Treatment of Patients with Myxoid / Round Cell Liposarcoma

Abstract

The present invention provides a method for treating patients diagnosed with myxoid / round cell liposarcoma with anti-GPC3 therapeutic agents.The present invention also relates to quantifying GPC3 expression in tissue samples from patients diagnosed with myxoid / round cell liposarcoma by immunostaining assay, and identifying GPC3 expression levels that correlate with patient selection for administration of anti-GPC3 therapeutic agents.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 279,797, filed November 16, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Sarcomas, including soft tissue sarcomas (STS), are a group of rare malignancies of mesenchymal origin that account for approximately 20% of all pediatric solid tumors and 1% of adult solid tumors (Abaricia & Hirbe, 2018; Hui, 2016). Liposarcomas (LPS), the most common type of STS, are malignancies that arise from adipose tissue and affect adipogenesis. Myxoid / round cell liposarcoma (MRCLS) accounts for approximately 30% of LPS and tends to occur in a slightly younger age group, with a peak incidence in the fourth decade.

[0003] Standard treatment for MRCLS consists of surgical resection of localized primary disease. MRCLS is known to be more sensitive to radiation therapy and cytotoxic chemotherapy compared to other LPS subtypes. However, approximately 40% of patients will relapse despite adequate local treatment. Nevertheless, treatment options for patients with MRCLS remain scarce, and new therapeutic options are needed for this high medical need indication. Summary of the Invention

[0004] The present disclosure is based, at least in part, on the unexpected discovery that glypican 3 (GPC3) can serve as a diagnostic biomarker and therapeutic target for MRCLS. To better understand GPC3-positive tumor prevalence and prioritize indications for anti-GPC3 therapy, we investigated the expression profile of GPC3 in solid tumor biopsies from cancer patients, and using a new scoring rule to accurately and reliably quantify GPC3 expression levels across multiple tissue samples and scoring pathologists, we identified a subgroup of LPS (i.e., MRCLS) as a patient population that expresses relatively high GPC3. This finding was unexpected given the complexity of the staining patterns observed in various tumor tissue sections stained with anti-GPC3 antibody (GC33), suggesting that MRCLS may be prioritized and / or selected for treatment with anti-GPC3 therapeutic agents.

[0005] Thus, the disclosure in some aspects features a method of treating a patient diagnosed with myxoid / round cell liposarcoma, the method comprising administering an anti-glypican-3 (GPC3) therapeutic to the patient. In some embodiments, the patient is selected for treatment by diagnosing myxoid / round cell liposarcoma. In some instances, the myxoid / round cell liposarcoma expresses GPC3.

[0006] In some embodiments, patients diagnosed with myxoid / round cell liposarcoma are selected by immunostaining, for example, by immunohistochemistry (IHC) staining. In some examples, patients diagnosed with myxoid / round cell liposarcoma are selected if they have a cytoplasmic / membrane H-score of greater than 30.

[0007] In some embodiments, the patient is (a) obtaining a tissue section from a tumor biopsy sample, the section having a thickness of between 3 μm and 15 μm; (b) immunostaining by IHC, preferably with an antibody that specifically binds to GPC3, more specifically with the antibody GC33; (c) determining a cytoplasmic / membrane H-score; and (d) selecting patients with an H-score greater than 30 for treatment.

[0008] In some embodiments, the selection can include immunostaining, for example immunohistochemical staining of GPC3 in tumor samples from patients.GPC3 expression level can be determined and compared with a predetermined threshold level of GPC3 expression.If the patient has a GPC3 expression level equal to or greater than a predetermined threshold level, the patient is selected for treatment.

[0009] In some embodiments, the therapeutic agent comprises an anti-GPC3 binding domain, e.g., an anti-GPC3 antibody, e.g., a full-length antibody or functional fragment thereof that retains binding to GPC3. In some cases, the therapeutic agent may comprise an anti-GPC3 antibody, an anti-GPC3 antibody-drug conjugate, an anti-GPC3 antibody-radionuclide conjugate, or a fusion protein of an anti-GPC3 antibody or antibody derivative that binds to GPC3 with an anti-CD3 binding domain or an immunostimulatory polypeptide. In some examples, the therapeutic agent comprises a genetically engineered hematopoietic cell that expresses an anti-GPC3 chimeric receptor polypeptide (CAR), which is (a) an extracellular binding domain that binds to GPC3; (b) a transmembrane domain; (c) a cytoplasmic signaling domain.

[0010] In some instances, the hematopoietic cells may further exogenously express a gene that improves the viability and / or function of the hematopoietic cells in a solid tumor microenvironment. In some cases, the hematopoietic cells may have improved glucose uptake activity compared to wild-type hematopoietic cells of the same type, but the hematopoietic cells exogenously express a glucose importing polypeptide. In some instances, the glucose importing polypeptide is a glucose transporter (GLUT) or a sodium-glucose cotransporter (SGLT). Examples include, but are not limited to, GLUT1, GLUT3, GLUT1 S226D, SGLT1, SGLT2, GLUT8, GLUT8 L12A L13A, GLUT11, GLUT7, and GLUT4.

[0011] In some cases, hematopoietic cells may have a regulated Krebs cycle compared to the same type of wild-type hematopoietic cells, but the hematopoietic cells exogenously express a Krebs cycle regulator polypeptide. In some examples, the Krebs cycle regulator is an enzyme that catalyzes a reaction in the Krebs cycle. Examples include, but are not limited to, isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), or phosphoglycerate dehydrogenase (PHGDH). In some examples, the Krebs cycle regulator is an enzyme that uses a Krebs cycle metabolite as a substrate, such as glutamate-oxaloacetate transaminase (GOT) or phosphoenolpyruvate carboxykinase 1 (PCK1). Alternatively, the Krebs cycle regulator may be an enzyme that converts a precursor to a Krebs cycle metabolite. Examples include, but are not limited to, phosphoserine aminotransferase (PSAT1), glutamate dehydrogenase (GDH1), glutamate-pyruvate transaminase 1 (GPT1), or glutaminase (GLS).

[0012] In some cases, hematopoietic cells may have enhanced intracellular lactate concentrations compared to wild-type hematopoietic cells of the same type, while the hematopoietic cells exogenously express a lactate-regulating polypeptide. In some examples, the lactate-regulating polypeptide is a monocarboxylate transporter (MCT), such as MCT1, MCT2, or MCT4. In other examples, the lactate-regulating polypeptide is an enzyme involved in lactate synthesis, such as lactate dehydrogenase A (LDHA). Alternatively, the lactate-regulating polypeptide is a polypeptide that inhibits a pathway that competes with lactate synthesis substrates, such as pyruvate dehydrogenase kinase 1 (PDK1).

[0013] In any of the methods disclosed herein, the extracellular antigen-binding domain is a single-chain antibody fragment (scFv) that binds to GPC3. In some examples, the scFv is derived from a GC33 antibody. In one example, the scFv can comprise (e.g., consist of) the sequence of SEQ ID NO:2. In some examples, the anti-GPC3 CAR polypeptide can comprise a CD28 costimulatory domain in combination with a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof (e.g., SEQ ID NO:4). Alternatively, the anti-GPC3 CAR polypeptide can comprise a 4-1BB costimulatory domain (e.g., SEQ ID NO:5) in combination with a CD8 transmembrane domain, a CD8 hinge domain, or a combination thereof (e.g., SEQ ID NO:3). Alternatively or in addition, the anti-GPC3 CAR polypeptide can comprise a cytoplasmic signaling domain of (c), which can be the cytoplasmic domain of CD3ζ, preferably SEQ ID NO:7, or FcεR1γ. In a particular example, the anti-GPC3 CAR can comprise the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9.

[0014] Any of the hematopoietic cells disclosed herein can be natural killer (NK) cells, macrophages, neutrophils, eosinophils, or T cells. In some examples, the hematopoietic cells are T cells. In some cases, the expression of endogenous T cell receptors, endogenous major histocompatibility complexes, endogenous beta-2-microglobulin, or combinations thereof, is inhibited or eliminated in such T cells. In some examples, the hematopoietic cells can be derived from peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs). In some examples, the hematopoietic cells are autologous to the patient. In other examples, the hematopoietic cells are allogeneic to the patient.

[0015] In some embodiments, the hematopoietic cell may comprise a nucleic acid or a set of nucleic acids, e.g., a DNA molecule or a set of DNA molecules, which collectively comprise (a) a first nucleotide sequence encoding a glucose import polypeptide, a Krebs cycle regulating polypeptide, and / or a lactate regulating polypeptide, and (b) a second nucleotide sequence encoding a chimeric antigen receptor polypeptide. In some examples, the hematopoietic cell may comprise a nucleic acid comprising both the first nucleotide sequence and the second nucleotide sequence. In some cases, the nucleic acid may comprise a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence. The third nucleotide sequence may encode a ribosome skipping site, an internal ribosome entry site (IRES), or a second promoter. In some examples, the third nucleotide sequence encodes a ribosome skipping site, e.g., a P2A peptide. In some examples, the nucleic acid or set of nucleic acids may be comprised within a vector or a set of vectors, e.g., an expression vector or a set of expression vectors. In certain examples, the vector or set of vectors comprises one or more viral vectors, e.g., lentiviral vectors or retroviral vectors.

[0016] In some embodiments, at least about 5×10 per kg 4 anti-GPC3-CAR T cells are administered to the patient. In some instances, about 5×104 ~Approx. 1×10 12 anti-GPC3-CAR T cells / kg will be administered to the patient.

[0017] In some embodiments, the therapeutic agent may comprise an anti-GPC3 targeting polypeptide or polypeptide fusion, such as an anti-GPC3 antibody, an anti-GPC3 bispecific or multispecific protein, or an anti-GPC3 antibody-drug conjugate.

[0018] In some embodiments, administration of the anti-GPC3 therapeutic agent is effective to achieve stable disease according to RECIST (e.g., RECIST 1.1) as measured by computed tomography (CT) scan. Alternatively, or in addition, administration of the anti-GPC3 therapeutic agent achieves an objective response according to RECIST (e.g., RECIST 1.1) as measured by computed tomography (CT) scan.

[0019] Any of the methods disclosed herein may further include administering at least one immunomodulatory agent to the patient, either in parallel or sequentially with the therapeutic agent. In some embodiments, the immunomodulatory agent may be an immune checkpoint inhibitor or an immune stimulating cytokine. Alternatively, or in addition, the method may further include subjecting the patient to a lymphodepletion therapy, which may include cyclophosphamide, fludarabine, or a combination thereof.

[0020] Also within the scope of the present disclosure are anti-GPC3 therapeutic agents (e.g., those disclosed herein) for use in treating patients diagnosed with myxoid / round cell liposarcoma, as well as the use of any of the anti-GPC3 therapeutic agents disclosed herein for the manufacture of a medicament for use in treating patients diagnosed with myxoid / round cell liposarcoma.

[0021] Additionally, the present disclosure also provides a method for diagnosing a patient with myxoid / round cell liposarcoma or for selecting a patient for treatment of the disease, the method comprising: (a) obtaining a tissue section from a tumor biopsy sample obtained from a potential patient, the section having a thickness of between 3 μm and 15 μm; (b) immunostaining by IHC, preferably with an antibody that specifically binds to GPC3, more specifically with the antibody GC33; (c) determining a cytoplasmic / membrane H-score; and (d) diagnosing the potential patient as having or suspected of having myxoid / round cell liposarcoma based on the H-score.

[0022] In some cases, an H-score of greater than 30 indicates the onset of disease. In some cases, patients with an H-score of greater than 30 may be selected for treatment.

[0023] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as the appended claims. [Brief description of the drawings]

[0024] [Figure 1] Images of FFPE tissue sections of myxoid / round cell liposarcoma stained by IHC with antibody GC33 are shown, with boxes indicating specific fields viewed at (A) 1x, (B) 4x, and (C) 20x magnification. Cells within each field were scored as 1+ for weak staining, 2+ for moderate staining, and 3+ for strong staining. In (C), examples of 1+, 2+, and 3+ are shown in boxes. [Diagram 2] Shown are images of IHC staining with antibody GC33 on healthy FFPE tissue sections of (A) breast, (B) heart, (C) stomach, and (D) kidney at 20x magnification. [Diagram 3]Shown are images of IHC staining with antibody GC33 on FFPE tissue sections of (A) smooth muscle (negative control; H score-0), various types of cancer, namely (B) hepatocellular carcinoma (H score-280), (C) non-small cell lung cancer (H score-260), (D) Merkel cell carcinoma (H score-130), and (E) liposarcoma (H score-120) at 20x magnification. All stained areas show both membranous and cytoplasmic GPC3 staining. [Figure 4] Shown are images of IHC staining with antibody GC33 on FFPE tissue sections of liposarcoma subtypes: (A) myxoid / round cell liposarcoma (H score-140), (B) pleomorphic liposarcoma (H score-0), (C) well-differentiated liposarcoma (H score-0), and (D) mixed liposarcoma (H score-0) at 20x magnification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention: [Table 1]

[0026] definition The terms "administration" and "treatment" as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell as well as contact of a reagent to a fluid (where the fluid is in contact with the cell). "Administration" and "treatment" also refer to in vitro and ex vivo treatments, such as in vitro and ex vivo treatments of a cell with a reagent, diagnostic, binding compound, or another cell. As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural history of the individual or cell being treated, and can occur before or during the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of a disease or a condition or symptom thereof, delaying the onset of a disease or condition, alleviating a condition or symptom of a disease, reducing any direct or indirect pathological consequences of a disease, reducing the rate of disease progression, improving or alleviating a disease condition, and achieving remission or improved prognosis.

[0027] The term "antibodies" or "antibodies", also referred to as "immunoglobulins" (Ig), generally comprises four polypeptide chains, two heavy (H) and two light (L) chains, and thus is a multimeric protein, or includes their equivalent Ig homologues (e.g., camelid antibodies that comprise only heavy chains, single domain antibodies (sdAbs) or nanobodies that can be derived from either the heavy or light chains). The term "antibody" includes antibody-based binding proteins, modified antibody formats that retain target binding ability. The term "antibody" also includes full-length functional mutants, variants, or derivatives thereof (including, but not limited to, murine, chimeric, humanized, and fully human antibodies) that retain the essential epitope binding characteristics of the Ig molecule and include dual specific, bispecific, multispecific, and dual variable domain Igs. Ig molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) and allotype. Ig molecules can also be mutated, for example, to enhance or decrease affinity for Fcγ receptors or neonatal Fc receptors (FcRn).

[0028] As used herein, the term "antibody fragment" refers to a molecule that contains at least one polypeptide chain derived from an antibody that is not full-length and exhibits target binding. Antibody fragments are capable of binding to the same epitope or target as the corresponding full-length antibody. Antibody fragments include: (i) Fab fragments, which are monovalent fragments consisting of a variable light (VL) domain, a variable heavy (VH) domain, a constant light (CL) domain, and a constant heavy 1 (CH1) domain; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region (reduction of the F(ab')2 fragments generates two Fab' fragments with free sulfhydryl groups); (iii) the heavy chain portion of the Fab (Fa) fragment, which consists of the VH and CH1 domains; (iv) variable fragment (Fv) fragments, which consist of the VL and VH domains of a single arm of an antibody; (v) domain antibody (dAb) fragments, which contain a single variable domain; (vi) isolated complementarity determining regions (CDRs); (vii) single chain Fv fragments. (viii) diabodies: bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but which are paired with complementary domains on another chain to generate two antigen-binding sites by using a linker that is too short to allow pairing between the two domains on the same chain; (ix) linear antibodies: (x) dual variable domain immunoglobulins, which comprise a pair of tandem Fv segments (VH-CH1-VH-CH1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding regions; and (xi) other non-full-length portions of immunoglobulin heavy and / or light chains, alone or in any combination, or mutants, variants, or derivatives thereof.

[0029] As used herein, the term "antibody-based binding protein" may refer to any protein that contains at least one antibody-derived VH, VL, or CH immunoglobulin domain in the context of other non-immunoglobulin or non-antibody derived components. Such antibody-based proteins include, but are not limited to, (i) Fc fusion proteins of binding proteins that include receptors or receptor components with all or part of an immunoglobulin CH domain, (ii) binding proteins in which the VH and / or VL domains are attached to alternative molecular scaffolds, or (iii) molecules in which immunoglobulin VH, and / or VL, and / or CH domains are combined and / or assembled in a manner not normally found in naturally occurring antibodies or antibody fragments.

[0030] The term "antibody-drug conjugate" or "ADC" refers to an antibody or antibody fragment to which a toxin (or drug) is linked. In an ADC, the toxin is conjugated to the antibody or antibody fragment by a cleavable or non-cleavable linker.

[0031] The term "anti-GPC3 therapeutic agent" refers to a therapeutic agent that targets GPC3. Desirable or beneficial effects include (a) inhibition of further growth or spread of cancer cells, (b) killing of cancer cells, (c) inhibition of cancer recurrence, (d) alleviation, reduction, alleviation, or inhibition of cancer-related symptoms (such as pain), or reduction in the frequency of symptoms, and (e) improvement of patient survival. Targeted therapeutic agents include binding moieties that specifically bind to GPC3 antigens expressed on tumor cells. Some non-limiting examples of anti-GPC3 therapeutic agents include genetically modified cells with chimeric antigen receptor polypeptides, anti-GPC3 antibodies, and / or antibody-drug conjugates.

[0032] The term "auxiliary diagnosis" is used herein to refer to a method that aids in making a clinical decision regarding the presence, degree, or other nature of a particular type of symptom or condition of cancer, such as LPS or non-LPS. Diagnosis of cancer, such as LPS, or its subtype, such as MRCLS, may be made according to any protocol that one of skill in the art would use. The term "bright-field type image" or "virtual stain image" (VSI) refers to an image of a biological sample that simulates that of an image obtained from a bright-field staining protocol. The image has similar contrast, intensity, and color scheme as a bright-field image. This allows features within the biological sample to be characterized, including but not limited to features of the nucleus, epithelium, stroma, or any type of extracellular matrix material, as if a bright-field staining protocol had been used directly on the biological sample.

[0033] The terms "cancer," "cancerous," "tumor," or "malignant" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, gastrointestinal (ductal) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, prostate cancer, thyroid cancer, melanoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bladder cancer, and sarcoma.

[0034] The term "chemical agent" may include one or more chemicals capable of modifying the fluorophore or the cleavable linker (if present) between the fluorophore and the binding agent. The chemical agent may be in contact with the fluorophore in the form of a solid, solution, gel, or suspension. Suitable chemical agents useful for modifying the signal include agents that modify pH (e.g., acids or bases), electron donors (e.g., nucleophiles), electron acceptors (e.g., electrophiles), oxidizing agents, reducing agents, or combinations thereof.

[0035] The term "chimeric antigen receptor" or "CAR" refers to an artificial antigen receptor engineered to be expressed in immune effector cells, specifically bind to cell surface antigens, and activate such immune effector cells with one or more signaling molecules. If the immune effector cells are T cells, activation can result in cell killing, proliferation, and / or cytokine production (Jena et al., 2010). CARs can be used as a therapy involving adoptive cell transfer. Hematopoietic cells, e.g., PBMCs, are removed from the patient and engineered to express the CAR. CARs can be expressed specifically for tumor-associated antigens mediated by an extracellular antigen-binding domain, e.g., scFv, where recognition is independent of human leukocyte antigen (HLA) presentation, or engineered T cell receptors that still recognize HLA-presented peptides (Zhang & Wang, 2019). In the present invention, narrowly defined CARs with an antigen-binding domain (e.g., single chain variable fragment (scFv)) that can bind to tumor-associated antigens independent of HLA presentation are preferred. CARs further comprise an intracellular activation domain, a transmembrane domain, and optionally a hinge domain. The specificity of the CAR can be derived from the ligand of the receptor (e.g., a peptide).

[0036] The term "cleavable linker" may be designed to be cleaved extracellularly in the tumor environment or intracellularly in the lysosome. Cleavable linkers take advantage of different conditions of reducing power or enzymatic degradation that may be present either outside or inside the target cell. In some embodiments of the antibody-drug conjugates, the cleavable linker may be a dipeptide (e.g., valine-citrulline and alanine-alanine).

[0037] The term "fluorescent marker" refers to a fluorophore that selectively stains a particular subcellular compartment. Examples of suitable fluorescent markers (and, where applicable, their target cells, subcellular compartments, or cellular components) are well known in the art.

[0038] The term "fluorophore" refers to a compound that emits light (at a different wavelength) when excited by exposure to light of a particular wavelength. The terms "fluorescence", "fluorescence", or "fluorescent signal" all refer to the emission of light by an excited fluorophore. Fluorophores may be described in terms of their emission profile, or "color". For example, green fluorophores (e.g., Cy3, FITC, and Oregon Green) may be characterized by their emission in wavelengths generally ranging from 515 to 540 nanometers. Red fluorophores (e.g., Texas Red, Cy5, and tetramethylrhodamine) may be characterized by their emission in wavelengths generally ranging from 590 to 690 nanometers. Examples of fluorophores are well known in the art (WO2011 / 138462A1; (Giepmans et al., 2006; Zhang et al., 2002).

[0039] The term "binder" refers to a biological molecule that can bind to one or more targets in a biological sample. A binder can specifically bind to a target. Suitable binders can include one or more of natural or modified peptides, proteins (e.g., antibodies, affibodies, or aptamers), nucleic acids (e.g., polynucleotides, DNA, RNA, or aptamers), polysaccharides (e.g., lectins, sugars), lipids, enzymes, enzyme substrates or inhibitors, ligands, receptors, antigens, haptens, and the like. Suitable binders can be selected depending on the sample to be analyzed and the targets available for detection. For example, the target in the sample can include a ligand and the binder can include a receptor, or the target can include a receptor and the probe can include a ligand. Similarly, the target can include an antigen and the binder can include an antibody or antibody fragment, or vice versa.

[0040] The term "in situ" generally refers to events occurring in situ (e.g., an intact organ or tissue) or a representative segment of an organ or tissue. In some embodiments, in situ analysis of targets can be performed on cells from various sources, including organisms, organs, tissue samples, or cell cultures. In situ analysis provides contextual information that may be lost when targets are removed from their site of origin. Thus, in situ analysis of targets describes the analysis of probes bound to targets located within whole cells or tissue samples, regardless of whether the cell membrane is fully intact or partially intact when the probe bound to the target remains within the cell. Furthermore, the methods disclosed herein can be used to analyze targets in situ in fixed or unfixed cells or tissue samples.

[0041] The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition. For example, "diagnosis" may refer to the identification of a particular type of sarcoma. "Diagnosis" may also refer to the classification of a particular subtype of LPS.

[0042] The term "homology" refers to the sequence similarity between two polypeptide sequences when they are optimally aligned. If a position in both of the two compared sequences is occupied by the same amino acid monomer subunit, for example, if a position in the light chain CDR of two different Abs is occupied by alanine, then the two Abs are homologous at that position. The percentage of homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared, multiplied by 100. For example, if 8 out of 10 positions in two sequences are identical or homologous when the sequences are optimally aligned, the two sequences are 80% homologous. In general, the comparison is performed when the two sequences are aligned to give the maximum percentage homology. For example, the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match between the respective sequences over the entire length of each reference sequence.

[0043] As used herein, the term "monoclonal antibody" refers to a substantially homogeneous antibody population, i.e., the antibody molecules constituting the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of different antibodies with different amino acid sequences in their variable domains (especially their CDRs), often specific for different epitopes. The modified "monoclonal" refers to the character of the antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring production of the antibody by any particular method.

[0044] The terms "multispecific", "multispecific antigen-binding", and / or "multispecific molecule" are interchangeable. They comprise a first antigen-binding domain and a second antigen-binding domain, each of which binds to a different molecule, each of which is referred to as a target molecule. The target molecule may be an internalizing effector protein. As used herein, the term "simultaneous binding" means that, in the context of a multispecific antigen-binding molecule, the multispecific antigen-binding molecule contacts both the target molecule (T) and the internalizing effector protein (E) under physiologically relevant conditions for at least a period of time to promote physical binding between T and E. The binding of the multispecific antigen-binding molecule to the T and E components may be sequential. For example, the multispecific EP 3,722,318 A1 antigen-binding molecule may first bind to T and then to E, or first bind to E and then to T. In any event, as long as both T and E are bound by the multispecific antigen-binding molecule for a period of time (regardless of the order of binding), the multispecific antigen-binding molecule is considered to "bind simultaneously" to T and E for the purposes of this disclosure. Without being bound by theory, it is believed that the enhanced inactivation of T is caused by the internalization and degradative rerouting of T within the cell due to physical binding to E. Thus, the multispecific antigen-binding molecules of the present invention are useful for inactivating and / or reducing the activity and / or extracellular concentration of a target molecule without directly blocking or antagonizing the function of the target molecule. A multispecific molecule can be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with each other. As would be known to one of skill in the art, a multispecific molecule or any of its variants can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques).

[0045] The term "modified antibody format" as used herein encompasses antibody-drug conjugates (ADCs), polyalkylene oxide modified scFvs, monobodies, diabodies, camelid antibodies, domain antibodies, bi-, tri-, or multispecific antibodies, IgA or two IgG structures joined by a J chain and secretory component, shark antibodies, New World monkey framework and non-New World monkey CDRs, an IgG4 antibody with the hinge region removed, an IgG with two additional binding sites incorporated into the CH3 domain, antibodies with Fc regions altered to enhance or reduce affinity for the Fc gamma receptor, dimerized constructs comprising CH3, VL, and VH, and the like.

[0046] The term "non-cleavable linker" refers to a linker that requires the ADC to be internalized, and the antibody-linker component needs to be degraded by lysosomal proteases for the toxin to be released. The conjugation of the linker to the antibody can also vary. Conjugation can depend on the presence of lysine and cysteine ​​residues in the polypeptide structure of the antibody as the point of conjugation. The reactive group on the linker can be conjugated to the side chain of a lysine residue, for example, via the formation of an amide or amidine bond. Conjugation via a cysteine ​​residue requires partial reduction of the antibody. Alternatively, site-specific enzymatic conjugation can be used. This requires enzymes that can react with the antibody and induce site- or amino acid sequence-specific modifications. Peptide sequences recognized by these enzymes may have to be inserted into the engineered antibody or fragment to be conjugated. Enzymes that have been used for such purposes are sortases, transglutaminases, galactosyltransferases, sialyltransferases, and tubulin tyrosine ligases. A review of ADC linker conjugation and toxins can be found in (Ponziani et al., 2020). A review of toxin conjugation to antibody fragments can be found in (Aguiar et al., 2018). The type of linker used to conjugate the toxin to the antibody or antibody fragment and the method of conjugation can determine the drug-to-antibody ratio (DAR).

[0047] As used herein, the term "oligonucleotide" refers to a short, single-stranded polynucleotide that is at least about 7 nucleotides long and less than about 250 nucleotides long. Oligonucleotides may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides.

[0048] The term "pharmaceutical acceptable" refers to molecular entities and other components of such compositions that are physiologically tolerated and typically do not cause adverse reactions when administered to a mammal (e.g., human). Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, particularly humans. "Acceptable" means that the carrier is compatible with the active components of the composition (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) and does not adversely affect the subject to whom the composition is administered. Any of the pharmaceutical compositions used in the present methods may include pharmaceutical acceptable carriers, excipients, or stabilizers in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphate, citric acid, and other organic acids, antioxidants (including ascorbic acid and methionine), preservatives, low molecular weight polypeptides, proteins (such as serum albumin, gelatin, immunoglobulins, etc.), amino acids, hydrophobic polymers, monosaccharides, disaccharides, and other carbohydrates, metal complexes, and / or non-ionic surfactants.

[0049] The term "polynucleotide" or "nucleic acid" is used interchangeably herein and refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component as described in the art (see, for example, WO2013 / 148448).

[0050] The term "primary anti-GPC3 antibody" refers to an antibody that specifically binds to GPC3 (e.g., GC33) in tissue sections and is generally the first antibody used in immunostaining assays (e.g., immunohistochemistry and immunofluorescence) of GPC3 expression in tumor samples.

[0051] As used herein, the term "sample" refers to a composition obtained or derived from a patient, including cells and / or other molecular entities that are characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological properties.

[0052] The term "secondary antibody" refers to an antibody that specifically binds to a primary anti-GPC3 antibody, thereby forming a bridge between the primary antibody and a subsequent detection reagent (if present) in an immunostaining assay for GPC3 expression (e.g., IHC and IF, or in situ hybridization).

[0053] The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, cynomolgus monkey, and human). "Patient" or "subject" refers to any single subject for whom therapy is desired or who participates in a clinical trial, epidemiological study, or is used as a control, including humans, as well as mammalian veterinary patients, such as mice, rats, and cynomolgus monkeys. As used herein, the term "patient" refers to a human or non-human animal. Typically, the terms "subject," "individual," and "patient" may be used interchangeably herein with respect to a subject. Thus, a "patient" includes a human or non-human mammal undergoing treatment and / or diagnosis of a disease, such as cancer.

[0054] The term "tissue sample" refers to a collection of similar cells obtained from a subject's tissue. The source of the tissue sample can be solid tissue from fresh, frozen, and / or preserved tissue samples. A tissue sample can also be primary or cultured cells or cell lines taken from an individual and / or derived from an individual. A tissue sample can contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. A tissue sample can also be a fluid isolated from a subject. In non-limiting aspects, examples of such samples include plasma, serum, spinal fluid, lymphatic fluid, whole blood or any blood fraction, blood products, blood cells, tumors, any sample obtained by lavage (e.g., a sample derived from the bronchus), and samples of components that constitute cell cultures in vitro.

[0055] The term "therapeutic agent" refers to a chemical compound or biological molecule useful in the treatment of cancer. Classes of therapeutic agents include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, and antibodies and fusion proteins that block ligand / receptor signaling in any biological pathway that supports tumor maintenance and / or growth. Therapeutic agents useful in the treatment methods of the invention include cytostatic agents, cytotoxic agents, antibody-drug conjugates, chimeric antigen receptor polypeptides, and immunotherapeutic agents.

[0056] The term "therapeutically effective amount" refers to an amount of a therapeutic agent effective to "treat" cancer in a subject or mammal by achieving at least one positive therapeutic effect, such as, for example, a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell invasion into peripheral organs, and a reduction in the rate of tumor metastasis or tumor growth. A positive therapeutic effect in cancer can be measured in several ways (see Weber, 2009).

[0057] The term "tissue section" refers to a single portion or part of a tissue sample, for example a thin slice of tissue cut from a normal tissue or tumor sample.

[0058] The term "toxin" refers to cytotoxic and / or cytostatic agents that may be based on synthetic, plant, fungal, or bacterial molecules. Cytotoxic or cytostatic means that they inhibit the proliferation of cells, especially malignant cells, typically due to their increased metabolic turnover, and / or inhibit the replication of cells and / or kill cells.

[0059] The term "treat" or "treating" refers to the administration, either internally or externally, of a composition containing a therapeutic agent (e.g., any of the antibodies or antigen-binding fragments of the present invention) to a subject or patient having or suspected of having one or more disease symptoms for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population by inducing regression of such symptoms or inhibiting progression to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") may vary depending on factors such as the disease state, age, and weight of the patient, as well as the ability of the drug to elicit a desired response in the subject. Whether a disease symptom is alleviated can be assessed by any clinical measurement typically used by a physician or other skilled health care provider to assess the severity or progression of the condition.

[0060] Detailed Description Sarcomas are a group of rare malignant tumors of mesenchymal origin that account for approximately 20% of all pediatric solid tumors and 1% of adult solid tumors (Abaricia & Hirbe, 2018; Hui, 2016). Sarcomas are broadly classified into (i) soft tissue sarcomas (STS) and (ii) osteosarcomas. STS have an incidence of approximately 3.4 cases per 100,000, with a median age of diagnosis of 59 years (according to the Surveillance, Epidemiology, and End Results Program of the National Cancer Institute, USA). Osteosarcoma is even rarer, accounting for approximately 0.2% of all cancer diagnoses (Hui, 2016). Liposarcoma (LPS) is a malignant tumor that arises from adipose tissue and affects fat differentiation. It is the most common type of STS, accounting for 17-25% of all newly diagnosed adult sarcomas (Dodd, 2012; Henze & Bauer, 2013; Singhi & Montgomery, 2011). The World Health Organization (WHO) classifies LPS into four histological subtypes: (i) atypical lipoma-like tumor (ALT) / well-differentiated LPS (WDLPS; 40–45% of all LPS; low grade, 5-year survival rate of 93%), (ii) dedifferentiated LPS (DDLPS; high grade, 5-year survival rate of 45%), (iii) myxoid LPS (MLPS; low grade, but 10% of patients develop metastases and 10-year survival rate of 60%) / round cell liposarcoma LPS (RCLPS; 30–35% of all LPS; high grade), myxoid / round cell liposarcoma LPS (MRCLS), and (iv) pleomorphic LPS (PLP); <15% of all LPS; high grade with poor prognosis). The fifth subtype, known as mixed LPS, consists of a histological combination of one or more subtypes. Both WDLPS and DDLPS are nowadays classified together as they share the same underlying genetic alterations and exhibit similar clinical features (Amer et al., 2020; Henze & Bauer, 2013; Jo & Fletcher, 2014).

[0061] Myxoid / round cell liposarcoma (MRCLS) account for approximately 30% of LPS and tend to occur in a slightly younger age group, with a peak incidence in the fourth decade. These tumors preferentially develop in the lower extremities within the thigh or popliteal cavity (75%), whereas they rarely develop in the retroperitoneum. Overall, local recurrence rates reported for MRCLS range from 15% to 30%. Some studies have reported a 20% to 40% risk of distant metastasis. Interestingly, MRCLS has an unusual metastatic pattern compared to DDL and other STS, with metastases to other soft tissue sites, intraperitoneal / retroperitoneal spaces or bone being more common (66%), and a lower rate of isolated pulmonary metastases (34%). Disease-specific mortality rates reported for MRCLS range from 12% to 30%. Cytogenetic and molecular analyses characterize MRCLS as a recurrent reciprocal translocation, t(12;16)(q13;p11), which results in FUS-DDIT3 gene fusion in more than 95% of cases. Therapies targeted at inhibiting these fusion proteins are being developed for the treatment of MRCLS (Lee et al., 2018).

[0062] Standard treatment for MRCLS includes surgical resection of localized primary disease. In patients with advanced or metastatic disease, MRCLS is known to be more sensitive to radiation therapy and cytotoxic chemotherapy compared to other LPS subtypes. However, despite adequate local treatment, approximately 40% of patients relapse. Chemotherapy is usually administered for advanced or unresectable disease. Typically, this includes doxorubicin, alone or in combination with ifosfamide, as first-line therapy, and trabectedin, as second-line therapy. Studies performed with doxorubicin-based regimens in MRCLS have shown an overall response rate of 45-50%. Trabectedin has proven to be highly active in MRCLS. Today, trabectedin is approved as a second-line therapy for STS and plays an important role in the so-called "histology-driven" medical therapy of STS. In MRCLS, trabectedin can obtain a response rate in the 50% range, as assessed by standard dimensional criteria, and a 6-month progression-free survival (PFS) in the 80% range. However, this is true for two drugs, doxorubicin and trabectedin. If the tumor is resistant to these drugs, there are currently no other medical options with significant activity in MRCLS patients (Regina & Hettmer, 2019; Sanfilippo et al., 2013). The tyrosine kinase inhibitors pazopanib and suntinib have been evaluated, but their use alone could not be warranted in the treatment of MRCLS. Recently, immunotherapy regimens using genetically modified T cells have been initiated for patients with MRCLS (see, e.g., ClinicalTrials.gov Identifier: NCT03450122 and ClinicalTrial.gov identifier NCT03399448) (Abaricia & Hirbe, 2018; Lee et al., 2018; Regina & Hettmer, 2019; Suarez-Kelly et al., 2019). Nevertheless, treatment options for patients with inoperable or metastatic MRCLS remain scarce, and new therapeutic options are needed for this high medical need indication.

[0063] Herein, it is reported that due to the complexity of staining patterns observed in various tumor tissue sections stained with anti-GPC3 antibody (GC33), new scoring rules were required to accurately and reliably quantify GPC3 expression levels across multiple tissue samples and pathologists performing the scoring. Also, when investigating the expression profile of GPC3 in solid tumor biopsies from cancer patients to better understand GPC3-positive tumor prevalence and prioritize indications for anti-GPC3 therapy, surprisingly, a subgroup of LPS (i.e., MRCLS) was identified as a relatively high GPC3 expression patient population, suggesting that this indication (i.e., MRCLS) should be prioritized and / or selected for treatment with anti-GPC3 therapeutic agents.

[0064] Glypican-3 (GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, and SGBS1) is an oncofetal tumor antigen that is an attractive target for anti-GPC3 therapy due to its highly restricted expression in normal tissues and high prevalence in several adult and pediatric solid tumors (Ho & Kim, 2011). Expression of GPC3 has been observed in various human cancers, such as ovarian, renal, colon, pancreatic, liver, and melanoma. Therefore, approaches that quantify and identify eligible patient subpopulations that would benefit from anti-GPC3 therapy are important for success.

[0065] The present disclosure is based on the surprising discovery that MRCLS as a niche subtype of LPS frequently shows increased positive GPC3 expression. The frequency of GPC3 expression in this subtype of LPS patients allows the selection of MRCLS patients with high unmet medical needs to be treated with anti-GPC3 therapeutic agents that are likely to provide clinical benefit. Thus, one aspect of the present disclosure features a method of treating a patient diagnosed with MRCLS, comprising administering an anti-glypican-3 (GPC3) therapeutic agent to the patient. Such patents may be identified by any of the diagnostic methods disclosed herein for detecting the presence of GPC3 in tumor tissue samples.

[0066] In one embodiment, patients are selected for treatment by diagnosing MRCLS. Liposarcoma has not been described for high GPC3 expression, nor has any subgroup of it been described. The inventors have surprisingly identified MRCLS as a patient group with relatively high GPC3 expression that allows such targeted GPC therapy to be successful, and use a scoring rule based on GC33 antibody to reliably and accurately quantify GPC3 expression across multiple tissue samples to find such GPC3 expression in tumors. Due to the relatively high positive rate of GPC3 expression in MRCLS patients, stratification of patients for GPC3 expression may not be required to achieve substantial response rates with anti-GPC3 therapeutic agents. MRCLS is generally diagnosed by imaging modalities such as CT or MRI, followed by histological evaluation, mainly after matoxylin & eosin staining on FFPE tissue sections of biopsy samples. Histologically, MRCLS is characterized as a multinodular mass composed of signet ring lipoblasts with a myxoid matrix of hyaluronic acid and low central cellularity and increased peripheral cellularity of spindle or round cells with a delicate plexiform capillary network. As these tumors lose differentiation, they develop areas of increased cellularity. Secondly, the majority of patients diagnosed with MRCLS have a reciprocal translocation t(12;16)(q13;p11) resulting in FUS-DDIT3(CHOP), so the diagnosis can be confirmed with high confidence using, for example, FISH for DDIT3(CHOP) on FFPE sections (Fritchie et al., 2012). Another non-limiting example for diagnosing MRCLS patients is by IHC staining for NY-ESO-1 (Hemminger & Iwenofu, 2013).

[0067] In the present invention, a preferred example of a biological sample used for detecting the expression level of GPC3 in tissue includes a preparation derived from a subject. The preparation derived from a subject is preferably a tissue obtained from a subject, more preferably a tissue of a patient with MRCLS. The expression level of GPC3 in a patient with MRCLS can be determined by immunostaining and / or in situ hybridization. In a preferred embodiment, a patient selected for treatment of MRCLS expresses GPC3. In a non-limiting aspect, the present invention can also provide a method for determining the effectiveness of an anti-GPC3 therapeutic agent, or a method for determining the continuation of an anti-GPC3 therapeutic agent from the concentration of free GPC3, as well as a method for determining the expression level of GPC3 detected in tissue by the method described below.

[0068] Methods of immunostaining and in situ hybridization are well known in the art (see Lu et al., 2021; Wang et al., 2018; Zhou et al., 2018; WO2006 / 006693; WO2009 / 116659; WO2013 / 148448; WO2014 / 165422; WO2014 / 097648). Any of the diagnostic assays may have common procedural steps.

[0069] I. Identification of MRCLS patients for treatment with anti-GPC3 therapeutic agents In some aspects, the present disclosure features a method for diagnosing patients with myxoid / round cell liposarcoma (MRCLS) suitable for treatment with any of the anti-GPC3 therapeutics (e.g., anti-GPC3 CAR-T cell therapy) disclosed herein. Briefly, a tumor biopsy sample can be taken from a candidate patient and examined for the presence of GPC3 and / or the level of GPC3 in the biopsy sample, for example, via an immunostaining assay. A tumor biopsy sample showing the presence of GPC3 or a particular level of GPC3 can be identified, and the patient from whom the biopsy sample is obtained can be identified as a patient suitable for treatment with an anti-GPC3 therapy (e.g., one disclosed herein). In some embodiments, a fixed tissue sample can be used in the diagnostic assays disclosed herein. Alternatively, a fluid sample can be used.

[0070] Furthermore, the level of GPC3 can also be used as a biomarker to assess the efficacy of any of the therapeutic methods disclosed herein. Thus, the present disclosure also provides a method for assessing the therapeutic efficacy of MRCLS patients undergoing or about to undergo treatment for MRCLS (e.g., any of the anti-GPC3 therapies disclosed herein). The level of GPC3 in a suitable biological sample from an MRCLS patient can be measured using any of the assay methods disclosed herein or known in the art. The efficacy of GPC3-targeted drug therapy for MRCLS patients disclosed herein can be determined before the patient starts anti-GPC3 therapeutic agents or before continuing anti-GPC3 therapy. For example, a physician can use, for example, the GPC3 expression score disclosed herein as a guide in determining how to treat a patient diagnosed with a type of MRCLS that is susceptible to treatment with an anti-GPC3 therapeutic agent. In some cases, a physician may use a diagnostic test in any of the methods disclosed above to determine GPC3 expression in a tumor tissue sample removed from a patient prior to the start of treatment with an anti-GPC3 therapeutic agent and / or other chemotherapeutic agent, but it is contemplated that a physician may prescribe a subsequent test at any time after an individual has been administered a first dose of an anti-GPC3 therapeutic agent.

[0071] A. Diagnostic Assays Using Fixed Tissue Samples (i) Sampling and preparation of tissue sections Tumor biopsies from MRCLS patients are used to prepare stained tissue sections for scoring GPC3 expression. Biopsies are typically taken from subjects before starting treatment with anti-GPC3 therapeutic agents. Additionally, biopsies can be taken during treatment to confirm GPC3 positivity or observe upregulation of its expression. Thus, tumor samples can be taken from subjects over a period of time. Tumor samples can be obtained by a variety of procedures, including but not limited to surgical resection, aspiration, or biopsy. In some embodiments, the tissue sample can be first fixed and then dehydrated with an ascending series of alcohols, infiltrated, and embedded in paraffin or other sectioning media so that the tissue sample can be sectioned. In alternative embodiments, the tissue sample can be sectioned and then fixed. In some embodiments, the tissue sample can be embedded in paraffin and processed. Neutral buffered formalin, glutaraldehyde, buine, or paraformaldehyde are non-limiting examples of fixatives. In a preferred embodiment, the tissue sample is fixed in formalin. In some embodiments, the fixed tissue sample is also embedded in paraffin to prepare a formalin-fixed and paraffin-embedded (FFPE) tissue sample. Examples of paraffin include, but are not limited to, Paraplast, Broloid, and Tissuemay.

[0072] It is understood that multiple sections of a single tissue sample may be prepared and analyzed according to the present invention. Each tissue section has a thickness of 3 μm to 15 μm, preferably 3 μm to 8 μm. In one embodiment, the tissue sections are obtained from a tumor biopsy sample, and the sections have a thickness of 3 μm to 15 μm. In one embodiment, an IHC assay of GPC3 expression in a tumor sample used FFPE fixed tissue sections with a thickness of 5 μm. In another embodiment, an ISH assay of GPC3 expression in a tumor sample used FFPE fixed tissue sections with a thickness of 6 μm. In some embodiments, the scoring process of the present invention is performed on FFPE tissue sections with a thickness of about 3 μm to 8 μm, preferably 5 μm, which are mounted and dried on a microscope slide.

[0073] (ii) Anti-GPC3 antibody used for immunostaining As used herein, the primary antibody is an anti-GPC3 antibody (mouse monoclonal AbGC33; catalog number 790-4564; Ventana) that was used for immunostaining in both IHC and IF. The GC33 antibody is specifically directed against the heparan sulfate proteoglycan GPC3. The anti-GPC3 antibody shows preferential binding to human GPC3 compared to other antigens, but this specificity does not require absolute binding specificity. An anti-hGPC3 antibody is considered specific for human GPC3 if its binding determines the presence of human GPC3 in a sample without causing undesirable results, such as false positives, in an IHC diagnostic assay. An antibody or binding fragment thereof useful as a primary antibody in the processes and methods of the present invention binds to human GPC3 with an affinity that is at least 2-fold, preferably at least 10-fold, more preferably at least 20-fold, and most preferably at least 100-fold higher than the affinity for any non-GPC3 protein. Tissue sections of tumor samples from human subjects can be scored for GPC3 expression using any anti-hGPC3 Ab that produces essentially the same staining results in FFPE or frozen tissue sections of tumor samples from humans as produced by the GC33 Ab.

[0074] Typically, an anti-GPC3 Ab or antigen-binding fragment useful for scoring human GPC3 expression by IHC assay will exhibit the same degree of specificity for human GPC3 as the GC33 antibody and retain at least 80%, 85%, 90%, 95%, or 100% of its binding affinity for human GPC3 when the affinity is expressed on a molar basis. It is also contemplated that an anti-GPC3 antibody or antigen-binding fragment useful in the present invention may contain conservative or non-conservative amino acid substitutions from GC33 Ab or GC33 that do not substantially alter its binding specificity or affinity.

[0075] (iii) Diagnostic testing of GPC3 expression by immunostaining The present invention has identified a niche subtype MRCLS within the LPS population with enhanced GPC3 expression. It further provides a process for scoring GPC3 expression in MRCLS tumor tissue sections that have been immunostained with anti-GPC3 antibodies in IHC or IF assays. In one embodiment, patients diagnosed with MRCLS are selected for treatment by immunostaining by IHC, preferably with an antibody that specifically binds to GPC3, more specifically with antibody GC33. The results of these scoring processes can be used to select patients for treatment with anti-GPC3 therapeutic agents.

[0076] An IHC or IF assay typically begins with antigen retrieval, which may differ in terms of reagents and methods. Examples of antigen retrieval processes are well known in the art (see, for example, Leong, 1996). In some embodiments, protease treatment is used for antigen retrieval. In a preferred embodiment, paraffin-embedded (FFPE) tissue sections are subjected to a heat-induced antigen retrieval process. Both IHC and IF can be used in direct or indirect assays. In a direct IHC or IF assay, the binding of an antibody to a target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody.

[0077] In one embodiment, the primary anti-GPC3 antibody, which preferably specifically binds to GPC3 in tissue sections, is GC33. This is generally the first antibody used in the immunostaining assay (e.g., IHC and IF) of GPC3 expression in tumor samples. In one embodiment, the primary antibody is the only antibody used in the IHC assay. If the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorescent substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with either different epitopes or the same epitope on the primary antibody. The secondary antibody specifically binds to the primary anti-GPC3 antibody, thereby forming a bridge between the primary antibody and the subsequent detection reagent, if present, in the immunostaining assay (e.g., IHC and IF or in situ hybridization) of GPC3 expression. In one embodiment, the secondary antibody is generally the second antibody used in the IHC assay of GPC3 expression in tumor samples.

[0078] The primary and / or secondary antibodies used in IHC or IF will typically be labeled with a detectable moiety. In some embodiments, the primary antibody is linked to a detectable label (e.g., paramagnetic ions, radioisotopes, fluorescent dyes, and NM detectables) and the slide is evaluated for GPC3 staining using an appropriate imaging device. In other embodiments, the immune complex between GPC3 and the primary antibody can be detected using a second binding agent linked to a detectable label. The second binding agent is preferably a secondary antibody, which is applied to the slide at a concentration and for a period sufficient to allow the formation of secondary immune complexes. The slide is then typically washed to remove any non-specifically bound secondary antibodies and detect the label in the secondary immune complexes. The secondary antibody can be labeled using avidin, streptavidin, or biotin, which are independently labeled with a detectable moiety such as a fluorescent dye (stain), a luminescent dye, or a non-fluorescent dye. Many labels are available, which can generally be categorized as (a) radioisotopes, (b) colloidal gold particles, and (c) fluorescent or chemiluminescent labels. Examples of detectable moieties are disclosed extensively in WO2013 / 148448. Some examples include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, phycoerythrin, phycocyanin, or commercially available fluorophores (e.g., SPECTRUM ORANGE® and SPECTRUM GREEN®, and / or derivatives of any one or more of the above).

[0079] A variety of enzyme-substrate labels are available, and US 4,275,149 provides a review of some of these. Enzymes generally catalyze a chemical change in a chromogenic substrate that can be measured using a variety of techniques. For example, enzymes can catalyze a color change in a substrate, which can be evaluated under a bright field microscope. In one embodiment, the expression of GPC3 in MRCLS is evaluated in an IHC chromogenic assay under a bright field microscope, preferably with a scanner (Lecia or Ventana, e.g., Ventana DP 200 scanner, Aperio AT2). In another embodiment, the expression of GPC3 in MRCLS is evaluated in an IF assay under a fluorescent microscope, preferably with a fluorescent scanner. Examples of fluorescent microscopes include, but are not limited to, inverted microscopes, compound microscopes, stereo microscopes, polarizing microscopes, preferably confocal microscopes (Leica), and scanning microscopes (Leica and Ventana, e.g., Ventana DP 200 scanner).

[0080] Alternatively, the enzyme may change the fluorescence or chemiluminescence of the substrate. Techniques for quantifying the change in fluorescence are described above. The chemiluminescent substrate can be electronically excited by a chemical reaction and then emit light that can be measured (e.g., using a chemiluminometer) or donate energy to a fluorescent acceptor. Non-limiting examples of enzyme labels include luciferase (e.g., firefly luciferase and bacterial luciferase; US4,737,456; WO2013 / 095896A1), luciferin, peroxidase (e.g., horseradish peroxidase (HRP)), alkaline phosphatase, β-galactosidase, lactoperoxidase, microperoxidase, and the like. In some embodiments, the label is indirectly conjugated to the antibody. Those skilled in the art will recognize various techniques for achieving this. For example, an antibody can be conjugated with biotin, any of the four broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Techniques for conjugating enzymes to antibodies are described in O'Sullivan & Marks, 1981.

[0081] Numerous enzyme-substrate combinations are available to those skilled in the art. For general reviews of these, see US 4,275,149 and US 4,318,980. Examples of enzyme-substrate combinations include: (i) horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate (hydrogen peroxidase oxidizes dye precursors such as 3,3'diaminobenzidine (DAB) which produces a brown end product, 3-amino-9-ethylcarbazole (AEC) which forms a rose end product upon oxidation, 4-chloro-l-napthol (CN) which precipitates as a blue end product, and p-phenylenediamine dihydrochloride / pyrocatecol, orthophenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB) which produce blue to black products); (ii) alkaline phosphatase (AP) and para-nitrophenyl phosphate, naphthol AS-MX phosphate, Fast Red TR and Fast Blue BB, naphthol AS-BI phosphate, naphthol AS-TR phosphate, 5-bromo-4-chloro-3-indoxyl phosphate (BCIP), Fast Red LB, Fast Garnet GBC, nitro blue tetrazolium (NBT), and iodonitrotetrazolium violet (INT); (iii) β-D-galactosidase (β-D-Gal) and a chromogenic substrate (e.g., p-nitrophenyl-PD-galactosidase) or a fluorogenic substrate (e.g., 4-methylumbelliferyl-PD-galactosidase).

[0082] Any method known in the art for conjugating antibody molecules to the various moieties may be used, including those described in David & Reisfeld, 1974; Nygren, 1982; Pain & Surolia, 1981.

[0083] (iv) Immunostaining scoring process After completing the staining process of the tissue sections of MRCLS patients, the slides are analyzed for GPC3 staining, either by a human (e.g., a pathologist) or by a computer programmed to distinguish between specific and non-specific staining results. This analysis can be performed by directly observing the slides through a microscope at low, medium (e.g., 10-20x), and high (e.g., 40-63x) magnifications, or by observing high-resolution images of the slides taken at low, medium, and high magnifications. Typically, low and medium magnifications are used for detection and overview of stained tumor cells. Typically, medium and high magnifications are used to examine individual tumor cells to estimate the number and intensity of viable cells that show at least partial localization of GPC3 to the cell membrane (apical and circumferential) and cytoplasmic staining. Canalicular staining patterns were also recorded. In a preferred embodiment, an H-score is assigned to each stained tissue section that is IHC assayed. The H-score involves (i) estimating four separate percentages of cells with no staining, weak staining (+1), moderate staining (+2), and strong staining (+3) across all viable GPC3 stained tumor cells in all sections examined, where a cell must have at least partial membrane and / or cytoplasmic staining to be included in the weak, moderate, or strong staining percentages, the sum of the four percentages equaling 100, and (ii) inputting the estimated percentages into the formula 1*(percentage of tumor cells with 1+ staining intensity)+2*(percentage of tumor cells with 2+ staining intensity)+3*(percentage of tumor cells with 1+ staining intensity) and assigning the result of the formula to the tissue section as an H-score. The H-score combines components of staining intensity and the percentage of positive cells, which range from 0 to 500, preferably 0 to 300. A cutoff of total (cytoplasmic and membrane) H-score >30 in tumor cells is used to determine the positive / negative status of specimens stained with the GPC3 (GC33) IHC assay.

[0084] Such staining procedures can be used to select patients by the presence of the target antigen GPC3 for treatment with GPC3 therapeutic agents. In one embodiment, MRCLS patients are selected by determining a cytoplasmic / membrane H-score. In some embodiments, the pre-defined threshold for GPC3 expression in MRCLS tissue samples is between 30 and 300. Thus, in a preferred embodiment, a method for treating patients diagnosed with MRCLS is by immunostaining, preferably immunohistochemistry (IHC) staining, preferably with a cytoplasmic / membrane H-score of greater than 30. In another embodiment, MRCLS patients are selected for treatment with an H-score of greater than 30.

[0085] In some embodiments, the individuals involved in the preparation and analysis of tissue sections by IHC assay do not know the identity of the subject whose sample is being tested, i.e., the sample received by the laboratory is made anonymous in some manner before being sent to the laboratory. For example, the sample may simply be identified by a number or some other code ("sample ID"), and the results of the IHC assay are reported to the party requesting the test using the sample ID. In a preferred embodiment, the association between the subject's identity and the subject's tissue sample is known only to the individual or the individual's physician.

[0086] In some embodiments, after the test results are obtained, the diagnostic laboratory generates a test report that may include any one or more of the following results: the tissue sample was positive or negative for GPC3 expression based on a threshold H score. The test report may also include guidance on how to interpret the results to predict whether the subject is likely to respond to an anti-GPC3 therapeutic agent. For example, in one embodiment, the patient's tumor is from an MRCLS, and if the H score is equal to or greater than the cutoff threshold, the test report may indicate that the patient has a GPC3 expression score that correlates with a response or better response to treatment with an anti-GPC3 therapeutic agent, whereas if the H score is less than the cutoff threshold, the test report may indicate that the patient has a GPC3 expression score that may correlate with no response or poor response to treatment with an anti-GPC3 therapeutic agent. In a preferred embodiment, a method of treating a patient diagnosed with MRCLS, wherein the selection comprises immunostaining, preferably immunohistochemical staining, of GPC3 in a tumor sample from the patient, the GPC3 expression level is determined and compared to a predefined threshold level of GPC3 expression, and if the patient has a GPC3 expression level equal to or greater than the predefined threshold level, the patient is selected for treatment.

[0087] (v) Diagnostic testing of GPC3 expression by in situ hybridization Another approach to assess the expression of GPC3 in MRCLS, either to assist or additionally complement the immunostaining assay, is by in situ hybridization assay. Sample specimen collection and tissue section preparation are similar to those disclosed above for immunostaining assay. The present invention generally includes embodiments relating to methods applicable for analytical, diagnostic, or prognostic applications, which individually and / or combine chromogenic or immunofluorescent detection with chromogenic or fluorescent-based nucleic acid analysis. The disclosed methods generally relate to the detection and correlation of different types of targets (i.e., proteins and / or nucleic acids) from a single biological sample. In some embodiments, methods are disclosed that use the same detection channel to detect multiple targets of the same type (i.e., proteins and / or nucleic acids, respectively). In such embodiments, correlations are drawn between multiple different types of targets.

[0088] In one embodiment, the target may comprise a nucleic acid and the binder may comprise a complementary nucleic acid. In some embodiments, both the target and the binder may comprise proteins capable of binding to each other. In some embodiments, the method of detecting multiple targets in a biological sample comprises sequentially detecting the targets in the biological sample. The method generally comprises detecting a first target in the biological sample, optionally modifying a signal from the first target, and detecting a second target in the biological sample. The method may further comprise repeating the steps of modifying the signal from the first or second target, followed by detecting a different target in the biological sample, and so on. Detailed methods for designing and performing in situ hybridization assays are well known in the art. It may be used with one long stretch of oligonucleotides or may include short stretches of oligonucleotides as probes (see WO2013 / 148448; (Wang et al., 2012)). Overall, the assay consists of: (a) Developing a target nucleic acid sequence, which is a sequence of interest contained in a nucleic acid molecule (e.g., GPC3) in a biological sample. The nucleic acid molecule may be present in the nucleus (e.g., chromosomal DNA) or in the cytoplasm (e.g., mRNA) of cells of the biological sample. In some embodiments, the nucleic acid molecule is not essentially present on the surface of the biological sample, and the biological sample may have to be treated to make the nucleic acid molecule accessible by the probe. In some embodiments, the analysis may provide information about the expression level of the GPC3 gene in the biological sample. In certain embodiments, the target nucleic acid sequence comprises a sequence that is part of the gene sequence that codes for GPC3. In other embodiments, the target nucleic acid sequence does not comprise a sequence that is part of the gene sequence that codes for GPC3. Thus, the target nucleic acid sequence may comprise a sequence that is part of the gene sequence that codes for a protein different from the target protein. (b) Probes - used to detect target nucleic acid sequences as defined above. It is desirable for the probe to specifically bind to a region of a nucleic acid molecule that contains a sequence of interest (e.g., GPC3). Thus, in some embodiments, the probe is GPC3 sequence specific. A sequence specific probe may comprise a nucleic acid, and the probe may be capable of recognizing a particular linear arrangement of nucleotides or derivatives thereof. In some embodiments, the linear arrangement may comprise consecutive nucleotides or derivatives thereof, each of which may bind to a corresponding complementary nucleotide in the probe. In alternative embodiments, the sequence may not be contiguous, as there may be one, two or more nucleotides that may not have a corresponding complementary residue on the probe. Suitable examples of probes include, but are not limited to, DNA or RNA oligonucleotides or polynucleotides, peptide nucleic acid (PNA) sequences, locked nucleic acid (LNA) sequences, or aptamers. In some embodiments, suitable probes may comprise nucleic acid analogs such as dioxygenin dCTP, biotin dCTP, 7-azaguanosine, azidothymidine, inosine, or uridine. In some embodiments, the probe may comprise a nucleic acid probe, a peptide nucleic acid probe, a locked nucleic acid probe, or an mRNA probe.

[0089] The length of the probe may determine the specificity of binding. In some embodiments, hybridization of a smaller probe may be more specific than that of a longer probe, since a longer probe may be more susceptible to mismatches and may continue to bind to the nucleic acid depending on the conditions. The probe may further comprise additional nucleic acid sequences (e.g., spacer, head, and / or tail sequences). In some embodiments, the probe may have a length ranging from about 4 nucleotides to about 12 nucleotides, about 12 nucleotides to about 25 nucleotides, about 25 nucleotides to about 50 nucleotides, about 50 nucleotides to about 100 nucleotides, about 100 nucleotides to about 250 nucleotides, about 250 nucleotides to about 500 nucleotides, or about 500 nucleotides to about 1000 nucleotides. In some embodiments, the probe may have a length ranging greater than about 1000 nucleotides. In one embodiment, the designed GPC3-targeting riboprobe is 1 to 1000 bp in length, preferably 300 to 700 bp. In another embodiment, the designed GPC3 targeting probe comprises multiple short aptamers of 1-100 bp in length, preferably 15-30 bp. In another embodiment, the designed GPC3 targeting probe further comprises an additional nucleic acid sequence.

[0090] The nucleic acid (e.g., mRNA) recovery process may then include treatments well known in the art (see (Chen et al., 2004; Leong, 1996; Patil et al., 2005; Wang et al., 2012)). Generally, indirect assays are used in ISH. In a typical indirect assay, sense or antisense nucleic acids may be labeled (e.g., for digoxigenin and FITC). Unconjugated primary probes and / or aptamers bind to the target nucleic acid sequence. The secondary antibody used in ISH will typically be labeled with a detectable moiety. The labeled secondary antibody binds to the primary probe, the secondary antibody is conjugated to an enzyme label, and a chromogenic or fluorogenic substrate is added to provide visualization of the antigen (e.g., HRP-conjugated anti-DIG). Numerous labels, enzyme substrates, and detection by microscope / scanner are available, and examples within immunostaining are listed and disclosed above. In some embodiments, the label is indirectly conjugated to the antibody. Those skilled in the art will be aware of various techniques for achieving this. In the case of a primary antibody, signal amplification occurs because several secondary antibodies may react with either different or the same epitopes. Alternatively, when multiple probes are used, each of the probes may be conjugated to a different fluorophore or enzyme, thereby allowing detection of the primary probe within the assay.

[0091] In certain embodiments, the biological sample may include a MRCLS tumor tissue sample that may be subjected to ISH using a probe. In some embodiments, the MRCLS tissue sample may be subjected to ISH in addition to immunofluorescence (IF) to obtain desired information about the tissue sample. In some embodiments, such nucleic acids (e.g., DNA) may be directly chemically labeled using appropriate chemistry for this purpose.

[0092] Methods for detection of nucleic acid sequences, such as hybridization, are well known. In certain embodiments, specific nucleic acid sequences are detected by FISH, polymerase chain reaction (PCR) (or variations of PCR, such as in situ PCR), RCA (rolling circle amplification) or PRINS (primed in situ labeling), as disclosed in detail in WO2013 / 148448. In an exemplary embodiment, specific nucleic acid sequences are detected by FISH. A preferred ISH assay uses the commercially available RNAscope fluorescent multiplex ACD Biotechnie™, as disclosed in Wang et al. (2012). Target nucleic acid sequences can be analyzed by their presence, absence, expression, or amplification levels. Protein expression data and nucleic acid analysis data can be further compared to provide a combined data set.

[0093] B. Diagnostic Assays Using Fluid Samples In the present invention, GPC3 expression analysis for MRCLS patients can be performed by various methods on fluid samples. In a non-limiting example, GPC3 concentration can be measured in serum or plasma isolated from MRCLS patients. In a preferred embodiment, GPC3 concentration is measured in serum or plasma isolated from patients diagnosed with MRCLS by ELISA. For example, human glypican-3 ELISA kit (BioMosaics Inc.) is used to quantify free GPC3 in whole serum. Examples of preferred methods for assaying free GPC3 include immunological methods using antibodies that can bind to epitopes present in GPC3, and are disclosed in WO2006 / 006693, WO2009 / 116659, WO2014 / 097648, (Hippo et al., 2004).

[0094] Further, in another non-limiting example, GPC3 concentration can be non-invasively assessed in liquid biopsies containing circulating tumor DNA (ctDNA) or cell-free DNA (cfDNA) or circulating RNA (ctRNA, e.g., microRNA), circulating tumor cells (CTCs), and / or extracellular vesicles (EVs, e.g., exosomes) (see Maravelia et al., 2021). In one embodiment, the circulating cells of the MRCLS patient, preferably tumor cells, can be obtained by non-invasive methods. In another embodiment, the circulating cells of the MRCLS patient are isolated based on density, preferably by Ficoll-paque. In another embodiment, the isolated circulating cells, preferably tumor cells, are stained with the anti-GPC3 antibody GC33. In a preferred embodiment, the isolated CTCs are stained with the anti-GPC3 antibody GC33, preferably by flow cytometry and / or immunostaining. In another embodiment, the CTCs are subjected to in situ hybridization with an anti-GPC3 probe. ctDNA or ctRNA can be isolated directly from circulating cells, for example, for CTCs, or from liquid biopsy samples of MRCLS patients. In one embodiment, the step of confirming the mutation or expression change (increase or decrease) of GPC3 in the biological sample isolated from the MRCLS patient is preferably by qRT-PCR, next-generation sequencing method, digital PCR, digital droplet PCR, etc. However, it is not limited to this, if it is a common method used to analyze the sequence of ctDNA or measure the amount of ctDNA. In another embodiment of the present invention, the method can provide information about MRCLS regarding the overall disease progression, such as diagnosis, recurrence, and advanced stage, which helps to determine further treatment with anti-GPC3 therapeutic agents. In addition, the present invention can include the steps of (a) extracting circulating tumor DNA (ctDNA) from a biological sample of an MRCLS patient to which an anti-GPC3 therapeutic agent is administered, (b) administering the anti-GPC3 therapeutic agent, and (c) extracting circulating tumor DNA (ctDNA) from the same MRCLS patient to which an anti-GPC3 therapeutic agent is administered.Examples of preferred methods for assaying CTCs or ctDNA are disclosed in WO20150 / 58079, WO2016 / 179530, WO2020 / 112566, (Cree et al., 2017, Ge et al., 2021, Ono et al., 2015, Yi et al., 2021).

[0095] Any of the detection agents (eg, anti-GPC3 antibodies) disclosed herein can also be used in diagnostic assays performed on fluid samples.

[0096] II. Treatment of MRCLS with anti-GPC3 therapeutic agents MRCLS has an intermediate risk, with approximately one-third of patients developing metastases and ultimately dying from their tumors. Another feature that distinguishes MRCLS from other types of liposarcoma and most other soft tissue sarcomas is their tendency to metastasize to other soft tissue sites, including the trunk and extremities, retroperitoneum, chest wall, pleura, and pericardium. Histologically, MRCLS resection specimens are classified as purely myxoid or myxoid with round cell component. Tumors within the purely myxoid subgroup exhibit a wide morphological spectrum in terms of cellularity and lipid differentiation. The round cell component is considered either as a well-circumscribed nodule or a gradual transition from the cellular areas of myxoid liposarcoma. The round cell component is defined as highly cellular areas with primitive round cells with increased nuclear-cytoplasmic ratios and usually prominent nucleoli. According to the Trojani grading system, pure myxoid liposarcoma is grade 2, whereas tumors with prominent round cell component are grade 3 (Haniball et al., 2011). MRCLS is associated with an unusual pattern of metastasis to bones of the spine and other soft tissues (e.g., retroperitoneum, extremities, and axilla), whereas other soft tissue sarcomas tend to metastasize to the lungs, whereas other sites are typically involved in advanced stages of the disease. Furthermore, extrapulmonary metastases have only been observed in a minority of MRCLS patients (Asano et al., 2012).

[0097] In some aspects, the present disclosure provides a method for treating MRCLS patients with anti-GPC3 therapeutic agents such as those provided herein. In some embodiments, the method for treating MRCLS patients with anti-GPC3 therapeutic agents may be staged as grade 1. In another embodiment, the method for treating MRCLS patients with anti-GPC3 therapeutic agents may be staged as grade 2. In another embodiment, the method for treating MRCLS patients with anti-GPC3 therapeutic agents may be staged as grade 3. Furthermore, in a preferred embodiment, the method for treating MRCLS patients with anti-GPC3 therapeutic agents may be staged as progressive and unresectable. In some examples, the anti-GPC3 therapeutic agent is an anti-GPC3 CAR-T cell disclosed herein. In some embodiments, patients who have received the anti-GPC3 therapy disclosed herein may be identified using any of the diagnostic methods disclosed herein.

[0098] In some embodiments, the physician may consider whether to treat the patient with a medicament indicated for patients with MRCLS tumors testing positive for GPC3 expression. In one embodiment, the therapeutic agent comprises an anti-GPC3 binding domain, preferably an anti-GPC3 antibody or a functional fragment thereof that retains binding to GPC3, and preferably the therapeutic agent comprises administration of an anti-GPC3 antibody, an anti-GPC3 antibody-drug conjugate, an anti-GPC3 antibody-radionuclide conjugate, or a fusion protein of an anti-GPC3 antibody or antibody derivative that binds to GPC3 with an anti-CD3 binding domain or an immunostimulatory polypeptide.

[0099] When determining how to use the GPC3 test results in the treatment of an individual patient, the physician may also take into account other relevant circumstances, such as the stage of MRCLS being treated, the patient's age, weight, sex, genetic background, race, etc., including inputting a combination of these factors and the test results into a model that guides the physician in selecting a therapy and / or treatment regimen with that therapy. Some non-limiting examples of anti-GPC3 therapeutic agents include chimeric antigen receptor polypeptides, antibody-drug conjugates, bispecifics and multispecifics. Anti-GPC3 therapeutic agents may be administered intravenously, intradermally, intraperitoneally, and / or encapsulated, preferably as an oral composition.

[0100] A. Anti-GPC3 therapeutic agents Any therapeutic agent that targets GPC3 can be used in the methods disclosed herein.In some embodiments, as disclosed herein, the anti-GPC3 therapeutic agent for use in treating MRCLS can be an anti-GPC3 antibody.Alternatively, the anti-GPC3 therapeutic agent can be hematopoietic cells, such as anti-GPC3 chimeric antigen receptor (CAR) and immune cells (e.g., T cells) that express such.

[0101] (i) Anti-GPC3 antibody In a non-limiting embodiment, examples of anti-GPC3 antibodies that can be used as anti-GPC3 therapeutic agents of the present invention include antibody-drug conjugates (ADCs) (WO2007 / 137170) including 1G12 antibodies (WO2003 / 100429) conjugated with cytotoxic toxins (sold by BioMosaics Inc. under catalog number B0134R). Other examples of anti-GPC3 ADCs are disclosed in WO2017 / 196764 and CN110577600. Antigen-binding molecules are conjugated to these compounds via suitable linkers, etc.

[0102] In an alternative non-limiting embodiment, examples of anti-GPC3 antibodies include humanized anti-GPC3 antibodies described in WO2006 / 006693, WO2009 / 041062, and WO2013 / 070468.

[0103] In a further alternative non-limiting aspect, examples of anti-GPC3 antibodies include bispecific antibodies, such as antibodies targeting GPC3 and ASGPR1 (WO2016 / 086813) and antibodies targeting GPC3 and CD40 (WO2020 / 230901).

[0104] (ii) anti-GPC3 CAR and genetically modified hematopoietic cells expressing it In other embodiments, the anti-GPC3 therapeutic agent disclosed herein may be an anti-GPC3 chimeric antigen receptor (CAR), and a hematopoietic cell, such as an immune cell (e.g., a T cell) expressing such an anti-GPC3 CAR, for example, as described in WO2015 / 172341, CN105949324, and WO2016 / 049459.

[0105] The CAR polypeptides described herein are used in cell-based immunotherapy. The CAR polypeptides described herein may comprise an extracellular domain comprising an scFv with binding affinity to GPC3, a transmembrane domain, and a CD3ζ cytoplasmic signaling domain. In some embodiments, the CAR polypeptides described herein may comprise, from N-terminus to C-terminus, an extracellular antigen binding domain, a transmembrane domain, optionally one or more costimulatory domains (e.g., CD28 costimulatory domain, 4-1BB costimulatory signaling domain, OX40 costimulatory signaling domain, CD27 costimulatory signaling domain, or ICOS costimulatory signaling domain; SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22), and a CD3ζ cytoplasmic signaling domain.

[0106] Alternatively or in addition, the CAR polypeptides described herein may contain two or more costimulatory signaling domains, which may be linked to each other or separated by a cytoplasmic signaling domain. The extracellular antigen binding domain, the transmembrane domain, the optional costimulatory signaling domain, and the cytoplasmic signaling domain in a CAR polypeptide may be linked to each other directly or via a peptide linker. In some embodiments, any of the CAR polypeptides described herein may include a signal sequence at the N-terminus.

[0107] In some examples, the modified hematopoietic cells may express a chimeric receptor polypeptide that binds to GPC3. Such an anti-GPC3 CAR may include (a) an extracellular target binding domain that binds to GPC3, (b) a transmembrane domain, and (c) a cytoplasmic signaling domain (e.g., a cytoplasmic domain that includes an immunoreceptor tyrosine-based activation motif (ITAM)). In some examples, (c) is located at the C-terminus of the chimeric receptor polypeptide. In some cases, the chimeric polypeptide may further include at least one costimulatory signaling domain. In other cases, the chimeric receptor polypeptide may not include a costimulatory signaling domain. In other cases, the CAR polypeptide may not include a costimulatory signaling domain. Any of the CAR polypeptides described herein may further include a hinge domain located at the C-terminus of (a) and the N-terminus of (b). In other examples, the chimeric receptor polypeptide may not include any hinge domain. In one embodiment, the extracellular antigen-binding domain is a single chain antibody fragment (scFv) that binds to GPC3, preferably the scFv is derived from the GC33 antibody. In some examples, the scFv has the sequence of SEQ ID NO: 2. In some embodiments, the (b) transmembrane domain in the CAR can be a single-pass transmembrane protein, such as CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16A, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, and FGFR2B (SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, respectively). CD16A is the polymorphic variant CD16 158F (SEQ ID NO: 16 and SEQ ID NO: 17) and CD16 158V(SEQ ID NO:18 and SEQ ID NO:18) (Arriga et al., 2020). Alternatively, the transmembrane domain of (b) can be a non-naturally occurring hydrophobic protein segment. In some embodiments, at least one costimulatory signaling domain of the CAR polypeptide described herein is a co-stimulatory signaling domain of, for example, 4-1BB, CD28 ... LL→GG Variants may be of the costimulatory molecules OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, and CD2 (SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, respectively).

[0108] In some examples, at least one costimulatory signaling domain is a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain. In some cases, one of the costimulatory signaling domains is a CD28 costimulatory signaling domain, and the other costimulatory domain can be a 4-1BB costimulatory signaling domain, an OX40 costimulatory signaling domain, a CD27 costimulatory signaling domain, or an ICOS costimulatory signaling domain. Specific examples include CD28 and 4-1BB, or CD28 LL→GG The chimeric receptor polypeptides include, but are not limited to, variants and 4-1BB. Alternatively, any of the chimeric receptor polypeptides may not include any costimulatory signaling domain. In some embodiments, the CAR polypeptide may further include (i) a CD28 costimulatory domain (SEQ ID NO: 6) in combination with a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof (SEQ ID NO: 4), or (ii) a 4-1BB costimulatory domain in combination with a CD8 transmembrane domain, a CD8 hinge domain, or a combination thereof (SEQ ID NO: 3), preferably SEQ ID NO: 5, and more preferably, the CAR polypeptide comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

[0109] In one embodiment, one or more costimulatory signaling domains, one of which may be a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain. The CAR polypeptide is configured such that, when expressed on a host cell, the extracellular antigen binding domain is located outside the cell to bind to a target molecule, and the CD3ζ cytoplasmic signaling domain is located inside the cell to signal into the cell. The costimulatory signaling domain may be located in the cytoplasm to trigger activation and / or effector signaling. In some embodiments, the cytoplasmic signaling domain of (c) in any of the CAR polypeptides described herein may be the cytoplasmic domain of CD3ζ (SEQ ID NO: 7) or FcεR1γ.

[0110] In some embodiments, the hinge domain of the CAR polypeptide described herein can be that of CD28, CD16A, CD8α, or IgG, as applicable. In other examples, the hinge domain is a non-naturally occurring peptide. For example, the non-naturally occurring peptide is an extended recombinant polypeptide (XTEN) or (Gly4Ser) n It may be a polypeptide, where n is an integer between 3 and 12, inclusive. In some examples, the hinge domain is a short segment that may contain up to 60 amino acid residues.

[0111] In particular examples, the CAR polypeptides described herein can comprise (i) a CD28 costimulatory domain or a 4-1BB costimulatory domain, and (ii) a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof. In further particular examples, the CAR polypeptides described herein can comprise (i) a CD28 costimulatory domain or a 4-1BB costimulatory domain, (ii) a CD8 transmembrane domain, a CD8 hinge domain, or a combination thereof. [Table 2]

[0112] For example, a CAR polypeptide can comprise an amino acid sequence selected from SEQ ID NO: 8 and SEQ ID NO: 9. Exemplary CAR polypeptide amino acid sequences are provided in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0113] In some embodiments, hematopoietic cells expressing anti-GPC3 CARs may further express or overexpress factors (e.g., exogenous factors) that affect glucose metabolism in hematopoietic cells, such as immune cells (see WO2020 / 0370666, WO2020 / 097346, and WO2020 / 010110, the relevant disclosures of each of which are incorporated herein by reference for the subject matter and purposes referenced herein). Such factors can be used to divert or redirect glucose metabolites from the glycolytic pathway in hematopoietic cells, such as immune cells.

[0114] In some embodiments, redirection of glucose metabolites from the glycolytic pathway can be achieved by expressing (e.g., overexpressing) one or more factors (e.g., proteins or nucleic acids) such as those described herein in hematopoietic cells (e.g., T cells or natural killer cells). Such engineered hematopoietic cells are expected to have enhanced metabolic activity, e.g., in low glucose, low amino acid, low pH, and / or low oxygen environments (e.g., tumor microenvironment), compared to native hematopoietic cells of the same type. Thus, hematopoietic cells, such as HSCs or immune cells, that co-express one or more factors (e.g., polypeptides or nucleic acids) that redirect glucose metabolites from the glycolytic pathway in hematopoietic cells and chimeric receptor polypeptides will exhibit superior biological activity, e.g., cell proliferation, activation (e.g., increased cytokine production, e.g., production of IL-2 or IFNγ), cytotoxicity, and / or in vivo anti-tumor activity, in the presence of a CAR (e.g., under low glucose, low amino acid, low pH, and / or low oxygen conditions).

[0115] Thus, provided herein are modified (e.g., genetically modified) hematopoietic cells (e.g., hematopoietic stem cells, immune cells such as, for example, T cells or natural killer cells) that have a regulated Krebs cycle, particularly, for example, in low glucose, low amino acid, low pH, and / or hypoxic conditions, as compared to native hematopoietic cells of the same type. The modified hematopoietic cells may express or overexpress a Krebs cycle regulating polypeptide. In some embodiments, the Krebs cycle regulating polypeptide may be an enzyme that catalyzes a reaction of the Krebs cycle. Examples include, but are not limited to, isocitrate dehydrogenase (IDH), such as IDH1 or IDH2, malate dehydrogenase (MDH), such as MDH1 or MDH2, or phosphoglycerate dehydrogenase (PHGDH). In other embodiments, the Krebs cycle regulating polypeptide is an enzyme that uses a Krebs cycle metabolite as a substrate. Examples include, but are not limited to, glutamate-oxaloacetate transaminase (GOT) (also known as aspartate transaminase or aspartate aminotransferase), such as GOT1 (e.g., SEQ ID NO: 13) or GOT2 (e.g., SEQ ID NO: 12), or phosphoenolpyruvate carboxykinase 1 (PCK1). In yet other embodiments, the Krebs cycle regulating polypeptide is an enzyme that converts a precursor to a Krebs cycle metabolite. Examples include, but are not limited to, phosphoserine aminotransferase (PSAT1), glutamate dehydrogenase (GDH1), glutamate-pyruvate transaminase 1 (GPT1), or glutaminase (GLS). In a particular example, the polypeptide that redirects glucose metabolites from the glycolysis pathway used in any of the modified hematopoietic cells, such as immune cells, can be GOT2, as previously disclosed in WO2020 / 037066.Other embodiments may be modified hematopoietic cells such as immune cells having glucose importing polypeptides such as glucose transporters (GLUT1, GLUT3) as disclosed in WO2020 / 010110, or lactate modulators such as monocarboxylate transporters (MCT1, MCT2, MCT4) as disclosed in WO2020 / 051493.

[0116] In some instances, hematopoietic cells co-expressing an anti-GPC3 CAR and a factor that affects glucose metabolism may have improved glucose uptake activity compared to wild-type hematopoietic cells of the same type. In some instances, hematopoietic cells may exogenously express a glucose import polypeptide, such as a glucose transporter (GLUT) or a sodium-glucose cotransporter (SGLT). Examples include, but are not limited to, GLUT1, GLUT3, GLUT1 S226D, SGLT1, SGLT2, GLUT8, GLUT8 L12A L13A, GLUT11, GLUT7, and GLUT4.

[0117] In other examples, hematopoietic cells that co-express anti-GPC3 CAR and a factor that affects glucose metabolism may have a regulated Krebs cycle compared to wild-type hematopoietic cells of the same type. In some cases, hematopoietic cells may exogenously express a Krebs cycle regulating polypeptide. In some examples, the Krebs cycle regulator may be an enzyme that catalyzes a reaction in the Krebs cycle. Examples include, but are not limited to, isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), or phosphoglycerate dehydrogenase (PHGDH). In other cases, the Krebs cycle regulating polypeptide may be an enzyme that uses a Krebs cycle metabolite as a substrate. Examples include, but are not limited to, glutamate-oxaloacetate transaminase (GOT) or phosphoenolpyruvate carboxykinase 1 (PCK1). In still other cases, the Krebs cycle regulating polypeptide may be an enzyme that converts a precursor to a Krebs cycle metabolite. Examples include, but are not limited to, phosphoserine aminotransferase (PSAT1), glutamate dehydrogenase (GDH1), glutamate-pyruvate transaminase 1 (GPT1), or glutaminase (GLS).

[0118] In yet another example, hematopoietic cells co-expressing anti-GPC3 CAR and a factor that affects glucose metabolism may have enhanced intracellular lactate concentration compared to wild-type hematopoietic cells of the same type. In some cases, hematopoietic cells may exogenously express lactate-regulating polypeptides. In some examples, the lactate-regulating polypeptide may be a monocarboxylate transporter (MCT), preferably MCT1, MCT2, or MCT4. In some examples, the lactate-regulating polypeptide may be an enzyme involved in lactate synthesis, such as lactate dehydrogenase A (LDHA). In yet another example, the lactate-regulating polypeptide may be a polypeptide that inhibits a pathway that competes with lactate synthesis substrates, such as pyruvate dehydrogenase kinase 1 (PDK1).

[0119] The Krebs cycle regulatory polypeptide may be a naturally occurring polypeptide from a mammalian Krebs cycle regulatory polypeptide, such as one derived from a suitable species (e.g., human or non-human primate). Such naturally occurring polypeptides are known in the art and can be obtained, for example, by using any of the above amino acid sequences as a query to search a publicly available gene database, such as GenBank. The Krebs cycle regulatory polypeptide used in the present disclosure may share at least 85% (e.g., 90%, 95%, 97%, 98%, 99% or more) sequence identity with any of the exemplary proteins GOT1 (SEQ ID NO: 13) and GOT2 (SEQ ID NO: 12), preferably GOT2 (SEQ ID NO: 12).

[0120] To construct hematopoietic cells expressing anti-GPC3 CAR and optionally any of the glucose import polypeptides described herein, expression vectors for stable or transient expression of glucose import polypeptides and / or chimeric receptor polypeptides can be made through conventional methods described herein and introduced into immune host cells. For example, the nucleic acid encoding the glucose import polypeptide and / or chimeric receptor polypeptide can be cloned into one or two suitable expression vectors, such as a viral vector operably linked to a suitable promoter. In some cases, the coding sequences of the chimeric receptor polypeptide and the glucose import polypeptide are on two separate nucleic acid molecules and can be cloned into two separate vectors, which can be introduced simultaneously or sequentially into a suitable host cell.

[0121] Alternatively, the coding sequences of the chimeric receptor polypeptide and the glucose importer polypeptide can be on one nucleic acid molecule and cloned into one vector. The coding sequences of the chimeric receptor polypeptide and the glucose importer polypeptide can be operably linked to two different promoters so that the expression of the two polypeptides is controlled by different promoters. Alternatively, the coding sequences of the chimeric receptor polypeptide and the glucose importer polypeptide can be operably linked to one promoter so that the expression of the two polypeptides is controlled by a single promoter. A suitable sequence can be inserted between the coding sequences of the two polypeptides so that the two separate polypeptides can be translated from a single mRNA molecule. Such sequences, for example, IRES or ribosomal skipping sites, are well known in the art.

[0122] The nucleic acid and vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and be ligated with a ligase. Alternatively, a synthetic nucleic acid linker can be ligated to the ends of the nucleic acid encoding the glucose importer polypeptide and / or the chimeric receptor polypeptide. The synthetic linker can contain a nucleic acid sequence that corresponds to a specific restriction site in the vector. The choice of expression vector / plasmid / viral vector will depend on the type of host cell for expression of the glucose importer polypeptide and / or the chimeric receptor polypeptide, but must be suitable for integration and replication in eukaryotic cells.

[0123] Various promoters can be used for the expression of the glucose importer polypeptide and / or chimeric receptor polypeptide described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTR (e.g., Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR), simian virus 40 (SV40) early promoter, human EF1-alpha promoter, or herpes simplex tk virus promoter. Additional promoters for the expression of glucose importer polypeptide and / or chimeric receptor polypeptide include any constitutively active promoter in hematopoietic cells. Alternatively, any regulatable promoter can be used so that its expression can be regulated in hematopoietic cells.

[0124] In addition, the vector may contain some or all of the following: a selectable marker gene, such as a neomycin gene or a kanamycin gene for selection of stable or transient transfectants in a host cell; an enhancer / promoter sequence from the immediate early gene of human CMV for high level transcription; an intron sequence from the human EF1-α gene; a transcription termination signal and an RNA processing signal from SV40 for mRNA stability; an SV40 polyomavirus origin of replication and Co1E1 for proper episomal replication; an internal ribosome binding site (IRESe); a versatile multiple cloning site; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a "suicide switch" or "suicide gene" that, when activated, kills cells harboring the vector (e.g., HSV thymidine kinase or inducible caspase, such as iCasp9); and a reporter gene for assessing expression of the glucose import polypeptide and / or the chimeric receptor polypeptide.

[0125] Suitable vectors and methods for generating vectors containing transgenes are known and available in the art. Examples of the preparation of vectors for the expression of glucose importer polypeptides and / or chimeric receptor polypeptides can be found, for example, in US2014 / 0106449, which is incorporated herein by reference in its entirety. Any of the vectors containing a nucleic acid sequence encoding a glucose importer polypeptide and / or chimeric receptor polypeptide described herein are within the scope of the present disclosure. Such vectors or the sequences encoding glucose importer polypeptides and / or chimeric receptor polypeptides contained therein can be delivered to a host cell, such as a host hematopoietic cell, by any suitable method. Methods for delivering vectors to hematopoietic cells are well known in the art and can include DNA electroporation, RNA electroporation, transfection using reagents such as liposomes, or viral transduction (e.g., retroviral transduction, such as lentiviral transduction).

[0126] In some embodiments, vectors for expression of the glucose import polypeptide and / or chimeric receptor polypeptide are delivered to the host cell by viral transduction (e.g., retroviral transduction, such as lentiviral transduction). Exemplary methods for viral delivery include, but are not limited to, recombinant retroviruses (e.g., WO90 / 07936, WO94 / 03622, WO93 / 25698, WO93 / 25234, WO93 / 11230, WO93 / 10218, and WO91 / 02805, US5,219,740, and US4,777,127, GB2,200,651, and EP0345242, alphavirus-based vectors, and adeno-associated virus (AAV) vectors (e.g., WO94 / 12649, WO93 / (See US Pat. No. 5,399,346, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655). In some embodiments, the vector for expression of the glucose import polypeptide and / or chimeric receptor polypeptide is a retrovirus. In some embodiments, the vector for expression of the glucose import polypeptide and / or chimeric receptor polypeptide is a lentivirus. Examples of references describing retroviral transduction include US Pat. No. 5,399,346, (Mann US 4,650,764, US 4,980,289, (Markowitz et al., 1988), US 5,124,263, WO 95 / 07358, (Kuo et al., 1993). WO 95 / 07358 describes highly efficient transduction of primary B lymphocytes. See also WO 2016 / 040441 A1 (hereby incorporated by reference for the purposes and subject matter referenced herein).

[0127] In examples where the vector encoding the glucose import polypeptide and / or the chimeric receptor polypeptide is introduced into the host cell using a viral vector, the hematopoietic cells can be infected and the viral particles carrying the vector can be produced by any method known in the art, for example, as can be found in WO1991 / 002805A2, WO1998 / 009271A1, and US6,194,191. The viral particles can be recovered from the cell culture supernatant and isolated and / or purified before contacting the viral particles with the hematopoietic cells. In other cases, the nucleic acid encoding the glucose import polypeptide and the nucleic acid encoding the chimeric receptor polypeptide can be cloned into the same expression vector. Polynucleotides for the expression of the chimeric receptor polypeptide and the glucose import polypeptide (including vectors in which such polynucleotides are operably linked to at least one regulatory element) are also within the scope of the present disclosure. Non-limiting examples of useful vectors of the present disclosure include viral vectors such as, for example, retroviral vectors (including gamma retroviral vectors), adeno-associated viral vectors (AAV vectors), and lentiviral vectors. In some cases, the nucleic acid encoding the glucose importer polypeptide and / or the chimeric receptor polypeptide can be delivered into the host cell via a transposon. In some cases, the encoding nucleic acid can be delivered into the host cell via gene editing, for example, by CRISPR, TALEN, ZFN, or meganuclease.

[0128] In some cases, the nucleic acid described herein may contain two coding sequences, one encoding a chimeric receptor polypeptide described herein, and the other encoding a polypeptide that can enhance glucose import (i.e., a glucose import polypeptide). The nucleic acid containing two coding sequences described herein may be configured such that the polypeptides encoded by the two coding sequences can be expressed as independent (and physically separate) polypeptides. To achieve this goal, the nucleic acid described herein may contain a third nucleotide sequence located between the first coding sequence and the second coding sequence. This third nucleotide sequence may, for example, encode a ribosome skipping site. A ribosome skipping site is a sequence that impairs normal peptide bond formation. This mechanism results in the translation of an additional open reading frame from one messenger RNA. This third nucleotide sequence may, for example, encode a P2A, T2A, or F2A peptide (see, for example, Kim et al., 2011). As a non-limiting example, an exemplary P2A peptide may have the amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 14). In another embodiment, the third nucleotide sequence may encode an internal ribosome entry site (IRES). An IRES is an RNA element that allows translation initiation in an end-independent manner and also allows translation of additional open reading frames from one messenger RNA. Alternatively, the third nucleotide sequence may encode a second promoter that controls expression of a second polypeptide. The third nucleotide sequence may also encode multiple ribosome skipping sequences, IRES sequences, additional promoter sequences, or combinations thereof. In some examples, the nucleic acid or set of nucleic acids is included within a vector or set of vectors, which may be an expression vector or set of expression vectors (e.g., a viral vector, such as a lentiviral vector or a retroviral vector).A nucleic acid set or vector set refers to a group of two or more nucleic acid molecules or two or more vectors, each encoding one of the polypeptides of interest (i.e., a polypeptide or nucleic acid that redirects glucose metabolites from the glycolytic pathway and a CAR polypeptide). Any of the nucleic acids described herein are within the scope of this disclosure.

[0129] The hematopoietic cells described herein may be immune cells expressing glucose importing polypeptides, and may be natural killer cells, monocytes / macrophages, neutrophils, eosinophils, or T cells. Furthermore, the hematopoietic cells, preferably immune cells, may be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, or tissue (e.g., spleen, lymph nodes, thymus, stem cells, or tumor tissue). Alternatively, the hematopoietic cells may be derived from stem cells, such as hematopoietic stem cells and induced pluripotent stem cells (iPSCs). Suitable sources for obtaining the desired type of host cells will be apparent to those skilled in the art. In some embodiments, the hematopoietic cells, preferably immune cells, are derived from PBMCs, which may be obtained from a patient (e.g., a human patient) in need of the treatment described herein. As non-limiting examples, anti-CD3, anti-CD28 antibodies, IL-2, IL-15, phytohemagglutinin, or engineered artificial stimulator cells or particles may be used for the expansion of T cells. In preferred embodiments, in some instances, the immune cell is a T cell in which expression of an endogenous T cell receptor, an endogenous major histocompatibility complex, an endogenous beta-2-microglobulin, or a combination thereof has been inhibited or eliminated. The hematopoietic cells described herein that express glucose metabolites and, optionally, factors (e.g., polypeptides or nucleic acids) that redirect the chimeric receptor polypeptide can be hematopoietic stem cells or their progeny. In some embodiments, the hematopoietic cells can be immune cells such as natural killer cells, monocytes / macrophages, neutrophils, eosinophils, or T cells.

[0130] In some embodiments, the hematopoietic cells are natural killer (NK) cells, macrophages, neutrophils, eosinophils, or T cells, preferably, the hematopoietic cells are T cells and have inhibited or eliminated expression of endogenous T cell receptor, endogenous major histocompatibility complex, endogenous beta-2-microglobulin, or a combination thereof, and / or the hematopoietic cells are derived from peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs), preferably, the hematopoietic cells are autologous to the patient.

[0131] Any of the genetically modified hematopoietic cells (e.g., HSCs or immune cells) described herein can include a nucleic acid or set of nucleic acids, which collectively include (a) a first nucleotide sequence encoding a factor (e.g., a polypeptide or nucleic acid) that redirects glucose metabolism, and (b) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide. The nucleic acid or set of nucleic acids is a DNA and / or RNA molecule or a set of DNA and / or RNA molecules. In some cases, the hematopoietic cell includes a nucleic acid that includes both the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the coding sequence of the factor (e.g., a polypeptide or nucleic acid) that redirects glucose metabolism is upstream of the coding sequence of the CAR polypeptide. In some embodiments, the coding sequence of the CAR polypeptide is upstream of the coding sequence of the factor that redirects glucose metabolism. Such a nucleic acid may further comprise a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, the third nucleotide sequence encoding a ribosome skipping site (e.g., a P2A peptide), an internal ribosome entry site (IRES), or a second promoter.

[0132] In some embodiments, a hematopoietic cell can comprise a nucleic acid or set of nucleic acids (e.g., a DNA molecule or set of DNA molecules), which collectively: (a) a first nucleotide sequence encoding a glucose importer polypeptide, a Krebs cycle regulator polypeptide, and / or a lactate regulator polypeptide; and (b) a second nucleotide sequence encoding a chimeric antigen receptor polypeptide. In some examples, the hematopoietic cell comprises a nucleic acid comprising both the first nucleotide sequence and the second nucleotide sequence. In some examples, the nucleic acid may further comprise (c) a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence. The third nucleotide sequence may encode a ribosome skipping site or may comprise an internal ribosome entry site (IRES) or a second promoter. In one example, the third nucleotide sequence encodes a ribosome skipping site. In one particular example, the ribosome skipping site is a P2A peptide.

[0133] In some embodiments, the nucleic acid or nucleic acid set may be included within a vector or a set of vectors. In some examples, the vector or vector set may be an expression vector or a set of expression vectors. In other examples, the vector or vector set may include one or more viral vectors, such as lentiviral vectors or retroviral vectors.

[0134] B. Pharmaceutical Compositions Any of the anti-GPC3 therapeutic agents disclosed herein may be formulated into a pharma- ceutical composition for use in the therapeutic methods disclosed herein. In some embodiments, the pharmaceutical composition may include a suitable carrier, buffer, and / or excipient in addition to the anti-GPC therapeutic agent. Preferably, the pharmaceutical composition includes any of the hematopoietic cells described herein and a pharma- ceutical acceptable carrier and excipient.

[0135] The phrase "pharmacologically acceptable" as used in connection with compositions of the present disclosure refers to molecular entities and other components of such compositions that are physiologically tolerated and typically do not cause adverse reactions when administered to a mammal (e.g., human). Preferably, as used herein, the term "pharmacologically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, particularly humans. "Acceptable" means that the carrier is compatible with the active components of the composition (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) and does not adversely affect the subject to whom the composition is administered. Any of the pharmaceutical compositions used in the present methods may include pharmaceutically acceptable carriers, excipients, or stabilizers in the form of a lyophilized formulation or an aqueous solution.

[0136] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphate, citric acid, and other organic acids, antioxidants (including ascorbic acid and methionine), preservatives, low molecular weight polypeptides, proteins (such as serum albumin, gelatin, immunoglobulins, etc.), amino acids, hydrophobic polymers, monosaccharides, disaccharides, and other carbohydrates, metal complexes, and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20 th See Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0137] The pharmaceutical compositions of the present disclosure may also contain one or more additional active compounds as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Non-limiting examples of possible additional active compounds include, for example, IL-2, as well as various agents known in the art and listed below in the discussion of combination therapy.

[0138] C. Treatment of MRCLS Further provided herein is a method for inhibiting cells expressing GPC3 (e.g., reducing the number of such cells, blocking cell proliferation, and / or suppressing cell activity) in a subject who may have or is suspected of having MRCLS. The method may include administering to a subject in need thereof a hematopoietic cell population as described herein, the hematopoietic cell population being capable of co-expressing a glucose metabolite and a factor (e.g., a polypeptide or nucleic acid) that redirects a CAR polypeptide. The subject (e.g., a human patient, such as a human patient suffering from cancer) may have been or is being treated with an anti-cancer therapy (e.g., an anti-cancer agent). In some examples, at least some of the cells expressing the target antigen are located in a low glucose environment, a low amino acid (e.g., low glutamate) environment, a low pH environment, and / or a low oxygen environment, e.g., a tumor microenvironment.

[0139] The methods described herein can include introducing into a subject a therapeutically effective amount of an antibody and a therapeutically effective amount of engineered hematopoietic cells, such as immune cells (e.g., T cells or NK cells), where the immune cells co-express a gene that improves the viability and / or functionality of the hematopoietic cells in a solid tumor microenvironment of the present disclosure and a CAR polypeptide of the present disclosure. In some examples, the immune cells are autologous. In other examples, the immune cells are allogeneic. In any of the methods described herein, the hematopoietic cells can be activated, expanded, or both ex vivo. In some cases, the immune cells include T cells that are activated in the presence of one or more of an anti-CD3 antibody, an anti-CD28 antibody, IL-2, IL-15, phytohemagglutinin, and engineered artificial stimulator cells or particles. In other cases, the hematopoietic cells include natural killer cells activated in the presence of one or more of 4-1BB ligand, anti-4-1BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-12, IL-21, and K562 cells, engineered artificial stimulator cells or particles.

[0140] In some embodiments, the hematopoietic cells are administered to a subject in an amount effective to inhibit cells expressing a target antigen by at least 20% and / or at least 2-fold, e.g., an amount effective to inhibit cells expressing a target antigen by 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more. The efficacy of the cell-based immunotherapy described herein may be assessed by any method known in the art and will be apparent to a skilled medical professional. For example, the efficacy of the cell-based immunotherapy may be assessed by the subject's survival rate or tumor or cancer burden in the subject or a tissue or sample thereof. In some embodiments, the hematopoietic cells are administered to a subject in need of treatment in an amount effective to enhance the efficacy of the cell-based immunotherapy by at least 10% and / or at least 2-fold, e.g., an amount effective to enhance the efficacy of the immunotherapy by 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more, compared to the efficacy using the same type of hematopoietic cells that do not express a glucose importing polypeptide.

[0141] In any of the compositions or methods described herein, the hematopoietic cells (e.g., NK and / or T cells) can be autologous to the subject, i.e., the hematopoietic cells can be obtained from a subject in need of treatment. Alternatively, the host cells are allogeneic, i.e., the cells are obtained from a first subject, genetically engineered as described herein, and administered to a second subject of the same species but different from the first subject. Either the autologous or allogeneic hematopoietic cells can be activated and / or expanded ex vivo prior to delivery to the subject.

[0142] According to the present disclosure, patients receive approximately 10 4 ~10 10The patient may be treated by injecting hematopoietic cells, such as T lymphocytes or NK cells, comprising a therapeutically effective dose of the glucose import polypeptide and / or CAR polypeptide of the present disclosure, in the range of 10 or more cells (cells / kg). Infusions can be repeated as frequently and as many times as the patient can tolerate, until the patient no longer responds to treatment, e.g., until progressive disease is diagnosed. Appropriate infusion doses and schedules vary from patient to patient, but can be determined by the treating physician of a particular patient. In a preferred embodiment, at least about 5×10 per kg of hematopoietic cells are injected. 4 anti-GPC3-CAR T cells are administered to selected patients with MRCLS, preferably about 5×10 4 pieces~approx. 1×10 12 anti-GPC3-CAR cells / kg will be administered to patients with MRCLS.

[0143] The efficacy of the compositions or methods described herein may be evaluated by any method known in the art and will be apparent to a skilled medical professional. For example, the efficacy of the compositions or methods described herein may be evaluated by the subject's survival rate or cancer burden in the subject or in a tissue or sample thereof. In some embodiments, the compositions and methods described herein may be evaluated based on the safety or toxicity of the treatment in the subject (e.g., administration of GPC3-targeted hematopoietic cells, antibody-drug conjugates, bispecific or multispecific targeting GPC3 described herein), for example, by the subject's overall health and / or the presence of adverse events or serious adverse events. In one embodiment, administration of the anti-GPC3 therapeutic agent is effective to reduce tumor size by at least 10% as measured by computed tomography (CT) scan. In another embodiment, administration of the anti-GPC3 therapeutic agent is effective when stable disease by RECIST (e.g., RECIST 1.1) is achieved, i.e., when the sum of the total tumor diameters can increase by 19% or decrease by 29% without new measurable lesions. Preferably, an objective response according to RECIST (e.g., RECIST 1.1) is achieved, i.e., the total tumor diameter is reduced by 30% or more without new measurable lesions.Preferably, the tumor is staged by computed tomography (CT) scan.In another embodiment, the resectable tumor is staged histologically.

[0144] In some examples, the subject treated by the methods described herein is a human patient suffering from MRCLS staged as grade 1, grade 2, grade 3, e.g., metastatic MRCLS or progressive unresectable MRCLS. In one embodiment, the GPC3-specific CAR-T is administered to a patient suffering from MRCLS staged as grade 1. In another embodiment, the GPC3-specific CAR-T is administered to a patient suffering from MRCLS staged as grade 2. In yet another embodiment, the GPC3-specific CAR-T is administered to a patient suffering from MRCLS staged as grade 3. In a preferred embodiment, the GPC3-specific CAR-T is administered to a patient suffering from MRCLS, e.g., metastatic MRCLS or progressive unresectable MRCLS.

[0145] Also within the scope of the present disclosure is the use of the anti-GPC3 therapeutic agents described herein for treating MRCLS, and for the manufacture of a medicament for the intended medical treatment thereof.

[0146] In some embodiments, engineered hematopoietic cells expressing genes that improve hematopoietic cell viability and / or function in the solid tumor microenvironment of the present disclosure can be derived from natural hematopoietic cells specific for MRCLS cells (e.g., MRCLS cells). Such engineered hematopoietic cells (e.g., tumor infiltrating lymphocytes or TILs) do not co-express any chimeric receptor polypeptide and can be used to destroy target disease cells, e.g., MRCLS cells. These engineered TILs expressing the genes that improve viability and / or functionality, but not the chimeric receptor, can be used with bispecific antibodies that can bind to target tumor cells and TILs (BiTEs).

[0147] Additionally, the compositions and methods described herein may be utilized in combination with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, etc., preferably with established standard treatments for MRCLS, e.g., doxorubicin, ifosfamide, trabectedin, as disclosed in Abaricia & Hirbe, 2018, Lee et al., 2018, Regina & Hettmer, 2019, Sanfilippo et al., 2013, Suarez-Kelly et al., 2019. Such therapy may be administered simultaneously or sequentially (in any order) with the immunotherapy according to the present disclosure.

[0148] When co-administered with additional therapeutic agents, additive or synergistic effects may reduce the suitable therapeutically effective dose of each agent. The treatments of the present disclosure can be combined with other immunomodulatory therapies, such as, for example, therapeutic vaccines (including but not limited to, GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to, agents that block CTLA-4, PD-1, LAG-3, TIM-3, etc.), or activators (including but not limited to, agents that enhance 41BB, OX40, etc.). Non-limiting examples of other therapeutic agents useful in combination with the immunotherapy methods of the present disclosure include: (i) antiangiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), prolactin (16 kD fragment), angiostatin (38 kD fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, and Carmeliet & Co. Jain, 2000, (ii) VEGF antagonists or VEGF receptor antagonists, such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinase, and any combination thereof, and (iii) chemotherapeutic compounds, such as pyrimidine analogues (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine), purine analogues, folate antagonists, and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine));Antiproliferative / antimitotic agents, including natural products such as the vinca alkaloids (vinblastine, vincristine, and vinorelbine), taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbines, epidipodophyllocytoxies (etoposide and teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, dow norubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (VP16); dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracillin antibiotics such as , mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine systemically and eliminates cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melaphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazene-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors);Fibrinolytic agents (e.g., tissue plasminogen activator, streptokinase, urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (brefeldin); immunosuppressants (cyclosporine, tacrolimus (FK-506)), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); AKT inhibitors (e.g., MK-2206 2HC1, Perifosine (KRX-0401), GSK690693, Ipatasertib (GDC-0068), AZD5363, Uprosertib, Afuresertib, or Triciribine; mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, and irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors;

[0149] In one embodiment, the method further comprises administering at least one immunomodulatory agent to the patient in parallel or sequential with the therapeutic agent, preferably the immunomodulatory agent is an immune checkpoint inhibitor or an immune stimulating cytokine. Immune checkpoint inhibitors are expected to remove inhibitory signals in the microtumor environment that may negatively interfere with the mode of action of the anti-GPC3 therapeutic agent with hematopoietic cells expressing CAR (e.g., immune cells such as T cells or NK cells), thereby reducing the activity of hematopoietic cells, thereby reducing or blocking their activity. In some embodiments, the method further comprises administering a lymphodepleting treatment, preferably selected from cyclophosphamide and fludarabine. Such lymphodepleting treatment is preferably applied prior to the infusion of hematopoietic cells expressing CAR, to allow for greater T cell expansion of the infused cells (Shank et al., 2017).

[0150] The details of one or more embodiments of the disclosure are set forth in the description below. Other features and advantages of the disclosure will become apparent from the detailed description of some embodiments and the appended claims.

[0151] General Technology The practice of the present disclosure 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 the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell Culture (RIFreshney, ed. 1987), Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Black-well, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan et al., eds.,1991)、Short Protocols in Molecular Biology(Wiley and Sons,1999)、Immunobiology(CAJaneway and P.Travers,1997)、Antibodies(P.Finch,1997)、Antibodies: A Practice Approach(D.Catty.,ed.IRL). Press,1988-1989)、Monoclo-nal antibodies: a practical approach(P.Shepherd and C.Dean,eds.,Oxford University Press,2000)、Using antibodies:a laboratory manual(E.Harlow and D.Lane(Cold Spring Harbor Laboratory Press,1999). JDCapra,eds.Harwood Academic Publishers,1995);DNACloning:A Practical Approach,Volumes I and II(DNGlover ed.1985); SJHiggins,eds.(1984≫、Animal Cell Culture(RIFreshney,ed.(1986≫、Immobi-lized Cells and Enzymes(IRL Press,(1986≫)and B.Perbal,A Practical Guide To Molecular Cloning(1984) FMAusubel et al.(eds.) In this context.

[0152] Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the above description. Therefore, the following specific embodiments are to be construed as merely illustrative, and in no way limiting to the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein. EXAMPLES

[0153] The following examples are intended only to illustrate methods and embodiments according to the present invention, and therefore should not be construed as imposing limitations on the claims.

[0154] Example 1. IHC assay of FFPE tissue sections using anti-GPC3 monoclonal antibody GC33 IHC staining with antibodies specific for GPC3 was performed according to conventional protocols. Human biopsy specimens (tumor and healthy tissue), xenograft biopsy specimens (tumor and healthy tissue), and cell line specimens were fixed in neutral buffered 10% formalin solution for 24 hours and embedded in paraffin according to standard procedures. Standard specimen size was 0.5 cm x 1 cm x 1 cm. 5 μm thick tissue sections were cut with a microtome (Leica) and mounted on positively charged slides. Slides were air-dried and stored at room temperature throughout the study period. Tissue sections were deparaffinized in EZ prep (Ventana) followed by antigen retrieval with target retrieval solution (Ventana) in a hot water bath (98°C, 60 min). Endogenous peroxidase was blocked with primary peroxidase inhibitor (Ventana) for 5 min at room temperature. Sections were then incubated with primary anti-GPC3 antibody (antibody GC33; Ventana #790-4564) for 32 minutes, followed by development of enzyme activity (OptiView DAB detection kit, Ventana). Sections were counterstained with hematoxylin (Ventana) for 30 seconds at room temperature. Specificity of staining was determined using appropriate isotype controls. Images of whole tumor sections were acquired using a Leica Aperio AT2 scanner (Leica).

[0155] Example 2. IF assay of FFPE tissue sections with anti-GPC3 monoclonal antibody GC33. IF staining with antibodies specific for GPC3 is performed according to conventional protocols. Human biopsy specimens (tumor and healthy tissue), xenograft biopsy specimens (tumor and healthy tissue), and cell line specimens are fixed in neutral buffered 10% formalin solution for 24 hours and embedded in paraffin according to standard procedures. Standard specimen size is 0.5 cm x 1 cm x 1 cm. 5 μm thick tissue sections are cut with a microtome (Leica) and mounted on positively charged slides. Slides are air-dried and stored at room temperature throughout the duration of the study. Briefly, tissue sections are deparaffinized and rehydrated in a descending alcohol series (100, 96, 70, and 50%), followed by antigen retrieval using target retrieval solution (Leica) in a preheated water bath (97°C, 30 min). Sections are cooled to room temperature for 30 min. Sections are then treated with signal enhancer (Fisher Thermoscientific) for 30 minutes at room temperature, followed by blocking buffer for 60 minutes at room temperature. Anti-GPC3 antibody (antibody GC33; Ventana #790-4564) is applied for 2 hours at room temperature. Slides are then incubated with appropriate fluorophore-labeled secondary antibodies for 1 hour at room temperature. Finally, sections are treated with TrueBlack® Lipofuscin autofluorescence quencher quencher (Biotium) for 30 seconds and mounted with ProLong Gold antifade reagent containing DAPI (Thermo Fisher Scientific). Specificity of staining is determined using appropriate isotype controls. Images of whole tumor sections were acquired using a Leica Aperio AT2 scanner (Leica).

[0156] Example 3. IHC scoring of FFPE tissue sections from different cancer types for GPC3 expression Example 1 was scored for GPC3 expression using a detailed scoring process. The brief description of the figures lists the various scores assigned to each individual field of each tissue section for illustrative purposes only. However, the scoring process was performed by examining the entire tissue section on the slide, and in practice, the pathologist scored the slide for GPC3 expression by observing the tissue section on the slide at low, medium, and high magnification. Low and medium magnification were used to detect stained tumor cells. Medium and high magnification were used to examine individual tumor cells to estimate the number and intensity of viable tumor cells that show at least partial membrane and cytoplasmic staining. Each stained tissue section was assigned an H score. The H-score involved (i) estimating four separate percentages of cells with no staining, weak staining (+1), moderate staining (+2), and strong staining (+3) across all viable tumor cells in all stained tissue sections examined, where a cell must have at least partial membrane and / or cytoplasmic staining to be included in the weak, moderate, or strong staining percentages, with the sum of the four percentages equaling 100, and (ii) entering the estimated percentages into the formula 1*(percentage of tumor cells with 1+ staining intensity)+2*(percentage of tumor cells with 2+ staining intensity)+3*(percentage of tumor cells with 1+ staining intensity) and assigning the result of the formula to the tissue section as an H-score.

[0157] Example 4. IF scoring of FFPE tissue sections from different cancer types for GPC3 expression. Example 2 can be scored for GPC3 expression using a detailed scoring process. The brief description of the figures lists the various scores assigned to each individual field of each tissue section for illustrative purposes only. However, the scoring process is performed by examining the entire tissue section on the slide, and in practice, the pathologist scores the slide for the expression of GPC3 by observing the tissue section on the slide at low, medium, and high magnification. Low and medium magnification is used to detect stained tumor cells. Medium and high magnification is used to examine individual tumor nests to estimate the number and intensity of viable tumor cells that show at least partial membrane and cytoplasmic staining. Each stained tissue section is assigned an H score. The H-score involves (i) estimating four separate percentages of cells with no staining, weak staining (+1), moderate staining (+2), and strong staining (+3) across all viable tumor cells in all stained tissue sections examined, where cells must have at least partial membrane and / or cytoplasmic staining to be included in the weak, moderate, or strong staining percentages, the sum of the four percentages equaling 100, and (ii) inputting the estimated percentages into the formula 1*(percentage of tumor cells with 1+ staining intensity)+2*(percentage of tumor cells with 2+ staining intensity)+3*(percentage of tumor cells with 1+ staining intensity) and assigning the result of the formula to the tissue section as an H-score.

[0158] Example 5. ISH assay of FFPE tissue sections using a probe specific for GPC3. In situ hybridization staining of the target gene GPC3 can be performed according to conventional protocols (Wang et al., 2012). Human biopsy specimens (tumor and healthy tissue), xenograft biopsy specimens (tumor and healthy tissue), and cell line specimens are fixed in neutral buffered 10% formalin solution for 24 hours and embedded in paraffin according to standard procedures. Standard specimen size is 0.5 cm x 1 cm x 1 cm. Tissue sections of 6 μm thickness are cut with a microtome (Leica) and mounted on positively charged slides. The tissue quality of each sample is assessed by performing RNA hybridization to the mRNA of the housekeeping gene Ubiquitin C (UBC) according to conventional protocols (Wang et al., 2012). The protocol begins with air-drying the slides, which are then stored at room temperature throughout the duration of the study. Briefly, tissue sections are deparaffinized and rehydrated in a descending alcohol series (100, 96, 70, and 50%), followed by air drying at room temperature for 5 min. Slides are pretreated in prehybridization buffer at 40° C. for 30 min, followed by peroxidase quenching at room temperature for 10 min (ACD Biotechnie). Slides are immersed in target retrieval solution at 40° C. for 30 min, followed by protease treatment at 40° C. for 30 min. Slides are incubated with target probes and incubated at 40° C. for 2 h, followed by washing off excess probe in an appropriate buffer (ACD Biotechnie). Probe detection is performed by, for example, incubating in fast red solution at room temperature for 10 min in the case of chromogenic assays. Sections are counterstained with hematoxylin (Ventana) for 30 s at room temperature. The specificity of the staining is determined using appropriate isotype controls. Images of whole tumor sections are acquired using a Leica Aperio AT2 scanner (Leica).

[0159] Example 6. Cell-free DNA (cfDNA) isolation and analysis by targeted next-generation sequencing (NGS). Blood samples can be isolated pre- or post-operatively from suspected patients. Furthermore, they can be matched with patients with primary and / or secondary tumor tissue biopsies. Blood samples can be isolated and processed within 24 hours after collection. Blood is first centrifuged at 1700g for 10 minutes to separate plasma and blood cells. The separated plasma is centrifuged at 12,000g for another 10 minutes to remove cellular debris. Plasma is collected, aliquoted in 2ml per vial, and stored at -80°C until further processing. cfDNA is isolated from 440μl to 4ml (median 3.95ml) of plasma using the Circulating Nucleic Acid Kit (Qiagen) and subsequently eluted in 30μl of elution buffer. ctDNA concentration is determined by Qubit™ 1×dsDNA HS Assay Kit (Thermo Fisher scientific) using 2μl of ctDNA.

[0160] Sequencing can be performed by ion semiconductor sequencing on an Ion Torrent S5XL next-generation sequencing (NGS) system using a ctDNA assay with molecular barcodes loaded onto an Ion540 chip. Experiments are performed according to the manufacturer's protocol (Thermo Fisher Scientific / Life Technologies). Several ctDNA panels can be used to cover mutation hotspots in multiple genes associated with MRCLS (e.g., Oncomine™ Colon ctDNA Panel, Thermo Fisher Scientific / Life Technologies). Analysis and cutoffs are performed according to (Ge et al., 2021).

[0161] Example 7. Isolation of circulating tumor cell RNA (ctRNA) and analysis by RT-PCR. Circulating tumor cells (CTCs) can be isolated from 7.5 ml of whole blood based on density gradient. Equal volumes of whole blood and PBS are carefully mixed by inversion and layered on Ficoll-paque, followed by centrifugation at 400 g for 30 min at room temperature. CTCs are collected from the plasma layer (see Low & Wan Abas, 2015). RNA is extracted from CTCs according to the manufacturer's protocol (QIAGEN). Reverse transcription can be performed at 37° C. for 1 h using Invitrogen Superscript III reverse transcriptase and random hexamers (Invitrogen) as primers, followed by inactivation at 95° C. for 5 min. cDNA (5 μl) is used for the subsequent PCR reaction. GPC3-specific primers and PCR reactions are performed as described in (Wang et al., 2011).

[0162] array SEQ ID NO:1 - CD8α signal sequence TIFF2024545363000004.tif447 SEQ ID NO:2 - GPC3 scFv derived from GC33 TIFF2024545363000005.tif16150 SEQ ID NO:3 - CD8 hinge and transmembrane domain TIFF2024545363000006.tif9150 SEQ ID NO:4 - CD28 hinge and transmembrane domain TIFF2024545363000007.tif9150 SEQ ID NO: 5-4-1BB costimulatory domain TIFF2024545363000008.tif493 SEQ ID NO:6 - CD28 costimulatory domain TIFF2024545363000009.tif491 SEQ ID NO:7 - CD3 ζ signaling domain TIFF2024545363000010.tif9150 SEQ ID NO:8 - GPC3 CAR polypeptide (signal sequence shown in italics) (CD8α / GC33 scFv / CD8α-CD8α / 4-1BB / CD3ζ) TIFF2024545363000011.tif35160 SEQ ID NO:9 - Mature GPC3 CAR polypeptide (GC33 scFv / CD8α-CD8α / 4-1BB / CD3ζ) TIFF2024545363000012.tif29150 SEQ ID NO:10 - GPC3-CAR polypeptide (signal sequence in italics) (CD8α / GC33 scFv / CD28-CD28 / CD3ζ) - no stimulatory domain TIFF2024545363000013.tif34151 SEQ ID NO:11 - Mature GPC3-CAR polypeptide (GC33 scFv / CD28-CD28 / CD3ζ) - no stimulatory domain TIFF2024545363000014.tif29151 sequence number 12-GOT2 TIFF2024545363000015.tif29151 SEQ ID NO:13-GOT1 TIFF2024545363000016.tif30160 SEQ ID NO:14-P2A TIFF2024545363000017.tif443 SEQ ID NO:15 - GLUT1 TIFF2024545363000018.tif34151 SEQ ID NO:16 - CD16A 158F Polypeptides (signal sequences are shown in italics) TIFF2024545363000019.tif16150 SEQ ID NO:17 - Mature CD16A 158F Polypeptides TIFF2024545363000020.tif16150 SEQ ID NO:18 - CD16A 158V Polypeptides (signal sequences are shown in italics) TIFF2024545363000021.tif16150 SEQ ID NO:19 - Mature CD16A 158V TIFF2024545363000022.tif16150 SEQ ID NO:20 - OX-40 costimulatory domain TIFF2024545363000023.tif493 SEQ ID NO:21 - CD27 costimulatory domain TIFF2024545363000024.tif4106 SEQ ID NO:22 - ICOS costimulatory domain TIFF2024545363000025.tif475 SEQ ID NO:23 - GITR costimulatory domain TIFF2024545363000026.tif4129 SEQ ID NO:24 - HVEM costimulatory domain TIFF2024545363000027.tif4132 SEQ ID NO:25 - TIM1 costimulatory domain TIFF2024545363000028.tif4106 SEQ ID NO:26 - LFA-1 costimulatory domain TIFF2024545363000029.tif9150 SEQ ID NO:27 - CD2 costimulatory domain TIFF2024545363000030.tif13150 SEQ ID NO:28 - CD8α transmembrane domain TIFF2024545363000031.tif456 SEQ ID NO:29 - CD8 beta transmembrane domain TIFF2024545363000032.tif556 SEQ ID NO: 30-4-IBB transmembrane domain TIFF2024545363000033.tif463 SEQ ID NO:31 - CD28 transmembrane domain TIFF2024545363000034.tif467 SEQ ID NO:32 - CD34 transmembrane domain TIFF2024545363000035.tif457 SEQ ID NO:33 - CD4 transmembrane domain TIFF2024545363000036.tif458 SEQ ID NO:34 - FcεRIγ transmembrane domain TIFF2024545363000037.tif556 SEQ ID NO:35 - OX-40 transmembrane domain TIFF2024545363000038.tif456 SEQ ID NO:36 - CD3 ζ transmembrane domain TIFF2024545363000039.tif456 SEQ ID NO:37 - CD3ε transmembrane domain TIFF2024545363000040.tif465 SEQ ID NO:38 - CD3γ transmembrane domain TIFF2024545363000041.tif456 SEQ ID NO:39 - CD3 delta transmembrane domain TIFF2024545363000042.tif458 SEQ ID NO:40 - TCR-α transmembrane domain TIFF2024545363000043.tif454 SEQ ID NO:41 - CD32 transmembrane domain TIFF2024545363000044.tif460 SEQ ID NO:42 - CD64 transmembrane domain TIFF2024545363000045.tif456 SEQ ID NO:43 - VEFGR2 transmembrane domain TIFF2024545363000046.tif456 SEQ ID NO:44 - FAS transmembrane domain TIFF2024545363000047.tif447 SEQ ID NO:45 - FGFR2B transmembrane domain TIFF2024545363000048.tif456

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[0164] Other embodiments All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0165] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to adapt it to various applications and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the claims.

[0166] Equivalent Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) for which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments have been presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0167] As defined and used herein, all definitions should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0168] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, including, in some cases, the entire document.

[0169] As used in this specification and the claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly indicated otherwise.

[0170] As used in the present specification and claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present, whether related or unrelated to those elements specifically identified, other than the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may in one embodiment refer to only A (optionally including elements other than B), in another embodiment refer to only B (optionally including elements other than A), and in yet another embodiment refer to both A and B (optionally including other elements).

[0171] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including not only at least one element, but also one or more of the elements or list of elements. Only terms clearly indicated otherwise, such as "only one of" or "only one of," or, when used in the claims, "consisting of," refer to the inclusion of only one element of the elements or list of elements. In general, as used herein, the term "or" shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by an exclusive term, such as "either," "one of," "only one of," or "only one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0172] As used in this specification and claims, the phrase "at least one," with reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that there may optionally be elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one that includes multiple As, optionally with no B present (and optionally including elements other than B); in another embodiment to at least one that includes multiple Bs, optionally with no A present (and optionally including elements other than A); and in yet another embodiment to at least one that optionally includes multiple As (and optionally includes other elements), and optionally includes multiple Bs (and optionally includes other elements).

[0173] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes multiple steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

Claims

1. A pharmaceutical composition for treating a patient diagnosed with myxoid / round cell liposarcoma, comprising an anti-glypican-3 (GPC3) therapeutic agent.

2. 10. The pharmaceutical composition of claim 1, wherein the patient is selected for treatment by diagnosing myxoid / round cell liposarcoma.

3. 2. The pharmaceutical composition of claim 1, wherein the myxoid / round cell liposarcoma expresses glypican-3 (GPC3).

4. The pharmaceutical composition of claim 1 , wherein the patient diagnosed with myxoid / round cell liposarcoma is selected by immunohistochemistry.

5. The patient: (a) obtaining a tissue section from a tumor biopsy sample, wherein said section has a thickness of 3 μm to 15 μm; (b) immunostaining with an antibody that specifically binds to GPC3; (c) determining a cytoplasmic / membrane H-score; and (d) selecting patients with an H-score greater than 30 for said treatment; The pharmaceutical composition of claim 1, wherein the composition is diagnosed by a process comprising:

6. The pharmaceutical composition of claim 4, wherein the immunostaining comprises immunostaining of GPC3 in a tumor sample from the patient, wherein the GPC3 expression level is determined and compared with a predetermined threshold level of GPC3 expression, and if the patient has a GPC3 expression level equal to or greater than the predetermined threshold level, the patient is selected for treatment.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the therapeutic agent comprises an anti-GPC3 binding domain.

8. The pharmaceutical composition of claim 7, wherein the anti-GPC3 binding domain comprises an anti-GPC3 antibody or a functional fragment thereof that retains binding to GPC3.

9. The pharmaceutical composition according to any one of claims 1 to 6, wherein the therapeutic agent comprises an anti-GPC3 antibody, an anti-GPC3 antibody-drug conjugate, an anti-GPC3 antibody-radionuclide conjugate, or a fusion protein of an anti-GPC3 antibody or an antibody derivative that binds to GPC3 with an anti-CD3 binding domain or an immunostimulatory polypeptide.

10. The therapeutic agent comprises genetically engineered hematopoietic cells that express an anti-GPC3 chimeric receptor polypeptide (CAR), the CAR polypeptide comprising: (a) an extracellular binding domain that binds to GPC3; (b) a transmembrane domain; and (c) a cytoplasmic signaling domain; and The pharmaceutical composition according to any one of claims 1 to 6, comprising:

11. the hematopoietic cells i. has improved glucose uptake activity compared to wild-type hematopoietic cells of the same type, wherein the hematopoietic cells exogenously express a glucose import polypeptide; ii. have a regulated Krebs pathway compared to wild-type hematopoietic cells of the same type, wherein the hematopoietic cells exogenously express a Krebs pathway-regulating polypeptide, and / or iii. have an enhanced intracellular lactate concentration compared to wild-type hematopoietic cells of the same type, wherein the hematopoietic cells exogenously express a lactate-regulating polypeptide; The pharmaceutical composition of claim 10.

12. The pharmaceutical composition of claim 10, wherein the extracellular antigen-binding domain is a single-chain antibody fragment (scFv) that binds to GPC3.

13. the CAR polypeptide is (i) a CD28 costimulatory domain in combination with a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof; or (ii) a 4-1BB costimulatory domain in combination with a CD8 transmembrane domain, a CD8 hinge domain, or a combination thereof (SEQ ID NO: 3).

11. The pharmaceutical composition of claim 10, comprising:

14. The pharmaceutical composition of claim 10, wherein the cytoplasmic signaling domain in (c) is the cytoplasmic domain of CD3ζ or FcεR1γ.

15. the hematopoietic cells are natural killer (NK) cells, macrophages, neutrophils, eosinophils, or T cells, and expression of endogenous T cell receptors, endogenous major histocompatibility complexes, endogenous beta-2-microglobulin, or a combination thereof, is inhibited or eliminated; and / or The hematopoietic cells are derived from peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs). The pharmaceutical composition of claim 10.

16. The hematopoietic cells comprise a nucleic acid or set of nucleic acids, which collectively comprise: (a) a first nucleotide sequence encoding the glucose import polypeptide, the Krebs cycle regulatory polypeptide, and / or the lactate regulatory polypeptide; and (b) a second nucleotide sequence encoding the chimeric antigen receptor polypeptide.

11. The pharmaceutical composition of claim 10, comprising:

17. A pharmaceutical composition according to any one of claims 1 to 6, used in combination with at least one immunomodulatory agent, either in parallel or sequentially, in the treatment of a patient with myxoid / round cell liposarcoma.

18. A pharmaceutical composition described in any one of claims 1 to 6, for lymphocyte reduction treatment.

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