Method for detecting membrane-bound glypican 3

Abstract

The embodiment provides an anti-GPC3 antibody composition comprising an anti-GPC3 antibody, and a method of using such an antibody and composition for the prevention, diagnosis, and treatment of cancer.In one embodiment, a method for predicting the therapeutic effect of anti-GPC3 immunotherapy on a cancer characterized by the cancer cells expressing GPC3 comprises detecting the presence of the cells in a subject by immunohistochemical methods, and if the presence of the cells is detected, it is predicted that the anti-GPC3 immunotherapy has a therapeutic effect on the cancer of the subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 235,093, filed August 19, 2021.

[0002] The present invention relates to antibodies that target cancer cells that express glypican 3 on their cell surface, as well as compositions and methods of using such antibodies for the prevention, diagnosis, and treatment of such cancers. [Background technology]

[0003] Glypican 3 (GPC3) is a membrane-bound heparin sulfate proteoglycan that is overexpressed in approximately 70%-80% of hepatocellular carcinomas (HCCs), as well as yolk sac tumors, gastric cancer, colorectal cancer, non-small cell lung cancer, and thyroid cancer (Moek et al., 2018, The American Journal of Pathology, 8:9; 1973-1981), but is rarely expressed in normal healthy tissues. In this regard, GPC3 represents a promising tumor antigen target. However, GPC3 can be expressed not only on the membrane but also in the cytoplasm. Because certain promising therapies (e.g., chimeric antigen receptor T cell (CAR-T) therapy) are currently only capable of recognizing GPC3 on the cell surface, there is a need for therapies that specifically target GPC3 on the cell surface and diagnostic methods that can accurately assess GPC3 levels on tumor cell surfaces.

[0004] To date, only one commercially available GPC3 immunohistochemistry (IHC) in vitro diagnostic (IVD) assay is a qualitative assay using an anti-GPC3 mAb (1G12) specific for the C-terminus of GPC3 (Cell Marque™, Rocklin, CA). According to the assay specifications, the GPC3 antibody exhibits a diffuse and membranous staining pattern in neoplastic cells of HCC. Furthermore, the sensitivity of the 1G12 mAb is known to be low in tumor cell lines with low expression levels (Phung et al., 2012, mAbs Landes Bioscience, 4:5; 592-599). Therefore, based at least on the above, there is a need for a mAb that is more sensitive and capable of preferentially staining the cell membrane of GPC3-expressing tumor cells, particularly for use in IVD assays to evaluate membrane-bound GPC3. Summary of the Invention

[0005] The present invention addresses and overcomes the aforementioned shortcomings in the prior art by providing compositions and methods that improve the discrimination between membrane-bound and cytosolic GPC3 for more accurate diagnostic analysis and treatment. In some embodiments, the present invention provides anti-GPC3 antibodies, including fragments thereof, and methods of use thereof, for example, for the diagnosis, prevention, and / or treatment of cancer.

[0006] In one embodiment, the anti-GPC3 antibody of the present invention binds to a GPC3 epitope located in the C-terminal beta chain of GPC3. In one embodiment, the anti-GPC3 antibody comprises a heavy chain variable region comprising SEQ ID NO: 2 and a light chain variable region comprising SEQ ID NO: 4.

[0007] In one embodiment, the heavy chain of the anti-GPC3 antibody of the present invention comprises a complementarity-determining region (CDR) 1 set forth as SEQ ID NO: 6, a CDR2 set forth as SEQ ID NO: 8, and a CDR3 set forth as SEQ ID NO: 10.

[0008] In one embodiment, the light chain of the anti-GPC3 antibody of the present invention comprises CDR1 set forth as SEQ ID NO: 13, CDR2 set forth as SEQ ID NO: 15, and CDR3 set forth as SEQ ID NO: 17.

[0009] In one embodiment, the heavy chain of the anti-GPC3 antibody of the present invention comprises a complementarity-determining region (CDR) 1 set forth as SEQ ID NO: 6, a CDR2 set forth as SEQ ID NO: 8, and a CDR3 set forth as SEQ ID NO: 10, and the light chain of the anti-GPC3 antibody of the present invention comprises a CDR1 set forth as SEQ ID NO: 13, a CDR2 set forth as SEQ ID NO: 15, and a CDR3 set forth as SEQ ID NO: 17.

[0010] In one embodiment, the heavy chain of the anti-GPC3 antibody comprises CDR1, CDR2, and CDR3 set forth as amino acid residues 31 to 35, 50 to 66, and 99 to 105, respectively, of SEQ ID NO: 2, and the light chain of the antibody comprises CDR1, CDR2, and CDR3 set forth as amino acid residues 24 to 34, 50 to 56, and 89 to 97, respectively, of SEQ ID NO: 4.

[0011] In one embodiment, the present invention provides an anti-GPC3 antibody that competes for binding to GPC3 with an antibody comprising a heavy chain variable region comprising SEQ ID NO: 2 and a light chain variable region comprising SEQ ID NO: 4.

[0012] The anti-GPC3 antibodies of the present invention include, for example, monoclonal antibodies, antibody fragments including Fab, Fab', F(ab')2, and Fv fragments, diabodies, single-domain antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, and antibodies that competitively inhibit the binding to GPC3 of antibodies comprising a heavy chain variable region comprising SEQ ID NO: 2 and a light chain variable region comprising SEQ ID NO: 4.

[0013] In one embodiment, the anti-GPC3 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10.

[0014] In one embodiment, the anti-GPC3 antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17.

[0015] In one embodiment, the anti-GPC3 antibody is a chimeric, humanized, or human antibody.

[0016] In one embodiment, the anti-GPC3 antibody is a monoclonal antibody.

[0017] In one embodiment, the anti-GPC3 antibody is an antibody fragment.

[0018] In one embodiment, the anti-GPC3 antibody is a bispecific antibody.

[0019] In one aspect, the invention provides a method for diagnosing cancer in a subject, the method comprising detecting the presence of GPC3 on the cell surface of cells comprising said cancer in said subject or in a biological sample derived from said subject.

[0020] In one aspect, the present invention provides a method for determining the prognosis of a subject diagnosed with cancer, the method comprises detecting the presence of GPC3 expressed on the cell surface of the cells comprising the cancer in the subject or in a biological sample derived from the subject.In one embodiment, the method comprises detecting the presence of GPC3 in the subject or in a biological sample derived from the subject after the subject has been administered a therapeutic agent for treating cancer.In one embodiment, the therapeutic agent is a drug for treating cancer that comprises cancer cells that express GPC3 on the cell surface.

[0021] In one aspect, the present invention provides a method for predicting the therapeutic effect of anti-GPC3 immunotherapy on cancer.In one embodiment, the cancer is composed of cancer that expresses GPC3.In one embodiment, the method comprises detecting the presence of the cell, and if the cell is detected, it is predicted that the anti-GPC3 immunotherapy will have a therapeutic effect on the cancer of the subject.In one embodiment, the prediction is made before the subject receives any anti-GPC3 immunotherapy.In one embodiment, the prediction is made during the process of the subject receiving anti-GPC3 immunotherapy.

[0022] In one aspect, the invention provides nucleic acids encoding the GPC3 antibodies (or portion(s) thereof) of the invention.

[0023] In one aspect, the present invention provides a vector comprising DNA encoding any of the anti-GPC3 antibodies or portions thereof described herein. Host cells comprising any such vectors are also provided. For example, the host cells may be CHO cells, E. coli cells, or yeast cells. A process for producing any of the polypeptides described herein is further provided, comprising culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture. In one embodiment, the vector comprises SEQ ID NO: 1 and / or SEQ ID NO: 3 (Table 2).

[0024] In one aspect, the present invention provides a CAR-modified immune cell, preferably a CAR-T or CAR-NK cell, comprising a chimeric antigen receptor capable of binding to GPC3, preferably capable of binding to the beta chain of GPC3. In one aspect, the present invention provides a CAR-modified immune cell, preferably a CAR-T or CAR-NK cell, comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the light chain variable region of an anti-GPC3 antibody of the present disclosure and the heavy chain variable region of an anti-GPC3 antibody of the present disclosure.

[0025] In one aspect, the present invention provides CAR-modified immune cells (s) comprising an anti-GPC3 antibody, preferably CAR-T or CAR-NK cells. In one embodiment, the anti-GPC3 antibody is an antibody fragment. In one embodiment, the anti-GPC3 antibody is an scFv. In one embodiment, the modified T cells are αβ T cells. In one embodiment, the modified T cells are γδ T cells.

[0026] In one aspect, the present invention provides a pharmaceutical composition comprising an anti-GPC3 antibody and a pharmaceutically acceptable carrier. In another aspect, the present invention provides a pharmaceutical composition comprising a CAR-modified immune cell of the present invention, preferably a CAR-T or CAR-NK cell, and a pharmaceutically acceptable carrier. In one embodiment, the anti-GPC3 antibody is used in the form of an antibody-drug conjugate (ADC).

[0027] In one aspect, the present invention provides a method for producing an anti-GPC3 antibody. In one aspect, the present invention provides a method for producing the CAR-modified immune cells disclosed herein. In one embodiment, the present invention provides a method for producing an ADC comprising an anti-GPC3 antibody.

[0028] In one aspect, the present invention provides a method for preparing a medicament for the treatment of cancer. In one embodiment, the present invention is directed to the use of the anti-GPC3 antibodies disclosed herein for the preparation of a medicament useful for the treatment of conditions responsive to the anti-GPC3 antibodies.

[0029] In one aspect, the invention provides use of a nucleic acid of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, eg, cancer, a tumor and / or a cell proliferative disorder.

[0030] In one aspect, the invention provides use of an expression vector of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, e.g., cancer, a tumor and / or a cell proliferative disorder.

[0031] In one aspect, the invention provides use of a host cell of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, e.g., cancer, a tumor, and / or a cell proliferative disorder.

[0032] In one embodiment, the present invention provides an ADC comprising an anti-GPC3 antibody conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or an anti-GPC3 antibody conjugated to a radioisotope (i.e., a radioconjugate). In another embodiment, the present invention further provides a method for using the immunoconjugate. In one embodiment, the immunoconjugate comprises any of the above-described anti-GPC3 antibodies covalently linked to a cytotoxic agent or a detectable agent.

[0033] In one aspect, the present invention provides a method for inhibiting the proliferation or growth of cells expressing GPC3 on their cell surface, the method comprising contacting the cells with an anti-GPC3 antibody of the present invention or a CAR-modified immune cell, preferably a CAR-T or CAR-NK cell. In one embodiment, the anti-GPC3 antibody is used in the form of an ADC. In one embodiment, the proliferation or growth of the cells comprises a cell proliferative disorder. In one embodiment, the cell proliferative disorder is cancer.

[0034] In one aspect, the present invention provides a method for therapeutically treating a mammal having a cancerous tumor comprising cells expressing GPC3, the method comprising administering to the mammal a therapeutically effective amount of an antibody or CAR-modified immune cell(s) of the present invention, preferably CAR-T or CAR-NK cell(s), thereby effectively treating the mammal. In one embodiment, the mammal is a human subject. In one embodiment, the cancer is selected from the group consisting of liver cancer, ovarian cancer, lung cancer, Merkel cell carcinoma, and gastric (or stomach) cancer.

[0035] In one aspect, the present invention provides a method for inducing death of a cell expressing GPC3 on its cell surface, the method comprising contacting the cell with an anti-GPC3 antibody or CAR-modified immune cell(s) of the present invention, preferably CAR-T or CAR-NK cell(s). In one embodiment, the anti-GPC3 antibody is an ADC.

[0036] In yet another aspect, the present invention relates to a composition comprising the anti-GPC3 antibody described herein, in some embodiments, in combination with a carrier.Optionally, the carrier is a pharmaceutically acceptable carrier.In yet another aspect, the present invention relates to a composition comprising the CAR-modified immune cells described herein, preferably CAR-T or CAR-NK cells, in combination with a carrier.Optionally, the carrier is a pharmaceutically acceptable carrier.

[0037] Also provided herein are kits and methods for using same.

[0038] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0039] Embodiments are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings. [Brief explanation of the drawings]

[0040] [Figure 1A] 2 shows the results of a biolayer inferometry (BLI) binding assay performed using 204. [Figure 1B] 1 shows the results of a biolayer inferometry (BLI) binding assay performed using 1G12. [Figure 1C]1 shows the results of a biolayer inferometry (BLI) binding assay performed using GC33. [Figure 2A] Western blot detection of recombinant human (rh) GPC3 by 204 (left panel), GC33 (middle panel), and 1G12 (right panel) under reducing (R) and non-reducing (NR) conditions. [Figure 2B] 1 shows the results of Western blotting of 204 and 1G12 antibodies used to probe rhGPC3, rhGPC5, and rhGPC6 under reducing (R) and non-reducing (NR) conditions. [Figure 3] Western blot analysis of soluble native human GPC3 detected by 204. The samples examined were obtained as supernatants from tumor cell lines HepG2, NCI-H661, and Hep3B. [Figure 4] High-level schematic diagram of the major GPC3 isoform (isoform 2), showing the alpha chain, beta chain, furin cleavage site, GC33 immunogen, 1G12 immunogen, GC33 epitope, and potential 204 epitope. [Figure 5] FIG. 1 is another high-level schematic of GPC3 showing the ADAM10 cleavage site and the region of 204 binding compared to GC33. [Figure 6A] 1 shows a Coomassie-stained gel showing cleavage of rhGPC3 by ADAM10 and ADAM17. An approximately 12 kDa fragment is released upon cleavage of GPC3 by ADAM10. [Figure 6B] Western blot analysis of GPC3 cleaved by ADAM10 and ADAM17 probed with 204 and GC33 is shown. The approximately 12 kDa fragment described in Figure 6A is detected by GC33 but not by 204, indicating that the epitope for 204 lies between the furin cleavage site and the predicted ADAM10 site. [Figure 7] 1 shows a high-level exemplary optimized immunohistochemistry (IHC) method for use with the 204 antibody of the present disclosure. [Figure 8]Representative images from a tumor microarray (TMA) derived from human hepatocellular carcinoma (HCC) using the 204 antibody and the optimized protocol in Figure 7 are shown. Staining was assessed using a semi-quantitative membrane-bound H-score, as indicated. [Figure 9] Representative images of IHC experiments using 204 or 1G12 to detect GPC3 in lung squamous cell carcinoma and HCC using the optimized protocol in Figure 7 are shown along with the corresponding membrane-bound H-scores. [Figure 10] Figure 7 shows representative images of IHC experiments using 204 or 1G12 to probe healthy lung and liver tissue using the optimized protocol. [Figure 11] IHC images of tissues from HepG2 (GPC3hi) and PP5 (GPC3lo) tumors stained with 1G12 (0.5 μg / mL) or 204 (0.1 μg / mL) antibodies and visualized via 3,3'-diaminobenzidine (DAB) as a substrate for secondary antibody-conjugated horseradish peroxidase (HRP). Isotype controls are also shown. Human hepatocellular carcinoma (HCC) cell lines HepG2 (GPC3hi) and PP5 (GPC3lo) were subcutaneously implanted into NOD SCID mice, and tumors were harvested on days 24 and 31 after implantation for PP5 and HepG2, respectively. [Figure 12] 1 shows bar graphs quantifying IHC staining corresponding to the images in Figure 11. Quantification was performed on HepG2 tumors and two different PP5 tumors. The top panel of the bar graph shows the membrane-bound H-score, and the bottom panel of the bar graph shows the total H-score (cytoplasmic and membrane). [Figure 13] 1 shows a plot showing a direct comparison of membrane-bound H-scores obtained using 204 and 1G12 in IHC experiments on formalin-fixed, paraffin-embedded (FFPE) tumor blocks (top graph) and FFPE tumor cores from tissue microarrays (TMAs) for various cancers, including gastric cancer (adenocarcinoma), liver cancer (HCC), lung cancer (squamous cell carcinoma), and ovarian cancer (clear cell carcinoma). [Figure 14A]The distribution of membrane-bound GPC3 expression in HCC and SCCL is shown based on staining intensity using 204mAb and 1G12mAb for IHC. [Figure 14B] The distribution of membrane-bound GPC3 expression in HCC is shown based on staining intensity using 204mAb and 1G12mAb for IHC. [Figure 14C] The distribution of membrane-bound GPC3 expression in SCCLs is shown based on staining intensity using 204mAb and 1G12mAb for IHC. [Figure 14D] The distribution of the incidence of membrane-bound GPC3 in HCC and SCCL is shown based on the membrane-bound H-score using 204mAb and 1G12mAb for IHC. [Figure 14E] The distribution of membrane-bound GPC3 incidence in HCC is shown based on membrane-bound H-scores using 204mAb and 1G12mAb for IHC. [Figure 14F] The distribution of membrane-bound GPC3 in SCCLs is shown based on the membrane-bound H-score using 204mAb and 1G12mAb for IHC. [Figure 15] Images of membrane-bound GPC3 expression in FFPE tissue from xenograft tumor models using 204mAb compared with 1G12mAb are shown. Cell lines used for xenografting include Hep3B, HepG2, Huh-7, and PLC / PRF / 5. The inset (larger square) in each image corresponds to a higher-resolution image of the indicated area (smaller square). For reference, the membrane-bound H-score corresponding to each condition is shown. [Figure 16A] 16 shows a graph quantifying the IHC experiments of FIG. 15 in terms of membrane-bound H-scores. [Figure 16B] 16 shows a graph quantifying the IHC experiment of FIG. 15 in terms of % of medium vs. high membrane intensity. [Figure 17A] 1 is a table showing the scoring of tumors from xenograft mouse models as determined by IHC using 204mAb. [Figure 17B]1 is a table showing the scoring of tumors from xenograft mouse models as determined by IHC using 1G12 mAb. DETAILED DESCRIPTION OF THE INVENTION

[0041] In the following detailed description, references are made to the accompanying drawings that form a part hereof, and which show by way of illustration exemplary embodiments that may be practiced. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope thereof. Therefore, the following detailed description is not to be taken in a limiting sense.

[0042] Various operations may be described in sequence as multiple separate operations, in a manner that may be helpful in understanding the embodiments; however, the order of description should not be construed to imply that these operations are order dependent.

[0043] The description may use the terms "embodiment" or "embodiments," each of which may refer to one or more of the same or different embodiments. Furthermore, terms such as "comprising," "including," and "having" when used with respect to embodiments are synonymous and generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.).

[0044] With respect to the use of any plural and / or singular terms herein, those of skill in the art will be able to convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural arrangements may be explicitly set forth herein.

[0045] Before describing the present invention, it is to be understood that the present invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may 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, as the scope of the present invention will be limited only by the appended claims. Any embodiments or features of embodiments can be combined with each other, and such combinations are expressly encompassed within the scope of the present invention.

[0046] 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.

[0047] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of one in the art. Such techniques are fully explained in the literature, for example, in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (MJ Gait, ed., 1984), "Animal Cell Culture" (RI Freshney, ed., 1987), "Methods in Enzymology" (Academic Press, Inc.), "Current Protocols in Molecular Biology" (FMA Usubel et al., eds., 1987, and periodic updates), "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994), "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988), and "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0048] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0049] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0050] I. Definition For purposes of interpreting this specification, the following definitions shall apply, except that where appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any set forth definition conflicts with any document incorporated herein by reference, the definition set forth below shall control. 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.

[0051] As used herein, "about" when referring to a measurable value, e.g., an amount, a time period, etc., is meant to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value. Such variations are appropriate for carrying out the methods of the present disclosure. Furthermore, the recitation of numerical ranges includes any number encompassed by the range and / or any range of values ​​contained within the range. For example, a numerical range of 1 to 10 encompasses the range and further encompasses individual numbers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and ranges within the numerical range (e.g., 1-2, 1-4, 2-5, 3-7, 4-9, 5-10, etc.).

[0052] As used herein, "contacting" includes bringing at least two substances together in solution or in the solid phase.

[0053] As used herein, "glypican 3 (GPC3)" refers to a member of the glypican family of heparan sulfate (HS) proteoglycans that are attached to the cell surface by a glycosylphosphatidylinositol anchor (Filmus and Selleck, J Clin Invest 108:497-501, 2001). The GPC3 gene encodes a core protein of approximately 70 kD, which can be cleaved by furin to produce an N-terminal 40 kD fragment and a C-terminal 30 kD fragment. Two HS chains are attached to the C-terminal portion of GPC3. GPC3 and other glypican family proteins play a role in cell division and cell proliferation regulation. GPC3 is highly expressed in HCC and several other human cancers, including melanoma, lung squamous cell carcinoma, and ovarian clear cell carcinoma (Ho and Kim, Eur J Cancer 47(3):333-338, 2011), but is not expressed in normal tissues. GPC3 is also known as SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1 and GTR2-2.

[0054] There are four known isoforms of human GPC3 (isoforms 1 to 4). The nucleic acid and amino acid sequences of these four isoforms of GPC3 are known and include GenBank accession numbers NM_001164617 and NP_001158089 (isoform 1), NM_004484 and NP_004475 (isoform 2), NM_001164618 and NP_001158090 (isoform 3), and NM_001164619 and NP_001158091 (isoform 4). In some embodiments of the present disclosure, the antibodies disclosed herein bind to one or more of the four human GPC3 isoforms or conservative variants thereof.

[0055] As used herein, a "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, resulting from a sequence mutation involving the amino acid residue / position. For example, typical modifications include substitution of the residue (or position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more (generally less than five or three) amino acids adjacent to the residue / position, and deletion of the residue / position. An "amino acid substitution," or a variation thereof, refers to the replacement of an existing amino acid residue in a given (starting) amino acid sequence with a different amino acid residue. Generally and preferably, the modification results in a change in at least one physico-biochemical activity of the variant polypeptide compared to a polypeptide comprising the starting (or "wild-type") amino acid sequence. For example, in the case of an antibody, the altered physico-biochemical activity can be binding affinity, binding ability, and / or binding efficacy for a target molecule.

[0056] As used herein, the term "T lymphocyte" or "T cell" refers to an immune cell that expresses or has expressed CD3 (CD3+) and a T cell receptor (TCR+). T cells play a central role in cell-mediated immunity. T cells that have "expressed CD3 and TCR" have been engineered to eliminate cell surface expression of CD3 and / or TCR.

[0057] As used herein, the term "TCR" or "T cell receptor" refers to dimeric heterologous cell surface signaling proteins that form alpha-beta or gamma-delta receptors, or combinations thereof. αβTCRs recognize antigens presented by MHC molecules, whereas γδTCRs can recognize antigens independently of MHC presentation.

[0058] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encode cell surface antigen-presenting proteins in humans; these genes are called human leukocyte antigen (HLA) genes. The abbreviations MHC or HLA are used interchangeably herein.

[0059] As used herein, "activated" refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells that are undergoing cell division.

[0060] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or immunoreactive portions of intact immunoglobulins. Antibodies are usually tetramers of immunoglobulin molecules. Antibodies in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with polyepitopic specificity, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies exhibiting a desired biological activity), diabodies, single domain antibodies (sdAbs), Fv, Fab, and F(ab) exhibiting a desired biological or immunological activity, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Nat. Acad. Sci. USA 85:5879-5883; Bird et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; ah, 1988, Science 242:423-426).

[0061] An "antibody fragment" includes a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments, diabodies, linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ), single-chain antibody molecules, disulfide-linked fragment variable (dsFv), and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment contains the antigen-binding site of an intact antibody and thus retains the ability to bind to antigen. Also included among antibody fragments are portions of antibodies (and combinations of antibody portions, e.g., scFv) that can be used as targeting arms, e.g., against the GPC3 tumor epitope, in chimeric antigen receptors of CAR-T cells or CAR-NK cells. Such fragments are not necessarily proteolytic fragments, but rather portions of a polypeptide sequence that are capable of conferring affinity for a target.

[0062] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to readily crystallize. The Fab fragment consists of the entire L chain, plus the variable region domain (VH) of the H chain, and the first constant domain (CHI) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of antibodies yields a single large F(ab')2 fragment, which generally corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments in that they contain several additional residues at the carboxy terminus of the CHI 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) in the constant domain 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.

[0063] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain cell types.

[0064] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, noncovalent association. In single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in two-chain Fv species. The folding of these two domains generates six hypervariable loops (three loops each from the heavy and light chains) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0065] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, enabling the sFv to form the desired structure for antigen binding. For reviews of sFvs, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. In one embodiment, an scFv derived from an anti-GPC3 antibody is used as the targeting arm of the CAR-T or CAR-NK cells disclosed herein.

[0066] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., a population in which the individual antibodies comprising the population are identical except for possible naturally occurring mutations, which may be present in minor amounts. Monoclonal antibodies are highly specific to a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be produced using recombinant DNA methodologies in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0067] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions: three in the VH (HI, H2, H3) and three in the VL (LI, L2, L3). Several hypervariable region delineations are used and encompassed herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the terminus of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Residues in each of these hypervariable regions are shown below. Loop Kabat AbM Chothia Contact LI L24-L34 L24-L34 L24-L34 L30-L36 L2 L50-L56 L50-L56 L50-L56 L46-L55 L3 L89-L97 L89-L97 L89-L97 L89-L96 HI H31-H35B H26-H35B H26-H32..34 H30-H35B (Kabat numbering) HI H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H52-H56 H47-H58 H3 H95-H102 H95-H102 H95-H102 H93-H101

[0068] The hypervariable regions may comprise the following "extended hypervariable regions": 24-36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 (L3) in VL, and 26-35B (H1), 50-65, 47-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al. (supra) for each of these definitions.

[0069] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0070] The terms "Kabat variable domain residue numbering" or "Kabat amino acid position numbering," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of antibodies organized in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment of the sequence with a "standard" Kabat numbered sequence in the homologous region of that antibody's sequence.

[0071] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "Kabat EU index" refers to the numbering of residues in a human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the variable domain of an antibody refer to the numbering of residues according to the Kabat numbering system.

[0072] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0073] An antibody that "binds" to an antigen or epitope of interest is one that binds to the antigen or epitope with sufficient affinity that it is measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule relative to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity.

[0074] Antibodies that inhibit tumor cell growth are those that result in measurable growth inhibition of cancer cells. In one embodiment, an anti-GPC3 antibody is capable of inhibiting the growth of cancer cells that display the GPC3 tumor epitope. Preferred growth-inhibitory anti-GPC3 antibodies inhibit the growth of GPC3-expressing tumor cells by more than 20%, preferably about 20% to about 50%, and even more preferably more than 50% (e.g., about 50% to about 100%), compared to an appropriate control, which is usually tumor cells not treated with the antibody being tested (or treated with an isotype control).

[0075] Anti-GPC3 antibodies may (i) inhibit the growth or proliferation of cells to which they bind, (ii) induce the death of cells to which they bind, (iii) inhibit the desquamation of cells to which they bind, (iv) inhibit the metastasis of cells to which they bind, or (v) inhibit the angiogenesis of tumors containing cells to which they bind.

[0076] The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of an antigen. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native GPC3 polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. A method for identifying an antagonist of a GPC3 polypeptide may include contacting the GPC3 polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the GPC3 polypeptide.

[0077] The terms "anti-GPC3 antibody," "GPC3 antibody," and "antibody that binds to GPC3" are used interchangeably. The anti-GPC3 antibody is preferably capable of binding to GPC3 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent.

[0078] In one embodiment, anti-GPC3 antibody is used herein to specifically refer to an anti-GPC3 monoclonal antibody that comprises (i) a heavy chain variable domain of SEQ ID NO: 2 and / or a light chain variable domain of SEQ ID NO: 4 as shown in Table 2, or (ii) one, two, three, four, five, or six of the CDRs shown in Table 1.

[0079] An "isolated antibody" is one that has been identified and separated and / or recovered from components of its natural environment, which are materials that would interfere with therapeutic uses for the antibody, and which may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0080] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is usually approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) in the α and γ chains, and four CH domains in the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CHI) of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. The pairing of VH and VL forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0081] Light chains 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 their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH" or "VH." The variable domain of the light chain may be referred to as "VL" or "VL4." These domains are generally the most variable parts of antibodies and contain the antigen-binding sites.

[0082] The term "variable" refers to the fact that certain segments of variable domains vary significantly in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the 110-amino acid variable domain. Instead, the V region consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions," each 9-12 amino acids long. Naturally occurring heavy and light chain variable domains each contain four FRs that adopt a primarily β-sheet configuration connected by three hypervariable regions, which form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0083] An "intact" antibody is one that contains an antigen-binding site as well as a CL and at least heavy chain constant domains CHI, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

[0084] As used herein, the term "synthetic antibody" refers to an antibody made using recombinant DNA technology. The term should also be construed to mean an antibody made by synthesizing a DNA molecule encoding the antibody, which DNA molecule expresses the antibody protein, or an amino acid sequence defining the antibody, which DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0085] A "chimeric antibody" has framework residues derived from one species, e.g., human, and CDRs (which generally confer antigen binding) derived from another species, e.g., a murine antibody that specifically binds to GPC3.

[0086] A "human" antibody (also called a "fully human" antibody) is an antibody that contains human framework regions and all of the CDRs derived from a human immunoglobulin. In one example, the framework and the CDRs are derived from the same original human heavy and / or light chain amino acid sequence. However, a framework from one human antibody may be engineered to contain CDRs from a different human antibody. A "humanized" immunoglobulin is an immunoglobulin that contains human framework regions and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is referred to as the "donor," and the human immunoglobulin that provides the framework is referred to as the "acceptor." In one embodiment, in a humanized immunoglobulin, all of the CDRs are derived from the donor immunoglobulin. Constant regions need not be present, but if they are present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, all portions of a humanized immunoglobulin are substantially identical to the corresponding portions of a natural human immunoglobulin sequence, except, optionally, for the CDRs. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids removed from the donor framework. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Humanized immunoglobulins may be constructed by genetic engineering (see, e.g., U.S. Pat. No. 5,585,089).

[0087] The term "binding" in the context of binding of an antibody, Ig, or antibody-binding fragment to either an antigen or other molecule (e.g., a carbohydrate) generally refers to an interaction or association between at least two entities or molecular structures, e.g., an antibody-antigen interaction.

[0088] A "conservative" amino acid substitution is a substitution that does not substantially affect or reduce the affinity of a protein, such as an antibody, to GPC3. For example, a monoclonal antibody that specifically binds to GPC3 may contain at most about 1, at most about 2, at most about 5, at most about 10, or at most about 15 conservative substitutions and still specifically bind to a GPC3 polypeptide. The term "conservative variant" also includes the use of a substituted amino acid instead of an unsubstituted parent amino acid, provided that the antibody specifically binds to GPC3. A non-conservative substitution is a substitution that reduces the activity or binding to GPC3.

[0089] Conservative amino acid substitution tables providing functionally similar amino acids are well known to those of skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), serine (S), threonine (T), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0090] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may include either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including proteins or peptides, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response, therefore, encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. It will also be readily apparent that an antigen need not be encoded by a "gene" at all. It will be readily apparent that antigens may be generated, synthesized, or obtained from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0091] The term "epitope" includes any protein, lipid, or carbohydrate determinant capable of specific binding to an immunoglobulin or T-cell receptor (e.g., a specific antigen-binding site). Epitopic determinants usually consist of active surface groupings of molecules, such as amino acids, lipid, or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes with an equilibrium dissociation constant (Kd) of 10 or greater are known. -6 ~10 -12An antibody is said to specifically bind to an antigen when its binding affinity is within the range of 0.1 to 1.0. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions of an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are formed by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are formed by adjacent amino acid residues in a polypeptide chain.

[0092] As used herein, the term "chimeric antigen receptor (CAR)" may refer to, for example, an artificial T cell receptor, a T-body, a single-chain immunoreceptor, a chimeric T cell receptor, or a chimeric immunoreceptor, and encompasses engineered receptors that graft artificial specificity onto specific immune effector cells. CARs may be used to confer the specificity of a monoclonal antibody to T cells, thereby enabling the generation of large numbers of specific T cells for use in, for example, adoptive cell therapy; in certain embodiments, for example, CARs direct the specificity of the cells to a tumor-associated antigen. In some embodiments, a CAR comprises an intracellular activation domain (which activates the T cell when the targeting moiety binds to a target cell, such as a target tumor cell), a transmembrane domain, and an extracellular domain that can vary in length and includes, for example, a tumor-antigen binding region associated with a disease or disorder. In certain aspects, a CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the transmembrane domain and endodomain of CD3-zeta. Other CAR design specificities can be derived from receptor ligands (e.g., peptides) or pattern recognition receptors, such as dectin. In certain cases, the spacing of the antigen recognition domain can be modified to reduce activation-induced cell death. In certain cases, the CAR includes additional domains for costimulatory signaling, such as CD3C, FcR, CD27, CD28, CD137, DAP10 / 12, and / or OX40, 4-1BB, 1COS, TLR (e.g., TLR2), etc. In some cases, molecules can be co-expressed with the CAR, including costimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally eliminate T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors. Furthermore, those skilled in the art will understand that the costimulatory domain need not be encoded solely by the full-length nucleotide sequence of the gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response.

[0093] The term "immunoconjugate" or "antibody-drug conjugate" (ADC) refers to the covalent attachment of an effector molecule to an antibody or functional fragment thereof. The effector molecule may be a detectable label or an immunotoxin. Specific, non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, e.g., PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof, or other poisons that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically cause death through liver toxicity. PE and DT, however, can be modified for use as immunotoxins by removing the toxin's natural targeting component (such as domain Ia of PE and the B chain of DT) and replacing it with a different targeting moiety, e.g., an antibody.

[0094] As used herein, the term "anti-tumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" may also be manifested primarily by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the development of tumors.

[0095] The term "therapeutically effective amount" refers to that amount of a compound that elicits the biological or medical response in a tissue, system, or subject that is desired by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes that amount of a composition that, when administered, is sufficient to prevent the onset of, or alleviate to some extent, one or more signs or symptoms of the disorder or disease (e.g., solid cancer) being treated. The therapeutically effective amount may vary depending on the composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0096] As used herein, the term "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0097] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0098] As used herein, the term "pharmaceutically acceptable" refers to a relatively non-toxic substance, including but not limited to salts, carriers, or diluents, that does not abrogate the biological activity or properties of the compound. That is, the substance may be administered to an individual without causing undesired biological effects or interacting adversely with any of the components of the composition in which it is contained.

[0099] "Encoding" refers to the inherent property of a nucleotide sequence of a particular polynucleotide, e.g., a gene, cDNA, or RNA sequence, to serve as a template for the synthesis of other polymers and macromolecules having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom in biological processes. Thus, a gene encodes a protein when transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, i.e., the nucleotide sequence which is identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of the gene or cDNA.

[0100] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form (e.g., a monoclonal antibody of the present disclosure) or may exist in a non-native environment, such as a host cell.

[0101] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs may contain introns.

[0102] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal suitable for the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0103] As used herein with respect to antibodies, the terms "specifically bind" or "specifically recognize" refer to an antibody that recognizes a specific antigen contained in a sample but does not substantially recognize or bind other molecules. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to various allelic forms of that antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binding" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) of the chemical species, e.g., an antibody recognizes and binds to a specific protein structure, rather than to proteins in general. If an antibody is specific for epitope "A," the presence of a molecule containing epitope A (i.e., free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction involving labeled "A" and the antibody.

[0104] In some embodiments, specific binding is at least about 1x10 -8 The binding may be characterized by an equilibrium dissociation constant equal to or less than M (e.g., a smaller value indicates stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0105] As used herein, "K D The term "(M)" refers to the dissociation equilibrium constant of a particular binding protein-ligand interaction. For example, K D K may also refer to the dissociation equilibrium constant between an antibody, Ig, or antibody-binding fragment and an antigen. D Since there is an inverse correlation between K and binding affinity, DThe smaller the value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to interact and thus a smaller K D Conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to interact and thus a larger K D The value of the dissociation equilibrium constant K D is equal to 1 / K.

[0106] As used herein, "k a " (M -1 x seconds -1 ) refers to the association rate constant of a particular protein-antigen (e.g., antibody-antigen) interaction.

[0107] As used herein, "k d ”(seconds -1 ) refers to the dissociation rate constant of a particular protein-antigen interaction (e.g., antibody-antigen).

[0108] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer, including small cell lung cancer, non-small cell lung cancer ("NSCLC," adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric (gastric or stomach) cancer, including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, and ovarian cancer (e.g., high-grade ovarian carcinoma, ovarian clear cell carcinoma). , liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney (or renal) cancer (e.g., renal cell carcinoma, nephroblastoma, or Wilms' tumor), prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, and head and neck cancer. Additional exemplary cancers include, but are not limited to, retinoblastoma, theca cell tumor, androgenic tumor, hepatoma, non-Hodgkin's lymphoma (NHL), hematological malignancies including multiple myeloma and acute hematological malignancies, endometrial or uterine cancer, endometriosis, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, hepatic carcinoma, anal cancer, penile cancer, nasopharyngeal cancer, laryngeal cancer, Kaposi's sarcoma, melanoma, skin cancer, schwannoma, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, and urinary tract cancer.

[0109] In a preferred embodiment, the cancer is liver cancer (e.g., HCC). In another preferred embodiment, the cancer is gastric or stomach cancer (e.g., adenocarcinoma). In another preferred embodiment, the cancer is lung cancer (e.g., squamous cell carcinoma). In another preferred embodiment, the cancer is ovarian cancer (e.g., ovarian clear cell carcinoma). The term "metastatic cancer" refers to a cancerous state in which cancer cells from the primary tissue are transmitted by blood or lymphatic vessels from the site of origin to one or more other sites in the body, causing one or more secondary tumors in one or more organs other than the primary tissue. A notable example is metastatic breast cancer.

[0110] As used herein, a "GPC3-associated cancer" refers to a cancer associated with overexpression of the GPC3 gene or gene product and / or associated with presentation of a GPC3 tumor epitope. Suitable control cells can be, for example, cells derived from an individual not suffering from cancer, or non-cancerous cells derived from a subject with cancer.

[0111] The methods include methods for treating a subject with cancer, particularly cancers associated with the expression of GPC3. The methods also include methods for regulating certain cell behaviors, particularly cancer cell behaviors, particularly GPC3 on the cell surface of cancer cells.

[0112] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0113] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0114] As used herein, the terms "prediction" and "forecast" are also synonymous. In a sense, the prediction or forecasting method allows the practitioner of the prediction / forecasting method of the present invention to select patients who are likely to respond to treatment with an anticancer drug, preferably the anti-GPC3 antibody of the present invention, or CAR-T or CAR-NK cells (usually, but not necessarily, before treatment).

[0115] A "solid tumor" as referred to herein is a tumor comprising a tumor mass of at least about 10 or at least about 100 tumor cells. The solid tumor may be a soft tissue tumor, a primary solid tumor, or a metastatic lesion. Examples of solid tumors relevant to the present disclosure include, but are not limited to, sarcomas, adenocarcinomas, and carcinomas of various organ systems, such as those affecting the liver, lung, gastrointestinal (e.g., colon), genitourinary tract (e.g., rectum, urothelial cells), etc. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer. Metastatic lesions of the aforementioned cancers may also be treated or prevented using the methods and compositions of the present invention.

[0116] In some embodiments, the solid tumor cells express or overexpress glypican 3 (GPC3). In some embodiments, the solid tumor cells are derived from a tumor cell line described herein (e.g., 204 monoclonal antibody) and / or a tumor cell line described in US 7,919,086, WO 2014 / 180306, WO 2018 / 019772, WO 2016 / 049459, WO 2003 / 000883, WO 2006 / 046751, WO 2007 / 047291, WO 2016 / 086813, WO 2016 / 047722, WO 2016 / 036973, WO 2020 / 072546, Cancer Res. 2008;68:9832-9838: Proc Natl Acad Sci USA. 2013 Mar 19;1 10(12):E The solid tumor cells express or overexpress an epitope of GPC3 specifically bound by an anti-GPC3 antibody, T cell receptor, or chimeric antigen receptor described in 1083-1. The contents of each of these are incorporated by reference in their entirety and for all purposes, and in particular with respect to the binding domain, antibody, antibody fragment, complementarity-determining region, polypeptide comprising the complementarity-determining region, nucleic acid encoding the complementarity-determining region, and epitope specificity, as well as assays for determining epitope specificity described herein. In some embodiments, the solid tumor cells express or overexpress an epitope of GPC3 specifically bound by the anti-GPC3 antibody GC33, 1G12, or 204. In some embodiments, the solid tumor expresses or overexpresses an HLA:peptide complex comprising a GPC3 fragment. In some embodiments, the HLA is a class I HLA, such as HLA-A2.

[0117] II. Compositions and Methods of the Invention A. Overview of anti-GPC3 antibodies In one aspect, the present invention provides anti-GPC3 antibodies, including fragments thereof, compositions containing them, and methods of using them for various purposes, including cancer treatment. In one aspect, the present invention provides antibodies that bind to the beta chain of GPC3 expressed on the surface of cells (e.g., tumor cells). Optionally, the antibodies are monoclonal antibodies, antibody fragments including Fab, Fab', F(ab')2, and scFv fragments, diabodies, single-domain antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, or antibodies that competitively inhibit the binding of anti-GPC3 antibodies to their respective antigen epitopes. The antibodies of the present invention can optionally be produced in CHO cells or bacterial cells, or by other means. For detection purposes, the anti-GPC3 antibodies of the present invention can be detectably labeled, bound to a solid support, etc.

[0118] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a heavy chain variable region comprising: EVQLQQSGPELVKPGASVKISCKTSGYTFTEYAMHWVKQSHGKSLEWIGGINPNNGVTTYNQRFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGLLWYAYWGQGTLVTVSA (SEQ ID NO: 2)

[0119] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a light chain variable region comprising: DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 4).

[0120] In one embodiment, an antibody that binds to GPC3 is provided, the antibody comprising a heavy chain variable region comprising SEQ ID NO:2 and a light chain variable region comprising SEQ ID NO:4.

[0121] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth as EYAMH (SEQ ID NO: 6).

[0122] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a heavy chain variable region comprising a CDR2 comprising the amino acid sequence set forth as GINPNNGVTTYNQRFKG (SEQ ID NO: 8).

[0123] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a heavy chain variable region comprising a CDR3 comprising the amino acid sequence set forth as GLLWYAY (SEQ ID NO: 10).

[0124] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth as KASQDINSYLS (SEQ ID NO: 13).

[0125] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a light chain variable region comprising a CDR2 comprising the amino acid sequence set forth as RANRLVD (SEQ ID NO: 15).

[0126] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a light chain variable region comprising a CDR3 comprising the amino acid sequence set forth as LQYDEFPLT (SEQ ID NO: 17).

[0127] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a heavy chain variable region comprising CDR1 set forth as SEQ ID NO:6, CDR2 set forth as SEQ ID NO:8, and CDR3 set forth as SEQ ID NO:10.

[0128] In one embodiment, an antibody that binds to GPC3 is provided, wherein the antibody comprises a light chain variable region comprising CDR1 set forth as SEQ ID NO: 13, CDR2 set forth as SEQ ID NO: 15, and CDR3 set forth as SEQ ID NO: 17.

[0129] In one aspect, an antibody that binds to GPC3 is provided, the antibody comprising a heavy chain variable region comprising CDR1 set forth as SEQ ID NO: 6, CDR2 set forth as SEQ ID NO: 8, and CDR3 set forth as SEQ ID NO: 10, and further comprising a light chain variable region comprising CDR1 set forth as SEQ ID NO: 13, CDR2 set forth as SEQ ID NO: 15, and CDR3 set forth as SEQ ID NO: 17. In one embodiment, the antibody of the invention comprising these sequences (in the combinations described herein) is a humanized or human antibody.

[0130] In one aspect, the present invention includes an anti-GPC3 antibody comprising (i) a heavy chain variable region comprising SEQ ID NO: 2, and / or (ii) a light chain variable region comprising SEQ ID NO: 4.

[0131] In some embodiments, these antibodies further comprise a human subgroup III heavy chain framework consensus sequence. In one embodiment of these antibodies, these antibodies further comprise a human kappa ι light chain framework consensus sequence.

[0132] In one embodiment, the anti-GPC3 antibody competes with an anti-GPC3 antibody comprising a heavy chain variable region comprising SEQ ID NO: 2 and a light chain variable region comprising SEQ ID NO: 4 for binding to tumor-presented GPC3 (e.g., presented by HCC cells).

[0133] A more comprehensive description of the anti-GPC3 antibodies encompassed by this disclosure is provided below.

[0134] B. Detection Methods The present invention relates to a method for determining the presence of GPC3 polypeptide in a sample suspected of containing the GPC3 polypeptide, the method comprises exposing the sample to an antibody that binds to the GPC3 polypeptide, and determining the binding of the antibody to the GPC3 polypeptide in the sample, and the presence of such binding indicates the presence of the GPC3 polypeptide in the sample.Optionally, the sample can contain cells (which may be cancer cells) that are suspected of expressing the GPC3 polypeptide.The antibody used in the method can optionally be detectably labeled, or be bound to a solid support, etc.

[0135] The binding of the anti-glypican 3 antibody to glypican 3 can be preferably detected by the immunohistochemistry (IHC) method disclosed herein, but it should be understood that the binding is not limited to IHC and can include methods commonly known to those skilled in the art. For example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), immunofluorescence, Western blotting, etc. may be used. Related methods are described in the general text "Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988."

[0136] Exemplary IHC Assays As discussed herein, there is a need for a diagnostic method capable of accurately assessing tumor cell surface GPC3 levels. This is because, at least in part, certain immunotherapies (e.g., CAR-T therapy) rely on the interaction between cell surface GPC3 expressed on tumor cells and a corresponding anti-GPC3 antibody or antigen-binding fragment. In this regard, the present disclosure provides an in vitro IHC method that relies on an anti-GPC3 monoclonal antibody, which exhibits advantages over the use of other prior art antibodies. Specifically, the IHC method disclosed herein is achieved by requiring the assay to exhibit several advantageous criteria. First, the disclosed IHC IVD assay is constrained to exhibit a substantial absence of nonspecific background. Nonspecific background in the context of IHC IVD can complicate the analysis and scoring of tissue sample staining, which can in turn lead to an inaccurate assessment of the occurrence (or lack thereof) of the detected target (e.g., membrane-bound GPC3 in the context of the present disclosure). Therefore, as disclosed herein and illustrated in the Examples, the IHC IVD assay of the present disclosure exhibits a substantial absence of nonspecific background signals. Second, the assay was constrained to exhibit a clear boundary at the cell surface, which advantageously allows for reliable scoring of the membrane-bound GPC3 staining. Third, the assay was constrained to exhibit a clear and distinct nuclear counterstaining. Fourth, the assay was constrained to rely on an antibody (204 described herein) that allows the assay to meet the above-mentioned criteria while also exhibiting high accuracy, sensitivity, and specificity, as well as low nanomolar affinity for GPC3, as disclosed and exemplified herein.

[0137] a. Tissue preparation As used herein, the term "tissue preparation" refers to a biological preparation obtained from an individual, body fluids (e.g., blood, serum, plasma, spinal fluid), tissue culture, tissue section, etc. Preferably, the tissue preparation is a preparation derived from a subject, for example, tissue obtained from the subject's tumor. Biopsy, a method known in the art, is preferably used to collect the tissue. In an example, the tissue preparation is liver tissue, lung tissue, stomach tissue, or ovarian tissue, although other tissue sources, including any tissue containing tumor cell(s) expressing GPC-3, are also within the scope of the present disclosure. As an exemplary illustration, biopsy can be used to collect liver tissue by inserting a long, thin needle directly into the subject's liver through the skin surface. The puncture site can be between the ribs in the lower right chest, although other sites are also within the scope of the present disclosure. The procedure includes, for example, using an ultrasound examination device to confirm the safety of the puncture site, then disinfecting the puncture site, anesthetizing the area from the skin to the liver surface, and finally making a small incision in the skin at the puncture site and puncturing it with a puncture needle. Although not specifically described, similar biopsy methods may be used to collect tissue from other body locations (e.g., lungs, gastrointestinal system, ovaries, etc.), and such methods will be readily understood by those skilled in the art.

[0138] The tissue preparations disclosed herein are observed under a microscope using transmitted light, and are therefore cut into thin sections to facilitate sufficient penetration of the light used in the microscope into the preparation. Before cutting into thin sections, the tissue preparation is fixed. Briefly, the tissue preparation to be fixed is cut into pieces of a size and shape suitable for preparing paraffin-embedded sections using a cutting tool (e.g., a scalpel). The pieces are then immersed in a fixative, which is a reagent used for fixation. The fixative used is preferably formalin, more preferably neutral buffered formalin. The concentration of the neutral buffered formalin is appropriately selected depending on the characteristics or physical properties of the tissue preparation. The concentration can be appropriately varied between 1 and 50%, preferably 5 and 25%, and more preferably 10 and 15% for use. The fixative in which the tissue preparation is immersed is appropriately degassed using a vacuum pump. The fixation is carried out by leaving the tissue preparation in the fixative under normal pressure and room temperature for several hours. The time required for fixation can be appropriately selected within the range of 1 hour to 7 days, preferably 2 hours to 3 days, more preferably 3 hours to 24 hours, and even more preferably 4 hours to 16 hours. The preparation thus fixed is then immersed in a phosphate buffer solution or the like for several hours (this time can be appropriately selected within the range of 2 hours to 48 hours, preferably 3 hours to 24 hours, and more preferably 4 hours to 16 hours).

[0139] Next, sections can be prepared from the fixed tissue preparation, preferably using frozen sectioning or paraffin sectioning. A preferred example of frozen sectioning involves freezing the tissue by adding OCT compound (Miles, Inc.) and cutting the frozen tissue into thin sections using a cryostat (a frozen section preparation device). In paraffin sectioning, the fixed tissue preparation is immersed in an embedding medium, which is then solidified to impart a uniform and appropriate hardness to the sections. Paraffin is preferably used as the embedding medium. The fixed tissue preparation is dehydrated using ethanol or a combination of an ethanol wash and a xylene wash. In one example, the tissue preparation is dehydrated by sequentially immersing it in 70% ethanol, 80% ethanol, and 100% ethanol. The immersion time and number of immersions can be appropriately selected from the ranges of 1 minute to 1 hour to several days, and from 1 to 3 times. Furthermore, the immersion may be performed at room temperature or at 4°C. When immersing at 4°C, a longer immersion time (e.g., overnight) is preferred. In another example, the tissue preparation is dehydrated by sequentially immersing in 95% EtOH and 100% EtOH. Again, the time required for each immersion step and the number of immersions may be appropriately selected within the ranges of 1 minute to 1 hour to several days and 1 to 3 times. Next, the liquid phase is replaced with xylene, and the tissue preparation is then embedded in paraffin. The time required for replacing the liquid phase with xylene may be appropriately selected within the ranges of several minutes to several hours. In this procedure, the replacement may be performed at room temperature or at 4°C. When replacing at 4°C, a longer replacement time (e.g., overnight) is preferred. The time required for the paraffin embedding and the number of times may be appropriately selected within the ranges of 1 hour to several hours and 1 to 4 times. In this procedure, the embedding may be performed at room temperature or at 4°C. Longer embedding times (e.g., overnight) are preferred when embedding at 4° C. Furthermore, the tissue preparation can be paraffin-embedded, preferably by using a paraffin embedding device (e.g., EG1160, Leica Microsystems) that automatically processes the paraffin embedding reaction.

[0140] The paraffin-embedded tissue preparation is then attached to a scaffold to form a "block," which is then cut into slices of the desired thickness, selected from 1 to 20 μm, using a microtome. The thin tissue sections thus cut are placed on a glass slide as a transparent support for adhesion. In this case, a glass slide coated with 0.01% poly-L-lysine (Sigma-Aldrich Co.) and dried to prevent the tissue sections from peeling off is preferably used. The attached tissue sections are then dried in air for an appropriate time, selected from several minutes to one hour.

[0141] In some further or alternative examples, the present disclosure employs the use of tissue microarrays (TMAs). In examples, TMAs are constructed by using a hollow needle to extract tissue cores (e.g., tubular sections) from a paraffin block (donor block, FFPE tissue) and transferring the cores to predetermined locations in a single paraffin block (e.g., recipient block). TMAs can be used on semi-automated platforms to compare control samples and test samples (e.g., positive controls, negative controls, test samples from various tissue regions or types) within a single constructed slide. By way of example, individual arrays can be constructed with 360 cores of 0.6 mm diameter, or 187 cores of 1 mm diameter, or 60 cores of 2 mm diameter, etc. Such examples are intended to be illustrative and non-limiting.

[0142] b. Antigen retrieval An exemplary method of the present invention restores the reactivity of antigens whose reactivity has been reduced by formalin fixation. In one example, protease-induced epitope retrieval (PIER) can be used. Briefly, this method involves digesting sections with a protease (e.g., trypsin, pepsin, etc.) prior to immunostaining. The type or source of the protease used in protease-induced epitope retrieval is not particularly limited, and commonly available proteases can be appropriately selected for use. Examples of preferred proteases include 0.05% pepsin in 0.01 N hydrochloric acid, 0.1% trypsin in Tris buffer (pH 7.6) containing 0.1% CaCl2, and 10 mM Tris-HCl buffer (pH 7.8) containing 10 mM EDTA and 0.5% SDS at a concentration of 1 to 50 μg / ml of protease K. Furthermore, when protease K is used, the pH of the reaction solution is appropriately selected from 6.5 to 9.5, and an SH reagent or a trypsin or chymotrypsin inhibitor may be used as appropriate. The protease included in the Histofine Her2 Kit (MONO) (Nichirei Bioscience) is also a specific example of a preferred protease. The protease-induced epitope retrieval is typically performed at 37°C. However, the reaction temperature can be appropriately changed within the range of 25°C to 50°C. When the protease-induced epitope retrieval is performed at 37°C, the reaction time is appropriately selected from, for example, 1 minute to 5 hours, such as 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, or 4 hours. After the PIER treatment is completed, the tissue preparation thus treated is washed with a washing buffer. PBS (phosphate-buffered saline) is preferably used as the washing buffer. Furthermore, Tris-HCl buffer may also be preferably used. The washing conditions typically include washing three times for 5 minutes at room temperature, although the washing time and temperature can be varied as appropriate.

[0143] In another exemplary method, the reactivity of an antigen whose reactivity has been reduced by formalin fixation is restored by heat-induced epitope retrieval (HIER). Specifically, high-temperature treatment using microwaves, boiling, or autoclaves hydrolyzes the antigen, allowing the epitope to bind to the antibody. When the boiling treatment is performed at an output of 780 W and the temperature of the solution is maintained at approximately 98°C, the time required for retrieval, including the boiling treatment, is appropriately selected from 5 to 60 minutes, for example, 10 minutes. The antigen retrieval treatment can be performed using 10 mM sodium citrate buffer, commercially available Diva Decloaker solution (Biocare Medical, LLC, Pacheo, CA), BOND Epitope Retrieval Solution 1 (ER1) or BOND Epitope Retrieval Solution 2 (ER2) (Leica Biosystems Richmond, Inc., Richmond, IL), or the like. Any buffer or aqueous solution is preferably used as long as the epitope of the antigen recognized by the anti-glypican 3 antibody acquires affinity for the antibody as a result of the retrieval process, so that membrane-bound GPC3 can be detected by the 204 antibody of the present disclosure and show a cell surface staining boundary without appreciable nonspecific background. After completion of the retrieval process, the tissue preparation thus treated is left at room temperature for 30 minutes, with the gradual addition of DI water until the slide cools.

[0144] c. Anti-glypican 3 antibody for use in IHC IVD assays A preferred anti-glypican 3 antibody for use in the IHC IVD method of the present invention is 204 or a portion thereof. The 204 antibody, described in detail in the Examples, is preferred compared to, for example, the GC33 antibody (WO 2006 / 006693) and the 1G12 antibody (WO 2003 / 100429). This is because the 204 antibody was unexpectedly and advantageously found to exhibit less nonspecific background staining, cell surface borders, and clear nuclear counterstaining with hematoxylin. Accordingly, IHC staining with 204 was found to frequently result in high membrane-bound H scores in tissue samples compared to 1G12 staining (see, e.g., Figure 9). This demonstrates that the 204 disclosed herein includes anti-GPC3 monoclonal antibodies that have higher sensitivity than the 1G12 antibody and are capable of preferentially staining the cell membrane of GPC3-expressing cells, a problem recognized in the art that needs to be solved (see, e.g., Phung et al., 2012, mAbs Landes Bioscience, 4:5; 592-599). As discussed in Example 1 below, anti-glypican 3 antibodies preferably used in the present invention were obtained by immunizing non-human animals with glypican 3 as an immunizing antigen. General methods for preparing such anti-glypican 3 antibodies are described in the Examples below and in WO 2003 / 100429 and WO 2006 / 006693.

[0145] In embodiments, the preferred antibody comprises a heavy chain of the antibody that comprises complementarity determining region (CDR)1 set forth herein as SEQ ID NO:6, a CDR2 set forth herein as SEQ ID NO:8, and a CDR3 set forth herein as SEQ ID NO:10, and a light chain of the antibody that comprises CDR1 set forth herein as SEQ ID NO:13, a CDR2 set forth herein as SEQ ID NO:15, and a CDR3 set forth herein as SEQ ID NO:17. In some embodiments, the preferred antibody comprises a heavy chain variable region (HCVR) set forth herein as SEQ ID NO:2 and a light chain variable region (LCVR) set forth herein as SEQ ID NO:4.

[0146] d. Reaction of tissue preparations with anti-glypican 3 antibodies The tissue preparation, optionally subjected to the above-mentioned antigen retrieval treatment, is reacted (i.e., contacted) with the anti-GPC3 antibody (e.g., 204) as the primary antibody. The reaction is carried out under conditions suitable for the anti-GPC3 antibody to specifically recognize the epitope of the antigen (e.g., GPC3), thereby forming an antigen-antibody complex.

[0147] The reaction is typically carried out overnight at 4°C or for 1 hour at 37°C. However, the reaction conditions can be varied as appropriate within a range suitable for the recognition of the antigen epitope by the antibody and the formation of an antigen-antibody complex. For example, the reaction temperature can be varied within a range of 4°C to 50°C, and the reaction time can be varied between 1 minute and 7 days. Longer reaction times are preferred for reactions at lower temperatures. After completion of the primary antibody reaction, the tissue preparation is washed with a washing buffer. PBS (phosphate-buffered saline) is preferably used as the washing buffer. Furthermore, Tris-HCl buffer can also be preferably used. The washing conditions typically include three 5-minute washes at room temperature. However, the washing time and temperature can be varied as appropriate.

[0148] The tissue preparation subjected to the primary antibody reaction is then reacted with a secondary antibody that recognizes the primary antibody. A secondary antibody pre-labeled with a labeling substance for visualizing the secondary antibody is usually used. Preferred examples of the labeling substance include fluorescent dyes such as FITC (fluorescein isothiocyanate), Cy2 (Amersham Biosciences), and Alexa488 (Molecular Probes, Inc.), enzymes such as peroxidase and alkaline phosphatase, and colloidal gold.

[0149] The reaction with the secondary antibody is carried out under conditions suitable for the formation of an antigen-antibody complex between the anti-GPC3 antibody and a secondary antibody that recognizes the anti-GPC3 antibody. The reaction is typically carried out at room temperature or 37°C for 30 minutes to 1 hour. However, the reaction conditions can be modified as appropriate within a range suitable for the formation of an antigen-antibody complex between the anti-GPC3 antibody and the secondary antibody. For example, the reaction temperature can be varied between 4°C and 50°C, and the reaction time can be varied between 1 minute and 7 days. Longer reaction times are preferred for reactions at lower temperatures. After completion of the secondary antibody reaction, the tissue preparation is washed with a washing buffer. PBS (phosphate-buffered saline) is preferably used as the washing buffer. Alternatively, Tris-HCl buffer can also be preferably used. The washing conditions typically include three 5-minute washes at room temperature. However, the washing time and temperature can be modified as appropriate.

[0150] Next, the tissue preparation subjected to the secondary antibody reaction is reacted with a substance for visualizing the labeling substance. When peroxidase is used as the labeling substance for the secondary antibody, the tissue preparation is incubated with a reaction solution obtained by mixing equal amounts of 0.02% aqueous hydrogen peroxide and a DAB (diaminobenzidine) solution adjusted to a concentration of 0.1% in 0.1 M Tris-HCl buffer (pH 7.2) immediately before incubation. In addition to DAB, color-developing substrates such as DAB-Ni and AEC+ (Agilent Technologies, Santa Clara, CA), and DAB sparkle (Biocare Medical, Pacheo, CA) can be appropriately selected. During the incubation process, the degree of color development is occasionally observed under a microscope. Once appropriate color development is confirmed, the visualization reaction is terminated by immersing the tissue preparation in PBS.

[0151] When alkaline phosphatase is used as a label for the secondary antibody, the tissue preparation is incubated with BCIP (5-bromo-4-chloro-3-indolylphosphate) / NBT (nitro blue tetrazolium) (Zymed Laboratories Inc., San Francisco, CA) substrate solution (0.4 mM NBT and 0.38 mM BCIP dissolved in 50 mM sodium carbonate buffer (pH 9.8) containing 10 mM MgCl2 and 28 mM NaCl). In addition to BCIP and NBT, Permanent Red, Fast Red, or Fuchsin+ (all Agilent) may also be used as appropriate. Prior to the incubation, the tissue preparation may be preincubated at room temperature for 1 minute to several hours with 0.1 M Tris-HCl buffer (pH 9.5) containing 1 mM levamisole chloride (an inhibitor of endogenous alkaline phosphatase, Nacalai Tesque, Inc., Kyoto, Japan), 0.1 M sodium chloride, and 50 mM magnesium chloride. During the incubation period, the tissue preparation is occasionally observed under a microscope. When precipitation of the final reaction product, purple formazan, is observed, the reaction is terminated by washing the tissue preparation with water or adding TBS containing 2% polyvinyl alcohol. The tissue preparation is then washed with TBST (TBS containing 0.1% Tween 20). When colloidal gold is used to label the secondary antibody, the colloidal gold is visualized by silver enhancement, which involves attaching metallic silver to the gold particles. Silver enhancement methods are commonly known to those skilled in the art.

[0152] In embodiments, detection of the desired antibody-antigen complex can also be combined with nuclear staining. For example, nuclear staining can be performed using hematoxylin, which stains nuclear components including heterochromatin and nucleoli. As a representative example, CAT hematoxylin (Biocare Medical, Pacheo, CA) can be used for histological demonstration of nuclear staining. Commonly used hematoxylin solutions are mordanted with aluminum, typically using aluminum alum (aluminum ammonium sulfate) as the mordant salt. Because aluminum salts are not themselves oxidizing agents, exposure of the hematoxylin solution to air or chemicals is required to convert the hematoxylin to hematein. Adding acid to an alum-hematoxylin solution (e.g., CAT hematoxylin) is thought to enhance the selectivity of the stain for the nucleus and counteract the rapid oxidizing effect of chemical oxidants. This latter function allows the solution to maintain an equilibrium between some hematoxylin and hematein, ensuring good staining. Glycerol tends to stabilize the system against peroxidation and helps prevent rapid evaporation.

[0153] When any fluorescent dye, such as FITC (fluorescein isothiocyanate), Cy2 (Amersham Biosciences, Amersham, UK), or Alexa488 (Molecular Probes, Inc., Eugene, OR), is used as a labeling substance for the secondary antibody, the visualization substance reaction step is not necessary. The light emitted by irradiation with light of the excitation wavelength of the fluorescent substance can be appropriately detected using a fluorescence microscope.

[0154] In an exemplary embodiment, an in vitro immunoassay for detecting the presence of GPC3-expressing cells in a subject comprises the following steps: (a) providing a tissue preparation from the subject as a formalin-fixed, paraffin-embedded section and attaching the formalin-fixed, paraffin-embedded section to a transparent support; (b) subjecting the tissue preparation to a deparaffinization treatment; (c) optionally subjecting the tissue preparation to an antigen retrieval treatment; and (d) contacting the anti-GPC3 antibody with the tissue preparation under conditions sufficient to form a complex between the anti-GPC3 antibody and GPC3 present in the cell membrane of the cells in the tissue preparation treated in step (c). and (e) detecting the presence of the complex by immunohistochemistry, wherein if the complex is present, the subject is diagnosed with a GPC3-expressing tumor, and the anti-GPC3 antibody is a monoclonal antibody that specifically binds to an epitope of the beta chain of GPC3, and the heavy chain of the anti-GPC3 antibody comprises a complementarity-determining region (CDR) 1 set forth as SEQ ID NO: 6, a CDR2 set forth as SEQ ID NO: 8, and a CDR3 set forth as SEQ ID NO: 10, and the light chain of the antibody comprises a CDR1 set forth as SEQ ID NO: 13, a CDR2 set forth as SEQ ID NO: 15, and a CDR3 set forth as SEQ ID NO: 17. In an example, the GPC3-expressing tumor is selected from the group consisting of hepatocellular carcinoma, non-small cell lung cancer, ovarian clear cell carcinoma, and gastric cancer. In an example, the heavy chain of the anti-GPC3 antibody has a heavy chain variable region (HCVR) set forth as SEQ ID NO: 2. In an example, the light chain of the anti-GPC3 antibody has a light chain variable region (LCVR) set forth as SEQ ID NO: 4. In some examples, the anti-GPC3 antibody is 204, which specifically recognizes an epitope on the beta chain of GPC3, which epitope is different from the epitope specifically recognized by 1G12, which is further different from the epitope specifically recognized by GC33. In some examples, the antigen retrieval treatment is based on the heat-induced epitope retrieval (HIER) method. In some examples, the HIER method includes heating the tissue preparation of step (c) to 105-115°C for a time period of 10-20 minutes, preferably, the tissue preparation is heated to 110°C for 15 minutes. In some examples, the antigen retrieval treatment is also or alternatively based on the protease-induced epitope retrieval (PIER) method.In examples using the PIER method, the protease used in the PIER method is selected from the group consisting of pepsin, trypsin, and protease K. In examples, detecting the presence of the complex using immunohistochemistry comprises an enzymatic reaction. In examples, step (e) further comprises contacting the tissue preparation of step (d) with a secondary antibody conjugated to horseradish peroxidase (HRP) enzyme and visualizing the complex via oxidation of 3,3'-diaminobenzidine by hydrogen peroxide in a reaction catalyzed by HRP. In examples, detecting the presence of the complex further comprises scoring the amount of the detected complex. In some examples, the scoring is performed by a pathologist. In some examples, detecting the presence of the complex is performed via digitization, and the scoring is automated based on the digitization of the detected complex. In some examples, the scoring further includes specifying the staining intensity of the complex detected via immunohistochemistry using an integer scale from 0 (negative) to 3+, recording the percentage of cells that stain positively at each intensity level, and calculating a membrane-bound H-score based on the percentage of cells that stain positively at each intensity level.

[0155] e. Automation of IHC IVD assays The IHC IVD assays disclosed herein may be performed manually or automated. Relevant examples of automated systems capable of performing the IHC IVD assays of the present disclosure include, but are not limited to, Intellipath FLX® (Biocare Medical, Pacheo, CA), Autostainer Link 48 (Agilent Technologies, Santa Clara, CA), and the BOND-III Fully Automated IHC Staining System (Leica Biosystems, Richmond, IL).

[0156] f. Classification of GPC3-expressing tissues and prediction of therapeutic efficacy It is known that GPC3 can release its N-terminal portion into serum, for example, during digestion in cancerous tissue (e.g., liver cancer tissue) (WO 2004 / 022739).Therefore, it is not expected that an antibody that reacts with the N-terminal portion of GPC3 can bind to the C-terminal portion of the GPC3 polypeptide that remains fixed on the cell surface after digestion.As described in the following examples, the 204 antibody is advantageous for use in the IHC IVD assay disclosed herein, at least in part due to its ability to specifically recognize the C-terminal portion of GPC3 that remains fixed on the cell membrane after digestion in tumor tissue.

[0157] Anti-GPC3 antibodies are known to be useful in the treatment and prevention of liver cancer (see, e.g., WO 2004 / 022739), and there is evidence of anti-tumor activity conferred by cells expressing CAR constructs that specifically bind to epitopes within GPC3 expressed on the surface of solid tumor cells (see, e.g., WO 2020 / 072546). Because immunotherapies relying on antibodies, CARs, etc. function by binding to cell-surface GPC3, it is desirable for any prediction of therapeutic efficacy to primarily account for the expression of membrane-bound GPC3. In other words, if the therapeutic efficacy of therapeutic anti-GPC immunotherapy (e.g., antibodies, CARs, etc.) against GPC3-expressing tumor cells (e.g., solid tumor cells) is predicted depending on whether the epitope to which the anti-GPC3 targeting agent binds is present in the GPC3-expressing cells, it is desirable for this method to rely on an anti-GPC3 targeting agent that specifically binds to the C-terminal portion of GPC3 that remains anchored to the cell membrane. As discussed herein and illustrated in the Examples, the preferred 204 antibody for use in the IHC IVD method of the present disclosure specifically recognizes the C-terminal portion of GPC3 and preferentially binds to GPC3 expressed on the cell surface of tumor cells. Therefore, the 204 antibody and its use in the IHC IVD method disclosed herein are advantageous in that the results of the assay can be applied to predict the therapeutic efficacy of anti-GPC3 agents, including, but not limited to, anti-GPC3 antibodies, cells expressing anti-GPC3 CAR(s), and the like.

[0158] Classification of GPC3-expressing cells / tissues relies on a scoring system based on one or more of staining intensity and membrane-bound H-score, but is not necessarily limited to these parameters. Briefly, staining intensity in the IHC IVD assay of the present disclosure is scored using a semiquantitative integer scale from 0 (negative) to 3 (or "3+"). The percentage of positively stained cells at each intensity level is recorded. Scoring is preferably based on the localization of GPC3 to the cell membrane (apical and peripheral), but in some instances, any cytoplasmic staining can also be accounted for. The H score is calculated as a value between 0 and 300 and is defined as follows: 1x (percentage of cells staining at intensity 1+) + 2x (percentage of cells staining at intensity 2+) + 3x (percentage of cells staining at intensity 3+) = H score. A higher H score indicates a greater predicted therapeutic effect of a therapeutic anti-GPC3 therapy (e.g., anti-GPC3 immunotherapy) on GPC3-expressing tumor cells. In some examples, the scoring may be performed by a certified pathologist. Additionally or alternatively, it is within the scope of the present disclosure that the scoring may be automated. For example, the present disclosure provides for digitization of the difference in the degree and pattern of microscopic detection of antigen-antibody complexes resulting from GPC3 and anti-GPC3 antibodies (e.g., 204). In examples where the scoring is performed by a pathologist and / or the scoring is digitized, the test sample may be normalized to a control sample, such as an isotype control sample or a similar tissue preparation lacking expression of GPC3 on the cell surface.

[0159] g. Diagnostic and / or therapeutic methods based on IHC IVD assays The IHC IVD method of the present disclosure is useful for diagnosing a patient as having a cancer containing corresponding cells that express GPC3 on the cell membrane.The IHC IVD method of the present disclosure is further useful for determining whether to treat a patient with an anti-GPC3 therapy (e.g., antibody-based immunotherapy, CAR-based immunotherapy, etc.) if the patient has not yet received anti-GPC3 therapy.The IHC IVD method is also useful for determining whether to continue treatment of a patient with anti-GPC3 therapy if the patient has already received anti-GPC3 therapy for some time.In some examples, the dose and / or administration interval of the anti-GPC3 therapy can be adjusted (e.g., the dose can be increased or decreased, the administration interval can be extended or shortened, etc.) according to the results of the IHC IVD assay disclosed herein. Thus, the IHC IVD assays disclosed herein can be used to diagnose a patient as having a particular cancer, i.e., a solid tumor that expresses GPC3 on the surface of the cells comprising the tumor, and can also be used as a means of monitoring a patient's response to cancer treatments, including but not limited to, cancer treatments that specifically target GPC3 on the surface of cancerous cells.

[0160] Thus, in one aspect, the present invention provides a method for determining the presence of GPC3 in a sample suspected of containing GPC3, the method comprising exposing the sample to an antibody of the present invention and determining binding of the antibody to GPC3 in the sample, wherein binding of the antibody to GPC3 in the sample indicates the presence of the protein in the sample. In one embodiment, the sample is a biological sample (e.g., a tissue preparation). In a further embodiment, the biological sample comprises liver cancer cells. In one embodiment, the biological sample is derived from a mammal suffering from or suspected of suffering from a liver cancer disorder and / or a liver cancer cell proliferative disorder. In a further embodiment, the biological sample comprises ovarian cancer cells. In one embodiment, the biological sample is derived from a mammal suffering from or suspected of suffering from an ovarian cancer disorder and / or an ovarian cancer cell proliferative disorder. In a further embodiment, the biological sample comprises gastric cancer (adenocarcinoma) cells. In one embodiment, the biological sample is from a mammal suffering from or suspected of suffering from a gastric (or stomach) disorder and / or a gastric (or stomach) cell proliferative disorder. In a further embodiment, the biological sample comprises lung cancer cells. In one embodiment, the biological sample is from a mammal suffering from or suspected of suffering from a squamous cell carcinoma disorder and / or a lung cancer cell proliferative disorder. In one embodiment, the biological sample comprises skin cells. In a further embodiment, the biological sample is from a mammal suffering from or suspected of suffering from Merkel cell carcinoma or melanoma.

[0161] In one aspect, a method for diagnosing a cell proliferative disorder associated with (i) an increase in GPC3-expressing cells, such as liver cancer cells, ovarian cancer cells, lung cancer cells, or gastric or stomach cancer cells, or (ii) an increase in GPC3 expression in tumors is provided. In one embodiment, the method comprises contacting test cells of a biological sample (e.g., a tissue preparation) with an anti-GPC3 antibody of the present disclosure, detecting the binding of the antibody to GPC3 to determine the level of antibody bound to the test cells of the sample, and comparing the level of antibody bound to cells of a control sample, wherein the level of antibody bound is normalized to the number of GPC3-expressing cells in the test sample and the control sample, and a higher level of antibody bound in the test sample compared to the control sample indicates the presence of a cell proliferative disorder associated with cells expressing GPC3.

[0162] In one aspect, a method for predicting the therapeutic effect of anti-GPC3 immunotherapy on cancer is provided, wherein the cancer is characterized by the cancer cells expressing GPC3, and the method comprises detecting the presence of the cells in a subject via the IVD IHC assay disclosed herein.In an embodiment, if the complex between the anti-GPC3 antibody and GPC3 expressed on the membrane of the cancer cell is detected, the anti-GPC3 immunotherapy is predicted to have a therapeutic effect on the cancer of the subject.In an embodiment, the method for predicting therapeutic effect is carried out before the subject receives any anti-GPC3 immunotherapy.In some embodiments, the method for predicting therapeutic effect is carried out while the subject is already receiving anti-GPC3 immunotherapy.

[0163] h. Related Detection Schemes and Assay Methods Although the present invention focuses on IHC methods using the anti-GPC3 antibodies disclosed herein, it is understood that the anti-GPC3 antibodies of the present invention may be used in any known assay method, such as ELISA, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, pp. 147-158, CRC Press, Inc.).

[0164] Detection labels can be useful for localizing, visualizing, and quantifying binding or recognition events. The labeled antibodies of the present invention can detect cell surface GPC3. Another use of detectably labeled antibodies is in bead-based immunocapture methods, which involve conjugating beads with fluorescently labeled antibodies and detecting fluorescent signals upon ligand binding. A similar binding detection method uses surface plasmon resonance (SPR) to measure and detect antibody-antigen interactions. Detection labels, such as fluorescent dyes and chemiluminescent dyes (Briggs et al. (1997) "Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1:1051-1058), provide a detectable signal and are generally applicable to labeling antibodies, preferably having the following properties: (i) the labeled antibody should produce a very high signal with low background so that small amounts of antibody can be detected with high sensitivity in both cell-free and cell-based assays, and (ii) the labeled antibody should be photostable so that the fluorescent signal can be observed, monitored, and recorded without significant bleaching. For applications involving membrane or cell surface binding of labeled antibodies, particularly to living cells, the label preferably (iii) has good water solubility to achieve effective conjugate concentrations and detection sensitivity, and (iv) is non-toxic to living cells so as not to disrupt their normal metabolic processes or result in premature cell death.

[0165] Direct quantification of cellular fluorescence intensity and fluorescently labeled events, such as cell surface binding of peptide-dye conjugates, may be performed with a system (FMAT® 8100 HTS system, Applied Biosystems, Foster City, Calif.) that automates mix-and-read, non-radioactive assays with live cells or beads (Miraglia, “Homogeneous cell- and bead-based assays for high throughput screening using fluorometric microvolume assay technology,” (1999) J. of Biomolecular Screening 4:193-204). Uses of labeled antibodies also include cell surface receptor binding assays, immunocapture assays, fluorescence-linked immunosorbent assays (FLISA), caspase cleavage (Zheng, "Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo," (1998) Proc. Natl. Acad. Sci. USA 95:618-23, US 6,372,907), apoptosis (Vermes, "A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein-labeled Annexin V," (1995) J. Immunol. Methods 184:39-51), and cytotoxicity assays. Fluorescence microanalysis techniques can be used to identify up- or down-regulation by molecules that target the cell surface (Swartzman, "A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology", (1999) Anal. Biochem. 271:143-51).

[0166] The labeled antibodies of the present invention are useful as imaging biomarkers and probes for various biomedical and molecular imaging methods and techniques, such as (i) MRI (magnetic resonance imaging), (ii) microCT (computed tomography), (iii) SPECT (single photon emission computed tomography), (iv) PET (positron emission tomography) Chen et al. (2004) Bioconjugate Chem. 15:41-49, (v) bioluminescence, (vi) fluorescence, and (vii) ultrasound. Immunoscintigraphy is an imaging technique in which a radiolabeled antibody is administered to an animal or human patient and images are obtained at the site in the body where the antibody is localized (US 6,528,624). Imaging biomarkers can be objectively measured and evaluated as indicators of normal biological processes, pathological processes, or pharmacological responses to therapeutic interventions.

[0167] Methods for labeling peptides are well known. Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc. Brinkley, 1992, Bioconjugate Chem. 3:2, Garman, (1997) Non-Radioactive Labeling: A Practical Approach, Academic Press, London, Means (1990) Bioconjugate Chem. 1:2, Glazer et al. al(1975) Chemical Modification of Proteins. Laboratory Techniques in Biochemistry and Molecular Biology (TSWork and E. Work, Eds.) American Elsevier Publishing Co., New York, Lundblad, RLand Noyes, CM (1984) Chemical Reagents for Protein Modification, Vols. I and II, CRC Press, New York, Pfleiderer, G. (1985) “Chemical Modification of Proteins”,Modern Methods in See Protein Chemistry, H. Tschesche, Ed., Walter DeGryter, Berlin and New York, and Wong (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.), De Leon-Rodriguez et al (2004) Chem. Eur. J. 10:1149-1155, Lewis et al (2001) Bioconjugate Chem. 12:320-324, Li et al (2002) Bioconjugate Chem. 13:110-115, Mier et al (2005) Bioconjugate Chem. 16:240-237.

[0168] The labeled antibody of the present invention can also be used as an affinity purification agent.In this process, the labeled antibody is immobilized on a solid phase, such as Sephadex resin or filter paper, using methods well known in the art.The immobilized antibody is contacted with a sample containing the antigen to be purified, and then the support is washed with a suitable solvent that substantially removes all substances in the sample except for the antigen to be purified that is bound to the immobilized polypeptide variant.Finally, the support is washed with another suitable solvent, such as a glycine buffer solution at pH 5.0, to release the antigen from the polypeptide variant.

[0169] In one aspect, the anti-GPC3 antibody of the present invention binds to the same epitope of GPC3 bound by another GPC3 antibody. In another embodiment, the GPC3 antibody of the present invention binds to the same epitope of GPC3 bound by a fragment (e.g., a Fab fragment) of a monoclonal antibody comprising the variable domains of SEQ ID NO:2 and SEQ ID NO:4, or a chimeric antibody comprising the variable domains of a monoclonal antibody comprising the sequences of SEQ ID NO:2 and SEQ ID NO:4 and a constant domain derived from IgG1.

[0170] C. General Description of Anti-GPC3 Antibodies Included in the Disclosure In one embodiment, the present invention provides anti-GPC3 antibodies that can be used herein as diagnostic and / or therapeutic agents. Exemplary antibodies include polyclonal, monoclonal, chimeric, humanized, and human antibodies.

[0171] 1. Polyclonal antibodies Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species being immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or RTST=C=NR (where R and R are different alkyl groups).

[0172] Animals are immunized against the antigen, immunogenic conjugate, or derivative by mixing, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted subcutaneously at multiple sites with Freund's complete adjuvant containing 1 / 2 to 1 / 10 the original amount of peptide or conjugate. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates can also be produced in recombinant cell culture as protein fusions. Aggregating agents such as alum are also suitably used to enhance the immune response.

[0173] 2. Monoclonal antibodies Monoclonal antibodies (mAbs) to an antigen of interest can be prepared using any technique known in the art, including, but not limited to, the hybridoma technique first described by Kohler and Milstein (1975, Nature 256, 495-497), the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Selected lymphocyte antibody method (SLAM) (Babcook, JS, et al., A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci USA, 1996. 93(15):7843-8) and (McLean GR, Olsen OA, Watt IN, Rathanaswami P, Leslie KB, Babcook JS, Schrader JW. Recognition of human cytomegalovirus by human primary immunoglobulins identifies an innate foundation to an adaptive immune response. J Immunol. 2005 Apr 15;174(8):4768-78). Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Hybridomas producing mAbs used in the present invention can be grown in vitro or in vivo.

[0174] Monoclonal antibodies may be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, the lymphocytes are isolated and then fused with a myeloma cell line using an appropriate fusing agent, such as polyethylene glycol, to form hybridoma cells (Coding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0175] The hybridoma cells thus prepared are seeded and grown in an appropriate medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells (also called the fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0176] Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to selective media that select against the unfused parent cells. Preferred myeloma cell lines are mouse myeloma lines, such as MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and SP-2 and derivatives, such as X63-Ag8-653 cells, available from the American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984), and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0177] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0178] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980).

[0179] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Coding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Additionally, the hybridoma cells may be grown in vivo as ascites tumors in animals, for example, by i.p. injection of the cells into mice.

[0180] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures, such as affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0181] DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the murine antibody). The hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA can be placed into an expression vector, which can then be transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, resulting in the synthesis of the monoclonal antibody in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Rev. 130:151-188 (1992).

[0182] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990), Clackson et al., Nature, 352:624-628 (1991), and Marks et al., J. Mol. Biol., 222:581-597 (1991), which describe the isolation of murine and human antibodies, respectively, using phage libraries. Further publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), and combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0183] The DNA encoding the antibody can be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy and light chain constant domains (CH and CO sequences) for the homologous murine sequences (U.S. Pat. No. 4,816,567, and Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can replace the antibody constant domains, or they can replace the variable domains of one antigen-binding site of an antibody, creating a chimeric bivalent antibody containing an antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0184] 3. Chimeric, Humanized, and Human Antibodies In some embodiments, the anti-GPC3 antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0185] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from human antibody sequences. Optionally, a humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0186] The anti-GPC3 antibodies of the present invention may further include humanized or human antibodies. Humanized forms of non-human (e.g., mouse or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues of the recipient's complementarity-determining regions (CDRs) are replaced with residues from CDRs of non-human species (donor antibodies), such as mouse, rat, or rabbit, that have the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibodies may also contain residues found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, the humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0187] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, and they are usually taken from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and colleagues [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)] by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Such "humanized" antibodies are therefore chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0188] The choice of human variable domains, both light and heavy, used in the generation of the humanized antibody is crucial if the antibody is intended for therapeutic use in humans in order to reduce antigenicity and the HAMA response (human anti-mouse antibody). The reduction or elimination of the HAMA response is a key aspect of the clinical development of suitable therapeutic agents. See, e.g., Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985), 135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res. (1990), 50:1495. As described herein, the present invention provides antibodies that have been humanized to reduce or eliminate the HAMA response. These antibody variants can also be obtained using conventional methods known in the art, some of which are further described below. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The sequence of the human V domain that is closest to that of the rodent is identified, and the human framework regions (FRs) therein are adopted for the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).

[0189] For example, an amino acid sequence from an antibody described herein can serve as a starting (parent) sequence for diversifying the framework and / or hypervariable sequence(s). The selected framework sequence to which the starting hypervariable sequence is linked is referred to herein as the acceptor human framework. The acceptor human framework can be from or derived from a human immunoglobulin (the VL and / or VH region thereof), but preferably the acceptor human framework is from or derived from a human consensus framework sequence, as such frameworks have been demonstrated to be minimally or non-immunogenic in human patients.

[0190] If the acceptor is derived from a human immunoglobulin, optionally, human framework sequences selected based on their homology to the donor framework sequence may be selected by aligning the donor framework sequence with various human framework sequences in a collection of human framework sequences, and the most homologous framework sequence may be selected as the acceptor.

[0191] In one embodiment, the human consensus framework herein is from or is derived from a VH subgroup III and / or VL kappa subgroup I consensus heavy chain framework sequence.

[0192] Although the acceptor may be identical in sequence to a selected human framework sequence, whether from a human immunoglobulin or a human consensus framework, the present invention contemplates that the acceptor sequence may contain pre-existing amino acid substitutions relative to the human immunoglobulin sequence or human consensus framework sequence. These pre-existing substitutions are preferably minimal, typically only 4, 3, 2, or 1 amino acid mismatch relative to the human immunoglobulin sequence or consensus framework sequence.

[0193] Hypervariable region residues of a non-human antibody are incorporated into the VL and / or VH acceptor human framework. For example, residues corresponding to Kabat CDR residues, Chothia hypervariable loop residues, Abm residues, and / or contact residues may be incorporated. Optionally, the following extended hypervariable region residues are incorporated: 24-34 (LI), 50-56 (L2), and 89-97 (L3), 26-35B (HI), 50-65, 47-65, or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3).

[0194] While the "incorporation" of hypervariable region residues is discussed herein, it will be understood that this can be achieved in a variety of ways. For example, a nucleic acid encoding a desired amino acid sequence can be generated by mutating a nucleic acid encoding a murine variable domain sequence so that its framework residues are changed to acceptor human framework residues, or by mutating a nucleic acid encoding a human variable domain sequence so that the hypervariable domain residues are changed to non-human residues, or by synthesizing a nucleic acid encoding the desired sequence, etc.

[0195] As described herein, hypervariable region graft variants can be generated by Kunkel mutagenesis of nucleic acids encoding the human acceptor sequences, using separate oligonucleotides for each hypervariable region. Kunkel et al., Methods Enzymol. 154:367-382 (1987). Routine techniques can be used to introduce appropriate changes within the framework and / or hypervariable regions to correct and re-establish proper hypervariable region-antigen interactions.

[0196] Thus, in one embodiment, the invention provides humanized antibodies that induce, and / or are expected to induce, a substantially lower level of human anti-mouse antibody response (HAMA) in a host subject compared to antibodies comprising the sequences of SEQ ID NOs: 2 and 4. In another example, the invention provides humanized antibodies that induce, and / or are expected to induce, minimal, or do not induce, and / or are expected to not induce, a human anti-mouse antibody response (HAMA). In one example, the antibodies of the invention induce an anti-mouse antibody response that is at or below a clinically acceptable level.

[0197] A humanized antibody of the invention may comprise one or more human and / or human consensus non-hypervariable region (e.g., framework) sequences in its heavy and / or light chain variable regions. In some embodiments, one or more additional modifications are present within the human and / or human consensus non-hypervariable region sequences. In one embodiment, the heavy chain variable domain of an antibody of the invention comprises a human consensus framework sequence, which in one embodiment is a subgroup III consensus framework sequence. In one embodiment, an antibody of the invention comprises a variant subgroup III consensus framework sequence modified at at least one amino acid position.

[0198] As is known in the art and described in more detail herein, the amino acid positions / boundaries delineating an antibody hypervariable region can vary depending on the context and the various definitions known in the art (as described below). Some positions within a variable domain can be considered hybrid hypervariable positions because these positions can be considered within a hypervariable region under one set of criteria, but outside of a hypervariable region under a different set of criteria. One or more of these positions can also be found in an extended hypervariable region (further defined below). The present invention provides antibodies containing modifications at these hybrid hypervariable positions. In one embodiment, these hypervariable positions include one or more of positions 26-30, 33-35B, 47-49, 57-65, 93, 94, and 101-102 in the heavy chain variable domain. In one embodiment, these hybrid hypervariable positions include one or more of positions 24-29, 35-36, 46-49, 56, and 97 in the light chain variable domain. In one embodiment, an antibody of the invention comprises a consensus framework sequence of a human variant human subgroup modified at one or more hybrid hypervariable positions.

[0199] The antibodies of the present invention can comprise any suitable human or human consensus light chain framework sequence, so long as the antibody exhibits the desired biological properties (e.g., desired binding affinity). In one embodiment, the antibodies of the present invention comprise at least a portion (or all) of the framework sequence of a human κ light chain. In one embodiment, the antibodies of the present invention comprise at least a portion (or all) of the framework consensus sequence of human κ subgroup I.

[0200] Phage(mid) display (also referred to as phage display in some contexts herein) can be used as a convenient and rapid method for generating and screening many different potential variant antibodies in libraries generated by sequence randomization. However, other methods for making and screening modified antibodies are available to those skilled in the art.

[0201] Phage(mid) display technology has provided a powerful tool for generating and selecting novel proteins that bind to ligands, e.g., antigens. Phage(mid) display technology allows for the generation of large libraries of protein variants that can be rapidly sorted for sequences that bind to target molecules with high affinity. Nucleic acids encoding variant polypeptides are generally fused to nucleic acid sequences encoding viral coat proteins, such as gene III or gene VIII proteins. Monovalent phagemid display systems have been developed in which the nucleic acid sequence encoding the protein or polypeptide is fused to a nucleic acid sequence encoding a portion of the gene III protein. (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In monovalent phagemid display systems, the gene fusion is expressed at low levels, and wild-type gene III protein is also expressed, thereby maintaining the infectivity of the particles. Methods for generating peptide libraries and screening those libraries are disclosed in many patents (e.g., U.S. Patent No. 5,723,286, U.S. Patent No. 5,432,018, U.S. Patent No. 5,580,717, U.S. Patent No. 5,427,908, and U.S. Patent No. 5,498,530).

[0202] Libraries of antibodies or antigen-binding polypeptides have been prepared by several methods, including modifying a single gene by inserting random DNA sequences or cloning a family of related genes. Methods for displaying antibodies or antigen-binding fragments using phage(mid) display are described in U.S. Patent Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727. The libraries are then screened for the expression of antibodies or antigen-binding proteins with the desired properties.

[0203] Methods for substituting optimal amino acids into template nucleic acids are well established in the art, some of which are described herein. For example, hypervariable region residues can be substituted using the Kunkel method. See, for example, Kunkel et al., Methods Enzymol. 154:367-382 (1987).

[0204] The sequence of the oligonucleotide contains one or more codon sets designed for the hypervariable region residues to be altered. A codon set is a set of different nucleotide triplet sequences used to encode the desired variant amino acids. Codon sets can be represented using symbols to represent specific nucleotides or equimolar mixtures of nucleotides, as shown below according to the IUB code. IUB Code G Guanine A Adenine T thymine C Cytosine R (A or G) Y (C or T) M (A or C) K (G or T) S (C or G) W (A or T) H (A or C or T) B (C ​​or G or T) V (A or C or G) D (A or G or T)H N (A or C or G or T)

[0205] For example, in the codon set DVK, D can be the nucleotide A, G, or T, V can be A, G, or C, and K can be G or T. This codon set can represent 18 different codons and can encode the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

[0206] Oligonucleotide or primer sets can be synthesized using standard methods. The set of oligonucleotides can be synthesized, for example, by solid-phase synthesis, and contains sequences that represent all possible combinations of nucleotide triplets provided by the codon set and encode the desired amino acid group. The synthesis of oligonucleotides with "degeneracy" of selected nucleotides at specific positions is well known in the art. Such a set of nucleotides with a specific codon set can be synthesized using a commercially available nucleic acid synthesizer (e.g., available from Applied Biosystems, Foster City, Calif.) or can be obtained commercially (e.g., from Life Technologies, Rockville, Md.). Thus, a set of synthesized oligonucleotides with a specific codon set usually contains multiple oligonucleotides with different sequences, the differences being determined by the codon set within the overall sequence. The oligonucleotides used in accordance with the present invention have sequences that allow hybridization to a nucleic acid template of the variable domain and may also contain restriction enzyme sites for cloning purposes.

[0207] In one method, nucleic acid sequences encoding variant amino acids can be created by oligonucleotide-mediated mutagenesis. This technique is well known in the art and is described by Zoller et al. Nucleic Acids Res. 10:6487-6504 (1987). Briefly, nucleic acid sequences encoding variant amino acids are created by hybridizing an oligonucleotide set encoding a desired codon set to a DNA template, where the template is a single-stranded form of a plasmid containing the nucleic acid template sequence of the variable region. After hybridization, DNA polymerase is used to synthesize the entire second complementary strand of the template, which consequently incorporates the oligonucleotide primer and contains the codon set provided by the oligonucleotide set.

[0208] Generally, oligonucleotides of at least 25 nucleotides in length are used.The optimal oligonucleotide has 12-15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) that code for the mutation(s).This ensures that the oligonucleotide can be properly hybridized to the single-stranded DNA template molecule.The oligonucleotide can be easily synthesized using techniques known in the art, for example, by Crea et al., Proc.Nat'l.Acad.Sci.USA, 75:5765 (1978).

[0209] The DNA template is generated by a vector derived from either a bacteriophage M13 vector (the commercially available M13mp18 and M13mp19 vectors are suitable) or a vector containing the single-stranded phage origin of replication described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, the DNA to be mutated can be inserted into one of these vectors to generate a single-stranded template.

[0210] The production of single-stranded templates is described in sections 4.21-4.41 of Sambrook et al., supra. To modify the native DNA sequence, the oligonucleotide is hybridized to the single-stranded template under appropriate hybridization conditions. A DNA polymerizing enzyme, usually T7 DNA polymerase or the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis. A heteroduplex molecule is thus formed, with one strand of DNA encoding a mutant form of gene 1 and the other strand (the original template) encoding the native, unmodified sequence of gene 1. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote, such as E. coli JM101. After the cells are grown, they are plated on agarose plates and screened using an oligonucleotide primer radiolabeled with 32-phosphate to identify bacterial colonies containing the mutated DNA.

[0211] The method just described may be modified to create a homoduplex molecule in which both strands of the plasmid contain the mutation(s). The modification is as follows: The single-stranded oligonucleotide is annealed to the single-stranded template described above. A mixture of three deoxyribonucleotides, namely, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTT), is mixed with a modified thiodeoxyribocytosine called dCTP-(aS) (available from Amersham). This mixture is added to the template-oligonucleotide complex. After adding DNA polymerase to this mixture, a DNA strand identical to the template except for the mutated base is generated. Furthermore, this new DNA strand contains dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion.

[0212] After nicking the double-stranded heteroduplex template strand with an appropriate restriction enzyme, the template strand can be digested with ExoIII nuclease or another appropriate nuclease past the region containing the mutagenesis site(s). The reaction is then stopped, leaving a molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell.

[0213] As mentioned above, the sequence of the oligonucleotide set is of sufficient length to hybridize to the template nucleic acid and may, but need not, include a restriction enzyme site. The DNA template can be generated by a vector derived from one of the bacteriophage M13 vectors or a vector containing the single-stranded phage replication origin described by Viera et al., Meth. Enzymol., 153:3 (1987). Therefore, the DNA to be mutated must be inserted into one of these vectors to generate a single-stranded template. Production of single-stranded templates is described in sections 4.21-4.41 of Sambrook et al., supra.

[0214] According to another method, antigen binding can be restored by selecting repaired hypervariable regions during antibody humanization (see Application No. 11 / 061,841, filed February 18, 2005). The method involves incorporating non-human hypervariable regions into an acceptor framework and further introducing one or more amino acid substitutions in one or more hypervariable regions without modifying the acceptor framework sequence. Alternatively, the introduction of the one or more amino acid substitutions can be accompanied by modification of the acceptor framework sequence.

[0215] According to another method, a library can be generated by providing upstream and downstream oligonucleotide sets, each set having multiple oligonucleotides of different sequences, the different sequences being established by the codon sets provided within the sequences of the oligonucleotides. The upstream and downstream oligonucleotide sets, along with variable domain template nucleic acid sequences, can be used in a polymerase chain reaction to generate a "library" of PCR products. The PCR products can be referred to as "nucleic acid cassettes" because they can be fused to other related or unrelated nucleic acid sequences, such as viral coat proteins and dimerization domains, using established molecular biology techniques.

[0216] The PCR primer sequences include one or more codon sets designed for solvent-accessible and highly diverse positions in the hypervariable regions. A codon set is a set of different nucleotide triplet sequences used to encode desired variant amino acids. Antibody selectants that meet the desired criteria, selected through appropriate screening / selection steps, can be isolated and cloned using standard recombinant techniques.

[0217] It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve the desired antibody characteristic, e.g., increased affinity for the target antigen(s). Generally, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0218] Various forms of humanized anti-GPC3 antibodies are contemplated. For example, the humanized antibody may be an antibody fragment, such as Fab. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

[0219] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice is said to result in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993), Jakobovits et al., Nature, 362:255-258 (1993), Bruggemann et al., Year in Immuno. 7:33 (1993), U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all GenPharm), 5,545,807, and WO 97 / 17852.

[0220] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553

[1990] ) can be used to produce human antibodies and antibody fragments in vitro from gene repertoires of immunoglobulin variable (V) domains from unimmunized donors. According to this technology, antibody V domain genes are cloned in frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage recapitulate some of the properties of B cells. Phage display can be carried out in a variety of formats, as reviewed, for example, in Johnson, Kevin S., and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene fragments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993), a repertoire of V genes from unimmunized human donors can be constructed, and antibodies against a diverse array of antigens (including self-antigens) can be isolated. See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0221] As noted above, human antibodies may also be generated in vitro by activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0222] In another embodiment, the antibody of the present disclosure is a human monoclonal antibody. Such human monoclonal antibodies against GPC3 can be generated using transgenic or transchromosomic mice that possess parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include those referred to herein as HuMAb Mouse™ and KM Mouse™, respectively, and are collectively referred to herein as "human Ig mice." The HuMAb Mouse™ (Medarex, Inc.) contains a human immunoglobulin gene minilocus encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, with targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg, et al. (1994) Nature 368(6474):856-859). Thus, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation, resulting in the generation of high-affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93; and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764:536-546).The preparation and use of HuMAb Mouse™ and the genomic modifications carried by such mice are described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, L et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591, and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are specifically incorporated herein by reference in their entireties. Further, U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, all to Lonberg and Kay; U.S. Patent No. 5,545,807, Surani et al.; PCT Publication Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962, all to Lonberg and Kay. Kay, and PCT Publication No. WO 01 / 14424, Korman et al. In another embodiment, the human antibodies of the disclosure can be propagated using mice carrying human immunoglobulin sequences in transgenes and transchromosomes, e.g., mice carrying a human heavy chain transgene and a human light chain transchromosome. This mouse is referred to herein as the "KM Mouse™" and is described in detail in PCT Publication No. WO 02 / 43478 by Ishida et al.

[0223] Additionally, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-GPC3 antibodies of the present disclosure. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used, and such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584 and 6,162,963 to Kucherlapati et al.

[0224] Furthermore, alternative chromosomal transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise the anti-GPC3 antibodies of the present disclosure. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice," can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, cattle carrying human heavy and light chain transchromosomes have been described in the art (e.g., Kuroiwa et al. (2002) Nature Biotechnology 20:889-894 and PCT Application No. WO 2002 / 092812), and can be used to raise the anti-GPC3 antibodies of the present disclosure.

[0225] 4. Antibody fragment In certain situations, it is advantageous to use antibody fragments rather than whole antibodies: the smaller size of the fragments may allow for rapid clearance and improved access to solid tumors.

[0226] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli, allowing for the facile production of large amounts of these fragments. Antibody fragments can also be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with extended in vivo half-lives containing salvage receptor-binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 93 / 16185, U.S. Pat. No. 5,571,894, and U.S. Pat. No. 5,587,458. Fv and sFv are the only species with intact combining sites devoid of constant regions, and thus are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins can be constructed to provide fusion of an effector protein to either the amino or carboxy terminus of the sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody", eg, as described in US Pat. No. 5,641,870.

[0227] In one embodiment, scFv derived from an anti-GPC3 antibody is used in a CAR-modified immune cell, preferably a CAR-T or CAR-NK cell, as disclosed herein. Anti-GPC3 antibody fragments include portions of anti-GPC3 antibodies (and combinations of anti-GPC3 antibody portions, e.g., scFvs) that can be used as targeting arms for the GPC3 tumor epitope in the chimeric antigen receptor of CAR-T or CAR-NK cells. Such fragments are not necessarily proteolytic fragments, but rather portions of a polypeptide sequence that can confer affinity for a target.

[0228] 5. Bispecific antibodies Bispecific antibodies are antibodies with binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the GPC3 protein described herein. Other such antibodies may combine a GPC3-binding site with a binding site for another protein. Alternatively, the anti-GPC3 arm can be combined with an arm that binds to a trigger molecule on leukocytes, such as a T cell receptor molecule (e.g., CD3), or an Fc receptor (FcyR) for IgG, such as FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16), to concentrate and localize cellular defense mechanisms to the GPC3-expressing cells. Bispecific antibodies can also be used to localize cytotoxic drugs to GPC3-expressing cells. These antibodies have a GPC3-binding arm and an arm that binds to a cytotoxic drug (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate, or a radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab')2 bispecific antibodies).

[0229] WO 96 / 16673 describes a bispecific anti-ErbB2 / anti-FcYRIII antibody, and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2 / anti-FcyRI antibody. A bispecific anti-ErbB2 / Fca antibody is shown in WO 98 / 02463. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2 / anti-CD3 antibody. Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, with the two chains having different specificities (Millstein et al., Nature 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a mixture of 10 possible different antibody molecules, of which only one has the correct bispecific structure.

[0230] Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93 / 08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).

[0231] 6. Effector function operation It may be desirable to modify the antibody of the invention with respect to effector function, for example to enhance the antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody.

[0232] Alternatively, or additionally, cysteine ​​residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992). Homodimeric antibodies with improved anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, antibodies with dual Fc regions can be engineered, which may thereby have improved complement lysis and ADCC capabilities. Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). To extend the serum half-life of the antibody, a salvage receptor binding epitope may be incorporated into the antibody (e.g., antibody fragment), as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is involved in extending the in vivo serum half-life of the IgG molecule.

[0233] D. Specific Methods for Producing Antibodies 1. Screening for anti-GPC3 antibodies with desired properties Techniques for producing antibodies that bind to GPC3 polypeptides have been described above. For example, the present invention provides a method for producing a GPC3 antibody (which includes full-length and fragments thereof, as defined herein), comprising expressing a recombinant vector of the present invention encoding the antibody (or fragment) in a suitable host cell and recovering the antibody. If desired, antibodies with certain biological properties may be further selected.

[0234] The growth inhibitory effect of the anti-GPC3 antibodies of the present invention can be assessed by methods known in the art, for example, using cells that express GPC3 polypeptide endogenously or after transfection of the GPC3 gene. For example, appropriate tumor cell lines and GPC3-transfected cells can be treated with various concentrations of the anti-GPC3 monoclonal antibodies of the present invention for several days (e.g., 2-7 days) and analyzed by staining with crystal violet or MTT or other colorimetric assays. Another method for measuring proliferation is by comparing the incorporation of 3H-thymidine by cells treated with or without the anti-GPC3 antibodies of the present invention. After treatment, the cells are harvested, and the amount of radioactivity incorporated into DNA is quantified using a scintillation counter. An appropriate positive control includes treating a selected cell line with a growth inhibitory antibody known to inhibit the growth of that cell line. In vivo tumor cell growth inhibition can be determined by various methods known in the art. The tumor cells can overexpress GPC3 polypeptide. The anti-GPC3 antibody inhibits cell proliferation of GPC3-expressing tumor cells by about 25-100%, more preferably about 30-100%, even more preferably about 50-100% or 70-100%, in vitro or in vivo compared to untreated tumor cells, and in one embodiment, at an antibody concentration of about 0.5-30 μg ml. Growth inhibition can be measured in cell culture at antibody concentrations of about 0.5-30 μg ml or about 0.5 nM-200 nM, where growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. An antibody is growth inhibitory in vivo if administration of the anti-GPC3 antibody at a dose of about 1 μg / kg to about 100 mg / kg body weight results in a reduction in tumor size or tumor cell proliferation within about 5 days to 3 months, preferably within about 5-30 days, of the initial administration of the antibody.

[0235] To select anti-GPC3 antibodies that induce cell death, loss of membrane integrity, as indicated by, for example, propidium iodide (PI), trypan blue, or 7AAD uptake, can be assessed against controls. PI uptake assays can be performed in the absence of complement and immune effector cells. GPC3 polypeptide-expressing tumor cells are incubated with medium alone or medium containing an appropriate anti-GPC3 antibody (e.g., approximately 10 μg / ml). The cells are incubated for 3 days. After each treatment, the cells are washed and divided into 12 x 75 tubes (1 ml per tube, 3 tubes per treatment group) with 35 mm strainers to remove cell clumps. PI (1 C^g / ml) is then added to the tubes. Samples can be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). An anti-GPC3 antibody that induces a statistically significant level of cell death, as determined by PI uptake, can be selected as an anti-GPC3 antibody that induces cell death.

[0236] To screen for antibodies that bind to the epitope of the GPC3 polypeptide bound by the antibody of interest, a conventional cross-blocking assay, such as that described in *Antibodies, A Laboratory Manual*, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine whether a test antibody binds to the same site or epitope as a known anti-GPC3 antibody. Alternatively, or in addition, epitope mapping can be performed using methods known in the art. For example, the antibody sequence can be mutated, e.g., by alanine scanning, to identify contact residues. The mutated antibody is first tested for binding with a polyclonal antibody to ensure correct folding. In a different method, peptides corresponding to different regions of the GPC3 polypeptide can be used in a competition assay with the test antibody or with an antibody bearing a characterized or known epitope.

[0237] Briefly, in one aspect, the present disclosure provides a method for identifying an epitope of an agent that can be used in the disclosed IHC IVD assay and / or any other method disclosed herein. In some embodiments, the agent is 204. The epitope can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in a unique spatial conformation. Epitopes can be formed from both contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents.

[0238] Epitope mapping can be performed to identify linear or nonlinear discontinuous amino acid sequence(s), i.e., epitopes, that are recognized by an activator of interest, e.g., the 204 antibody. A general approach for epitope mapping may require the expression, generally in a heterologous expression system, of the full-length polypeptide sequence recognized by the antibody or ligand of interest, as well as various fragments, i.e., truncated forms of the polypeptide sequence. These various recombinant polypeptide sequences or fragments thereof (e.g., fused to an N-terminal protein (e.g., GFP)) can then be used to determine whether the antibody or ligand of interest is capable of binding to one or more of the truncated forms of the polypeptide sequence. Iterative truncation and generation of recombinant polypeptide sequences with overlapping amino acid regions can be used to identify the region of the polypeptide sequence recognized by the antibody of interest (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996)). This method relies on the ability of the drug (for example, the antibody of interest) to bind to the sequence regenerated from an epitope library, for example, a synthetic peptide array on a membrane support, an epitope library derived from a combinatorial phage display peptide library.This epitope library thus provides a variety of possibilities for antibody screening.Furthermore, site-directed mutagenesis can be carried out to target one or more residues of the epitope, or random Ala scanning can be carried out to confirm the identity of the epitope.

[0239] A library of epitopes can be created by synthetically designing various possible modifications of GPC3 as cDNA constructs and expressing them in an appropriate system. For example, multiple GPC3 gene segments (e.g., various sequences corresponding to the N-terminal alpha chain, various sequences corresponding to the C-terminal beta chain, etc.) can be synthetically designed. Alternatively, selected sequences can be ordered as synthetic genes and cloned into an appropriate vector. In other cases, various GPC3 sequences can be amplified from total RNA extracted from human normal and malignant tissues, preferably malignant tissues in which GPC3 is expressed at higher levels than normal tissues.

[0240] The host system can be any suitable expression system, such as 293 cells, insect cells, or a suitable in vitro translation system. The multiple possible recombinations of synthetically designed GPC3 gene segments transfected into the host system can provide, for example, more than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 possible GPC3 pairing combinations. Binding of an agent to one of the epitopes in the above-described library can be detected by contacting the library epitope with a labeled antibody, e.g., 204, and detecting a signal from the label.

[0241] For epitope mapping, computational algorithms have also been developed, and these have been shown to map conformationally discontinuous epitopes.Conformational epitopes can be identified by determining the spatial conformation of amino acids, for example, by methods including X-ray crystallography and two-dimensional nuclear magnetic resonance.Some epitope mapping methods, such as X-ray analysis of crystals of antigen-antibody complexes, can provide atomic resolution of epitopes.In other cases, computational combinatorial methods for epitope mapping can be used to model potential epitopes based on the sequence of the antibody, for example, the 204 antibody.In such cases, the antigen-binding portion of the antibody is sequenced, and a computational model is used to reconstruct the antibody and predict its potential binding site.

[0242] In some cases, the present disclosure provides a method for identifying an epitope of GPC3 specifically recognized by 204 or an antigen-binding fragment thereof, the method comprising: (a) preparing a library of epitopes from GPC3; (b) contacting the epitope library with the 204 antibody or an antigen-binding fragment thereof; and (b) identifying the amino acid sequence of at least one epitope in the epitope library that is bound by the antibody. In one example, the antibody is bound to a solid support. The epitope library may include sequences corresponding to continuous and discontinuous epitopes of GPC3. In some cases, the epitope library includes fragments from GPC3 ranging from about 10 amino acids to about 30 amino acids in length, from about 10 amino acids to about 20 amino acids in length, or from about 5 amino acids to about 12 amino acids in length. In some cases, the 204 antibody or an antigen-binding fragment thereof is labeled, and the label is a radioactive molecule, a luminescent molecule, a fluorescent molecule, an enzyme, or biotin.

[0243] A high-level epitope mapping study relying on Western blotting and protease cleavage of GPC3 is described in Example 2 below. Briefly, after protease cleavage of GPC3 (e.g., a disintegrin and metalloprotease, or ADAM), samples are subjected to Western blotting analysis using an antibody with a known GPC3 epitope (e.g., GC33) and an antibody with an unknown GPC3 epitope (e.g., 204). Specific recognition of protease-cleaved GPC3 fragments by different antibodies can provide information about the potential binding sites of the target antibodies and can be used as a starting point for the epitope mapping method described above.

[0244] Additionally, candidate antibodies may be screened for functionality using one or more of the following: in vivo screening for inhibition of metastasis, inhibition of chemotaxis by in vitro methods (e.g., Huntsman et al. US2010 / 0061978, incorporated herein by reference in its entirety), inhibition of angiogenesis, inhibition of tumor growth, and reduction in tumor size.

[0245] 2. Specific Library Screening Methods The anti-GPC3 antibodies of the present invention can be generated using combinatorial libraries to screen for antibodies with the desired activity(ies). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are generally described in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ), and in certain embodiments, Lee et al. (2004) J. Mol. Biol. 340:1073-1093.

[0246] In principle, synthetic antibody clones are selected by screening a phage library containing phages displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles. Any of the anti-GPC3 antibodies of the present invention can be obtained by designing an antigen screening procedure suitable for selecting the desired phage clone, followed by constructing a full-length anti-GPC3 antibody clone using the Fv sequence from the desired phage clone and the appropriate constant region (Fc) sequence described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3.

[0247] In certain embodiments, the antigen-binding domain of an antibody is formed from two variable (V) regions of approximately 110 amino acids, one each from the light (VL) and heavy (VH) chains, both of which display three hypervariable loops (HVRs) or complementarity-determining regions (CDRs). The variable domains can be functionally displayed on phage either as single-chain Fv (scFv) fragments in which the VH and VL are covalently linked via a short, flexible peptide, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994), or as Fab fragments in which they are each fused to a constant domain and interact noncovalently. As used herein, scFv-encoding phage clones and Fab-encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones."

[0248] Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies against a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J., 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions to achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0249] In certain embodiments, filamentous phage are used to display antibody fragments by fusion to the minor coat protein, pill. The antibody fragments can be displayed as single-chain Fv fragments in which the VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, as described, for example, in Marks et al., J. Mol. Biol., 222:581-597 (1991), or as Fab fragments in which one chain is fused to pill and the other is secreted into the bacterial host cell periplasm, where assembly of the Fab coat protein structure becomes displayed on the phage surface by displaying some of the wild-type coat protein, as described, for example, in Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991).

[0250] Generally, nucleic acids encoding antibody gene fragments are obtained from immune cells collected from humans or animals. If a library biased toward anti-GPC3 clones is desired, a subject is immunized with GPC3 to generate an antibody response, and splenocytes and / or circulating B cells, other peripheral blood lymphocytes (PBLs), are collected for library construction. In a preferred embodiment, a human antibody gene fragment library biased toward anti-GPC3 clones is obtained by generating an anti-GPC3 antibody response in a transgenic mouse with a functional human immunoglobulin gene array (and lacking a functional endogenous antibody-producing system), such that GPC3 immunization generates B cells that produce human antibodies against GPC3. The generation of human antibody-producing transgenic mice is described below.

[0251] Further enrichment of the anti-GPC3-reactive cell population can be achieved by using screening procedures suitable for the isolation of B cells expressing GPC3-specific membrane-bound antibodies, for example, by GPC3 affinity chromatography or by adsorption of cells to fluorochrome-labeled GPC3 followed by cell separation using flow-activated cell sorting (FACS).

[0252] Alternatively, the use of splenocytes and / or B cells or other PBLs from unimmunized donors increases the representation of the possible antibody repertoire and allows the construction of antibody libraries using any animal species (human or non-human) to which GPC3 is not antigenic. For libraries incorporating in vitro antibody gene construction, stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments. Immune cells of interest can be obtained from a variety of animal species, including humans, mice, rats, rabbits, wolves, dogs, cats, pigs, cows, horses, and birds.

[0253] Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered and amplified from the cells of interest. For rearranged VH and VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes, followed by polymerase chain reaction (PCR) with primers matching the 5' and 3' ends of the rearranged VH and VL genes to generate a diverse V gene repertoire for expression, as described by Orlandi et al. (1989) and Ward et al., Nature, 341:544-546 (1989). The V genes can be amplified from cDNA and genomic DNA using a back primer at the 5' end of the exon encoding the mature V domain and a forward primer within the J-segment, as described by Orlandi et al. (1989) and Ward et al., Nature, 341:544-546 (1989). However, for amplification from cDNA, the back primer can also be based in the leader exon as described by Jones et al., Biotechnol., 9:88-89 (1991), and the forward primer can be based within the constant region as described by Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated into the primers as described by Orlandi et al. (1989) or Sastry et al. (1989). In certain embodiments, library diversity is maximized by amplifying all available VH and VL sequences present in the immune cell nucleic acid sample using PCR primers targeted to each V gene family, e.g., as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or Orum et al., Nucleic Acids Res., 21:4491-4498 (1993).For cloning of the amplified DNA into an expression vector, rare restriction sites can be introduced into the PCR primers either as tags at one end as described by Orlandi et al. (1989) or by further PCR amplification with tagged primers as described by Clackson et al., Nature, 352:624-628 (1991).

[0254] Synthetically rearranged V gene repertoires can be derived in vitro from V gene segments. Most human VH gene segments have been cloned, sequenced (as reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)) and mapped (as reported in Matsuda et al., Nature Genet., 3:88-94 (1993)), and these cloned segments (containing all major conformations of the H1 and H2 loops) can be used to generate diverse VH gene repertoires with PCR primers encoding H3 loops of various sequences and lengths, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can be generated by concentrating all sequence diversity in a single, long H3 loop, as described by Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-4461 (1992). Human VK and vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23:1456-1461 (1993)) and can be used to generate synthetic light chain repertoires. Synthetic V gene repertoires encode antibodies with considerable structural diversity based on the range of VH and VL folds and the lengths of L3 and H3. After amplification of DNA encoding V genes, germline V gene segments can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0255] Antibody fragment repertoires can be constructed by combining VH and VL gene repertoires in several ways. Each repertoire can be generated in a different vector, and the vectors can be recombined in vitro, e.g., as described by Hogrefe et al., Gene, 128:119-126 (1993), or in vivo by combinatorial infection, e.g., the loxP system described by Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993). This in vivo recombination approach exploits the two-chain nature of Fab fragments to overcome the library size limitation imposed by the transformation efficiency of E. coli. Naive VH and VL repertoires are cloned separately, i.e., one into a phagemid and the other into a phage vector. These two libraries are then combined by phage infection of phagemid-containing bacteria, so that each cell contains a different combination, and the library size is limited only by the number of cells present (approximately 10 clones). Both vectors contain in vivo recombination signals, and the VH and VL genes are recombined into a single replicon and co-packaged into phage virions. These large libraries have good affinity (Kd -1 about 10 -8 The present invention provides a large number of diverse antibodies to M.

[0256] Alternatively, the repertoires can be cloned sequentially into the same vector, e.g., as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or cloned after assembly by PCR, e.g., as described in Clackson et al., Nature, 352:624-628 (1991). PCR assembly can also be used to link VH and VL DNA with DNA encoding a flexible peptide spacer to form single-chain Fv (scFv) repertoires. In yet another technique, "in situ PCR assembly" is used to combine VH and VL genes in lymphocytes by PCR, and then the repertoire of linked genes is cloned, as described in Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992).

[0257] Antibodies produced by naive libraries (either natural or synthetic) may have moderate affinity (Kd-1 approximately 10-10 M-1), but affinity maturation can be reproduced in vitro by constructing and reselecting from secondary libraries as described by Winter et al. (1994), supra. For example, mutations can be randomly introduced in vitro using error-prone polymerases (as reported in Leung et al., Technique, 1:11-15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 226:889-896 (1992) or Gram et al., Proc. Natl. Acad. Sci USA, 89:3576-3580 (1992). Furthermore, affinity maturation can be performed in selected individual Fv clones by randomly mutating one or more CDRs, for example, using PCR with primers containing random sequences spanning the CDRs of interest, and screening for clones with higher affinity. WO 9607754 (published March 14, 1996) describes a method for directing mutagenesis in the complementarity-determining regions of immunoglobulin light chains to create a library of light chain genes. Another effective approach is to recombine VH or VL domains selected by phage display with a repertoire of naturally occurring V domain variants obtained from unimmunized donors, as described by Marks et al., Biotechnol., 10:779-783 (1992), and screen for higher affinity through several rounds of chain reshuffling. This technique allows for the generation of approximately 10 -9 It becomes possible to produce antibodies and antibody fragments with affinities of M or less.

[0258] The screening of this library can be achieved by various techniques known in the art.For example, GPC3 can be used to coat the well of adsorption plate, or be expressed in the host cell that is fixed on adsorption plate, or be used in cell sorting, or be conjugated with biotin to be captured on streptavidin-coated beads, or be used in any other method for panning phage display library.

[0259] The phage library sample is contacted with immobilized GPC3 under conditions suitable for binding of the adsorbent to at least a portion of the phage particles. Typically, conditions, including pH, ionic strength, temperature, etc., are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then eluted with acid, e.g., as described by Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or alkali, e.g., as described by Marks et al., J. Mol. Biol., 222:581-597 (1991), or by GPC3 antigen competition, e.g., as described in Clackson et al., Nature, 352:624-628 (1991). Phages can be enriched 20- to 1,000-fold in a single round of selection. The enriched phages can then be grown in bacterial culture and subjected to additional rounds of selection.

[0260] The efficiency of selection depends on many factors, including the dissociation rate during washing and whether multiple antibody fragments on a single phage can simultaneously bind to the antigen. Antibodies with high dissociation rates (and weak binding affinity) can be retained by using short washing times, multivalent phage display, and a high coating density of antigen on the solid phase. This high density not only stabilizes the phage through multivalent interactions, but also favors the rebinding of dissociated phage. The selection of antibodies with low dissociation rates (and good binding affinity) can be facilitated by using long washing times and monovalent phage display, as described in Bass et al., Proteins, 8:309-314 (1990) and WO 92 / 09690, and by using a low coating density of antigen, as described in Marks et al., Biotechnol., 10:779-783 (1992).

[0261] Selection between phage antibodies with different affinities for GPC3 is possible, even if they only have slightly different affinities. However, random mutation of a selected antibody (e.g., as performed in some affinity maturation techniques) can generate many variants, most of which bind to the antigen and some of which have high affinity. By limiting GPC3, rare high-affinity phages can be selected by competition. To retain all high-affinity variants, phages can be incubated with excess biotinylated GPC3, but the biotinylated GPC3 is used at a molar concentration lower than the desired molar affinity constant for GPC3. The high-affinity binding phages can then be captured by streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows for the selection of phages based on the binding affinity of the antibody to be selected, with sensitivity that allows the isolation of mutant clones with only two-fold lower affinity from a large excess of phages with lower affinity. The conditions used to wash the solid phase-bound phages can also be manipulated to differentiate based on dissociation rate.

[0262] Anti-GPC3 clones may also be selected based on activity. In certain embodiments, the present invention provides anti-GPC3 antibodies that bind to live cells that naturally express GPC3. In one embodiment, the present invention provides anti-GPC3 antibodies that block the binding between a GPC3 ligand and GPC3 but not between a GPC3 ligand and a secondary protein. Fv clones corresponding to such anti-GPC3 antibodies can be selected by (1) isolating anti-GPC3 clones from the phage library described above and, optionally, amplifying the population of isolated phage clones by growing the population in a suitable bacterial host; (2) selecting GPC3 and secondary proteins for which blocking and non-blocking activities are desired, respectively; (3) adsorbing the anti-GPC3 phage clones to immobilized GPC3; (4) using an excess amount of the secondary protein to elute any unwanted clones that recognize GPC3 binding determinants that overlap or are shared with those of the secondary protein; and (5) eluting the clones that remain adsorbed after step (4). Optionally, the desired blocking / non-blocking properties can be further enriched by repeating the selection procedures described herein one or more times.

[0263] DNA encoding the hybridoma-derived monoclonal antibodies or phage-displayed Fv clones of the present invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the desired heavy and light chain coding regions from a hybridoma or phage DNA template). After isolation, the DNA can be placed into an expression vector, which is then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, resulting in the synthesis of the desired monoclonal antibody in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256 (1993) and Pluckthun, Immunol. Revs, 130:151 (1992).

[0264] DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy and / or light chain constant regions (e.g., suitable DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full-length or partial-length heavy and / or light chains. It will be understood that constant regions of any isotype, including those of IgG, IgM, IgA, IgD, and IgE, can be used for this purpose, and that such constant regions can be obtained from any human or animal species. Fv clones derived from variable domain DNA of one animal (e.g., human) species and then fused to constant region DNA of another animal species to form "hybrid" full-length heavy and / or light chain coding sequence(s) are included in the definition of "chimeric" and "hybrid" antibodies as used herein. In certain embodiments, Fv clones derived from human variable DNA are fused to human constant region DNA to form full-length or partial-length human heavy and / or light chain coding sequence(s).

[0265] DNA encoding an anti-GPC3 antibody derived from a hybridoma can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences derived from the hybridoma clone (e.g., as described by Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). DNA encoding an antibody or fragment derived from a hybridoma or Fv clone can be further modified by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, "chimeric" or "hybrid" antibodies having the binding specificity of an antibody derived from an Fv clone or hybridoma clone of the present invention can be prepared.

[0266] 3.Generation of antibodies using CAR T cells The anti-GPC3 antibodies of the present invention can be generated using a CAR T cell platform to screen for antibodies with the desired activity(ies). Chimeric antigen receptors (CARs) consist of an extracellular antigen-recognition domain (usually a single-chain variable fragment (scFv) antibody) linked to a transmembrane and cytoplasmic signaling domain. Alvarez-Vallina, L, Curr Gene Ther 1:385-397 (2001). CAR recognition converts tumor-associated antigens (TAAs) expressed on the cell surface into recruitment points for effector function, addressing the purpose of major histocompatibility complex-dependent activation of effector cells. First-generation CARs were constructed by fusing an scFv-based TAA-binding domain with a cytoplasmic signaling domain, usually derived from either the zeta chain of the T cell receptor (TCR) / CD3 complex or the gamma chain associated with some Fc receptors. Gross, G. et al., Proc Natl Acad Sci USA 86:10024-10028 (1989). Second-generation CARs (CARv2) have also been developed, which contain the signaling region of TCR ζ in tandem with signaling domains from the T cell costimulatory receptors CD28, 4-lBB (CD137), or OX40 (CD134). Sanz, L. et al., Trends Immunol 25:85-91 (2004). Upon antigen encounter, the genetically engineered CAR interaction triggers effector function and can mediate tumor cell lysis. The utility and efficacy of this CAR approach have been demonstrated in various animal models and in ongoing clinical trials using CAR-based engineered T lymphocytes for the treatment of cancer patients. Lipowska-Bhalla, G. et al., Cancer Immunol Immunother 61:953-962 (2012). CARs enable the targeting of effector cells to any natural extracellular antigen for which a suitable antibody exists. Engineered cells can be targeted not only to proteins but also to structures such as carbohydrate and glycolipid tumor antigens.Mezzanzanica, D. et al., Cancer Gene Ther 5:401-407 (1998), Kershaw, M.H. et al., Nat Rev Immunol 5:928-940 (2005).

[0267] Current methods for generating recombinant antibodies are primarily based on the use of purified proteins. Hoogenboom, HR et al., Nat Biotechnol 23:1105-1116 (2005). However, a mammalian cell-based antibody display platform that exploits the functionality of T lymphocytes has recently been described. Alonso-Camino et al., Molecular Therapy Nucleic Acids (2013) 2, e93. Displaying antibodies on the surface of T lymphocytes as part of CAR-mediated signaling could ideally link antigen-antibody interactions to a clear change in cell phenotype due to surface expression of activation markers. Alonso-Camino, V. et al., PLoS ONE 4:e7174 (2009). By using an scFv-based CAR that recognizes a TAA, combining CAR-mediated activation with fluorescence-activated cell sorting (FACS) of CD69+ T cells revealed that surface binding agents for the TAA were expressed in at least 10% of cells after two rounds. 3 It was demonstrated that this enabled the isolation of a 2-fold enrichment factor, resulting in a homogenous population of T cells expressing a TAA-specific CAR. Alonso-Camino, V, et al., PLoS ONE 4:e7174 (2009).

[0268] E. Anti-GPC3 Antibody Variants and Modifications 1. Variant In addition to the anti-GPC3 antibodies described herein, it is contemplated that anti-GPC3 antibody variants can be prepared. Anti-GPC3 antibody variants can be prepared by introducing appropriate nucleotide changes into the coding DNA and / or synthesizing the desired antibody or polypeptide. Those skilled in the art will understand that amino acid changes can alter the post-translational processes of the anti-GPC3 antibody, such as changing the number or position of glycosylation sites or changing membrane anchoring properties.

[0269] Variations of the anti-GPC3 antibodies described herein can be made using, for example, any of the techniques and guidelines for conservative and non-conservative mutations, such as those described in U.S. Patent No. 5,364,934. Variations can be the substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide, resulting in a change in amino acid sequence compared to the native sequence of the antibody or polypeptide. Optionally, the variation is achieved by substituting at least one amino acid with any other amino acid in one or more domains of the anti-GPC3 antibody. Guidance for determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity can be found by comparing the sequence of the anti-GPC3 antibody with that of a known homologous protein molecule and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can result in one amino acid being replaced with another amino acid with similar structural and / or chemical properties, e.g., a conservative amino acid substitution, such as the substitution of serine for leucine. Insertions or deletions can optionally range from about 1 to 5 amino acids. Permissible variations can be determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting variants for the activity exhibited by the full-length or mature native sequence.

[0270] Anti-GPC3 antibody fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, compared with full-length native antibodies or proteins. Certain fragments lack amino acid residues that are not essential for the desired biological activity of the anti-GPC3 antibody.

[0271] Anti-GPC3 antibody fragments can be prepared by any of a number of conventional techniques. The desired peptide fragment may also be chemically synthesized. An alternative approach involves generating antibody or polypeptide fragments by enzymatic digestion, for example, by treating the protein with an enzyme known to cleave the protein at sites defined by specific amino acid residues, or by digesting the DNA with an appropriate restriction enzyme, and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding the desired antibody or polypeptide fragment by polymerase chain reaction (PCR). Oligonucleotides defining the desired termini of the DNA fragment are used as the 5' and 3' primers in the PCR. Preferably, the anti-GPC3 antibody fragment shares at least one biological and / or immunological activity with the native anti-GPC3 antibody disclosed herein.

[0272] In certain embodiments, conservative substitutions of interest are shown under the heading of preferred substitutions in Table 1. If such substitutions result in a change in biological activity, more substantial changes, indicated as exemplary substitutions in Table 1 or further described below with reference to amino acid classes, are introduced and the products screened. [Table 1]

[0273] Substantial modifications in the function or immunological identity of the anti-GPC3 antibody are achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the region of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are classified based on common side chain properties: (1) Hydrophobic: norleucine, met, ala, val, leu, ile, (2) Neutral hydrophilicity: cys, ser, thr, (3) Acidic: asp, glu, (4) basic: asn, gin, his, lys, arg; (5) residues that affect chain orientation: gly, pro, and (6) Aromatics: trp, tyr, phe.

[0274] Non-conservative substitutions entail exchanging a member of one of these classes for another. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

[0275] The variations can be produced using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction-selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)], or other known techniques can be performed on cloned DNA to produce the anti-GPC3 antibody variant DNA.

[0276] Amino acid scanning analysis can also be used to identify one or more amino acids along a continuous sequence. Preferred scanning amino acids include relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically the preferred scanning amino acid within this group because it excludes the side chain beyond the beta carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244:1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Furthermore, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (WH Freeman & Co., NY); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield sufficient variants, an isoteric amino acid can be used.

[0277] Any cysteine ​​residues not involved in maintaining the proper conformation of the anti-GPC3 antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to the anti-GPC3 antibody to improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment. A particularly preferred type of substitutional variant involves substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development have improved biological properties compared to the parent antibody from which they were generated. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the GPC3 polypeptide. Such contact and adjacent residues are candidates for substitution using the techniques detailed herein. Once such variants are generated, the panel of variants can be screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0278] Nucleic acid molecules encoding amino acid sequence variants of the anti-GPC3 antibody are prepared by various methods known in the art, including, but not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared variant or non-variant forms of the anti-GPC3 antibody.

[0279] 2. Qualification Covalent modification of anti-GPC3 antibody is included in the scope of the present invention.One type of covalent modification includes reacting the target amino acid residue of anti-GPC3 antibody with an organic derivatizing agent that can react with selected side chain or N-terminal or C-terminal residue of the anti-GPC3 antibody.Derivatization with bifunctional agents is useful, for example, for crosslinking anti-GPC3 antibody to a water-insoluble support matrix or surface for use in the method for purifying anti-GPC3 antibody, and vice versa. Commonly used cross-linking agents include, for example, l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g., with 4-azidosalicylic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3,3′-dithiobis(succinimidyl propionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane, and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0280] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains [TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0281] Another type of covalent modification of an anti-GPC3 antibody within the scope of the present invention involves altering the native glycosylation pattern of the antibody or polypeptide. For purposes herein, "altering the native glycosylation pattern" is intended to mean deleting one or more carbohydrate moieties found in a native-sequence anti-GPC3 antibody (either by removing underlying glycosylation sites or by deglycosylating by chemical and / or enzymatic means) and / or adding one or more glycosylation sites not present in the native-sequence anti-GPC3 antibody. Furthermore, this term includes qualitative changes in the glycosylation of the native protein, including changes in the identity and proportion of the various carbohydrate moieties present.

[0282] Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are 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 creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0283] Addition of glycosylation sites to the anti-GPC3 antibody is conveniently achieved by modifying the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The modification may also be performed by adding or substituting one or more serine or threonine residues to the original anti-GPC3 antibody sequence (for O-linked glycosylation sites). The amino acid sequence of the anti-GPC3 antibody can optionally be modified at the DNA level, particularly by mutating the DNA encoding the anti-GPC3 antibody at a preselected base to generate a codon that translates into the desired amino acid.

[0284] Another way to increase the number of carbohydrate moieties on the anti-GPC3 antibody is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, for example, in WO 87 / 05330 published September 11, 1987, and Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981). Removal of carbohydrate moieties from the anti-GPC3 antibody can be accomplished chemically or enzymatically, or by mutation of codons encoding amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exoglycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0285] F. Preparation of anti-GPC3 antibody The following description mainly relates to the production of anti-GPC3 antibodies by culturing cells transformed or transfected with a vector containing nucleic acid encoding the anti-GPC3 antibody.Of course, it is contemplated that alternative methods well known in the art can be used to prepare anti-GPC3 antibodies.For example, the appropriate amino acid sequence, or a portion thereof, can be produced by direct peptide synthesis using solid-phase technology (see, for example, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)).In vitro protein synthesis can be performed manually or automatically.Automated synthesis can be achieved, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) and following the manufacturer's instructions. Various portions of the anti-GPC3 antibody may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-GPC3 antibody.

[0286] 1. Isolation of DNA encoding anti-GPC3 antibody The DNA encoding anti-GPC3 antibody can be obtained from a cDNA library prepared from tissues that are believed to have the mRNA of the anti-GPC3 antibody and express it at detectable levels.Therefore, the DNA of human anti-GPC3 antibody can be conveniently obtained from a cDNA library prepared from human tissues.The gene encoding the anti-GPC3 antibody can also be obtained from a genomic library or by known synthetic procedures (for example, automated nucleic acid synthesis).

[0287] The library can be screened with a probe (such as an oligonucleotide of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening of the cDNA or genomic library with the selected probe can be performed using standard procedures, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means for isolating the gene encoding the anti-GPC3 antibody is to use PCR (Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0288] The technique for screening a cDNA library is well known in the art. The oligonucleotide sequence selected as a probe should be of sufficient length and should be sufficiently distinct so as to minimize false positives. The oligonucleotide is preferably labeled so that it can be detected when hybridized to the DNA in the library to be screened.

[0289] Labeling methods are well known in the art and include the use of radiolabels such as 32P-labeled ATP, biotinylation, or enzyme labeling. Hybridization conditions, including moderate and high stringency, are provided in Sambrook et al., supra. Sequences identified by such library screening methods can be compared and aligned with other known sequences available in public databases, such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions or across the full-length sequence of the molecule can be identified using methods known in the art and described herein.

[0290] Nucleic acids having protein-coding sequences may be obtained by screening a selected cDNA or genomic library using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse transcribed into cDNA.

[0291] 2. Host Cell Selection and Transformation Host cells are transfected or transformed with the expression or cloning vectors described herein for producing anti-GPC3 antibodies and cultured in conventional nutrient media modified as needed for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Culture conditions, such as medium, temperature, pH, etc., can be selected by those skilled in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0292] Methods for transfection of eukaryotic cells and transformation of prokaryotic cells (which refers to the introduction of DNA into a host, where the DNA can replicate either extrachromosomally or as a chromosomal integrant) are known to those skilled in the art, such as CaCl2, CaP04, liposome-mediated, polyethylene glycol / DMSO, and electroporation. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride, as described in Sambrook et al., supra, or electroporation, are generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used to transform certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89 / 05859, published June 29, 1989. For mammalian cells lacking such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be used. General aspects of mammalian cell host system transfection are described in U.S. Patent No. 4,399,216. Transformation into yeast is typically performed according to the methods of Van Solingen et al., J. Bac, 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, can also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0293] Suitable host cells for cloning or expressing the DNA contained in the vectors herein include prokaryote, yeast, or higher eukaryote cells.

[0294] a. Prokaryotic host cells Suitable prokaryotes include, but are not limited to, archaebacteria and eubacteria, such as gram-negative or gram-positive bacteria, for example, Enterobacteriaceae, such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446), E. coli XI 776 (ATCC 31,537), E. coli strain W3110 (ATCC 27,325), and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae, e.g., Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillus, e.g., B. subtilis and B. licheniformis (e.g., B. licheniformis 41P, disclosed in DD 266,710, published April 12, 1989), Pseudomonas, e.g., P. aeruginosa, Rhizobia, Vitreoscilla, Paracoccus, and Streptomyces. These examples are illustrative and not limiting. The W3110 strain is a particularly preferred host or parent host because it is a common host strain for fermentation of recombinant DNA products. Preferably, the host cell secretes minimal amounts of proteolytic enzymes.For example, strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219, ATCC Deposit No. 27,325) may be modified to introduce genetic mutations into genes encoding proteins endogenous to the host. Examples of such hosts include E. coli W3110 strain 1A2, which contains the complete tonA genotype; E. coli W3110 strain 9E4, which has the complete tonA ptr3 genotype; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete tonA ptr3 phoA E 15(argF-lac) 169 degP ompT kanr genotype; and E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete tonA ptr3 phoA E 15(argF-lac) 169 degP ompT rbs7 ilvG genotype. E. coli W3110 strain 37D6 with kanr, E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin-resistant degP deletion mutation, E. coli W3110 strain 33D3 with the genotype W3110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompTA(nmpc-fepE)degP41 kanR (U.S. Pat. No. 5,639,635), and the E. coli strain disclosed in U.S. Pat. No. 4,946,783, issued Aug. 7, 1990, which has a mutant periplasmic protease. Other strains and their derivatives, such as E. coli 294 (ATCC 31,446), E. coli B, E. uililambda 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), are also suitable. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the above-mentioned bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, it is necessary to select an appropriate bacterium taking into account the replicability of the replicon in the bacterial cell.For example, E. coli, Serratia, or Salmonella species may be suitably used as hosts when well-known plasmids, such as pBR322, pBR325, pACYC177, or pKN410, are used to provide the replicon. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated into the cell culture. Alternatively, in vitro methods of cloning, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0295] Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. Full-length antibodies have a long half-life in the blood. Production in E. coli is faster and more cost-effective. For expression of antibody fragments and polypeptides in bacteria, see, for example, US Pat. No. 5,648,237 (Carter et al.), US Pat. No. 5,789,199 (Joly et al.), and US Pat. No. 5,840,523 (Simmons et al.), which describes translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion. These patents are incorporated herein by reference. After expression, the antibody can be isolated from the E. coli cell paste in a soluble fraction and purified, for example, through a protein A or G column depending on the isotype. Final purification can be performed, for example, similar to the process for purifying antibodies expressed in CHO cells.

[0296] b.Eukaryotic host cells In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-GPC3 antibody-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other examples include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140

[1981] ; EP 139,383 published May 2, 1985), Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio / Technology, 9:968-975 (1991)), such as K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacterid., 154(2):737-742

[1983] ), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906, Van den Berg et al., Bio / Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus, yarrowia (EP 402,226), Pichia pastoris (EP 183,070, Sreekrishna et al., J. Basic Microbiol.,28:265-278

[1988] ), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263

[1979] ), Schwanniomyces, e.g. occidentalis (EP 394,538 published October 31, 1990), and filamentous fungi such as Neurospora, Penicillium, Tolypocladium (WO 91 / 00357 published January 10, 1991), as well as Aspergillus hosts such as A. nidulans (Ballance et al.Examples of suitable methylotrophic yeasts include A. niger (Kelly and Hynes, EMBO J., 4:475-479

[1985] ), A. niger (Kelly and Hynes, EMBO J., 4:475-479

[1985] ), and A. niger (Kelly and Hynes, Biochem. Biophys. Res. Commun., 112:284-289

[1983] ; Tilburn et al., Gene, 26:205-221

[1983] ; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474

[1984] ). Methylotrophic yeasts are suitable herein and include, but are not limited to, yeasts capable of growing on methanol selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are examples of this class of yeast can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0297] Suitable host cells for expressing glycosylated anti-GPC3 antibodies can be obtained from multicellular organisms. Examples of invertebrate cells include insect cells, such as Drosophila S2 and Spodoptera Sf9, and plant cells, such as cotton, corn, potato, soybean, petunia, tomato, and tobacco cell cultures. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used as viruses herein in the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0298] However, vertebrate cells have received the most attention, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines are monkey kidney CV1 transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney (293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney (BHK, ATCC CCL 10), Chinese hamster ovary (CHO)-DHFR (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney (CV1 ATCC CCL 70), African green monkey kidney (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT) 060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad.Sci.383:44-68 (1982)), MRC 5 cells, FS4 cells, and human hepatoma cell line (Hep G2).

[0299] Host cells are transformed with the above-described expression or cloning vectors for producing anti-GPC3 antibodies and cultured in conventional nutrient media modified as necessary for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0300] 3. Selection and Use of Replicable Vectors For recombinant production of an antibody of the invention, the nucleic acid (e.g., cDNA or genomic DNA) encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains). Many vectors are available. The choice of vector will depend in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.

[0301] The vector may be in the form of, for example, a plasmid, cosmid, virus particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. Generally, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art. The GPC3 can be produced recombinantly directly or as a fusion polypeptide with a heterologous polypeptide, which can be a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence may be a component of the vector or may be part of the DNA encoding the anti-GPC3 antibody that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence may be, for example, the yeast invertase leader, the alpha-factor leader (including the Saccharomyces and Kluyveromyces a-factor leaders, the latter of which is described in U.S. Pat. No. 5,010,182), or the acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179, published April 4, 1990), or a signal described in WO 90 / 13646, published November 15, 1990. In mammalian cell expression, mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or related species, and viral secretory leaders, may be used to direct protein secretion.

[0302] a. Prokaryotic host cells Polynucleotide sequences encoding polypeptide components of antibodies of the present invention can be obtained using standard recombinant techniques. Desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizers or PCR technology. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many available vectors known in the art can be used for the purposes of the present invention. Selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of a heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides.

[0303] Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are generally used in connection with these hosts. Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, as well as marking sequences that allow for phenotypic selection in transformed cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thus providing an easy means for identifying transformed cells, and is suitable for most Gram-negative bacteria; the 2μ plasmid origin is suitable for yeast; and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages can also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., US Pat. No. 5,648,237.

[0304] Additionally, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophages such as λOEM™-11 can be utilized to generate recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0305] The expression vectors of the present invention may contain two or more promoter-cistron pairs, one encoding each of the polypeptide components. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that controls its expression. Prokaryotic promoters are generally divided into two classes: inducible promoters and constitutive promoters. Inducible promoters are promoters that initiate elevated levels of transcription of the cistron under their control in response to changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

[0306] Numerous promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the light or heavy chain by removing the promoter from its native DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both the native promoter sequence and many heterologous promoters can be used to direct amplification and / or expression of the target gene. In some embodiments, heterologous promoters are used because they generally increase transcription of the expressed target gene and improve yield compared to the native target polypeptide promoter.

[0307] Promoters recognized by a variety of potential host cells are well known. Suitable promoters for use in prokaryotic hosts include the PhoA promoter, β-galactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, tryptophan (trp) promoter systems [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)] or trc promoter. Promoters used in bacterial systems also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the anti-GPC3 antibody. However, other promoters functional in bacteria (such as other known bacterial or phage promoters) are equally suitable. Their nucleotide sequences have been published, enabling one skilled in the art to operably ligate them to cistrons encoding the target light and heavy chains using linkers or adapters to provide any necessary restriction enzyme sites (Siebenlist et al. (1980) Cell 20:269).

[0308] In one aspect of the present invention, each cistron in the recombinant vector contains a secretory signal sequence component that directs membrane transport of the expressed polypeptide. Generally, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for purposes of the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize or process the native signal sequence of the heterologous polypeptide, the signal sequence is substituted with a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the present invention, the signal sequence used in both cistrons of the expression system is the STII signal sequence or a variant thereof.

[0309] In another embodiment, production of immunoglobulins according to the present invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of a secretory signal sequence within each cistron. In this regard, immunoglobulin light and heavy chains are expressed, folded, and assembled to yield functional immunoglobulins in the cytoplasm. Certain host strains (e.g., E. coli trxB- strains) provide cytoplasmic conditions favorable for disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0310] The present invention provides an expression system in which the ratio of expressed polypeptide components can be adjusted to maximize the yield of secreted, properly assembled antibodies of the invention. Such adjustment is achieved, at least in part, by simultaneously adjusting the translation strength of the polypeptide components. One technique for adjusting translation strength is disclosed in Simmons et al., U.S. Patent No. 5,840,523, which uses variants of intracistron translation initiation regions (TIRs). For a given TIR, a range of amino acid or nucleic acid sequence variants can be created with varying translation strengths, thus providing a convenient means for adjusting this factor to the desired expression level of a particular chain. TIR variants can be generated by conventional mutagenesis techniques, resulting in codon changes that can alter the amino acid sequence; however, silent changes in the nucleotide sequence are preferred. Alterations to the TIR can include, for example, altering the number or spacing of Shine-Dalgarno sequences in conjunction with altering the signal sequence. One method for generating variant signal sequences is to generate a "codon bank" at the beginning of the coding sequence that does not alter the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be achieved by changing the nucleotide at the third position of each codon; furthermore, some amino acids, such as leucine, serine, and arginine, occupy multiple first and second positions, which can add complexity to the creation of the bank. This mutagenesis method is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol 4 151-158.

[0311] Preferably, a set of vectors is generated with various TIR strengths for each cistron contained therein. This limited set provides a comparison of the expression level of each chain under various combinations of TIR strengths and the yield of the desired antibody product. TIR strength can be determined by quantifying the expression level of a reporter gene, as described in detail in Simmons et al., U.S. Patent No. 5,840,523. Based on the comparison of translation strengths, desired individual TIRs are selected to be combined in the expression vector construct of the present invention.

[0312] b.Eukaryotic host cells The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0313] (1) Signal sequence component Vectors for use in eukaryotic host cells may also contain a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For mammalian cell expression, mammalian signal sequences and viral secretory leaders, such as the herpes simplex gD signal, are available. The DNA of such a precursor region is ligated in reading frame to DNA encoding the antibody.

[0314] (2) Origin of replication Generally, the origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may usually only be used because it contains the early promoter.

[0315] (3) Selective gene component Expression and cloning vectors usually contain a selection gene, also known as a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement an auxotrophic deficiency, or (c) supply essential nutrients unavailable from complex media, e.g., a gene encoding D-alanine racemase for Bacilli.

[0316] One example of a selection scheme utilizes a drug to arrest growth of the host cell. Cells successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0317] Examples of suitable selectable markers for mammalian cells are those that allow the identification of cells capable of incorporating the nucleic acid encoding the anti-GPC3 antibody, such as DHFR or thymidine kinase, metallothionein-I and -II (preferably primate metallothionein gene), adenosine deaminase, ornithine decarboxylase, etc. When wild-type DHFR is used, an appropriate host cell is a CHO cell line (e.g., ATCC CRL-9096) lacking DHFR activity, prepared and grown as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, host cells transformed or co-transformed with a DNA sequence encoding an antibody, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), (particularly wild-type hosts containing endogenous DHFR) can be selected by growing the cells in medium containing a selection agent for the selectable marker, e.g., an aminoglycoside antibiotic, such as kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[0318] A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, e.g., ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

[0319] (4) Expression of promoter components The cloning vector usually contains a promoter to direct mRNA synthesis operably linked to the nucleic acid sequence encoding the anti-GPC3 antibody. Promoters recognized by a variety of potential host cells are well known.

[0320] Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription begins. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence, which may be a signal for addition of a poly(A) tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

[0321] Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0322] Other yeast promoters that are inducible promoters with the added advantage of transcription controlled by growth conditions are the promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

[0323] Transcription of the anti-GPC3 antibody from the vector in mammalian host cells is controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published July 5, 1989), adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40), heterologous mammalian promoters such as the actin promoter or immunoglobulin promoter, and heat shock promoters, provided that such promoters are compatible with the host cell system.

[0324] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate-early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. An improvement on this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), for expression of human β-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as the promoter.

[0325] (5) Enhancer element components Transcription of DNA encoding the anti-GPC3 antibody in higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers from eukaryotic viruses are typically used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982), for a discussion of enhancing elements for activating eukaryotic promoters. The enhancer may be spliced ​​into the vector at a position 5' or 3' to the anti-GPC3 antibody-encoding sequence, but is preferably located at a site 5' from the promoter.

[0326] (6) Transcription termination component Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells derived from other multicellular organisms) also contain sequences necessary for the termination of transcription and stabilization of mRNA. Such sequences are commonly obtained from the 5'-untranslated region, and occasionally from the 3'-untranslated region, of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-GPC3 antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vector disclosed therein. Further methods, vectors, and host cells suitable for the synthesis of anti-GPC3 antibodies in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981), Mantei et al., Nature, 281:40-46 (1979), EP 117,060, and EP 117,058.

[0327] 4. Host Cell Culturing The host cells used to produce the anti-GPC3 antibody of the present invention may be cultured in a variety of media.

[0328] a. Prokaryotic host cells Prokaryotic cells used to produce the polypeptides of the present invention are grown in a medium known in the art that is suitable for culturing the selected host cells. An example of a suitable medium is Luria Broth (LB) containing necessary nutritional supplements. In some embodiments, the medium also contains a selection substance selected based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells that express an ampicillin resistance gene.

[0329] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may also be included at appropriate concentrations, introduced alone or in mixture with other supplements or media, such as complex nitrogen sources. Optionally, the media may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol, and dithiothreitol.

[0330] The prokaryotic host cells are cultured at an appropriate temperature. For example, for growth of E. coli, preferred temperatures range from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, and even more preferably about 30°C. The pH of the medium can be any pH in the range of about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably about 6.8 to about 7.4, more preferably about 7.0.

[0331] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter. In one embodiment of the present invention, the PhoA promoter is used to control transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is CRAP medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147). Various other inducers may be used depending on the vector construct used, as known in the art. In one embodiment, the expressed polypeptide of the present invention is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery usually involves disruption of the microorganism, generally by means such as osmotic shock, sonication, or lysis. After cell disruption, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therein. The cells may be removed from the culture, and the culture supernatant may be filtered and concentrated for further purification of the produced protein. The expressed polypeptide may be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0332] In one embodiment of the present invention, antibody production is carried out in large quantities by a fermentation process. Various large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a volume of at least 1000 liters, preferably between about 1,000 and 100,000 liters. These fermentors use agitation impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermentors with a volume of approximately 100 liters or less, which can range from about 1 liter to about 100 liters.

[0333] In fermentation processes, induction of protein expression typically begins after cells have been grown under appropriate conditions to a desired density, e.g., an OD550 of approximately 180-220, at which point the cells are in early stationary phase. Various inducers may be used, as known in the art and as described above, depending on the vector construct used. Cells may be grown for a short period of time before induction. Cells are typically induced for approximately 12-50 hours, although longer or shorter induction times may also be used.

[0334] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptides of the present invention. For example, to improve the proper assembly and folding of secreted antibody polypeptides, the host prokaryotic cells can be co-transformed with an additional vector overexpressing a chaperone protein, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (a peptidyl prolyl cis,trans isomerase with chaperone activity). The chaperone protein has been demonstrated to promote the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274:19601-19605, Georgiou et al., U.S. Patent No. 6,083,715, Georgiou et al., U.S. Patent No. 6,027,888, Bothmann and Pluckthun (2000) J.Biol.Chem.275:17100-17105, Ramm and Pluckthun (2000) J.Biol.Chem.275:17106-17113, Arie et al. (2001) Mol.Microbiol.39:199-210.

[0335] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient in proteolytic enzymes can be used for the present invention. For example, host cell strains can be modified to introduce genetic mutation(s) in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available and are described, for example, in Joly et al. (1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; and Hara et al., Microbial Drug Resistance, 2:63-72 (1996). In one embodiment, an E. coli strain that is deficient in a proteolytic enzyme and transformed with a plasmid that overexpresses one or more chaperone proteins is used as a host cell in the expression system of the present invention.

[0336] b.Eukaryotic host cells Commercially available media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma, are suitable for culturing the host cells. Further, references to Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 90 / 03430, and WO 90 / 03430 are also included. Any of the media described in U.S. Pat. No. 87 / 00195, or Reissued U.S. Pat. No. 30,985 may be used as the culture medium for the host cells. Any of these media may be supplemented, as needed, with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., will be those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0337] 5. Detection of gene amplification / expression Gene amplification and / or expression can be measured directly in a sample by, for example, conventional Southern blotting, Northern blotting to quantify mRNA transcription [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization using appropriately labeled probes based on the sequences provided herein. Alternatively, antibodies capable of recognizing specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, may be used. The antibodies can then be labeled and assayed. In the assay, the duplex is bound to a surface, and the presence of antibody bound to the duplex can be detected after duplex formation on the surface. Alternatively, gene expression can be measured by immunological methods to directly quantify expression of gene products, such as immunohistochemical staining of cells or tissue sections and assays of cell culture or body fluids. Antibodies useful for immunohistochemical staining and / or assay of sample fluids may be monoclonal or polyclonal and may be prepared in any mammal. Conveniently, the antibodies may be prepared against native-sequence GPC3 polypeptides, against synthetic peptides based on the DNA sequences provided herein, or against exogenous sequences fused to GPC3 DNA and encoding specific antibody epitopes.

[0338] 6. Purification of Anti-GPC3 Antibody Anti-GPC3 antibody forms can be recovered from culture medium or host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells used to express anti-GPC3 antibodies can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysis agents.

[0339] It may be desirable to purify the anti-GPC3 antibody from recombinant cell proteins or polypeptides. The following procedures are examples of suitable purification procedures: fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or cation exchange resins such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, a protein A Sepharose column to remove contaminants such as IgG, and a metal chelate column to bind an epitope-tagged form of the anti-GPC3 antibody.

[0340] Various protein purification methods may be used, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology, 182 (1990), Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the specific anti-GPC3 antibody produced.

[0341] When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant of such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0342] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γι, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is often agarose, although other matrices are also available. Mechanically stable matrices, such as controlled-pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than those achievable with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered.

[0343] Following any preliminary purification step(s), the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5 to 4.5, preferably performed at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0344] G. Pharmaceutical Preparations The antibodies of the invention can be administered by any route appropriate to the condition being treated. They are typically administered parenterally, i.e., by injection, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally, and epidurally.

[0345] To treat these cancers, in one embodiment, the antibody is administered by intravenous infusion. The dose administered by infusion ranges from about 1 μg / m to about 10,000 μg / m. 2 , generally once a week for a total of 1, 2, 3 or 4 times. Alternatively, the dose range is about 1 μg / m 2 ~Approx. 1000μg / m 2 , about 1μg / m 2 ~about 800μg / m 2 , about 1μg / m 2 ~about 600μg / m 2 , about 1μg / m 2 ~about 400μg / m 2 , about 10μg / m 2 ~about 500μg / m 2 , about 10μg / m 2 ~about 300μg / m 2 , about 10μg / m 2 ~about 200μg / m 2 , and approximately 1 μg / m 2 ~about 200μg / m 2 The dose can be administered once a day, once a week, multiple times a week but less than once a day, multiple times a month but less than once a day, multiple times a month but less than once a week, once a month, or intermittently to alleviate or relieve the symptoms of the disease. Administration can be continued at any of the intervals disclosed until the symptoms of the tumor or cancer being treated are alleviated. Administration can be continued after symptom alleviation or alleviation is achieved, in which case such alleviation or alleviation is prolonged by continued administration.

[0346] The present invention also provides a method for treating breast cancer, comprising administering to a patient suffering from breast cancer a therapeutically effective amount of a humanized GPC3 antibody according to any one of the preceding embodiments. The antibody is typically administered at a dose of about 1 μg / m 2 ~about 1000mg / m 2The dose range is as follows:

[0347] In one aspect, the present invention further provides a pharmaceutical composition comprising at least one anti-GPC3 antibody of the present invention. In some embodiments, the pharmaceutical formulation comprises (1) an antibody of the present invention, and (2) a pharmaceutically acceptable carrier.

[0348] The therapeutic agent containing the anti-GPC3 antibody used according to the present invention is prepared in the form of a lyophilized preparation or an aqueous solution by mixing the antibody having the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) for storage. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, e.g., acetic acid, Tris, phosphoric acid, citric acid, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl parabens, e.g., methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, e.g., serum albumin, gelatin, or immunoglobulins. Examples of suitable surfactants include purine, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin, chelating agents such as EDTA, tonicifiers such as trehalose and sodium chloride, sugars such as sucrose, mannitol, trehalose, or sorbitol, surfactants such as polysorbates, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Pharmaceutical formulations to be used for in vivo administration are generally sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0349] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, in colloidal drug delivery systems. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0350] Sustained-release preparations can also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. If encapsulated immunoglobulins remain in the body for extended periods of time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, rational strategies for stabilization can be implemented. For example, if the aggregation mechanism is found to be the formation of intermolecular S—S bonds via thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling the moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0351] The antibody can be formulated in any form suitable for delivery to target cells / tissues. For example, the antibody can be formulated as an immunoliposome. Liposomes are vesicles composed of various types of lipids, phospholipids, and / or surfactants, which are useful for delivering drugs to mammals. The components of the liposome are generally arranged in a bilayer structure similar to the lipid arrangement of biological membranes. Liposomes containing the antibody can be prepared by methods known in the art, for example, as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985), Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980), U.S. Patent Nos. 4,485,045 and 4,544,545, and WO97 / 38731 published October 23, 1997. Liposomes with enhanced circulation time are disclosed in US Pat. No. 5,013,556.

[0352] Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes described in Martin et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide exchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81(19):1484 (1989).

[0353] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0354] H. Treatment with anti-GPC3 antibodies A variety of detection assays are available for identifying the expression of GPC3 in cancer. In one embodiment, the overexpression of GPC3 polypeptide can be analyzed by immunohistochemistry (IHC). Paraffin-embedded tissue sections from tumor biopsies are subjected to IHC assays to determine the GPC3 protein staining intensity standard. In a preferred embodiment, determining whether a cancer is suitable for treatment by the methods disclosed herein comprises detecting the presence of a GPC3 tumor epitope in a subject or a subject's sample.

[0355] Alternatively, or in addition, a FISH assay, such as INFORM® (distributed by Ventana, Arizona) or PATHVISION® (Vysis, Illinois), may be performed on formalin-fixed, paraffin-embedded tumor tissue to identify the degree, if any, of GPC3 overexpression in the tumor.

[0356] GPC3 overexpression or amplification can be assessed using in vivo detection assays, for example, by administering a molecule (e.g., an antibody) that binds to the molecule to be detected and is tagged with a detectable label (e.g., a radioisotope or fluorescent label), and externally scanning the patient for the location of the label. As described above, the anti-GPC3 antibodies of the present invention have various non-therapeutic applications. The anti-GPC3 antibodies of the present invention can be useful for staging cancers that express the GPC3 epitope (e.g., in radioimaging). The antibodies are also useful for purifying or immunoprecipitation of the GPC3 epitope from cells, for in vitro detection and quantification of the GPC3 epitope, for example, in ELISA or Western blot, and for killing and eliminating GPC3-expressing cells as a step in purifying other cells from a mixed cell population.

[0357] Currently, depending on the stage of cancer, cancer treatment includes one or a combination of the following therapies: surgery to remove cancerous tissue, radiation therapy, and chemotherapy. Anti-GPC3 antibody therapy may be particularly desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well, and in metastatic disease where the usefulness of radiation therapy is limited. The tumor-targeting anti-GPC3 antibodies of the present invention are useful for alleviating GPC3-expressing cancers after initial diagnosis or during the recurrence process of the disease.

[0358] The anti-GPC3 antibody is administered to a human patient according to known methods, for example, intravenously, e.g., as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intraarticularly, intrasynovially, intrathecally, orally, topically, or by inhalation. Intravenous or subcutaneous administration of the antibody is preferred.

[0359] The antibody compositions of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice, including factors to consider in this regard, including the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0360] For disease prevention or treatment, the dosage and administration method will be selected by a physician according to known criteria. The appropriate antibody dosage depends on the type of disease being treated (as defined above), the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatment history, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody is appropriately administered to the patient once or over a series of treatments. Preferably, the antibody is administered by intravenous infusion or subcutaneous injection. Depending on the type and severity of the disease, an initial candidate dose of antibody of about 1 μg / kg to about 50 mg / kg body weight (e.g., about 0.1 to 15 mg / kg / administration) may be administered to the patient, whether administered by one or more separate administrations or by continuous infusion. The administration regimen may include an initial loading dose of about 4 mg / kg of the anti-GPC3 antibody, followed by a weekly maintenance dose of about 2 mg / kg. However, other administration regimens may be useful. Typical daily dosages can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment is continued until a desired suppression of disease symptoms occurs. The progress of this treatment can be easily monitored by conventional methods and assays and based on criteria known to the physician or skilled artisan.

[0361] The anti-GPC3 antibodies of the present invention may be in various forms encompassed by the definition of "antibody" herein. Thus, the antibodies include full-length or intact antibodies, antibody fragments, native sequence antibodies or amino acid variants, humanized, chimeric, or fusion antibodies, and functional fragments thereof. In fusion antibodies, the antibody sequence is fused to a heterologous polypeptide sequence. The antibody may be modified in the Fc region to provide desired effector functions. As discussed in more detail in this section, naked antibodies bound to cell surfaces can induce cytotoxicity, for example, through antibody-dependent cellular cytotoxicity (ADCC), or by recruitment of complement in complement-dependent cytotoxicity, or by some other mechanism. Alternatively, certain other Fc regions may be used when it is desirable to eliminate or reduce effector functions to minimize side effects or therapeutic complications.

[0362] In one embodiment, the antibody (i) competes for binding to the same epitope as the antibody of the present invention, and / or (ii) substantially binds to the same epitope. In particular, antibodies having the biological characteristics of the anti-GPC3 antibodies of the present invention, including in vivo tumor targeting and any cell growth inhibition or cytotoxicity, are also contemplated.

[0363] The anti-GPC3 antibody is useful for treating cancers expressing GPC3 in mammals or alleviating one or more symptoms of the cancer. The cancer includes metastatic cancers of any of the cancers described herein. The antibody is capable of binding to at least a portion of cancer cells expressing the GPC3 epitope in the mammal. In a preferred embodiment, the antibody effectively destroys or kills GPC3-expressing tumor cells or inhibits the growth of such tumor cells after binding to the GPC3 epitope in vitro or in vivo. In another preferred embodiment, the antibody effectively (i) inhibits the growth or proliferation of cells to which it binds, (ii) induces the death of cells to which it binds, (iii) inhibits the desquamation of cells to which it binds, (iv) inhibits the metastasis of cells to which it binds, or (v) inhibits the angiogenesis of tumors containing cells to which it binds.

[0364] The present invention provides a composition comprising an anti-GPC3 antibody of the present invention and a carrier. The present invention also provides a formulation comprising an anti-GPC3 antibody of the present invention and a carrier. In one embodiment, the formulation is a therapeutic agent comprising a pharmaceutically acceptable carrier.

[0365] Another aspect of the present invention is isolated nucleic acids encoding the anti-GPC3 antibodies. Nucleic acids encoding both the heavy and light chains, particularly residues of the hypervariable regions, are included, including chains encoding native sequence antibodies, as well as variants, modifications, and humanized forms of the antibodies.

[0366] The present invention also provides a method for treating cancer that expresses GPC3 polypeptide in a mammal or alleviating one or more symptoms of the cancer, the method comprising administering a therapeutically effective amount of anti-GPC3 antibody to the mammal.The therapeutic composition of the antibody can be administered short-term (acute), chronically, or intermittently according to the instructions of a physician.Also provided is a method for inhibiting the growth of cells that express GPC3 polypeptide and a method for killing the cells.

[0367] The present invention also provides kits and products comprising at least one anti-GPC3 antibody. Kits comprising anti-GPC3 antibodies are used, for example, in GPC3 cell killing assays, purification or immunoprecipitation of GPC3 polypeptides from cells. For example, in the case of GPC3 isolation and purification, the kit can comprise an anti-GPC3 antibody bound to beads (e.g., Sepharose beads). Kits can be provided that contain antibodies for in vitro detection and quantification of GPC3, for example, in ELISA or Western blot or IHC assays (described in more detail herein). Such antibodies useful for detection can be labeled, for example, fluorescent or radioactive.

[0368] Effector function operation It may be desirable to modify the antibody of the invention with respect to effector function, for example to enhance the antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody.

[0369] Alternatively, or additionally, cysteine ​​residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992). Homodimeric antibodies with improved anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, antibodies with dual Fc regions can be engineered, which may thereby have improved complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). To extend the serum half-life of the antibody, a salvage receptor binding epitope may be incorporated into the antibody (particularly an antibody fragment), as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgGi, IgG2, IgG3, or IgG4) that is involved in extending the in vivo serum half-life of the IgG molecule.

[0370] CAR-modified immune cell therapy In certain embodiments, the present invention relates to compositions and methods for treating cancer, including, but not limited to, hematological malignancies and solid tumors. In certain embodiments, CAR-modified immune cells are used. CAR-T cells may be used therapeutically in patients with solid tumors arising from non-hematological tumors, such as breast, CNS, and skin malignancies. In certain embodiments, CAR-NK cells may be used therapeutically in patients with any one of several malignancies. In certain embodiments, the present invention relates to strategies for adoptive cell transfer of T cells or NK cells transduced to express a chimeric antigen receptor (CAR). CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., a tumor antigen) with, for example, a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cell immune activity.

[0371] In one embodiment, the present invention relates to the use of NK cells genetically modified to stably express a desired CAR. NK cells expressing a CAR are referred to herein as CAR-NK cells or CAR-modified NK cells. Preferably, the cells can be genetically modified to stably express an antibody binding domain on their surface to confer novel antigen specificity. Methods for generating CAR-NK cells are known in the art. See, for example, Glienke et al., "Advantages and applications of CAR-expressing natural killer cells," Front Pharmacol. 2015;6:21. Services for generating CAR-NK cells are commercially available. See, for example, Creative Biolabs Inc., 45-1 Ramsey Road, Shirley, NY 11967, USA.

[0372] In one aspect, the present invention relates to the use of T cells that are genetically modified to stably express a desired CAR. T cells that express a CAR are herein referred to as CAR-T cells or CAR-modified T cells. Preferably, the cells can be genetically modified to stably express an antibody binding domain on their surface, so as to confer new antigen specificity that is MHC-independent. In some cases, the T cells are genetically modified to stably express a CAR that combines the antigen recognition domain of a specific antibody with the intracellular domain of the CD3-zeta chain or FcyRI protein to form a single chimeric protein.

[0373] In one embodiment, the CAR of the present invention comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain. In one embodiment, a transmembrane domain naturally associated with one of the domains of the CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or a different surface membrane protein, in order to minimize interaction with other members of the receptor complex. In one embodiment, the transmembrane domain is the hinge domain of CD8a.

[0374] With respect to the cytoplasmic domain, the CAR of the present invention can be designed to contain the signaling domain of CD28 and / or 4-IBB by itself, or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR of the present invention. In one embodiment, the cytoplasmic domain of the CAR can be designed to further contain the signaling domain of CD3-zeta. For example, the cytoplasmic domain of the CAR can include, but is not limited to, the signaling modules of CD3-zeta, 4-1BB, and CD28, as well as combinations thereof. Thus, the present invention provides CAR T cells and methods of using them for adoptive therapy.

[0375] In one embodiment, the CAR T cells of the present invention can be generated by introducing a lentiviral vector containing a desired CAR, for example, a CAR containing anti-GPC3, the hinge and transmembrane domain of CD8a, and the signaling domains of human 4-1BB and CD3 zeta, into the cells. The CAR T cells of the present invention can replicate and persist for long periods in vivo, thereby resulting in sustained tumor control.

[0376] In one embodiment, the anti-GPC3 domain comprises a heavy chain variable region comprising: EVQLQQSGPELVKPGASVKISCKTSGYTFTEYAMHWVKQSHGKSLEWIGGINPNNGVTTYNQRFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGLLWYAYWGQGTLVTVSA (SEQ ID NO: 2)

[0377] In one embodiment, the anti-GPC3 domain comprises a light chain variable region comprising: DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 4)

[0378] In one embodiment, the anti-GPC3 domain comprises SEQ ID NO:2 and SEQ ID NO:4.

[0379] In one embodiment, the anti-GPC3 domain comprises an amino acid sequence selected from the group consisting of EYAMH (SEQ ID NO: 6), GINPNNGVTTYNQRFKG (SEQ ID NO: 8), and GLLWYAY (SEQ ID NO: 10).

[0380] In one embodiment, the anti-GPC3 domain comprises an amino acid sequence selected from the group consisting of KASQDINSYLS (SEQ ID NO: 13), RANRLVD (SEQ ID NO: 15), and LQYDEFPLT (SEQ ID NO: 17).

[0381] In one embodiment, the anti-GPC3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, and further comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17.

[0382] In one embodiment, the present invention relates to administering T cells genetically modified to express a CAR to treat patients with or at risk of having cancer using lymphocyte infusion. Preferably, autologous lymphocyte infusion is used for the treatment. Autologous PBMCs are collected from the patient in need of treatment, and the T cells are activated and expanded using methods known in the art and described herein, and then infused back into the patient.

[0383] The present invention also encompasses the treatment of malignant tumors or autoimmune diseases in which chemotherapy and / or immunotherapy in patients results in significant immunosuppression, thereby increasing the patient's risk of developing malignant tumors (e.g., CLL). The present invention encompasses the use of T cells (also referred to as CARTGPC3 T cells) expressing anti-GPC3 antibodies derived from a CAR containing both CD3-zeta and either the 4-IBB or CD28 costimulatory domains. The CARTGPC3 T cells of the present invention are capable of robust in vivo T cell proliferation and can establish memory cells specific for cells presenting the GPC3 tumor epitope. These memory cells persist at high levels in the blood and bone marrow for extended periods. The present invention provides chimeric antigen receptors (CARRs) comprising an extracellular domain and an intracellular domain. The extracellular domain comprises a target-specific binding element, otherwise referred to as an antigen-binding moiety. The intracellular or cytoplasmic domain comprises a costimulatory signaling region and a zeta chain moiety. The costimulatory signaling region refers to the portion of a CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules that remove antigen receptors or their ligands necessary for the efficient response of lymphocytes to antigens.

[0384] A spacer domain may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain of the polypeptide chain to either the extracellular or cytoplasmic domain. A spacer domain may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0385] antigen binding part In one embodiment, CAR of the present invention comprises a target-specific binding element, otherwise called antigen-binding portion or targeting arm.The antigen-binding portion used in the present invention can bind to the GPC3 polypeptide expressed on the surface of cancer cells.Therefore, the antigen-binding portion is selected to recognize the ligand that acts as the cell surface marker of the target cell associated with a specific pathology.

[0386] The CARs of the present invention are engineered to target cellular GPC3 by engineering an appropriate antigen-binding moiety that specifically binds to an epitope of GPC3.

[0387] Preferably, part of the antigen-binding portion of the CAR of the invention is an scFV or scFab, wherein the nucleic acid sequence of the scFV comprises the nucleic acid sequence(s) of one or more light chain CDRs and one or more heavy chain CDRs disclosed herein for the anti-GPC3 antibody, and the nucleic acid sequence of the scFab comprises the nucleic acid sequence(s) of one or more light chain CDRs and one or more heavy chain CDRs disclosed herein for the anti-GPC3 antibody.

[0388] Preferably, a portion of the antigen-binding portion in the CAR of the present invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of EYAMH (SEQ ID NO: 6), GINPNNGVTTYNQRFKG (SEQ ID NO: 8), and GLLWYAY (SEQ ID NO: 10). Preferably, a portion of the antigen-binding portion in the CAR of the present invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of KASQDINSYLS (SEQ ID NO: 13), RANRLVD (SEQ ID NO: 15), and LQYDEFPLT (SEQ ID NO: 17).

[0389] Preferably, a portion of the antigen-binding portion of the CAR of the present invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, and 10, and further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 17, and further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4.

[0390] In one embodiment, the portion of the antigen-binding portion of the CAR of the present invention is an scFv or scFab comprising an amino acid sequence having about 80%, 85%, 90%, or 95% identity to the above SEQ ID NOs.

[0391] Transmembrane domain Regarding the transmembrane domain, the CAR can be designed to include a transmembrane domain that is fused to the extracellular domain of the CAR.In one embodiment, the transmembrane domain that is naturally associated with one of the domains of the CAR is used.In some cases, the transmembrane domain can be selected or modified by amino acid substitution to prevent this domain from binding to the transmembrane domain of the same or different surface membrane protein, in order to minimize interaction with other members of the receptor complex.

[0392] The transmembrane domain can be derived from either natural or synthetic sources. If the origin is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region particularly used in the present invention can be derived from (i.e., at least the transmembrane region(s) thereof) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain can be synthetic, in which case it contains primarily hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0393] Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker. Preferably, the transmembrane domain in the CAR of the present invention is the CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 16 of U.S. Patent No. 9,102,760. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760. In another embodiment, the sequences disclosed in Table 2 of WO 2017 / 054089 are used.

[0394] In some cases, the transmembrane domain of the CAR of the present invention comprises the hinge domain of CD8a. In one embodiment, the hinge domain of CD8 comprises the nucleic acid sequence of SEQ ID NO: 15 of U.S. Patent No. 9,102,760. In one embodiment, the hinge domain of CD8 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 21 of U.S. Patent No. 9,102,760. In another embodiment, the hinge domain of CD8 comprises the amino acid sequence of SEQ ID NO: 21 of U.S. Patent No. 9,102,760. In another embodiment, the sequences disclosed in Table 2 of WO 2017 / 054089 are used.

[0395] Cytoplasmic domain The cytoplasmic domain, or intracellular signaling domain, of the CAR of the present invention is involved in activating at least one of the normal effector functions of the immune cell to which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits a signal for the effector function and instructs the cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used instead of the intact chain, as long as it transmits the signal for the effector function. The term intracellular signaling domain is therefore intended to include any truncated portion of the intracellular signaling domain that is sufficient to transmit the signal for the effector function.

[0396] Preferred examples of intracellular signaling domains for use in the CARs of the present invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act together to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same function.

[0397] It is known that signals generated solely through the TCR are insufficient for the complete activation of T cells, and that secondary or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0398] Examples of ITAMs comprising primary cytoplasmic signaling sequences that are particularly useful in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR of the present invention comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0399] In a preferred embodiment, the cytoplasmic domain of the CAR can be designed to include the signaling domain of CD3-zeta by itself or in combination with any other desired cytoplasmic signaling domain(s) useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of the CAR can include a portion of the CD3-zeta chain and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules that do not include antigen receptors or their ligands necessary for the efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0400] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be linked to each other randomly or in a defined order. Optionally, a short oligopeptide or polypeptide linker, preferably 2 to 10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0401] In one aspect, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-IBB. In yet another aspect, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0402] In one embodiment, the cytoplasmic domain of a CAR of the invention is designed to comprise the signaling domain of 4-IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-IBB comprises the nucleic acid sequence set forth in SEQ ID NO: 17 of U.S. Patent No. 9,102,760, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 18 of U.S. Patent No. 9,102,760. In another embodiment, the sequences disclosed in Table 2 of WO 2017 / 054089 are used. In one embodiment, the cytoplasmic domain of a CAR of the invention is designed to comprise the signaling domain of 4-IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-IBB comprises the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 23 of U.S. Patent No. 9,102,760, and the signaling domain of CD3-zeta comprises the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 of U.S. Patent No. 9,102,760. In another embodiment, the sequences disclosed in Table 2 of WO 2017 / 054089 are used.

[0403] In one embodiment, the cytoplasmic domain of the CAR of the invention is designed to comprise the signaling domain of 4-IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-IBB comprises the amino acid sequence set forth in SEQ ID NO: 23 of U.S. Patent No. 9,102,760, and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 24 of U.S. Patent No. 9,102,760. In another embodiment, the sequences disclosed in Table 2 of WO 2017 / 0540...

Claims

1. An isolated monoclonal antibody that binds to glypican 3 (GPC3), comprising: The isolated monoclonal antibody, wherein the heavy chain of the anti-GPC3 antibody comprises complementarity determining region (CDR) 1 set forth as SEQ ID NO: 6, CDR2 set forth as SEQ ID NO: 8, and CDR3 set forth as SEQ ID NO: 10, and the light chain of the antibody comprises CDR1 set forth as SEQ ID NO: 13, CDR2 set forth as SEQ ID NO: 15, and CDR3 set forth as SEQ ID NO:

17.

2. An isolated monoclonal antibody as described in claim 1, wherein the heavy chain of the antibody comprises CDR1, CDR2, and CDR3, respectively set forth as amino acid residues 31-35, 50-66, and 99-105 of SEQ ID NO: 2, and the light chain of the antibody comprises CDR1, CDR2, and CDR3, respectively set forth as amino acid residues 24-34, 50-56, and 89-97 of SEQ ID NO:

4.

3. 2. The isolated monoclonal antibody of claim 1, wherein the heavy chain of the antibody comprises SEQ ID NO: 2 and the light chain of the antibody comprises SEQ ID NO:

4.

4. The antibody (i) a variable heavy (VH) domain comprising the amino acid sequence of SEQ ID NO: 2; and 2. The isolated monoclonal antibody of claim 1, comprising: (ii) a variable light (VL) domain comprising the amino acid sequence of SEQ ID NO:

4.

5. The isolated monoclonal antibody of any one of claims 1 to 4, wherein the antibody is a chimeric, humanized, or human antibody.

6. The isolated monoclonal antibody of any one of claims 1 to 4, wherein the antibody is a bispecific antibody.

7. 3. The isolated monoclonal antibody of claim 1, wherein the antibody is an antibody fragment.

8. The antibody may be a Fab fragment, a Fab' fragment, or a F(ab)' fragment. ２ 8. The isolated monoclonal antibody of claim 7, which is a single-chain variable fragment (scFv), a single-chain variable fragment (scFv), or a disulfide-stabilized variable fragment (dsFv).

9. 1. A method for detecting GPC3 in a tissue preparation, comprising: contacting the tissue preparation with the isolated monoclonal antibody of claim 1 under conditions sufficient to form a complex between the isolated monoclonal antibody of claim 1 and GPC3 present in the plasma membrane of cells of the tissue preparation; and detecting binding of said antibody to said tissue preparation.

10. 10. The method of claim 9, wherein the tissue preparation comprises a tumor biopsy of hepatocellular carcinoma (HCC), melanoma, lung squamous cell carcinoma, Merkel cell carcinoma, or ovarian clear cell carcinoma.

11. The method of claim 9, wherein the monoclonal antibody is directly labeled.

12. moreover, contacting the tissue preparation with a second antibody that specifically binds to the monoclonal antibody; and 10. The method of claim 9, comprising detecting binding of the secondary antibody.

13. The method of any one of claims 9 to 12, wherein detecting binding of the antibody to the tissue preparation further comprises scoring the amount of the detected complex.

14. The method of claim 13, wherein the scoring is performed by a pathologist.

15. detecting the presence of the complex via digitization; The method of claim 13, wherein the scoring is automated based on digitization of the detected complexes.

16. 14. The method of claim 13, wherein said scoring further comprises specifying the staining intensity of the complex detected via immunohistochemistry using an integer scale of 0 (negative) to 3+, recording the percentage of cells that stain positively at each intensity level, and calculating a membrane-bound H-score based on the percentage of cells that stain positively at each intensity level.

17. A kit for predicting the therapeutic effect of anti-GPC3 immunotherapy on cancer, wherein the cancer is characterized in that cells of the cancer express GPC3, and the kit comprises the isolated monoclonal antibody of claim 1; A kit, comprising: a kit for detecting the presence of the cells in a subject via the method of claim 9; and a kit for predicting that the anti-GPC3 immunotherapy will have a therapeutic effect on the cancer in the subject if a complex between the anti-GPC3 antibody and GPC3 expressed on the membrane of the cancer cell is detected.

18. The kit of claim 17, wherein the kit is used before the subject receives any anti-GPC3 immunotherapy or is used in the course of already receiving the anti-GPC3 immunotherapy.

19. The kit of claim 17, wherein the anti-GPC3 immunotherapy comprises chimeric antigen receptor (CAR) T cell therapy or CAR NK cell therapy, and the CAR is designed to specifically recognize membrane-bound GPC3.

20. 18. The kit of claim 17, wherein the anti-GPC3 immunotherapy comprises an anti-GPC3 antibody.

21. An isolated nucleic acid molecule encoding the monoclonal antibody of claim 1.

22. 22. The isolated nucleic acid molecule of claim 21, wherein the nucleotide sequence encoding the heavy chain of the monoclonal antibody comprises SEQ ID NO: 1 and the nucleotide sequence encoding the light chain of the antibody comprises SEQ ID NO:

3.

23. 23. An expression vector comprising the isolated nucleic acid molecule of claim 21 or claim 22.

24. 24. An isolated host cell transformed with the expression vector of claim 23.

25. A bispecific antibody comprising the monoclonal antibody of claim 1.

26. An antibody-drug conjugate (ADC) comprising the isolated monoclonal antibody of claim 1.

27. A chimeric antigen receptor (CAR) comprising the antibody fragment of claim 7.

28. 28. A modified immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises the CAR of claim 27.

29. 29. The modified immune cell of claim 28, which is a modified T cell.

30. 30. The modified immune cell of claim 29, which is an αβ T cell or a γδ T cell.

31. 29. The modified immune cell of claim 28, which is a modified NK cell.

32. 29. A plurality of modified immune cells according to claim 28.

33. 28. An agent for inhibiting the growth of cells presenting a GPC3 epitope specifically recognized by the antibody of claim 1, comprising the isolated monoclonal antibody of claim 1, the modified immune cell(s) of claim 28, or the ADC of claim 26.

34. 29. A composition comprising a therapeutically effective amount of the isolated monoclonal antibody of any one of claims 1-4, the modified immune cell(s) of claim 28, or the ADC of claim 26, and a pharmaceutically acceptable carrier.

35. A drug for treating a subject having cancer, comprising the composition of claim 34, wherein the subject having cancer that expresses GPC3 is selected.

36. The method of claim 35, wherein the cancer is liver cancer, ovarian cancer, gastric cancer, Merkel cell carcinoma, or lung cancer.

37. Use of an antibody described in any one of claims 1 to 4 in the preparation of a medicament for the treatment of cancer.

38. Use of the modified immune cell(s) described in claim 28 in the preparation of a medicament for the treatment of cancer.

39. Use of the ADC described in claim 26 in the preparation of a medicament for the treatment of cancer.

40. Use of the CAR described in claim 27 in the preparation of a drug for the treatment of cancer.

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