comprises a polynucleotide encoding a polypeptide of interest, and the cassette and restriction sites are designed to facilitate insertion of the cassette in the proper reading frame for transcription and translation. An expression cassette can also include elements that promote expression of a polynucleotide encoding a polypeptide of interest in a host cell, e.g., in a mammalian host cell. Such elements can include, but are not limited to, a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and similar sequences.

[0028] An "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment include substances that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody will be purified (1) to greater than 90%, 95%, or 98% by weight of the antibody, e.g., greater than 99% by weight, as determined by the Lowry method; (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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will be absent. In some instances, isolated antibody will be prepared by at least one purification step.

[0029] The term "natural antibody" refers to an antibody in which the heavy and light chains of the antibody are produced and paired by the immune system of a multicellular organism. Examples of tissues that produce natural antibodies include the spleen, lymph nodes, bone marrow, and serum. For example, an antibody produced by antibody-producing cells isolated from a first animal immunized with an antigen is a natural antibody.

[0030] The term "humanized antibody" or "humanized immunoglobulin" refers to a non-human antibody (e.g., a mouse or rabbit antibody) that contains one or more amino acids (e.g., in a framework region, constant region, or CDR) substituted with an amino acid at the corresponding position from a human antibody. Generally, a humanized antibody results in a reduced immune response in a human host when compared to a non-humanized version of the same antibody. Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT Publication No. 91 / 09967; U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering, 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In certain embodiments, various framework substitutions are identified by modeling the interactions between CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies contemplated for use in the present invention are disclosed in U.S. Patent Nos. 5,750,078, 5,502,167, 5,705,154, 5,770,403, 5,698,417, 5,693,493, 5,558,864, 4,935,496 and 4,816,567, and PCT Publication Nos. 98 / 45331 and 98 / 45332.In certain embodiments, the subject rabbit antibodies may be humanized according to the methods set forth in U.S. Patent Application Publication Nos. 20040086979 and 20050033031. Accordingly, the antibodies described above may be humanized using a variety of methods well known in the art.

[0031] The term "chimeric antibody" refers to an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from variable and constant region genes of antibodies belonging to different species. For example, variable segments from genes derived from a mouse monoclonal antibody may be linked to human constant segments (e.g., gamma 1 and gamma 3). An example of a therapeutic chimeric antibody is a hybrid protein composed of variable or antigen-binding domains derived from a mouse antibody and constant or effector domains derived from a human antibody, although domains from other mammalian species may also be used.

[0032] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length. Unless specifically indicated otherwise, "polypeptide," "peptide," and "protein" can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The terms also include various fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and cognate leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant host cells); immunologically tagged proteins; and similar fusion proteins.

[0033] "Native amino acid sequence" or "originating amino acid sequence" are used interchangeably herein to refer to the amino acid sequence of a polypeptide before it is modified to include an altered amino acid residue.

[0034] The terms "amino acid analog," "artificial amino acid," and similar terms may be used interchangeably and include amino acid-like compounds similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as L-form amino acid analogs. In some instances, amino acid analogs share the backbone structure and / or side chain structure of one or more natural amino acids, but the difference(s) is one or more modified groups in the molecule. Such modifications may include, but are not limited to, substituting an atom (e.g., N) for a related atom (e.g., S), adding a group (e.g., methyl or hydroxyl) or atom (e.g., Cl or Br), deleting a group, substituting a covalent bond (e.g., a single bond instead of a double bond), or a combination thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids.

[0035] The term "amino acid side chain" or "side chain of an amino acid" and similar terms may be used to refer to a substituent attached to the α-carbon of an amino acid residue, including natural amino acids, artificial amino acids, and amino acid analogs. Amino acid side chains can also include amino acid side chains as described in connection with the modified amino acids and / or conjugates described herein.

[0036] The term "conjugated" generally refers to a chemical linkage (either covalent or non-covalent, usually covalent) that proximally associates one molecule of interest with a second molecule of interest. In some embodiments, the agent is selected from a half-life extending moiety, a labeling agent, and a therapeutic agent. For half-life extension, for example, it is possible that antibodies of the present disclosure may be modified to confer an improved pharmacokinetic profile (e.g., by PEGylation and hyperglycosylation, etc.). Modifications that can increase serum half-life are of interest.

[0037] The term "carbohydrate" and similar terms may be used to refer to monomeric units and / or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to smaller carbohydrates, such as monosaccharides, disaccharides, etc. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemical reaction), or absent (e.g., eliminated or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and may be adapted for use in the subject compounds and conjugates.

[0038] As used herein, the terms "treatment," "treating," and similar terms refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or may be therapeutic, in that a disease and / or adverse effects that may result from the disease are partially or completely cured. "Treatment," as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease, (b) inhibiting the disease, i.e., halting its development, and (c) relieving the disease, i.e., causing regression of the disease.

[0039] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0040] A "therapeutically effective amount" or "effective amount" refers to the amount of a subject anti-GPC3 Ab that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the anti-GPC3 Ab, the disease to be treated and its severity, and the age, weight, etc. of the subject.

[0041] "Biological sample" encompasses a variety of sample types obtained from an individual and can be used in diagnostic or monitoring assays. This definition includes blood samples and other liquid samples of biological origin, solid tissue samples, such as biopsies, or tissue cultures or cells derived from tissue cultures and the progeny of tissue cultures. This definition also includes samples that have been manipulated in any way after acquisition, such as by treatment with reagents, solubilization, or enrichment for certain components (e.g., polynucleotides). The term "biological sample" encompasses clinical samples, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, body fluids, and tissue samples. In some cases, the biological sample will contain liver cells.

[0042] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0043] When a range of values ​​is given, it is understood that each value between the upper and lower limit of that range (to one-tenth of the unit of the lower limit, unless the context clearly indicates otherwise), and any other stated or intervening value in that stated range, is encompassed within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and these are also encompassed within the scope of the invention, subject to any limit value subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0045] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an antibody" includes a plurality of such antibodies, a reference to "the CDR" includes a reference to one or more CDRs and equivalents thereof known to those skilled in the art; and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a premise for the use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or the use of "negative" limitations.

[0046] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0047] Detailed Description The present disclosure provides an antibody specific to GPC3. Nucleic acids encoding one or both variable chain polypeptides of the antibody of the present disclosure are also provided, as are cells containing such nucleic acids. Compositions, including pharmaceutical compositions in some instances, containing the antibody of the present disclosure are also provided. Methods of making and using the antibody of the present disclosure are also provided. In certain aspects, a method is provided comprising administering a therapeutically effective amount of an antibody of the present disclosure to an individual with a cell proliferative disorder, wherein the antibody is administered to the individual to enhance an immune response (e.g., a T cell response) against abnormally proliferating cells of the cell proliferative disorder. The antibodies of the present invention are useful in a variety of diagnostic and monitoring applications, which are also provided.

[0048] GPC3 antibody As summarized above, the present disclosure provides anti-GPC3 antibodies.

[0049] According to some aspects, the antibodies of the present disclosure specifically bind to GPC3 and compete for binding to GPC3 with the following antibodies: V containing the amino acid sequence GYTFTSYFLH (SEQ ID NO: 2) or the amino acid sequence GYTFTSYYMH (SEQ ID NO: 3) H CDR1 and V comprising the amino acid sequence IIDPPTGRTTYAQKFQG (SEQ ID NO: 4) H CDR2 and V comprising the amino acid sequence GNYGGRYFDY (SEQ ID NO: 5) H The variable heavy chain (V) comprises CDR3 and H ) polypeptide; and V containing the amino acid sequence RASQSISSYLN (SEQ ID NO: 6) L CDR1 and V comprising the amino acid sequence AASSLQS (SEQ ID NO: 7) L CDR2 and V comprising the amino acid sequence QQSYSTPLT (SEQ ID NO: 8) L A variable light chain (V) comprising CDR3 and L ) Polypeptides.

[0050] Any suitable approach can be used to determine whether a first antibody competes with a second antibody for binding to GPC3. Whether a first antibody "competes" with a second antibody for binding to a given compound can be easily determined using competitive binding assays known in the art. Competing antibodies can be identified, for example, by antibody competition assays. For example, a sample of a first antibody can be bound to a solid support. Then, a sample of a second antibody suspected of competing with the first antibody is added. One of these two antibodies is labeled. If the labeled antibody and the unlabeled antibody bind to different, separate sites on the compound surface, the labeled antibody will bind to the same level regardless of the presence of the suspected competing antibody. However, if the interaction sites are identical or overlapping, the unlabeled antibody will compete, reducing the amount of labeled antibody that binds to the antigen. If there is an excess of unlabeled antibody, the labeled antibody will hardly bind, if at all.

[0051] For purposes of this disclosure, a competing antibody is one that reduces the binding of an antibody to a compound by about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more. Details of the procedures for conducting such competitive assays are widely known in the art, and details can be found, for example, in Harlow and Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988, pp. 567-569, 1988, ISBN 0-87969-314-2). Such assays can be performed quantitatively using purified antibodies. A standard curve can be established by titrating one antibody against itself; i.e., the same antibody is used as both the label and the competitor. An unlabeled competing antibody can be titrated to determine whether it can inhibit the binding of the labeled antibody to the plate. The results may be plotted and the concentrations required to achieve the desired degree of binding inhibition may be compared.

[0052] According to some embodiments, the antibodies of the disclosure specifically bind to one or more of GPC3, e.g., human GPC3, rat GPC3, mouse GPC3, and cynomolgus GPC3, and include: V containing the amino acid sequence GYTFTSYFLH (SEQ ID NO: 2) or the amino acid sequence GYTFTSYYMH (SEQ ID NO: 3) H CDR1 and V comprising the amino acid sequence IIDPPTGRTTYAQKFQG (SEQ ID NO: 4) H CDR2 and V comprising the amino acid sequence GNYGGRYFDY (SEQ ID NO: 5) H The variable heavy chain (V) comprises CDR3 and H ) polypeptide; and V containing the amino acid sequence RASQSISSYLN (SEQ ID NO: 6) L CDR1 and V comprising the amino acid sequence AASSLQS (SEQ ID NO: 7) LCDR2 and V comprising the amino acid sequence QQSYSTPLT (SEQ ID NO: 8) L A variable light chain (V) comprising CDR3 and L ) Polypeptides.

[0053] In certain embodiments, an antibody of the present disclosure specifically binds to GPC3 and competes for binding to GPC3 with the following antibody: V comprising the amino acid sequence GYTFTSYFLH (SEQ ID NO: 2) or the amino acid sequence GYTFTSYYMH (SEQ ID NO: 3). H CDR1 and V comprising the amino acid sequence IIDPPTGRTTYAQKFQG (SEQ ID NO: 4) H CDR2 and V comprising the amino acid sequence GNYGGRYFDY (SEQ ID NO: 5) H The variable heavy chain (V) comprises CDR3 and H ) polypeptide; and V comprising the amino acid sequence RASQSISSYLN (SEQ ID NO: 6) L CDR1 and V comprising the amino acid sequence AASSLQS (SEQ ID NO: 7) L CDR2 and V comprising the amino acid sequence QQSYSTPLT (SEQ ID NO: 8) L A variable light chain (V) comprising CDR3 and L ) polypeptide, provided that said V H The polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:13, and / or L The polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence set forth in SEQ ID NO:10.

[0054] The subject anti-GPC3 antibodies can comprise: a) a heavy chain comprising a VH region having the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:13; and a light chain comprising a VL region having the amino acid sequence set forth in SEQ ID NO:10.

[0055] The antibodies of the present disclosure specifically bind to GPC3 and compete for binding to GPC3 with an antibody comprising the following heavy and light chain polypeptides: V comprising the amino acid sequence GYTFTSYFLH (SEQ ID NO: 2) or GYTFTSYYMH (SEQ ID NO: 3). H CDR1 and V comprising the amino acid sequence IIDPPTGRTTYAQKFQG (SEQ ID NO: 4) H CDR2 and V comprising the amino acid sequence GNYGGRYFDY (SEQ ID NO: 5) H a heavy chain polypeptide comprising CDR3 and a V chain comprising the amino acid sequence RASQSISSYLN (SEQ ID NO: 6); L CDR1 and V comprising the amino acid sequence AASSLQS (SEQ ID NO: 7) L CDR2 and V comprising the amino acid sequence QQSYSTPLT (SEQ ID NO: 8) L and a CDR3, wherein the heavy chain polypeptide comprises an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identical to the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:14, and / or the light chain polypeptide comprises an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identical to the amino acid sequence set forth in SEQ ID NO:12.

[0056] The subject anti-GPC3 antibodies may comprise a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:14, and / or a light chain comprising a VL region having the amino acid sequence set forth in SEQ ID NO:12.

[0057] The amino acid and nucleotide sequences of the anti-GPC3 antibodies disclosed herein are shown below: Amino acid sequence: CAT-07 variable heavy chain: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYFLHWVRQAPGQGLEWMGIIDPPTGRTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGNYGGRYFDYWGQGTLVTVSS (SEQ ID NO: 9) CDR1, CDR2 and CDR3 are underlined. The variable region framework is shown in bold.

[0058] CAT-07 heavy chain: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYFLHWVRQAPGQGLEWMGIIDPPTGRTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGNYGGRYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) CDR1, CDR2 and CDR3 are underlined. Variable region frameworks are shown in bold. Constant regions are shown in italics.

[0059] Variable heavy chain of CAT-07YM: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIDPPTGRTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGNYGGRYFDYWGQGTLVTVSS (SEQ ID NO: 13) CDR1, CDR2, and CDR3 are underlined. The variable region framework is shown in bold. The CDR1 sequence of the CAT-07YM antibody differs from the CDR1 sequence of the CAT-07 antibody by residues "YM."

[0060] CAT-07YM heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIDPPTGRTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGNYGGRYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) CDR1, CDR2, and CDR3 are underlined. The variable region framework is shown in bold. The constant region is shown in italics. The CDR1 sequence of the CAT-07YM antibody differs from the CDR1 sequence of the CAT-07 antibody by residues "YM."

[0061] CAT-07 variable light chain: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK (SEQ ID NO: 10) CDR1, CDR2 and CDR3 are underlined. The variable region framework is shown in bold.

[0062] CAT-07 light chain: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12) CDR1, CDR2 and CDR3 are underlined. Variable region frameworks are shown in bold. Constant regions are shown in italics.

[0063] Nucleotide sequence: CAT-07 Heavy DNA: The sequences encoding CDR1, CDR2 and CDR3 are underlined. The sequences encoding the variable region framework are shown in bold. The sequences encoding the constant region are shown in italics.

[0064] CAT-07YM Heavy DNA: The sequences encoding CDR1, CDR2 and CDR3 are underlined. The sequences encoding the variable region framework are shown in bold. The sequences encoding the constant region are shown in italics.

[0065] CAT-07 Light DNA: (SEQ ID NO: 17) The sequences encoding CDR1, CDR2 and CDR3 are underlined. The sequences encoding the variable region framework are shown in bold. The sequences encoding the constant region are shown in italics.

[0066] The antibodies of the invention find use in a variety of research, diagnostic and therapeutic applications, including for practicing any of the methods described in U.S. Patent Application Publication Nos. 20150132782 (A1), 20170233462 (A1), and 20150098941 (A1), the disclosures of which are incorporated by reference in their entireties for all purposes.

[0067] The subject antibodies specifically bind to a given GPC3 polypeptide, provided that the epitope comprises amino acid residues within the GPC3 antigen comprising the following amino acid sequence as set forth in SEQ ID NO:1: MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFV GEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVV EIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQK AARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDEDECIGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFLVH In certain embodiments, the subject antibodies specifically bind to one or more of human GPC3, rat GPC3, mouse GPC3, and cynomolgus monkey GPC3, and do not exhibit significant binding to other glypicans (e.g., glypican-1, glypican-2, glypican-5, and glypican-6, etc.).

[0068] The subject antibodies exhibit high affinity binding to GPC3. For example, the subject antibodies exhibit high affinity binding to GPC3. -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, or at least about 10 -12 M's or 10 -12 The subject antibodies bind to GPC3 with an affinity of greater than about 10 M. -7 M ~ about 10 -8 M, about 10 -8 M ~ about 10 -9 M, about 10 -9 M ~ about 10 -10 M, about 10 -10 M ~ about 10 -11 M's, or about 10 -11 M ~ about 10 -12 M's or 10 -12 It binds to an epitope present on the surface of GPC3 with an affinity greater than M.

[0069] The anti-GPC3 antibodies of the present disclosure can, in some cases, induce apoptosis in cells that express GPC3 on their cell surface.

[0070] A "GPC3 antigen" or "GPC3 polypeptide" can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to SEQ ID NO:1.

[0071] As used herein, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains (approximately 25 kD or 214 amino acids) are encoded by a variable region gene (approximately 110 amino acids) at the N-terminus and a kappa or lambda constant region at the C-terminus. Full-length immunoglobulin heavy chains (approximately 50 kD or 446 amino acids) are encoded by a variable region gene (approximately 116 amino acids) at the N-terminus and one of the other aforementioned constant region genes (e.g., gamma (encoding approximately 330 amino acids)) at the C-terminus. In some embodiments, a subject antibody comprises a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0072] In some embodiments, the subject antibodies do not comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain, but instead comprise antigen-binding fragments of a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain. In some embodiments, these antigen-binding fragments are contained in separate polypeptide chains, while in other embodiments, these antigen-binding fragments are contained within a single polypeptide chain. The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that can specifically bind to GPC3, as described above. Examples of binding fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment, which consists of the VH and CH1 domains; (iv) a Fv fragment, which consists of the VH and VL domains of only one arm of an antibody; (v) a dAb fragment, which consists of a VH domain; (vi) an isolated CDR; and (vii) a single-chain Fv (scFv), which is produced using recombinant means so that the VH and VL domains pair to form a monovalent molecule. (viii) diabodies, which consist of two scFvs whose VH and VL domains are linked in a manner that prevents pairing to form monovalent molecules, except that the VH of one scFv pairs with the VL domain of the other scFv to form divalent molecules; and (ix) diabodies, which consist of at least two antigen-binding regions, each region binding a different epitope. In some embodiments, a subject antibody fragment is a Fab fragment. In some embodiments, a subject antibody fragment is a single-chain antibody (scFv).

[0073] In some embodiments, the subject antibodies are recombinant or engineered antibodies, e.g., chimeric, humanized, deimmunized, or in vitro-generated antibodies. The terms "recombinant" or "engineered" antibodies, as used herein, are intended to include all antibodies prepared, expressed, generated, or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell, (ii) antibodies isolated from a recombinant combinatorial antibody library, (iii) antibodies isolated from an animal (e.g., a mouse) that is genetically modified for human immunoglobulin genes, or (iv) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant antibodies include humanized, CDR-grafted, chimeric, deimmunized, and in vitro-generated antibodies, and it is possible that such recombinant antibodies may comprise constant regions derived from human germline immunoglobulin sequences.

[0074] Full-length bispecific antibodies can be generated using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, for example, by introducing substitutions at the heavy chain CH3 interface in each half molecule to promote heterodimerization of two antibody half molecules with different specificities, either in vitro, in a cell-free environment, or by using coexpression. Fab arm exchange is the result of disulfide bond isomerization and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the originating monospecific antibody are reduced. The resulting free cysteine ​​in one of the originating monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue in the second originating monospecific antibody molecule, and simultaneously the CH3 domains of the originating antibodies are released and reformed by dissociation-association. The CH3 domain of the Fab arm can be engineered to favor heterodimerization over homodimerization; the resulting product is a bispecific antibody with two Fab arms or half-molecules, each binding a different epitope.

[0075] A "knob-in-hole" strategy (see, e.g., PCT Publication WO 2006 / 028936) may be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the boundaries of the CHS domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. Amino acids with small side chains (holes) are introduced into the heavy chain of an antibody that specifically binds to a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a second antigen. After coexpression of these two antibodies, heterodimers form as a result of the heavy chain with the "hole" preferentially interacting with the heavy chain with the "knob." Exemplary CH3 substitution pairs that form knobs and holes are as follows (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S / L368A / Y407V.

[0076] Other strategies may be used, such as substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface to promote heavy chain heterodimerization using electrostatic interactions, as described in U.S. Patent Application Publication Nos. 2010 / 0015133, 2009 / 0182127, 82010 / 028637, or 2011 / 0123532. In another strategy, heterodimerization may be promoted by the following substitutions (expressed as modified positions in the first CH3 domain of the first heavy chain / modified positions in the second CH3 domain of the second heavy chain), as described in U.S. Patent Application Publication Nos. 2012 / 0149876 or 2013 / 0195849: L351Y / F405A / Y407V / T394W. , T366I / K392M / T394W / F405A / Y407V, T366L / K392M / T394W / F405A / Y407V, L351Y / Y407A / T366A / K409F, L351Y / Y407A / T366V / K409F, Y407A / T366A / K409F, or T350V / L351Y / F405A / Y407V, T350V / T366L / K392L / T394W.

[0077] Single-chain bispecific antibodies are also provided. In some embodiments, the single-chain bispecific antibodies of the present disclosure are bispecific scFvs. Details regarding bispecific scFvs can be found, for example, in Zhou et al. (2017), J Cancer 8(18):3689-3696.

[0078] The subject antibodies can be humanized. See Queen et al., Proc. Natl. Acad. Sci. USA 86:10029 10033 (1989), U.S. Patent No. 5,530,101, U.S. Patent No. 5,585,089, U.S. Patent No. 5,693,761, WO 90 / 07861, and U.S. Patent No. 5,225,539. If constant region(s) are present, the constant region(s) can also be substantially or entirely derived from human immunoglobulin. Various methods for producing humanized antibodies are known in the art. See, e.g., U.S. Patent No. 7,256,273.

[0079] Substitution of mouse CDRs into a human variable domain framework can result in, for example, preserving their correct spatial orientation, with the human variable domain framework adopting the same or a similar conformation as the mouse variable framework from which the CDRs originated. This can be achieved by obtaining human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence identity with the mouse variable framework domain from which the CDRs were derived. Such heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be those of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).

[0080] Once the complementarity-determining regions of a mouse donor immunoglobulin and a suitable human acceptor immunoglobulin have been identified, the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody. Generally, substitution of human amino acid residues with mouse amino acid residues should be minimized, as the introduction of mouse residues increases the risk that the antibody will elicit a human anti-mouse antibody (HAMA) response in humans. Various art-recognized methods for determining immune response can be performed to monitor HAMA responses in specific patients or during clinical trials. Patients receiving a humanized antibody can undergo immunogenicity assessments at the start of treatment and throughout the course of treatment. HAMA responses are measured, for example, by detecting antibodies against the humanized therapeutic reagent in serum samples from patients using methods known to those skilled in the art, including surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA analysis. In many embodiments, the subject humanized antibodies do not substantially elicit a HAMA response in human subjects.

[0081] Certain amino acids from the human variable region framework residues are selected for substitution based on their potential effect on the conformation of the CDR and / or binding to the antigen. The artificial juxtaposition of the mouse CDR regions with the human variable framework regions can create artificial conformational constraints that, unless corrected by substitution of certain amino acid residues, can result in loss of binding affinity.

[0082] The selection of amino acid residues for substitution can be determined in part by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, various molecular models are generated starting from the solved structure of an immunoglobulin chain or its domain. The chains to be modeled are compared for amino acid sequence similarity with the chains or domains of the solved three-dimensional structure, and the chains or domains showing the greatest sequence similarity are selected as the starting point for constructing the molecular model. Chains or domains that share at least 50% sequence identity are selected for modeling, and preferably chains or domains that share at least 60%, 70%, 80%, 90% or more sequence identity are selected for modeling. The solved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chains or immunoglobulin domains being modeled and the amino acids in the starting structure. The modified structures are then assembled into composite immunoglobulins. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0083] CDR and framework regions are as defined by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991). An alternative structural definition has been proposed by Chothia et al., J. Mol. Biol. 196:901 (1987); Nature 342:878 (1989); and J. Mol. Biol. 186:651 (1989) (collectively referred to as "Chothia"). Amino acids present in murine antibodies may be selected for substitution into humanized antibodies when framework residues as defined by Kabat (supra) constitute structural loop residues as defined by Chothia (supra). Residues "adjacent to the CDR region" include amino acid residues immediately adjacent to one or more CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., CDRs as defined by Kabat or CDRs as defined by Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with amino acids in the CDRs and, if selected from the acceptor, distort the donor CDRs and reduce affinity. Moreover, adjacent amino acids may interact directly with the antigen (Amit et al., Science, 233:747 (1986)), and selecting these amino acids from the donor may be desirable to preserve all affinity-conferring antigen contacts in the original antibody.

[0084] In some embodiments, the subject antibodies comprise scFv multimers. For example, in some embodiments, the subject antibodies are scFv dimers (e.g., comprising two tandem scFvs (scFv2)), scFv trimers (e.g., comprising three tandem scFvs (scFv3)), scFv tetramers (e.g., comprising four tandem scFvs (scFv4)), or multimers of more than four scFvs (e.g., in tandem). The scFv monomers can be linked in tandem via a linker that is about 2 to about 10 amino acids in length, e.g., 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa in length. Suitable linkers include, for example, (Gly) x (wherein x is an integer from 2 to 10), and glycine-serine polymers, etc. Other suitable linkers are those discussed above.

[0085] In certain embodiments, the antibody is conjugated to the agent via a cleavable or non-cleavable linker. Linkers suitable for use with the subject antibodies include "flexible linkers." If present, the linker molecule is generally long enough to allow some flexible movement between the linked regions. Linker molecules are generally about 6 to 50 atoms in length. Linker molecules can also be, for example, aryl acetylene, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacids, amino acids, or combinations thereof. Other linker molecules capable of binding to polypeptides may be used in accordance with the present disclosure.

[0086] In some embodiments, the linker is a chemically labile linker, such as an acid-cleavable linker that is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of target cells (e.g., cancer cells). Chemically labile linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, and the like. In certain embodiments, the linker is an enzyme-labile linker, such as an enzyme-labile linker that is stable in the bloodstream but undergoes enzymatic cleavage by lysosomal proteases (e.g., cathepsin or plasmin) in the lysosomes of target cells (e.g., cancer cells) upon internalization into target cells. Enzyme-labile linkers include, but are not limited to, peptide bond-containing linkers, such as dipeptide-based linkers, for example, valine-citrulline (VC) linkers (e.g., maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linker, valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, etc.). Various chemically unstable linkers, enzyme-labile linkers, and non-cleavable linkers are known, and are described in detail in, for example, Ducry & Stump (2010), Bioconjugate Chem. 21:5-13; Nolting, B. (2013), Methods Mol Biol. 1045:71-100; Tsuchikama and An (2018), Protein & Cell 9(1):33-46; and elsewhere.

[0087] In some embodiments, each of the scFv monomers in a subject scFv multimer is humanized, as described above.

[0088] In some embodiments, the subject antibodies comprise an immunoglobulin constant region (e.g., an Fc region). If an Fc region is present, the Fc region can be a human Fc region. If a constant region is present, the antibody can contain both a light chain constant region and a heavy chain constant region. A suitable heavy chain constant region comprises a CH1 region, a hinge region, a CH2 region, a CH3 region, and a CH4 region. The antibodies described herein include antibodies with all types of constant regions, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. An example of a suitable heavy chain Fc region is a human Fc of isotype IgG1. The light chain constant region can be of the lambda or kappa type. The subject antibodies (e.g., the subject humanized antibodies) can comprise sequences from more than two classes or isotypes. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, or as separate heavy and light chains, or as Fab, Fab', F(ab')2 and Fv, or as single chain antibodies in which the heavy and light chain variable domains are linked by a spacer.

[0089] In some embodiments, anti-glypican-3 antibodies of the present disclosure may comprise one or more amino acid substitutions introduced into the Fc region. In some embodiments, the one or more amino acid substitutions may be at positions 239, 298, 326, 330, and 332 in the Fc region. In some embodiments, anti-glypican-3 antibodies of the present disclosure may comprise one or more of the following amino acid substitutions introduced into the Fc region: I332E; S239D / A330L / I332E; S239D / S298A / I332E; S239D / K326T / I332E; S239D / S298A / K326T / I332E; or S239D / A330L / I332E / D356E / L358M.

[0090] In some embodiments, the subject antibodies comprise a free thiol (—SH) group at the carboxyl terminus, where the free thiol group can be used to attach the antibody to a second polypeptide (e.g., another antibody, including the subject antibody), a scaffold, a carrier, etc.

[0091] In some embodiments, the subject antibodies comprise one or more non-naturally occurring amino acids. In some embodiments, the non-naturally occurring amino acids comprise a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. For suitable non-naturally occurring amino acids, see, e.g., U.S. Pat. No. 7,632,924. The inclusion of a non-naturally occurring amino acid allows for linkage to a polymer, a second polypeptide, a scaffold, or the like. For example, a subject antibody linked to a water-soluble polymer can be generated by reacting a water-soluble polymer (e.g., PEG) containing a carbonyl group with a subject antibody containing a non-naturally encoded amino acid containing an aminooxy group, a hydrazine group, a hydrazide group, or a semicarbazide group. As another example, a subject antibody linked to a water-soluble polymer can be generated by reacting a subject antibody containing an alkyne-containing amino acid with a water-soluble polymer (e.g., PEG) containing an azide moiety; in some embodiments, the azide or alkyne group is linked to the PEG molecule via an amide linkage. A "non-naturally encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids, or pyrrolysine, or selenocysteine. Other terms that may be used synonymously with the term "non-naturally encoded amino acid" are "unnatural amino acid," "artificial amino acid," "non-naturally occurring amino acid," and various hyphenated and non-hyphenated forms thereof. The term "non-naturally encoded amino acid" also includes, but is not limited to, various amino acids that arise by modification (e.g., post-translational modification) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but that are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0092] The present disclosure also provides anti-GPC3 antibodies with desired attached moieties (e.g., detectable labels, drugs, and half-life extending moieties). Antibody modification can be achieved by various synthetic and / or recombinant methods. The one or more moieties attached to the antibody can provide one or more of a wide variety of functions or characteristics. Exemplary moieties include detectable labels (e.g., dye labels (e.g., chromophores, fluorophores), biophysical probes (spin labels, nuclear magnetic resonance (NMR) probes), Förster resonance energy transfer (FRET)-type labels (e.g., at least one member of a FRET pair, including at least one member of a fluorophore / quencher pair), bioluminescence resonance energy transfer (BRET)-type labels (e.g., at least one member of a BRET pair), immunodetectable tags (e.g., FLAG, His(6), and the like); water-soluble polymers (e.g., PEGylation); purification tags (e.g., to facilitate isolation by affinity chromatography (e.g., binding of a FLAG epitope)); membrane localization domains (e.g., lipids or glycophosphatidylinositol (GPI)-type anchors); immobilization tags (e.g., to facilitate binding of a polypeptide to a surface, including selective binding); and drugs (e.g., to facilitate drug targeting, e.g., via binding of a drug to an antibody).

[0093] In some embodiments, the subject antibodies are linked (e.g., covalently linked) to a polymer (e.g., a polymer other than a polypeptide). Suitable polymers include, for example, biocompatible polymers, even water-soluble biocompatible polymers. Suitable polymers include synthetic polymers and naturally occurring polymers. Suitable polymers include, for example, substituted or unsubstituted linear or branched polyalkylene polymers, polyalkenylene polymers, or polyoxyalkylene polymers, or branched or unbranched polysaccharides (e.g., homopolysaccharides or heteropolysaccharides). Suitable polymers include, for example, ethylene vinyl alcohol copolymer (which is commonly known by the generic name EVOH or by the trade name EVAL); polybutyl methacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoesters; polyanhydrides; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphoesters; polyphosphoesterurethanes; poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(iminocarbonate); copoly(ether-esters) (e.g., poly(ethylene oxide)- poly(lactic acid) (PEO / PLA) copolymers); polyalkylene oxalates; polyphosphazenes; biomolecules, such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid; polyurethanes; silicones; polyesters; polyolefins; polyisobutylene and ethylene-alphaolefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketone; polyaromatic vinyls, such as polystyrene; polyvinyl esters, such as polyvinyl acetate;Copolymers of vinyl monomers with themselves and with olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; polyamides, such as nylon 66 and polycaprolactam; alkyd resins; polycarbonates; polyoxymethylene; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous Teflon; poly(ethylene glycol); and carboxymethyl cellulose.

[0094] Suitable synthetic polymers include substituted and unsubstituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), and derivatives thereof, such as substituted poly(ethylene glycol) (e.g., methoxypoly(ethylene glycol)) and derivatives thereof. Suitable naturally occurring polymers include, for example, albumin, amylose, dextran, glycogen, and derivatives thereof.

[0095] Suitable polymers can have an average molecular weight in the range of 500 Da to 50,000 Da, e.g., in the range of 5,000 Da to 40,000 Da, or in the range of 25,000 to 40,000 Da. For example, in some embodiments, when a subject antibody comprises a poly(ethylene glycol) (PEG) polymer or a methoxypoly(ethylene glycol) polymer, the PEG polymer or methoxypoly(ethylene glycol) polymer can have a molecular weight in the range of about 0.5 kilodaltons (kDa) to 1 kDa, about 1 kDa to 5 kDa, 5 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 40 kDa, or 40 kDa to 60 kDa.

[0096] As noted above, in some embodiments, a subject antibody is covalently linked to a PEG polymer. In some embodiments, a subject scFv multimer is covalently linked to a PEG polymer. Methods and reagents suitable for PEGylation of proteins are widely known in the art and can be found, for example, in U.S. Pat. No. 5,849,860. PEG suitable for conjugation to proteins is generally soluble in water at room temperature and has the general formula R(O-CH-CH). n OR, where R is hydrogen or a protecting group (e.g., an alkyl group or an alkanol group), and n is an integer of 1 to 1000. When R is a protecting group, the protecting group generally has 1 to 8 carbon atoms.

[0097] PEG conjugated to a subject antibody can be linear. PEG conjugated to a subject protein can also be branched. Branched PEG derivatives, such as those described in U.S. Pat. No. 5,643,575, "star PEGs," and multi-armed PEGs, such as those described in Shearwater Polymers, Inc.'s catalog "Polyethylene Glycol Derivatives 1997-1998." Various star PEGs are described in the art, including, for example, those described in U.S. Pat. No. 6,046,305.

[0098] The subject antibodies can be glycosylated; for example, the subject antibodies can comprise a covalently linked carbohydrate or polysaccharide moiety. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide provides a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Glycosylation can be achieved, for example, by recombinant production in a host cell possessing the desired glycosylation machinery.

[0099] Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). Alterations may also be made by adding, or substituting with, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). Similarly, removal of glycosylation sites can be accomplished by altering amino acids within the antibody's native glycosylation sites.

[0100] The subject antibodies can be covalently linked to a second moiety (e.g., lipids, polypeptides other than the subject antibodies, synthetic polymers, carbohydrates, etc.) using, for example, glutaraldehyde, homobifunctional crosslinkers, or heterobifunctional crosslinkers. Glutaraldehyde crosslinks polypeptides via their amino moieties. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl-reactive crosslinkers) contain two or more identical reactive moieties, and various homobifunctional crosslinkers can be used in a one-step reaction procedure in which the crosslinker is added to a solution containing a mixture of polypeptides to be linked. Homobifunctional NHS esters and imidoesters crosslink amine-containing polypeptides. At slightly alkaline pH, imidoesters react only with primary amines to form imidoamides, leaving the overall charge of the crosslinked polypeptides unaffected. Homobifunctional sulfhydryl-reactive crosslinkers include bismaleimidohexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propinoamidobutane (DPDPB).

[0101] Heterobifunctional crosslinkers have two or more different reactive moieties (e.g., amine-reactive moieties and sulfhydryl-reactive moieties), and one of the polypeptides is crosslinked via the amine-reactive moiety or the sulfhydryl-reactive moiety, and then the other polypeptide is reacted via the unreacted moiety. Numerous heterobifunctional haloacetyl-based crosslinkers are available, as are pyridyl disulfide-based crosslinkers. Carbodiimides are a classic example of heterobifunctional crosslinking reagents for coupling carboxyls to amines, thereby resulting in amide bonds.

[0102] The subject antibodies can be immobilized on a solid support. Suitable supports are widely known in the art and include, among others, commercially available column material, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, duracite, wells of reaction trays (e.g., multi-well plates), plastic tubes, and the like. Solid supports can comprise any of a variety of materials, including, for example, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. A variety of suitable methods for immobilizing the subject antibodies on a solid support are widely known and include, but are not limited to, ionic interactions, hydrophobic interactions, and covalent interactions. Solid supports can be soluble or insoluble, for example, in aqueous solutions. In some embodiments, suitable solid supports are generally insoluble in aqueous solutions.

[0103] The subject antibodies can, in some embodiments, comprise a detectable label. Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Suitable labels include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and similar fluorescent dyes), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C, or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase and other enzymes commonly used in enzyme-linked immunosorbent assays (ELISAs)), and colorimetric labels, such as colloidal gold or colored beads of glass or plastic (e.g., polystyrene, polypropylene, latex, etc.).

[0104] In some embodiments, the subject antibodies comprise an imaging agent or radioisotope, where the imaging agent or radioisotope is one that is suitable as a detectable label, e.g., for use in imaging, e.g., for use in imaging procedures performed on humans. Non-limiting examples of labels include radioisotopes, e.g., 1231 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium) and 67 Ga (gallium), and contrast agents such as gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotopes ( 153 Gd) are also available and suitable for imaging procedures in mammals other than humans.

[0105] The subject antibodies can be labeled using standard techniques. For example, the subject antibodies can be iodinated using chloramine T or 1,3,4,6-tetrachloro-3α,6α-dephenylglycouril. For fluorination, fluorine is added to the subject antibodies during synthesis by a fluoride ion displacement reaction. For reviews of protein synthesis using such radioisotopes, see Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244 (1999). The subject antibodies can also be labeled with imaging agents using standard techniques. For example, the subject antibodies can be labeled with gadolinium (Gd) by conjugating a low molecular weight Gd chelate, such as Gd-diethylenetriaminepentaacetic acid (GdDTPA) or Gd-tetraazacyclododecanetetraacetic acid (GdDOTA), to the antibody. See Caravan et al., Chem. Rev. 99:2293-2352 (1999), and Lauffer et al., J. Magn. Reson. Imaging, 3:11-16 (1985). The subject antibodies can be labeled with Gd, for example, by conjugating a polylysine-Gd chelate to the antibody. See, e.g., Curtet et al., Invest. Radiol., 33(10):752-761 (1998). Alternatively, the subject antibodies can be labeled with Gd by incubating biotinylated antibodies with paramagnetic polymeric liposomes containing a Gd chelator lipid in combination with avidin. See, e.g., Sipkins et al., Nature Med., 4:623-626 (1998).

[0106] Suitable fluorescent proteins that can be linked to the subject antibodies include green fluorescent protein from Aequoria victoria or variants or derivatives thereof, such as those described in U.S. Patent Nos. 6,066,476, 6,020,192, 5,985,577, 5,976,796, 5,968,750, 5,968,738, 5,958,713, 5,919,445, and 5,874,304; enhanced GFP, many of which are commercially available, e.g., from Clontech, Inc.; red fluorescent protein; yellow fluorescent protein; any of a variety of fluorescent and colored proteins obtained from species of Anthozoa, e.g., Matz et al. (1999), Nature 106:101-104, 1999; Biotechnol. 17:969-973; and similar fluorescent proteins.

[0107] The subject antibodies will, in some embodiments, comprise a "radiopaque" label, e.g., a label that allows for easy visualization, e.g., using x-rays. A variety of radiopaque materials are widely known to those skilled in the art. The most common radiopaque materials include iodide, bromide, or barium salts. Other radiopaque materials are also known, including, but not limited to, organobismuth derivatives (see, e.g., U.S. Pat. No. 5,939,045), radiopaque multiurethanes (see, e.g., U.S. Pat. No. 5,346,981), organobismuth complexes (see, e.g., U.S. Pat. No. 5,256,334), and radiopaque barium multimer complexes (see, e.g., U.S. Pat. No. 4,866,132).

[0108] The subject antibodies, in some embodiments, will be linked (e.g., covalently or non-covalently) to a fusion partner, e.g., a ligand, an epitope tag, a peptide, a protein other than an antibody, and the like. Suitable fusion partners include peptides and polypeptides that confer increased stability in vivo (e.g., increased serum half-life); peptides and polypeptides that provide for ease of purification; peptides and polypeptides that provide for secretion of the fusion protein from a cell; peptides and polypeptides that provide epitope tags, e.g., (His)n (e.g., 6His), and similar epitope tags; peptides and polypeptides that provide for secretion of the fusion protein from a cell; epitope tags, e.g., GST, hemagglutinin (HA; e.g., CYPYDVPDYA; SEQ ID NO: 35), FLAG (e.g., DYKDDD), and the like. DK; SEQ ID NO: 36), c-myc (e.g., CEQKLISEEDL; SEQ ID NO: 37), and similar epitope tags; peptides and polypeptides that provide a detectable signal, such as enzymes that produce a detectable product (e.g., β-galactosidase, luciferase), or proteins that are themselves detectable, such as green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and the like; peptides and polypeptides that provide for multimerization, such as multimerization domains (e.g., Fc portions of immunoglobulins, and the like); and similar fusion partners.

[0109] Fusions may also include affinity domains containing peptide sequences capable of interacting with binding partners useful for identification or purification (e.g., immobilized on a solid support). Contiguous single amino acids (e.g., histidines), when fused to a protein, can be used for one-step purification of the fusion protein by high-affinity binding to a resin column (e.g., nickel sepharose). Examples of affinity domains include His5 (HHHHH) (SEQ ID NO: 18), His6 (HHHHHH) (SEQ ID NO: 19), C-myc (EQKLISEEDL) (SEQ ID NO: 20), Flag (DYKDDDDK) (SEQ ID NO: 21), StrepTag (WSHPQFEK) (SEQ ID NO: 22), hemagglutinin, e.g., HA tag (YPYDVPDYA; SEQ ID NO: 23), glutathione-S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 24), Phe-His-His-Thr (SEQ ID NO: 25), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCREC Included are CARA (SEQ ID NO: 26), metal binding domains, e.g., zinc binding domains or calcium binding domains, such as calcium binding domains derived from calcium binding proteins (e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin), intein, biotin, streptavidin, MyoD, leucine zipper sequences, and maltose binding protein.

[0110] In some embodiments, the subject antibodies comprise polyamine modifications. The subject antibodies can be modified with either naturally occurring or synthetic polyamines. See, e.g., U.S. Patent No. 5,670,477. Useful naturally occurring polyamines include putrescine, spermidine, spermine, 1,3-deaminopropane, norspermidine, syn-homospermidine, thermine, thermospermine, caldopentamine, homocaldopentamine, and canavalmine. Putrescine, spermidine, and spermine are particularly useful. Synthetic polyamines can be represented by the empirical formula C X H Y N Z and can be a cyclic or acyclic, branched or unbranched hydrocarbon chain of 3 to 12 carbon atoms, consisting of: and further containing 1 to 6 NR or N(R)2 moieties, where R is H, (C1-C4) alkyl, phenyl, or benzyl. The polyamine can be linked to the antibody using any standard cross-linking method.

[0111] In some embodiments, the subject antibodies are modified to include a carbohydrate moiety, which can be covalently linked to the antibody. In some embodiments, the subject antibodies are modified to include a lipid moiety, which can be covalently linked to the antibody. Suitable lipid moieties include, for example, N-aliphatic acyl groups (e.g., N-lauroyl, N-oleoyl, etc.), fatty amines (e.g., dodecylamine, oleoylamine, etc.), and C3-C16 chain aliphatic lipids. See, e.g., U.S. Patent No. 6,638,513. In some embodiments, the subject antibodies are incorporated into liposomes.

[0112] When the anti-GPC3 antibody of the present disclosure contains a covalently linked heterologous moiety, the heterologous moiety can be linked directly to the heavy and / or light chain of the anti-GPC3 or via a linker. Suitable linkers can be readily selected and can be of various lengths, such as 1 to 20 amino acids (e.g., Gly), 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can also be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0113] Examples of flexible linkers include glycine polymers (G), glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 27) and (GGGS) n (SEQ ID NO: 28) (wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine polymers and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and can therefore serve as neutral tethers between components. Glycine polymers are of particular interest because glycine has significantly more access to phi-psi space than even alanine and is much less constrained than residues with longer side chains (Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO: 29), GGSGG (SEQ ID NO: 30), GSGSG (SEQ ID NO: 31), GSGGG (SEQ ID NO: 32), GGGSG (SEQ ID NO: 33), and GSSSG (SEQ ID NO: 34). Those skilled in the art will recognize that the design of peptides to be conjugated to any of the elements described above can include linkers that are flexible in whole or in part, such that the linker can include a flexible linker as well as one or more moieties that provide a less flexible structure.

[0114] Methods for modifying antibodies The antibodies of the present invention can be modified by any of a variety of methods to have a covalently attached heterologous moiety (e.g., a detectable label, a drug, etc.). The present disclosure provides an anti-GPC3 antibody conjugated to a moiety of interest; in this case, the anti-GPC3 antibody conjugated to a moiety of interest is referred to as an "anti-GPC3 antibody conjugate." The anti-GPC3 antibody conjugate of the present disclosure can include: 1) an Ig heavy chain constant region conjugated to a moiety of interest and an Ig light chain constant region conjugated to a moiety of interest; 2) an Ig heavy chain constant region conjugated to a moiety of interest and an Ig light chain constant region not conjugated to a moiety of interest; or 3) an Ig heavy chain constant region not conjugated to a moiety of interest and an Ig light chain constant region conjugated to a moiety of interest. The subject anti-GPC3 antibody conjugate can also include a VH and / or VL domain.

[0115] In one example, an antibody can be modified to contain a 2-formylglycine residue, which can serve as a chemical handle for attachment of a heterologous moiety. For example, the heavy and / or light chain constant regions of the anti-GPC3 antibodies of the present disclosure can be modified to contain an amino acid sequence of a sulfatase motif that can be converted by the action of 2-formylglycine generating enzyme (FGE) to contain 2-formylglycine (FGly). Such a sulfatase motif is also sometimes referred to herein as an FGE modification site. The action of FGE is directed in a sequence-specific manner in that FGE acts on a sulfatase motif located within an immunoglobulin polypeptide. A moiety of interest is provided as a component of a reactive partner for reaction with the aldehyde of the FGly residue of the converted aldehyde tag of the tagged Ig polypeptide. A wide range of commercially available reagents can be used to achieve attachment of a moiety of interest to the FGly residue of the aldehyde-tagged Ig polypeptide. For example, aminooxy, hydrazide, or thiosemicarbazide derivatives of many of the moieties of interest are suitable reactive partners and are readily available or can be generated using standard chemical methods.

[0116] For example, to attach a poly(ethylene glycol) (PEG) moiety to a tagged Ig polypeptide, aminooxy-PEG can be generated from monoamino-PEG and aminooxyglycine using standard protocols. The aminooxy-PEG can then be reacted with a converted (e.g., FGly-modified) aldehyde-tagged Ig polypeptide to prepare it for attachment of a PEG moiety. Delivery of a biotin moiety to the converted aldehyde-tagged polypeptide can be achieved using aminooxybiotin, biotin hydrazide, or 2,4-dinitrophenylhydrazine.

[0117] The minimum sulfatase motif of an aldehyde tag is typically 5 or 6 amino acid residues in length, and typically no more than 6 amino acid residues in length. The sulfatase motif provided in the Ig polypeptide is at least 5 or 6 amino acid residues in length and can be, for example, 5-16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, defining sulfatase motifs that are less than 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acid residues in length. In certain embodiments, the sulfatase motif used may be described by the following formula: X 1 Z 1 X 2 Z 2 X 3 Z 3 (I) (SEQ ID NO: 40) During the ceremony, Z 1 is a cysteine ​​or serine (which can also be represented by (C / S)); Z 2 is either a proline or an alanine residue (which can also be represented by (P / A)); Z 3 is a basic amino acid (e.g., arginine (R), which may also be lysine (K) or histidine (H) (usually lysine)), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), usually A, G, L, V, or I); X 1is present or absent, and when present can be any amino acid, but is usually an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., as opposed to an aromatic or charged amino acid), and is usually L, M, V, S, or T, more usually L, M, S, or V, except that when the sulfatase motif is present at the N-terminus of the target polypeptide, X 1 exists; and X 2 and X 3 can independently be any amino acid, but are typically aliphatic, polar, uncharged, or sulfur-containing amino acids (i.e., as opposed to aromatic or charged amino acids), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G. In one example, the aldehyde tag is of the formula: L(C / S)TPSR (SEQ ID NO: 35), e.g., LCTPSR (SEQ ID NO: 36) or LSTPSR (SEQ ID NO: 37). Thus, the present disclosure provides antibodies comprising an aldehyde-tagged Ig heavy chain and / or an aldehyde-tagged Ig light chain, wherein the aldehyde-tagged Ig antibody comprises a heavy and / or light chain Ig constant region amino acid sequence containing such a sulfatase motif.

[0118] Generally, the FGE used to facilitate the conversion of a cysteine ​​or serine in the sulfatase motif of the aldehyde tag of a target polypeptide to FGly is selected according to the sulfatase motif present in the aldehyde tag. The FGE can be native to the host cell in which the aldehyde-tagged polypeptide is expressed, or the host cell can be genetically modified to express an appropriate FGE. In some embodiments, it may be desirable to use a sulfatase motif compatible with human FGE and express the aldehyde-tagged protein in human cells that express FGE or in host cells (usually mammalian cells) that have been genetically modified to express human FGE. In general, FGEs suitable for use in generating FGly-modified antibodies can be obtained from naturally occurring sources or synthetically produced. For example, suitable FGEs can be derived from biological sources that naturally produce FGE or from biological sources that have been genetically modified to express a recombinant gene encoding FGE. Nucleic acids encoding numerous FGEs are also readily known in the art.

[0119] After the action of FGE on the sulfatase motif, Z1 is oxidized to generate a 2-formylglycine (FGly) residue. Furthermore, after both the FGE-mediated conversion and reaction with a reactive partner containing a moiety of interest, the FGly position in Z1 in the above formula is covalently linked to a moiety of interest (e.g., a detectable label, a water-soluble polymer, a polypeptide, a drug, etc.). Thus, the present disclosure provides an anti-GPC3 antibody modified to contain an FGly moiety, comprising an FGly-converted sulfatase motif of the following formula: X 1 (FGly)X 2 Z 2 X 3 Z 3 (SEQ ID NO: 41) During the ceremony, X 1is present or absent, and when present is any amino acid, except when the sulfatase motif is present at the N-terminus of the polypeptide, where X 1 exists; X 2 and X 3 are each independently any amino acid; Z 2 is either a proline or an alanine residue (which can also be represented by (P / A)); and Z 3 is a basic amino acid; and However, the FGly-modified anti-GPC3 antibody presents the FGly group on a solvent-accessible surface when in the folded state. In some embodiments, the FGly-converted sulfatase motif is of the formula L(FGly)TPSR (SEQ ID NO: 38).

[0120] As noted above, the subject anti-GPC3 antibodies modified to include an FGly moiety can be further modified to include a heterologous moiety of interest (e.g., a detectable label, a water-soluble polymer, a polypeptide, a drug, etc.) covalently linked to the anti-GPC3 antibody via the FGly moiety. Thus, the present disclosure provides an anti-GPC3 antibody conjugate (also referred to herein as an "anti-GPC3 conjugate") comprising the following sequence: X 1 (FGly')X 2 Z 2 X 3 Z 3 (I') (SEQ ID NO: 42) During the ceremony, FGly' is a 2-formylglycine residue with a covalently attached moiety; Z 2 is either a proline or an alanine residue (which can also be represented by (P / A)); Z 3is a basic amino acid (e.g., arginine (R), which may also be lysine (K) or histidine (H) (usually lysine)), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), usually A, G, L, V, or I); X 1 may be present or absent, and when present can be any amino acid, but is usually an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., as opposed to an aromatic or charged amino acid), and is usually L, M, V, S, or T, more usually L, M, or V, except that when the sulfatase motif is present at the N-terminus of the target polypeptide, X 1 exists; and X 2 and X 3 can independently be any amino acid, but are usually aliphatic, sulfur-containing, or polar uncharged amino acids (i.e., as opposed to aromatic or charged amino acids), usually S, T, A, V, G, or C, and more usually S, T, A, V, or G. In some embodiments, the motif is of the formula L(FGly')TPSR (SEQ ID NO: 39).

[0121] drugs In some cases, the anti-GPC3 antibody of the present disclosure comprises a drug covalently linked to the heavy and / or light chain of the antibody. "Drug" includes small molecule drugs, peptide drugs, and toxins (e.g., cytotoxins), etc.

[0122] "Small molecule drug," as used herein, refers to a compound (e.g., an organic compound) that exhibits a desired pharmaceutical activity and generally has a molecular weight of at most about 800 Da or at most 2000 Da, but can encompass molecules up to 5 kDa and can be as large as about 10 kDa. Small inorganic molecules refer to molecules that contain no carbon atoms, while small organic molecules refer to compounds that contain at least one carbon atom.

[0123] "Peptide drug," as used herein, refers to an amino acid-containing polymeric compound and is meant to encompass naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptidomimetics. Peptide drugs may be obtained by chemical synthesis or may be produced from genetically encoded sources (e.g., recombinant sources). Peptide drugs can vary in molecular weight, ranging from 200 Da to 10 kDa, or even larger.

[0124] In some cases, the drug is a toxin, for example, a cytotoxin. Ribosome-inactivating proteins (RIPs), a class of proteins ubiquitous in higher plants, are an example of such cytotoxins. RIPs are divided into type I and type II classes and are cytotoxic due to their activity as potent inhibitors of eukaryotic protein synthesis. Type I RIPSs are composed of a single peptide chain with ribosome-inactivating activity, while type II proteins are composed of an A chain (essentially equivalent to type I proteins) disulfide-linked to a B chain with cell-binding properties. The N-glycosidic bond of a specific adenine base is hydrolytically cleaved by RIPs in the highly conserved loop region of the 28S rRNA of eukaryotic ribosomes, thereby inactivating translation in eukaryotic cells. See, for example, U.S. Patent No. 5,744,580. Gelonin, dodecandrin, trichosanthin, tricokirin, bryodin, Mirabilis antiviral protein (MAP), barley ribosome-inactivating protein (BRIP), pokeweed antiviral protein (PAPS), saporins, luffins, and momordins are examples of type I RIPs, while ricin and abrin are examples of type II RIPS. Suitable cytotoxins include, but are not limited to, ricin, abrin, diphtheria toxin, Pseudomonas exotoxin (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, pokeweed antiviral protein (PAP), botulinum toxin, bryodin, momordin, and bouganin.

[0125] In some cases, the drug is a cancer chemotherapeutic agent.Cancer chemotherapeutic agents include non-peptide (i.e., non-proteinaceous) compounds that reduce the proliferation of cancer cells, including cytotoxic agents and cytostatic agents.Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant alkaloids (vinca alkaloids), and steroid hormones.Peptide compounds can also be used.

[0126] Suitable cancer chemotherapeutic agents include dolastatins and their active analogs and derivatives, and auristatins and their active analogs and derivatives. See, e.g., WO 96 / 33212, WO 96 / 14856, and U.S. Patent No. 6,323,315. For example, dolastatin 10 or auristatin PE can be included in the antibody-drug conjugates of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives (see, e.g., EP 1391213 and Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and duocarmycins and their active analogs and derivatives (including, e.g., synthetic analogs KW-2189 and CB1-TM1).

[0127] A variety of drugs that act to reduce cell proliferation are known in the art and are widely used. Such drugs include alkylating agents (such as nitrogen mustards), nitrosoureas, ethyleneimine derivatives, alkylsulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0128] Antimetabolites include folate analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0129] Suitable natural products and derivatives thereof (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, such as etoposide, teniposide, and the like; antibiotics, such as anthracyclines, daunorubicin hydrochloride (daunomycin, rubin), and the like. phenoxyzolidinone cyclopeptides such as dactinomycin; basic glycopeptides such as bleomycin; anthraquinone glycosides such as plicamycin (mithramycin); anthracenediones such as mitoxantrone; azirinopyrroloindole diones such as mitomycin; macrocyclic immunosuppressants such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like; and similar natural products and derivatives thereof.

[0130] Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0131] Various microtubule-affecting agents with antiproliferative activity are also suitable for use, including allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (NSC 332599), and the like. These include, but are not limited to, ritaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones (including but not limited to epothilone A, epothilone B, discodermolide); estramustine, nocodazole, and the like.

[0132] Hormone modulating agents and steroids (including synthetic analogs) suitable for use include, but are not limited to, corticosteroids such as prednisone, dexamethasone, and the like; estrogens and pregestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, and the like; and adrenocortical suppressants, aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex®. Estrogen stimulates proliferation and differentiation; therefore, compounds that bind to the estrogen receptor are used to block this activity.

[0133] Other suitable chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, and the like; urea-based compounds such as hydroxyurea; and hydrazine-based compounds such as N-methylhydrazine; epidophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, and the like. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF105685); Iressa® (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline), and the like.

[0134] Taxanes are suitable for use. "Taxanes" includes paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives, such as docetaxel, TAXOL™, TAXOTERE™ (formulations of docetaxel), the 10-desacetyl analog of paclitaxel, and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel) may be readily prepared using a variety of techniques known to those skilled in the art (WO 94 / 07882, WO 94 / 07881). see also WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076, U.S. Pat. Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529, and European Patent No. 590,267), or from various commercial sources, such as, for example, Sigma-Aldrich. Chemical Co. (St. Louis, Mo.) (T7402 from Taxus brevifolia, or T-1912 from Taxus yannanensis).

[0135] Paclitaxel should be understood to refer not only to the common chemically available forms of paclitaxel, but also to analogs and derivatives (e.g., TAXOTERE™ docetaxel, as noted above), and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0136] The term "taxane" also includes various known derivatives, including both hydrophilic and hydrophobic derivatives.Taxane derivatives include but are not limited to the galactose derivatives and mannose derivatives described in WO 99 / 18113; piperazino derivatives and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451 and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivatives described in U.S. Patent No. 5,415,869.Further included are various prodrugs of paclitaxel, including but not limited to the various prodrugs described in WO 98 / 58927, WO 98 / 13059 and U.S. Patent No. 5,824,701.

[0137] Antibody production method The subject antibodies can be produced by any known method, such as conventional synthetic methods for protein synthesis, recombinant DNA methods, and the like.

[0138] When the subject antibodies are single-chain polypeptides, they can be synthesized using standard chemical peptide synthesis techniques. When a polypeptide is chemically synthesized, the synthesis can be carried out via liquid phase or solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is bound to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence, is an example of a suitable method for chemically synthesizing the subject antibodies. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing the subject antibodies. Various techniques for solid-phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 (The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield et al., J. Am. Chem. Soc., 85: 2149-2156 (1963)); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10, and Camarero JA et al. 2005 Protein Pept Lett. 12: 723-8. Briefly, small, insoluble, porous beads are treated with functional units from which peptide chains are assembled. After repeated coupling / deprotection cycles, the free N-terminal amine of the bound solid phase is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which an additional amino acid may be attached. The peptide remains immobilized on the solid phase and undergoes a filtration process followed by cleavage.

[0139] Various standard recombinant methods can be used to produce the subject antibodies. For example, nucleic acids encoding the light chain variable region and heavy chain variable region, optionally linked to a constant region, are inserted into an expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure expression of immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated promoters or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. The expression control sequences can be eukaryotic promoter systems in vectors that can effect transformation of or transfection into eukaryotic host cells (e.g., COS or CHO cells). Once the vector is incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences, and the recovery and purification of the antibody.

[0140] Due to the degeneracy of the code, a variety of nucleic acid sequences can encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence can be produced by de novo solid-phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of a previously prepared variant of the desired polynucleotide. Oligonucleotide-mediated mutagenesis is an example of a suitable method for preparing substitution, deletion, and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, the target polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, a DNA polymerase is used to incorporate the oligonucleotide primer and synthesize an entire second, complementary strand of the template that encodes the selected alteration in the target polypeptide DNA.

[0141] Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selectable markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline-resistance, kanamycin-resistance, or neomycin-resistance) to permit detection of those cells transformed with the desired DNA sequences.

[0142] Escherichia coli is an example of a prokaryotic host cell that can be used to clone polynucleotides encoding the subject antibodies. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, as well as other Enterobacteriaceae, such as Salmonella spp., Serratia spp., and various Pseudomonas spp. In these prokaryotic hosts, expression vectors can also be made, which will typically contain expression control sequences (e.g., origins of replication) compatible with the host cell. In addition, many different well-known promoters will exist (e.g., the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or promoter systems from phage lambda, etc.). The promoter will typically control expression, optionally along with an operator sequence, and will also have ribosome binding site sequences, etc., for initiating and completing transcription and translation.

[0143] Other microorganisms, such as yeast, are also useful for expression. The genera Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells, in which case suitable vectors will have expression control sequences (e.g., promoters), origins of replication, and termination sequences, as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization, among others.

[0144] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) can also be used to express and produce the polypeptides of the present invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0145] Once synthesized (whether chemically or recombinantly), intact antibodies, dimers thereof, individual light and heavy chains, or other forms of the subject antibodies (e.g., scFv, etc.) can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)). The subject antibodies can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% or more pure, and can be free from contaminants such as cellular debris, macromolecules other than the subject antibodies, etc.

[0146] composition The present disclosure provides compositions comprising the subject antibodies. The subject antibody compositions can include, in addition to the subject antibodies, one or more of the following: salts, such as NaCl, MgCl, KCl, MgSO, etc.; buffering agents, such as Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizing agents; surfactants, such as non-ionic surfactants (e.g., Tween-20, etc.); protease inhibitors; glycerol; and similar components.

[0147] nucleic acid The present disclosure provides nucleic acids comprising nucleotide sequences encoding the subject antibodies, which can be operably linked to one or more regulatory elements (e.g., promoters, enhancers, etc.) that allow for expression of the nucleotide sequences in intended target cells (e.g., cells that are genetically engineered to synthesize the encoded antibody).

[0148] A variety of suitable promoter elements and enhancer elements are known in the art.For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc.For expression in eukaryotic cells, suitable promoters include, but are not limited to, the promoter and enhancer elements of light and / or heavy chain immunoglobulin genes; the immediate early promoter of cytomegalovirus; the thymidine kinase promoter of herpes simplex virus; the early and late SV40 promoters; the promoters present in the long terminal repeat sequences of retroviruses; the mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.

[0149] In some embodiments, for example, for expression in yeast cells, suitable promoters are constitutive promoters, such as the ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, and the like, or regulatable promoters, such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHO5 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art.

[0150] Suitable promoters for use in prokaryotic host cells include the RNA polymerase promoter of bacteriophage T7; the trp promoter; the lac operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter and similar promoters; the araBAD promoter; in vivo regulated promoters, such as the ssaG promoter or related promoters (see, e.g., U.S. Patent Application Publication No. 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991:173(1):86-93; Alpuche-Aranda et al., PNAS, 1992;89(21):10079-83), the nirB promoter (Harborne et al. (1992), Mol. Micro. 6:2805-2813), and similar promoters (e.g., Dunstan et al. (1999), Infect. Immun. 67:5133-5141; McKelvie et al. (2004), Vaccine 22:3243-3255; and Chatfield et al. (1992), Biotechnol. 10:888-892); sigma 70 promoters, such as the consensus sigma 70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); stationary phase promoters, such as the dps promoter and spv promoter; promoters from the pathogenic isolate SPI-2 (see, e.g., International Publication No. WO 96 / 17951); actA promoter (see, e.g., Shetron-Rama et al. al. (2002), Infect. Immun. 70:1087-1096); the rpsM promoter (see, e.g., Valdivia and Falkow (1996), Mol. Microbiol. 22:367); the tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989), In Saenger, W.and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al. (1984), Nucl. Acids Res. 12:7035); and similar promoters. Suitable strong promoters for use in prokaryotes such as E. coli include Trc, Tac, T5, T7 and P. Lambda Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation when contacted with lactose, thereby preventing the LacI repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al. (1983), Proc. Natl. Acad. Sci. USA 80:21-25).

[0151] The nucleotide sequence encoding the subject antibody can be present in an expression vector and / or a cloning vector. If the subject antibody comprises two separate polypeptides, the nucleotide sequences encoding these two polypeptides can be cloned into the same vector or separate vectors. Expression vectors can include selectable markers, origins of replication, and other features that provide for replication and / or maintenance of the vector.

[0152] Numerous suitable vectors and promoters are known to those of skill in the art, and many are commercially available for generating the subject recombinant constructs. The following vectors are provided as examples: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0153] Expression vectors generally contain convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker that is functional in the expression host may be present. Suitable expression vectors include viral vectors (e.g., vaccinia virus-based viral vectors; poliovirus-based viral vectors; adenovirus-based viral vectors (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655); adeno-associated virus-based viral vectors (see, e.g., Ali et al., al.,Hum Gene Ther 9:81 86,1998, Flannery et al.,PNAS 94:6916 6921,1997;Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863,1997;Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al. al.,Hum Gene Ther 10:641 648,1999;Ali et al.,Hum Mol Genet 5:591 594,1996;Srivastava, WO 93 / 09239;Samulski et al.,J.Vir.(1989)63:3822-3828;Mendelson et al. al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40-based viral vectors; herpes simplex virus-based viral vectors; human immunodeficiency virus-based viral vectors (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses (e.g., Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and similar viral vectors.

[0154] As noted above, the subject nucleic acids include nucleotide sequences encoding the subject antibodies. The subject nucleic acids can include nucleotide sequences encoding the anti-GPC3 heavy chain and the anti-GPC3 light chain, as described above.

[0155] In certain aspects, the nucleotide sequence encoding the anti-GPC3 heavy chain may comprise a nucleotide sequence having at least 50% identity (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to SEQ ID NO: 11 or SEQ ID NO: 14.

[0156] In certain aspects, the nucleotide sequence encoding the anti-GPC3 light chain may comprise a nucleotide sequence having at least 50% identity to SEQ ID NO: 12 (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity).

[0157] cell The present disclosure provides isolated genetically modified host cells (e.g., in vitro cells) that are genetically modified with a subject nucleic acid. In some embodiments, a subject isolated genetically modified host cell is capable of producing a subject antibody.

[0158] Suitable host cells include eukaryotic host cells, such as mammalian cells, insect host cells, yeast cells, etc., and prokaryotic cells, such as bacterial cells, etc. Introduction of the subject nucleic acids into host cells can be accomplished, for example, by calcium phosphate precipitation, DEAE-dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods.

[0159] Suitable mammalian cells include primary cells and immortalized cell lines, including human cell lines, non-human primate cell lines, and rodent (e.g., mouse, rat) cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, and CRL9096), Vero cells, NIH3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) 293 cells (ATCC No. CRL1573), and HLHepG2 cells.

[0160] Suitable yeast cells include Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp. Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp. sp.), Fusarium gramineum, Fusarium venenatum, Neurospora crassa, and Chlamydomonas reinhardtii.

[0161] Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus species, Salmonella species, and Shigella species. See, e.g., Carrier et al. (1992), J. Immunol. 148:1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995), Science 270:299-302. Examples of Salmonella strains that can be used in the present invention include, but are not limited to, Salmonella typhi (Salmonella typhi) and S. typhimurium (Salmonella typhi). Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, laboratory strains are non-pathogenic. Non-limiting examples of other suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, and Rhodococcus sp. In some embodiments, the host cell is Escherichia coli.

[0162] Pharmaceutical Composition The present disclosure provides compositions (including pharmaceutical compositions) comprising the subject antibodies. Generally, the formulations contain an effective amount of the subject antibodies. An "effective amount" refers to a dosage sufficient to bring about a desired result (e.g., a reduction in the number of cancerous cells). In some cases, the desired result is at least a reduction in the symptoms of a malignant tumor when compared to a control.

[0163] compound In the subject method, the subject antibody can be administered to the host using any convenient means that can produce the desired therapeutic or diagnostic effect.Therefore, the agent can be incorporated into various formulations for therapeutic administration.More specifically, the subject antibody can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable carrier or diluent, and can also be formulated into preparations in solid, semi-solid, liquid, or gaseous form (e.g., tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, aerosols, etc.).

[0164] In pharmaceutical dosage forms, the subject antibodies may be administered in the form of their pharmaceutically acceptable salts, or the subject antibodies may also be used alone or in appropriate association with other pharmaceutically active compounds, as well as in combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and in no way limiting.

[0165] For oral preparations, the subject antibodies can be used alone or in combination with suitable excipients for producing tablets, powders, granules, or capsules, such as conventional excipients (e.g., lactose, mannitol, corn starch, or potato starch, etc.), binders (e.g., crystalline cellulose, cellulose derivatives, gum arabic, corn starch, or gelatin, etc.), disintegrating agents (e.g., corn starch, potato starch, or sodium carboxymethylcellulose, etc.), lubricants (e.g., talc or magnesium stearate, etc.), and, if desired, diluents, buffers, wetting agents, preservatives, and flavoring agents.

[0166] The subject antibodies can be formulated into preparations for injection by dissolving, suspending, or emulsifying the antibodies in aqueous or non-aqueous solvents (e.g., vegetable oils or other similar oils, synthetic aliphatic acid glycerides, esters of higher fatty acids or propylene glycol, etc.) and, if desired, with conventional additives (e.g., solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizers, preservatives, etc.).

[0167] Pharmaceutical compositions containing the subject antibodies are prepared by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or isotonicity agents. Acceptable carriers, excipients, and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isopropyl alcohol, PEG-14 ... leucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants, such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0168] Pharmaceutical compositions can be in liquid form, freeze-dried form, or liquid form reconstituted from freeze-dried form, but freeze-dried preparations must be reconstituted with a sterile solution before administration.The standard procedure for reconstituting freeze-dried compositions is to add back a certain volume of pure water (typically equal to the volume removed during freeze-drying).However, solutions containing antibacterial agents may be used to prepare pharmaceutical compositions for parenteral administration; see also Chen (1992), Drug Dev Ind Pharm 18, 1311-54.

[0169] Exemplary antibody concentrations in the subject pharmaceutical compositions can range from about 1 mg / mL to about 200 mg / mL, or from about 50 mg / mL to about 200 mg / mL, or from about 150 mg / mL to about 200 mg / mL.

[0170] Aqueous antibody formulations may be prepared in pH buffered solutions, e.g., at a pH in the range of about 4.0 to about 7.0, or about 5.0 to about 6.0, or alternatively at a pH of about 5.5. Examples of buffers suitable for pHs within this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The buffer concentration can be about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending, for example, on the buffer and the desired tonicity of the formulation.

[0171] An isotonicity agent may be included in the antibody formulation to adjust the tonicity of the formulation. Exemplary isotonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be preferred. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is being compared (e.g., physiological saline solution or serum). The isotonicity agent may be used in an amount of about 5 mM to about 350 mM, e.g., 100 mM to 350 nM.

[0172] Surfactants may also be added to antibody formulations to reduce aggregation of the formulated antibody, minimize the formation of particulates in the formulation, and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactants can range from about 0.001% to about 1% w / v.

[0173] Cryoprotectants may also be added to protect unstable active ingredients (e.g., proteins) from destabilizing conditions during the freeze-drying process. For example, known cryoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). Cryoprotectants may be included in amounts of about 10 mM to 500 nM.

[0174] In some embodiments, a subject formulation comprises a subject antibody and one or more of the above-identified agents (e.g., surfactants, buffers, stabilizers, tonicity agents), and is essentially free of one or more preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, combinations thereof, etc.) In other embodiments, a preservative is included in the formulation, e.g., at a concentration ranging from about 0.001 to about 2% (w / v).

[0175] For example, a subject formulation can be a liquid or lyophilized formulation suitable for parenteral administration and can include from about 1 mg / mL to about 200 mg / mL of a subject antibody, from about 0.001% to about 1% of at least one surfactant, from about 1 mM to about 100 mM of buffering agent, optionally from about 10 mM to about 500 mM of stabilizer, and from about 5 mM to about 305 mM of tonicity agent, and has a pH of from about 4.0 to about 7.0.

[0176] As another example, a subject parenteral formulation is a liquid or lyophilized formulation comprising about 1 mg / mL to about 200 mg / mL of a subject antibody, 0.04% Tween 20 (w / v), 20 mM L-histidine, and 250 mM sucrose, and has a pH of 5.5.

[0177] As another example, a subject parenteral formulation comprises: 1) a lyophilized formulation comprising 15 mg / mL of a subject antibody, 0.04% Tween 20 (w / v), 20 mM L-histidine, and 250 mM sucrose, at a pH of 5.5; or 2) a lyophilized formulation comprising 75 mg / mL of a subject antibody, 0.04% Tween 20 (w / v), 20 mM L-histidine, and 250 mM sucrose, at a pH of 5.5; or 3) a lyophilized formulation comprising 75 mg / mL of a subject antibody, 0.02% Tween 20 (w / v), 20 mM L-histidine, and 250 mM sucrose, at a pH of 5.5. , 20 mM L-histidine, and 250 mM sucrose, having a pH of 5.5; or 4) a lyophilized formulation comprising 75 mg / mL of a subject antibody, 0.04% Tween 20 (w / v), 20 mM L-histidine, and 250 mM trehalose, having a pH of 5.5; or 6) a lyophilized formulation comprising 75 mg / mL of a subject antibody, 0.02% Tween 20 (w / v), 20 mM L-histidine, and 250 mM trehalose, having a pH of 5.5.

[0178] As another example, a subject parenteral formulation is: 1) a liquid formulation comprising 7.5 mg / mL of a subject antibody, 0.022% Tween 20 (w / v), 120 mM L-histidine, and 250-125 mM sucrose, having a pH of 5.5; or 2) a liquid formulation comprising 37.5 mg / mL of a subject antibody, 0.02% Tween 20 (w / v), 10 mM L-histidine, and 125 mM sucrose, having a pH of 5.5; or 3) a liquid formulation comprising 37.5 mg / mL of a subject antibody, 0.01% Tween 20 (w / v), 10 mM L-histidine, and 125 mM sucrose, having a pH of 5.5; or 4) a liquid formulation comprising 37.5 mg / mL of a subject antibody, 0.01% Tween 20 (w / v), 10 mM L-histidine, and 125 mM sucrose, having a pH of 5.5. a liquid formulation comprising an antibody, 0.02% Tween 20 (w / v), 10 mM L-histidine, and 125 mM trehalose, having a pH of 5.5; or 5) a liquid formulation comprising 37.5 mg / mL of the subject antibody, 0.01% Tween 20 (w / v), 10 mM L-histidine, and 125 mM trehalose, having a pH of 5.5; or 6) a liquid formulation comprising 5 mg / mL of the subject antibody, 0.02% Tween 20 (w / v), 20 mM L-histidine, and 250 mM trehalose. or 7) a liquid formulation comprising 75 mg / mL of a subject antibody, 0.02% Tween 20 (w / v), 20 mM L-histidine, and 250 mM mannitol, having a pH of 5.5; or 8) a liquid formulation comprising 75 mg / mL of a subject antibody, 0.02% Tween 20 (w / v), 20 mM L-histidine, and 140 mM sodium chloride, having a pH of 5.5; or 9) a liquid formulation comprising 150 mg / mL of a subject antibody, 0.02% Tween 20 (w / v), 20 mM L-histidine, and 140 mM sodium chloride, having a pH of 5.5. 10) a liquid formulation comprising 150 mg / mL of a subject antibody, 0.02% Tween 20 (w / v), 20 mM L-histidine, and 250 mM trehalose, having a pH of 5.5; or 11) a liquid formulation comprising 150 mg / mL of a subject antibody, 0.02% Tween 20 (w / v), 20 mM L-histidine, and 250 mM mannitol, having a pH of 5.5.5, or 12) a liquid formulation comprising 10 mg / mL of a subject antibody, 0.01% Tween 20 (w / v), 20 mM L-histidine, and 40 mM sodium chloride, having a pH of 5.5.

[0179] The subject antibodies can be utilized in aerosol formulations to be administered via inhalation. The subject antibodies can be formulated into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0180] Furthermore, the subject antibodies can be made into suppositories by mixing with various bases (e.g., emulsifying bases or water-soluble bases). The subject antibodies can be administered rectally via suppositories. Suppositories can include vehicles that melt at body temperature yet solidify at room temperature (e.g., cocoa butter, carbowax, polyethylene glycol, etc.).

[0181] Unit dosage forms for oral or rectal administration may be provided, such as syrups, elixirs, and suspensions, wherein each dosage unit (e.g., teaspoon, tablespoon, tablet, or suppository) contains a predetermined amount of a composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the subject antibodies in a composition as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.

[0182] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each containing a predetermined quantity of a compound of the invention calculated in an amount sufficient to produce a desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the subject antibodies can depend on the particular antibody employed and the effect to be achieved, as well as the pharmacodynamics associated with each antibody in the host.

[0183] Other modes of administration may also find use with the present invention. For example, the subject antibodies may be formulated into suppositories, and in some cases, into aerosol and intranasal compositions. For suppositories, the vehicle composition may include traditional binders and carriers, such as polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w / w), for example, from mixtures containing the active ingredient in the range of about 1% to about 2%.

[0184] Intranasal formulations will typically contain a vehicle that neither causes irritation to the nasal mucosa nor significantly interferes with ciliary function. Various diluents (e.g., water, saline solution, or other known substances) can be used with the present invention. Nasal formulations may also contain preservatives (e.g., but not limited to, chlorobutanol and benzalkonium chloride). A surfactant may be present to enhance absorption of the subject protein by the nasal mucosa.

[0185] The subject antibody can be administered as an injectable formulation. Typically, injectable compositions are prepared as liquid solutions or suspensions. Solid forms suitable for dissolving or suspending in liquid vehicles prior to injection may also be prepared. Preparations may also be emulsified, or the antibody may be encapsulated in liposome vehicles.

[0186] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, or pH buffering agents. Various practical methods for preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, Pennsylvania, 17th ed., 1985). In any case, the composition or formulation to be administered will contain a sufficient amount of the subject antibody to achieve the desired state in the subject being treated.

[0187] Pharmaceutically acceptable excipients (e.g., vehicles, adjuvants, carriers, or diluents, etc.) are readily available to the public. Moreover, a variety of pharmaceutically acceptable auxiliary substances (e.g., pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like) are readily available to the public.

[0188] In some embodiments, the subject antibodies are formulated in controlled-release formulations. Sustained-release preparations may be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. Potential loss of biological activity and potential changes in immunogenicity of antibodies contained in sustained-release preparations may be prevented by using appropriate additives, controlling the water content, and developing specific polymer matrix compositions.

[0189] Controlled release within the scope of the present invention can be construed to mean any one of a number of extended-release dosage forms. The following terms may be considered substantially equivalent to controlled release for purposes of the present invention: continuous release, controlled release, delayed release, depot, gradual release, extended release, programmed release, prolonged release, proportional release, prolonged release, repository, retard, slow release, interval release, sustained release, time coating, timed release, delayed action, extended action, layered time action, long-acting, prolonged action, repeated action, slow acting, sustained action, sustained action medication, and extended release. Further discussion of these terms can be found in Lesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

[0190] A wide variety of controlled release technologies cover a wide range of drug dosage forms, including, but not limited to, physical and chemical systems.

[0191] Physical systems include, but are not limited to, reservoir systems using rate-controlling membranes, such as microencapsulation, macroencapsulation, and membrane systems; reservoir systems without rate-controlling membranes, such as hollow fibers, ultraporous cellulose triacetate, and porous polymer substrates and foams; monolithic systems, including such systems physically dissolved in a non-porous, polymeric, or elastomeric matrix (e.g., non-erodible, erodible, environmentally permeable, and degradable), and materials physically dispersed in a non-porous, polymeric, or elastomeric matrix (e.g., non-erodible, erodible, environmentally permeable, and degradable); laminated structures containing a reservoir layer that is chemically similar or dissimilar to an outer controlling layer; and other physical methods, such as osmotic pumps, or adsorption onto ion exchange resins.

[0192] Chemical systems include, but are not limited to, chemical erosion of a polymer matrix (e.g., heterogeneous or homogeneous erosion) or biological erosion of a polymer matrix (e.g., heterogeneous or homogeneous). Further discussion of various categories of systems for controlled release can be found in Agis F. Kydonieus, Controlled Release Technologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

[0193] There are many controlled release drug formulations developed for oral administration.These include but are not limited to osmotic pressure controlled gastrointestinal delivery system; hydrodynamic pressure controlled gastrointestinal delivery system; membrane permeability controlled gastrointestinal delivery system, including microporous membrane permeability controlled gastrointestinal delivery device; gastric juice resistant intestinal targeted controlled release gastrointestinal delivery device; gel diffusion controlled gastrointestinal delivery system; and ion exchange controlled gastrointestinal delivery system, including cationic drug and anionic drug.More information about controlled release drug delivery system can be found in Yie W.Chien, Novel Drug Delivery Systems, 1992 (Marcel Dekker, Inc.).

[0194] Dosage The appropriate dosage can be determined by the attending physician or other qualified medical professional based on various clinical factors. As is well known in the medical field, the dosage for any single patient depends on many factors, including the patient's size, body surface area, age, the particular compound being administered, the patient's sex, the time and route of administration, general health, and other drugs being administered concomitantly. The subject antibodies may be administered in amounts between 1 ng / kg body weight and 20 mg / kg body weight per dose, e.g., between 0.1 mg / kg body weight and 10 mg / kg body weight, e.g., between 0.5 mg / kg body weight and 5 mg / kg body weight; however, doses below or above this exemplary range are contemplated, taking into account, among other factors, the foregoing. If the treatment regimen is a continuous infusion, the dosage can also be in the range of 1 μg to 10 mg per kilogram of body weight per minute.

[0195] Those of skill in the art will readily understand that dosage levels can vary as a function of the particular antibody, the severity of the symptoms, and the subject's susceptibility to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0196] Route of administration The subject antibodies are administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.

[0197] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined or adjusted, if desired, depending on the antibody and / or the desired effect. The subject antibody compositions can be administered in a single dose or in multiple doses. In some embodiments, the subject antibody compositions are administered orally. In some embodiments, the subject antibody compositions are administered by the inhalation route. In some embodiments, the subject antibody compositions are administered intranasally. In some embodiments, the subject antibody compositions are administered topically. In some embodiments, the subject antibody compositions are administered intracranially. In some embodiments, the subject antibody compositions are administered intravenously.

[0198] Agents can be administered to a host using any available conventional method and route suitable for the delivery of conventional drugs, including systemic or localized routes. Generally, routes of administration contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

[0199] Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intrahepatic, and intravenous routes, i.e., any route of administration other than via the digestive tract. Parenteral administration can be used to achieve systemic or local delivery of the subject antibodies. When systemic delivery is desired, administration typically involves topical or mucosal administration of the pharmaceutical preparation invasively or for systemic absorption.

[0200] The subject antibodies can also be delivered to a subject by enteral administration, including, but not necessarily limited to, oral delivery and rectal delivery (e.g., using a suppository).

[0201] By treatment is meant at least an amelioration of symptoms associated with the pathological condition afflicting the host, where amelioration is used broadly to indicate at least a reduction in the magnitude of a parameter (e.g., symptom) associated with the pathological condition (e.g., B-cell malignancy or B-cell-mediated autoimmune disorder, etc.) being treated. As such, treatment also includes situations in which the pathological condition, or at least the symptoms associated with the pathological condition, are completely suppressed, e.g., prevented from occurring, or arrested, e.g., terminated, such that the host no longer suffers from the pathological condition, or at least no longer suffers from the symptoms characterizing the pathological condition.

[0202] In some embodiments, the subject antibodies are administered by injection, eg, for systemic delivery (eg, intravenous infusion) or to a local site.

[0203] A variety of hosts (wherein the term "host" is used interchangeably herein with the terms "subject," "individual," and "patient") can be treated according to the subject methods. Generally, such hosts are "mammals" or "mammals," where these terms are used broadly to describe organisms within the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the host will be a human.

[0204] Kits are provided that contain a unit dose of the subject antibodies, e.g., in an oral or injectable dose. In such kits, in addition to the container containing the unit dose, there will be an informational package insert describing the use and associated benefits of the antibody in treating the pathological condition of interest. Preferred compounds and unit doses are those described hereinabove.

[0205] Treatment method The present disclosure provides a method for treating a disease or disorder associated with or caused by GPC3-positive cells, e.g., cancerous GPC3-positive cells, autoreactive GPC3-positive cells, Treatment of malignant tumors The present disclosure provides methods of treating malignancies, including solid tumors or hematological malignancies, which generally involve administering to an individual in need thereof (e.g., an individual having a malignancy) an effective amount of a subject antibody, alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy).

[0206] Malignant tumors include, for example, HCC, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, chronic lymphocytic leukemia, hairy cell leukemia, prolymphocytic leukemia, anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, cancer of unknown primary site (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, and vulvar cancer.

[0207] In some embodiments, an effective amount of a subject antibody is an amount that, when administered alone (e.g., in monotherapy), or in combination with one or more additional therapeutic agents (e.g., in combination therapy), whether in one or more doses, is effective to reduce the number of cancerous cells in an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, relative to the number of cancerous cells in an individual not treated with the antibody.

[0208] Combination therapy In some embodiments, the subject methods of treating malignancies involve administering a subject antibody and one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, cancer chemotherapeutic agents (as described above).

[0209] Suitable subjects for treatment Various subjects are suitable for treatment by the subject method. Suitable subjects include any individual with a malignant tumor (e.g., human); an individual (e.g., human) who has been diagnosed with a malignant tumor; an individual (e.g., human) who has had a malignant tumor and is at risk for the recurrence of the malignant tumor; an individual (e.g., human) who has been treated for a malignant tumor with a drug other than the subject anti-GPC3 antibody (e.g., treated with a cancer chemotherapy drug) and has not responded to the drug; or an individual (e.g., human) who has been treated for a malignant tumor with a drug other than the subject anti-GPC3 antibody (e.g., treated with a cancer chemotherapy drug) and has initially responded to the drug, but then has become unresponsive (e.g., relapse).

[0210] Detection Method The present disclosure provides various detection methods that involve using the subject antibodies. Detection methods include diagnostic, prognostic, and monitoring methods. The subject detection methods generally involve detecting GPC3-positive cells (e.g., cancerous cells).

[0211] In some embodiments, the subject methods are diagnostic methods, eg, for determining whether an individual has a malignant tumor.

[0212] In some embodiments, the subject methods are monitoring methods, e.g., an individual who has been diagnosed with a malignancy and is undergoing treatment for the disorder is monitored for response of the disorder to the treatment and / or progression / regression of the disorder.

[0213] In some cases, the subject detection method involves administering the detectably labeled anti-GPC3 antibody of the present disclosure to an individual and detecting the binding of the antibody to tissue in the individual.Detection can be achieved, for example, by magnetic resonance imaging or other suitable imaging techniques.

[0214] In another example, the subject detection method involves contacting a detectably labeled anti-GPC3 antibody of the present disclosure with a biological sample obtained from an individual and detecting binding of the antibody to a molecule in the biological sample.

[0215] Anti-GPC3 antibody can be directly or indirectly labeled.Indirect labeling includes the secondary antibody that contains detectable label, and in this case, the secondary antibody binds with the subject anti-GPC3 antibody.Other indirect labeling includes biotin, and in this case, the biotinylated anti-GPC3 antibody can be detected using avidin or streptavidin that contains detectable label.

[0216] Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Suitable labels include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and similar fluorescent dyes), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase and other enzymes commonly used in enzyme-linked immunosorbent assays (ELISAs)), and colorimetric labels, such as colloidal gold or colored beads of glass or plastic (e.g., polystyrene, polypropylene, latex, etc.).

[0217] In some embodiments, the subject antibodies comprise an imaging agent or radioisotope, where the imaging agent or radioisotope is one that is suitable for use in imaging, e.g., in imaging procedures performed on humans. Non-limiting examples of labels include radioisotopes, e.g., 1231 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium) and 67 Ga (gallium), and contrast agents such as gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotopes ( 153Radioisotopes (Gd) are also available and suitable for imaging procedures in mammals other than humans. The subject antibodies can be labeled using standard techniques. For example, the subject antibodies can be iodinated using chloramine T or 1,3,4,6-tetrachloro-3α,6α-dephenylglycouril. For fluorination, fluorine is added to the subject antibodies during synthesis by a fluoride ion displacement reaction. For reviews of protein synthesis using such radioisotopes, see Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244 (1999). The subject antibodies can also be labeled with imaging agents via standard techniques. For example, the subject antibodies can be labeled with Gd by conjugating a low molecular weight Gd chelate, such as Gd-diethylenetriaminepentaacetic acid (GdDTPA) or Gd-tetraazacyclododecanetetraacetic acid (GdDOTA), to the antibody. See Caravan et al., Chem. Rev. 99:2293-2352 (1999), and Lauffer et al., J. Magn. Reson. Imaging, 3:11-16 (1985). The subject antibodies can be labeled with Gd by conjugating, for example, a polylysine-Gd chelate to the antibody. See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998). Alternatively, the subject antibodies can be labeled with Gd by incubating biotinylated antibodies with paramagnetic polymeric liposomes containing a Gd chelator lipid in combination with avidin. See, e.g., Sipkins et al., Nature Med., 4:623-626 (1998).

[0218] Suitable fluorescent proteins that can be linked to the subject antibodies include green fluorescent protein from Aequoria victoria or variants or derivatives thereof, such as those described in U.S. Patent Nos. 6,066,476, 6,020,192, 5,985,577, 5,976,796, 5,968,750, 5,968,738, 5,958,713, 5,919,445, and 5,874,304; enhanced GFP, many of which are commercially available, e.g., from Clontech, Inc.; red fluorescent protein; yellow fluorescent protein; any of a variety of fluorescent and colored proteins obtained from species of Anthozoa, e.g., Matz et al. (1999), Nature 106:101-104, 1999; Biotechnol. 17:969-973; and similar fluorescent proteins.

[0219] kit The present disclosure provides kits (e.g., test kits) that include the subject antibodies. The subject kits are useful for performing the subject detection methods.

[0220] The subject kits can include one or more of the subject antibodies, nucleic acids encoding the subject antibodies, or cells containing the subject nucleic acids. The subject antibodies in the subject kits can be humanized. The subject kits can include a reagent for labeling the antibody. In some embodiments, the antibodies in the subject kits include a detectable label.

[0221] Other optional components of the kit include buffers, protease inhibitors, detectable labels, etc. Where the subject kits include the subject nucleic acids, the nucleic acids may also have restriction sites, multiple cloning sites, primer sites, etc. The various components of the kit may be present in separate containers, or certain compatible components may be pre-combined in a single container, as desired.

[0222] In addition to the components described above, the subject kits can include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are typically recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. As such, the instructions may be present in the kit as a package insert, such as on the labeling of the container of the kit or its components (i.e., on labeling associated with the packaging or separate packaging). In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium (e.g., a compact disc read-only memory (CD-ROM), a digital versatile disc (DVD), a diskette, etc.). In still other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0223] [Example] The following examples are written so as to provide those of ordinary skill in the art with a complete disclosure and description of how the present invention can be made and used, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation must be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used: e.g., bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular (intramuscular); ip, intraperitoneal (intraperitoneal); sc, subcutaneous (subcutaneous); etc. Commercially available reagents referred to in the examples were used according to manufacturer's instructions, unless otherwise indicated. The source of cells identified in the examples and throughout the specification by ECACC accession numbers is the European Collection of Cell Cultures (ECACC), Salisbury, UK. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting in scope.

[0224] [Example 1] CAT-07 monoclonal antibody CAT-07 is a human IgG1 kappa monoclonal antibody against human glypican-3. To determine aggregation, CAT-07-containing samples were analyzed using analytical size-exclusion chromatography (SEC; Tosoh #08541) with a mobile phase of 300 mM NaCl, 25 mM sodium phosphate (pH 6.8). Figure 1 shows that the CAT-07 monoclonal antibody is greater than 99% monomeric, as determined by size-exclusion chromatography (SEC).

[0225] To perform the ELISA, Maxisorp 96-well plates (Nunc) were coated with 1 μg / mL human glypican-3-His (R&D Systems) in PBS overnight at 4°C. The plates were blocked with casein buffer (ThermoFisher), and then CAT-07 was added to the plates in 11 two-fold dilutions starting at 150 ng / mL. The plates were incubated at room temperature for 2 h with shaking. After washing with 0.1% Tween-20 in PBS, bound analytes were detected with a donkey anti-human Fc-γ-specific horseradish peroxidase (HRP)-conjugated secondary antibody. The signal was visualized with Ultra TMB (Pierce) and stopped with 2N H2SO4. Absorbance at 450 nm was measured using a Molecular Devices SpectraMax M5 plate reader, and data were analyzed using GraphPad Prism. FIG. 2 shows that the CAT-07 monoclonal antibody binds to recombinant human glypican-3 protein as assessed by ELISA.

[0226] [Example 2] Binding specificity of CAT-07 monoclonal antibody To perform the ELISA, Maxisorp 96-well plates (Nunc) were coated overnight at 4°C with 1 μg / mL of His-tagged human glypican proteins (glypican-1, glypican-2, glypican-3, glypican-5, and glypican-6, all from R&D Systems) in PBS. The plates were blocked with casein buffer (ThermoFisher), and then CAT-07 was added to the plates in 11 two-fold dilutions starting at 150 ng / mL. The plates were incubated with shaking at room temperature for 2 h. After washing with 0.1% Tween-20 in PBS, bound analytes were detected with a donkey anti-human Fc-γ-specific horseradish peroxidase (HRP)-conjugated secondary antibody. The signal was visualized with Ultra TMB (Pierce) and stopped with 2N H2SO4. Absorbance at 450 nm was determined using a Molecular Devices SpectraMax M5 plate reader and data were analyzed using GraphPad Prism. Figure 3 shows that CAT-07 binds to glypican-3, but not other human glypican proteins, as assessed by ELISA.

[0227] To conduct species cross-reactivity studies, cell line reagents expressing human, cynomolgus monkey, rat, or mouse glypican-3 proteins were generated by stable transfection of CHO-K1 cells with a plasmid encoding the protein, followed by selection in hygromycin. The resulting cell pools were used for flow cytometry studies as follows: Cells were harvested using TrypLE (ThermoFisher), placed in PBS containing 2% heat-inactivated FBS, and incubated with 1 μg of CAT-07 per 100 μL of cells (0.5 e6 cells / test) on ice for 1 hour. Next, cells were washed once with PBS + 2% FBS and incubated with fluorescein-conjugated donkey anti-human Fc secondary reagent [F(ab)2 fragment, Jackson Immunoresearch] on ice for 30 minutes. After two washes with PBS + 2% FBS, cells were analyzed on a Becton Dickinson FACSCanto™ instrument using FACS Diva™ software. Reactivity to wild-type (untransfected cells) was also assessed. Data are reported as the increase in mean fluorescence intensity observed in transfected cells compared to untransfected cells (background). Figure 4. Flow cytometry data confirm that CAT-07 binds to glypican-3 proteins from a variety of species, including cynomolgus monkeys, rats, and mice.

[0228] While the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications, or substitutions of various equivalents, may be made without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. In one aspect, the present invention provides: [Item 1] The variable heavy chain (V) specifically binds to GPC3 and binds to GPC3 as follows:H ) polypeptide and variable light chain (V L ) Polypeptide: V containing the amino acid sequence GYTFTSYFLH (SEQ ID NO: 2) or the amino acid sequence GYTFTSYYMH (SEQ ID NO: 3) H CDR1 and V containing the amino acid sequence IIDPPTGRTTYAQKFQG (SEQ ID NO: 4) H CDR2 and V containing the amino acid sequence GNYGGRYFDY (SEQ ID NO: 5) H CDR3 and Heavy chain (V H ) polypeptide; and V containing the amino acid sequence RASQSISSYLN (SEQ ID NO: 6) L CDR1 and V containing the amino acid sequence AASSLQS (SEQ ID NO: 7) L CDR2 and V containing the amino acid sequence QQSYSTPLT (SEQ ID NO: 8) L CDR3 and Light chains (V L ) Polypeptide an antibody that competes with a second antibody comprising: [Item 2] The antibody that specifically binds to GPC3 has the following variable heavy chain (V H ) polypeptide and variable light chain (V L ) Polypeptide: V containing the amino acid sequence GYTFTSYFLH (SEQ ID NO: 2) or the amino acid sequence GYTFTSYYMH (SEQ ID NO: 3) H CDR1 and V containing the amino acid sequence IIDPPTGRTTYAQKFQG (SEQ ID NO: 4) H CDR2 and V containing the amino acid sequence GNYGGRYFDY (SEQ ID NO: 5) H CDR3 and Heavy chain (V H ) polypeptide; and V containing the amino acid sequence RASQSISSYLN (SEQ ID NO: 6) L CDR1 and V containing the amino acid sequence AASSLQS (SEQ ID NO: 7) L CDR2 and V containing the amino acid sequence QQSYSTPLT (SEQ ID NO: 8) L CDR3 and Light chains (V L ) Polypeptide Item 1. The antibody according to item 1, comprising: [Item 3] The above V H 3. The antibody of item 1 or item 2, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:9. [Item 4] The above V L 4. The antibody according to any one of items 1 to 3, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 10. [Item 5] 5. The antibody according to any one of items 1 to 4, which is a humanized antibody. [Item 6] 6. The antibody according to any one of items 1 to 5, which is a chimeric antibody. [Item 7] 7. The antibody according to any one of items 1 to 6, which is selected from the group consisting of IgG, Fv, single-chain antibody, scFv, Fab, F(ab')2, or Fab'. [Item 8] 7. The antibody according to any one of items 1 to 6, which is an IgG. [Item 9] 9. The antibody of item 8, which is an IgG1. [Item 10] 7. The antibody according to any one of items 1 to 6, which is a Fab. [Item 11] 7. The antibody according to any one of items 1 to 6, which is a single-chain antibody. [Item 12] 12. The antibody of item 11, which is an scFv. [Item 13] A bispecific antibody comprising a first antigen-binding domain that specifically binds to GPC3, wherein the first antigen-binding domain is a V H Polypeptides and V L 13. The antibody of any one of items 1 to 12, comprising a polypeptide. [Item 14] 14. The antibody of any one of items 1 to 13, which is detectably labeled. [Item 15] 15. The antibody of any one of items 1 to 14, comprising a covalently linked synthetic polymer other than a peptide. [Item 16] 16. The antibody of item 15, wherein the synthetic polymer is a poly(ethylene glycol) polymer. [Item 17] 17. The antibody of any one of items 1 to 16, comprising a covalently linked lipid or fatty acid moiety. [Item 18] 15. The antibody of any one of items 1 to 14, comprising a covalently linked polysaccharide or carbohydrate moiety. [Item 19] 19. The antibody of any one of items 1 to 18, comprising an imaging agent. [Item 20] 20. The antibody of any one of items 1 to 19, comprising an affinity domain. [Item 21] 21. The antibody according to any one of items 1 to 20, which is immobilized on a solid support. [Item 22] 22. The antibody of any one of items 1 to 21, comprising a covalently linked cytotoxin. [Item 23] 23. The antibody according to any one of items 1 to 22, comprising a constant region amino acid sequence comprising an amino acid sequence of a sulfatase motif. [Item 24] 23. The antibody of any one of items 1 to 22, comprising a constant region amino acid sequence comprising the amino acid sequence of a sulfatase motif, wherein the sulfatase motif is modified to include a 2-formylglycine (FGly) moiety. [Item 25] 25. The antibody of item 24, comprising a heterologous moiety covalently linked via the FGly moiety. [Item 26] 26. The antibody of item 25, wherein the heterologous moiety is selected from a drug, a toxin, a detectable label, a water-soluble polymer, and a synthetic peptide. [Item 27] The variable heavy chain (V) of the antibody according to any one of items 1 to 13 H ) polypeptide, variable light chain (V L ) polypeptide, or a nucleic acid encoding both. [Item 28] 28. The nucleic acid of item 27, wherein the antibody is a single-chain antibody and encodes the single-chain antibody. [Item 29] 9. The nucleic acid of item 8, wherein the single chain antibody is an scFv. [Item 30] 30. A recombinant expression vector comprising the nucleic acid according to any one of items 27 to 29, wherein the nucleic acid is operably linked to a transcriptional control element active in a eukaryotic cell. [Item 31] A cell comprising the nucleic acid according to any one of Items 27 to 29 or the expression vector according to Item 30. [Item 32] The nucleic acid is a V H V of the polypeptide and the antibody L 32. The cell of item 31, encoding a polypeptide. [Item 33] 33. The cell of item 32, wherein the antibody is a single chain antibody and the nucleic acid encodes the single chain antibody. [Item 34] 34. The cell of item 33, wherein the single-chain antibody is an scFv. [Item 35] The following nucleic acids: The variable heavy chain (V) of the antibody according to any one of items 1 to 13 H ) a first nucleic acid encoding a polypeptide; and The variable light chain (V L ) a second nucleic acid encoding a polypeptide Cells containing [Item 36] In the cell according to Item 35, a first expression vector comprising the first nucleic acid; and a second expression vector comprising the second nucleic acid; Cells containing [Item 37] Contains the following ingredients: The antibody according to any one of items 1 to 13; and an agent conjugated to said antibody A conjugate comprising: [Item 38] 38. The conjugate of item 37, wherein the agent is selected from the group consisting of a half-life extending moiety, a labeling agent, and a therapeutic agent. [Item 39] Contains the following ingredients: The variable heavy chain (V) of the antibody according to any one of items 1 to 13 H ) polypeptide, variable light chain (V L ) polypeptide, or both, fused thereto, Heterologous amino acid sequences and A fusion protein comprising: [Item 40] Contains the following ingredients: a) the antibody according to any one of items 1 to 13; and b) a pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising: [Item 41] Contains the following ingredients: a) the conjugate according to any one of items 37 to 38; and b) a pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising: [Item 42] Contains the following ingredients: a) the fusion protein according to item 40; and b) a pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising: [Item 43] 43. The pharmaceutical composition according to any one of items 40 to 42, further comprising a T cell activator. [Item 44] 44. The pharmaceutical composition of item 43, wherein the T cell activator is selected from the group consisting of an immune checkpoint inhibitor, a cytokine, and an antagonist of an inhibitory immunoreceptor. [Item 45] 45. The pharmaceutical composition according to any one of items 40 to 44, wherein the antibody is encapsulated in a liposome. [Item 46] 1. A method of treating a cell proliferative disorder in a subject, comprising: Administering a therapeutically effective amount of the pharmaceutical composition according to any one of items 40 to 45 to a subject suffering from a cell proliferative disorder. A method comprising:

Claims

1. An antibody that specifically binds to GPC3 or a fragment thereof that specifically binds to GPC3, comprising the following variable heavy chain (V Ｈ ) polypeptide and variable light chain (V Ｌ ) Polypeptide: V containing the amino acid sequence GYTFTSYFLH (SEQ ID NO: 2) Ｈ CDR1, and V containing the amino acid sequence IIDPPTGRTTYAQKFQG (SEQ ID NO: 4) Ｈ CDR2, and V containing the amino acid sequence GNYGGRYFDY (SEQ ID NO: 5) Ｈ CDR3 and A heavy chain (V Ｈ ) polypeptide; and V containing the amino acid sequence RASQSISSYLN (SEQ ID NO: 6) Ｌ CDR1, and V containing the amino acid sequence AASSLQS (SEQ ID NO: 7) Ｌ CDR2, and V containing the amino acid sequence QQSYSTPLT (SEQ ID NO: 8) Ｌ CDR3 and A light chain (V Ｌ ) Polypeptide The antibody or fragment thereof comprising:

2. The V Ｈ The antibody or fragment thereof of claim 1, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

9.

3. The V Ｌ The antibody or fragment thereof according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO:

10.

4. The antibody or fragment thereof according to any one of claims 1 to 3, which is a humanized antibody.

5. The antibody or fragment thereof according to any one of claims 1 to 3, which is a chimeric antibody.

6. The antibody or fragment thereof according to any one of claims 1 to 5, which is selected from the group consisting of IgG, Fv, single-chain antibody, scFv, Fab, F(ab')2 or Fab'.

7. The antibody or fragment thereof according to any one of claims 1 to 5, which is an IgG.

8. The antibody or fragment thereof of claim 7, which is IgG1.

9. The antibody or fragment thereof according to any one of claims 1 to 5, which is a Fab.

10. The antibody or fragment thereof according to any one of claims 1 to 5, which is a single-chain antibody.

11. The antibody or fragment thereof of claim 10, which is an scFv.

12. A bispecific antibody comprising a first antigen-binding domain that specifically binds to GPC3, wherein the first antigen-binding domain is a V as defined in any one of claims 1 to 3. Ｈ Polypeptides and V Ｌ An antibody or fragment thereof according to any one of claims 1 to 11, comprising a polypeptide.

13. The antibody or fragment thereof according to any one of claims 1 to 12, which is detectably labeled.

14. The antibody or fragment thereof according to any one of claims 1 to 13, comprising a covalently linked synthetic polymer other than a peptide.

15. The antibody or fragment thereof of claim 14, wherein the synthetic polymer is a poly(ethylene glycol) polymer.

16. The antibody or fragment thereof according to any one of claims 1 to 15, comprising a covalently linked lipid or fatty acid moiety.

17. The antibody or fragment thereof of any one of claims 1 to 13, comprising a covalently linked polysaccharide or carbohydrate moiety.

18. The antibody or fragment thereof according to any one of claims 1 to 17, comprising an imaging agent.

19. An antibody or fragment thereof according to any one of claims 1 to 18, comprising an affinity domain.

20. The antibody or fragment thereof according to any one of claims 1 to 19, immobilized on a solid support.

21. 21. The antibody or fragment thereof of any one of claims 1 to 20, comprising a covalently linked cytotoxin.

22. The antibody or fragment thereof according to any one of claims 1 to 21, comprising a constant region amino acid sequence comprising the amino acid sequence of a sulfatase motif.

23. 22. The antibody or fragment thereof according to any one of claims 1 to 21, comprising a constant region amino acid sequence comprising the amino acid sequence of a sulfatase motif, wherein the sulfatase motif is modified to contain a 2-formylglycine (FGly) moiety.

24. 24. The antibody or fragment thereof of claim 23, comprising a heterologous moiety covalently linked via the FGly moiety.

25. 25. The antibody or fragment thereof of claim 24, wherein the heterologous moiety is selected from a drug, a toxin, a detectable label, a water-soluble polymer, and a synthetic peptide.

26. The variable heavy chain (V) of the antibody or fragment thereof according to any one of claims 1 to 12 Ｈ ) polypeptide and said variable light chain (V Ｌ ) A nucleic acid encoding a polypeptide.

27. 27. The nucleic acid of claim 26, wherein the antibody is a single chain antibody and encodes the single chain antibody.

28. 28. The nucleic acid of claim 27, wherein the single chain antibody is an scFv.

29. A recombinant expression vector comprising the nucleic acid of any one of claims 26 to 28, wherein the nucleic acid is operably linked to a transcriptional control element that is active in a eukaryotic cell.

30. A cell comprising the nucleic acid of any one of claims 26 to 28 or the expression vector of claim 29.

31. The nucleic acid is a V Ｈ Polypeptides and V of the antibodies Ｌ 31. The cell of claim 30, encoding a polypeptide.

32. The cell of claim 31 , wherein the antibody is a single chain antibody and the nucleic acid encodes the single chain antibody.

33. The cell of claim 32, wherein the single-chain antibody is an scFv.

34. The following nucleic acids: The variable heavy chain (V) of the antibody or fragment thereof according to any one of claims 1 to 12 Ｈ a first nucleic acid encoding a .) polypeptide; and The variable light chain (V) of the antibody or fragment thereof according to any one of claims 1 to 12 Ｌ ) a second nucleic acid encoding a polypeptide Cells containing

35. 35. The cell of claim 34, a first expression vector comprising the first nucleic acid; and a second expression vector comprising the second nucleic acid; Cells containing

36. The following ingredients: The antibody or fragment thereof according to any one of claims 1 to 12; and an agent conjugated to said antibody A conjugate comprising:

37. 37. The conjugate of claim 36, wherein the agent is selected from the group consisting of a half-life extending moiety, a labeling agent, and a therapeutic agent.

38. The following ingredients: The variable heavy chain (V) of the antibody or fragment thereof according to any one of claims 1 to 12 Ｈ ) polypeptide and said variable light chain (V Ｌ ) polypeptide and, fused thereto, Heterologous amino acid sequences and A fusion protein comprising:

39. The following ingredients: a) an antibody or fragment thereof according to any one of claims 1 to 12; and b) a pharmaceutically acceptable carrier A pharmaceutical composition comprising:

40. The following ingredients: a) a conjugate according to any one of claims 36 to 37; and b) a pharmaceutically acceptable carrier A pharmaceutical composition comprising:

41. The following ingredients: a) the fusion protein of claim 38; and b) a pharmaceutically acceptable carrier A pharmaceutical composition comprising:

42. The pharmaceutical composition of any one of claims 39 to 41, further comprising a T cell activator.

43. 43. The pharmaceutical composition of claim 42, wherein the T cell activator is selected from the group consisting of an immune checkpoint inhibitor, a cytokine, and an antagonist of an inhibitory immunoreceptor.

44. The pharmaceutical composition of any one of claims 39 to 43, wherein the antibody is encapsulated in a liposome.

45. 45. The pharmaceutical composition of any one of claims 39 to 44 for use in a method for treating a cell proliferative disorder in a subject.

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