Methods and compositions for treating glioblastoma

Abstract

The present disclosure provides methods and compositions for treating glioblastoma (GBM) using a 5T4 targeting agent, such as a superantigen conjugate that includes an anti-5T4 antibody.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 178,054, filed April 22, 2021, the disclosure of which is incorporated by reference herein in its entirety for all purposes.

[0002] FIELD OF THEINVENTION The present disclosure relates generally to methods and compositions for treating glioblastoma in a subject, and more specifically, the present disclosure relates to methods and compositions for treating glioblastoma using 5T4 binding agents. [Background technology]

[0003] background According to the American Cancer Society, more than one million people are diagnosed with cancer each year in the United States. Cancer is a disease caused by the uncontrolled proliferation of cells that, once subjected to natural control mechanisms, are transformed into cancerous cells that continue to grow in an uncontrolled manner.

[0004] Glioblastoma multiforme (glioblastoma or GBM) is the most common and most aggressive malignant primary brain tumor in humans. GBM is a tumor of astrocytes, star-shaped glial cells that form neural networks and perform various important and active roles in the brain. GBM is highly lethal and characterized by excessive necrosis and a high rate of angiogenesis. Patients diagnosed with GBM have poor prognosis and survival rates. Treatment typically includes resection, chemotherapy or radiation. Median survival with standard of care (temozolomide) is 15 months with a 2-year survival of less than 25%. Survival without treatment is usually only a few months. Furthermore, the Human Protein Atlas states that malignant gliomas do not show membranous or cytoplasmic immunoreactivity for 5T4 (TPBG) expression (see world wide web proteinatlas.org / ENSG00000146242-TPBG / pathology).

[0005] Despite significant advances being made in the treatment and management of cancer, there remains a continuing need for new and effective therapies for treating and managing cancer, such as glioblastoma. Summary of the Invention

[0006] Summary of the Invention The present invention is based, in part, on the discovery that glioblastoma (GBM) tumor samples and cell lines exhibit significant levels of 5T4 expression, and that anti-cancer therapies comprising 5T4 targeting agents (e.g., superantigen conjugates comprising anti-5T4 antibodies) can be used to treat GBM.

[0007] Thus, the present invention provides a method of (i) reducing tumor volume in a subject having glioblastoma, (ii) killing tumor cells in a subject having glioblastoma, and / or (iii) treating glioblastoma in a subject in need of treatment, comprising administering to the subject an effective amount of a 5T4 targeting agent.

[0008] In certain embodiments of any of the foregoing methods, (i) at least 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% of the tumor cells in the subject, (ii) in the tumor in the subject, and / or (iii) in a tumor sample (e.g., a tumor tissue sample) from the subject exhibit cell membrane expression of 5T4 as measured by immunohistochemistry. The presence of membrane versus cytoplasmic expression of 5T4 is significant when using 5T4 as a target for targeted therapy of cancerous cells (e.g., GBM cells).

[0009] In certain embodiments of any of the foregoing methods, the 5T4 targeting agent is or comprises an antibody, a bispecific T cell engager (BiTE), an immune cell, or a vaccine. In certain embodiments, the 5T4 targeting agent is or comprises a superantigen conjugate comprising a superantigen covalently linked to an anti-5T4 antibody. The superantigen may comprise Staphylococcal enterotoxin A or an immunologically reactive variant and / or fragment thereof. In certain embodiments, the superantigen comprises the amino acid sequence of SEQ ID NO:3 or an immunologically reactive variant and / or fragment thereof. In certain embodiments, the anti-5T4 antibody comprises a heavy chain comprising amino acid residues 1-458 of SEQ ID NO:8 and a light chain comprising amino acid residues 1-214 of SEQ ID NO:9. In certain embodiments, the superantigen conjugate comprises a first protein chain comprising SEQ ID NO:8 and a second protein chain comprising SEQ ID NO:9.

[0010] In some embodiments of any of the foregoing methods, the 5T4 targeting agent is or comprises an immune cell (e.g., an isolated immune cell), which may be, for example, a T cell, a natural killer cell (NK), or a natural killer T cell (NKT). In some embodiments, the immune cell is a chimeric antigen receptor (CAR)-expressing immune cell.

[0011] In some embodiments of any of the above methods, the method further comprises administering to the subject an immunopotentiator, which may be, for example, a CTLA-4 system inhibitor or a PD-1 system inhibitor. In some embodiments, the PD-1 system inhibitor is a PD-1 or PD-L1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, for example, an anti-PD-1 antibody selected from nivolumab pembrolizumab and cemiplimab. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody, for example, an anti-PD-L1 antibody selected from atezolizumab, avelumab, and durvalumab.

[0012] These and other aspects and features of the present invention are described in the following detailed description and claims. [Brief description of the drawings]

[0013] Description of the drawings The present invention may be more fully understood with reference to the following drawings. [Figure 1] FIG. 1 is a sequence alignment showing the homologous A-E regions in certain wild-type and modified superantigens. [Diagram 2] FIG. 2 is a schematic representation of an exemplary superantigen conjugate, naptumomab estafenatox / ANYARA®. [Diagram 3] 3 is an amino acid sequence corresponding to an exemplary superantigen conjugate, naptumomab-estafenatox / ANYARA®, that comprises two protein chains. The first protein chain comprises residues 1-458 of SEQ ID NO:7 (see also SEQ ID NO:8) and comprises a chimeric 5T4 Fab heavy chain corresponding to residues 1-222 of SEQ ID NO:7 covalently linked via a GGP tripeptide linker corresponding to residues 223-225 of SEQ ID NO:7 and a SEA / E-120 superantigen corresponding to residues 226-458 of SEQ ID NO:7. The second chain comprises residues 459-672 of SEQ ID NO:7 (see also SEQ ID NO:9) and comprises a chimeric 5T4 Fab light chain. The two protein chains are held together by non-covalent interactions between the Fab heavy and light chains. [Figure 4] Figures 4A-4B are representative immunohistochemistry (IHC) images (A-0.5X; B-20X) of glioblastoma tumor sections stained with antibodies against either 5T4 (left) or control IgG antibody (right). In this tumor section, 20% of glioblastoma cells showed membrane 5T4 expression. The bar in the top left of each image indicates the image scale (A 2000 μm; B 100 μm). The box in Figure 4A indicates the area in the tumor section shown enlarged in Figure 4B. [Diagram 5]Figure 5 shows 5T4 expression on U-138 (GBM), U-87 (GBM) GMS-10 (GBM), Caki-2 (RCC; high 5T4 expression control) and OVCAR-3 (ovarian cancer; low 5T4 expression control) cell lines as measured by flow cytometry. FC indicates fold change in 5T4 antibody staining relative to isotype control antibody staining. [Figure 6A] Figure 6A-C are a series of dose-response curves showing the anti-cancer effect of naptumomab-estafenatox (NAP) in combination with T cells against glioblastoma (GBM) cell lines. GBM cells were treated with NAP (0-10 μg / ml) for 48 h and viable cells were counted. Dose-response curves are shown for three GBM cell lines: U-138 (Figure 6A), U-87 (Figure 6B) and GMS-10 (Figure 6C). The IC50 (NAP concentration that inhibits 50% of cancer cell viability) was found to be 0.5 ng / ml, 0.3 ng / ml and 0.95 ng / ml for U-138, U-87 and GMS-10, respectively. [Figure 6B] Figure 6A-C are a series of dose-response curves showing the anti-cancer effect of naptumomab-estafenatox (NAP) in combination with T cells against glioblastoma (GBM) cell lines. GBM cells were treated with NAP (0-10 μg / ml) for 48 h and viable cells were counted. Dose-response curves are shown for three GBM cell lines: U-138 (Figure 6A), U-87 (Figure 6B) and GMS-10 (Figure 6C). The IC50 (NAP concentration that inhibits 50% of cancer cell viability) was found to be 0.5 ng / ml, 0.3 ng / ml and 0.95 ng / ml for U-138, U-87 and GMS-10, respectively. [Figure 6C]Figure 6A-C are a series of dose-response curves showing the anti-cancer effect of naptumomab-estafenatox (NAP) in combination with T cells against glioblastoma (GBM) cell lines. GBM cells were treated with NAP (0-10 μg / ml) for 48 h and viable cells were counted. Dose-response curves are shown for three GBM cell lines: U-138 (Figure 6A), U-87 (Figure 6B) and GMS-10 (Figure 6C). The IC50 (NAP concentration that inhibits 50% of cancer cell viability) was found to be 0.5 ng / ml, 0.3 ng / ml and 0.95 ng / ml for U-138, U-87 and GMS-10, respectively. [Figure 7] Figure 7 is a series of viability curves showing the effect of anti-5T4 antibodies on the anti-cancer effect of naptumomab-estafenatox (NAP) on glioblastoma GMS-10 cell line. Fluorescently stained GMS-10 cells were treated with anti-5T4 Fab (0-50 μg / ml) for 24 hours to block 5T4 antigen. GMS-10 cells were then treated with NAP (0-0.005 μg / ml) and T cells were added at a 10:1 effector:target (T cells:GMS-10 cells) ratio for 48 hours. Cancer cell viability (%) was determined by FACS. Cancer cell viability (%) = (treated cell count / control cell count) x 100. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description The present invention is based in part on the discovery that glioblastoma (GBM) tumor samples and cell lines show significant levels of 5T4 expression (particularly membrane expression of 5T4), and that anti-cancer therapy using 5T4 targeting agents (e.g., superantigen conjugates with anti-5T4 antibodies) can be used to treat GBM. This finding was unexpected, since membrane and cytoplasmic immunoreactivity of 5T4 expression in malignant gliomas was considered negative (Human Protein Atlas, see above). As described in Example 1, it was surprisingly discovered that many (more than 80%) of the GBM tissue samples evaluated showed positive membrane 5T4 expression. Furthermore, it was also discovered that the 5T4 antigen is rapidly degraded in cellular and / or tissue samples. Without wishing to be bound by theory, it is possible that previous studies could not identify the significance of membrane-bound 5T4 given its instability ex vivo.

[0015] Thus, in one aspect, the present invention provides a method for reducing tumor volume in a subject with glioblastoma.In another aspect, the present invention provides a method for killing tumor cells in a subject with glioblastoma.In another aspect, the present invention provides a method for treating glioblastoma in a subject in need of treatment.In each of the above aspects, the method comprises administering to the subject an effective amount of a 5T4 targeting agent.

[0016] Also provided is a method for identifying a subject suitable for treatment with 5T4 targeting agent.The subject can be identified based on the expression (and expression level) of membrane 5T4 in cell samples and / or tissue samples taken from the subject.The expression and expression level of membrane 5T4 can be determined by various techniques in the art, such as immunohistochemistry test.Optionally, the subject with detectable level of membrane 5T4 is suitable for treatment with 5T4 targeting agent.

[0017] In some situations, at least 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of (i) tumor cells in a subject, (ii) tumor cells in a tumor in a subject, and / or (iii) tumor cells in a tumor sample (e.g., a tumor tissue sample) from the subject exhibit cell membrane expression of 5T4 as measured by immunohistochemistry and / or fluorescence activated cell sorting (FACS).

[0018] In certain embodiments, about 0.5% to about 80%, about 0.5% to about 60%, about 0.5% to about 40%, about 0.5% to about 20%, about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 0.5% to about 3%, about 0.5% to about 4%, about 0.5% to about 5%, about 0.5% to about 6%, about 0.5% to about 7%, about 0.5% to about 8%, about 0.5% to about 9%, about 0.5% to about 10 ... .5% to about 1%, about 1% to about 80%, about 1% to about 60%, about 1% to about 40%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 80%, about 2% to about 60%, about 2% to about 40%, about 2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 80%, about 3% to about 6 0%, about 3% to about 40%, about 3% to about 20%, about 3% to about 15%, about 3% to about 10%, about 3% to about 5%, about 3% to about 4%, about 4% to about 80%, about 4% to about 60%, about 4% to about 40%, about 4% to about 20%, about 4% to about 15%, about 4% to about 10%, about 4% to about 5%, about 5% to about 80%, about 5% to about 60%, about 5% to about 40%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 80%, about 10% to about 60%, about 10% to about 40%, about 10% to about 20%, about 10% to about 15%, about 15% to about 80%, about 15% to about 60%, about 15% to about 40%, about 15% to about 20%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 80%, about 40% to about 60% or about 60% to about 80% exhibit cell membrane expression of 5T4 as measured by immunohistochemistry and / or fluorescence activated cell sorting (FACS).

[0019] The level of 5T4 membrane expression exhibited by tumor cells can be measured, for example, by flow cytometry, for example, as described in Example 2 herein. In some embodiments, 5T4 membrane expression is quantified as the fold increase (also referred to as fold change or FC) in the binding of anti-5T4 antibody to tumor cells relative to an isotype control. For example, in some embodiments, there is about a 1.5-fold increase, about a 2-fold increase, about a 2.5-fold increase, about a 3-fold increase, about a 4-fold increase, about a 5-fold increase, about a 6-fold increase, about a 7-fold increase, about a 8-fold increase, about a 9-fold increase, about a 10-fold increase, about a 11-fold increase, about a 12-fold increase, about a 13-fold increase, about a 14-fold increase, about a 15-fold increase, about a 16-fold increase, about a 17-fold increase, about a 18-fold increase, about a 19-fold increase, about a 20-fold increase, or more than about a 20-fold increase in the binding of anti-5T4 antibody to tumor cells relative to an isotype control, for example, as measured by flow cytometry. In certain embodiments, (i) tumor cells in a subject, (ii) tumor cells in a tumor in a subject, and / or (iii) tumor cells in a tumor sample (e.g., a tumor tissue sample) from a subject exhibit about the same or higher levels of cell membrane expression of 5T4 as a low 5T4-expressing control, e.g., OVCAR-3, but about the same or lower levels of cell membrane expression of 5T4 as a high 5T4-expressing control, e.g., Caki-2.

[0020] In some embodiments, (i) tumor cells in a subject, (ii) tumor cells in a tumor in a subject, and / or (iii) tumor cells in a tumor sample (e.g., a tumor tissue sample) from a subject exhibit low membrane expression of 5T4, e.g., as measured by flow cytometry. For example, in some embodiments, the tumor cells exhibit lower or about the same level of membrane expression of 5T4 than a suitable low 5T4 expression control, e.g., OVCAR-3, and / or lower membrane expression of 5T4 than a suitable high 5T4 expression control, e.g., Caki-2.

[0021] In some embodiments, (i) tumor cells in a subject, (ii) tumor cells in a tumor in a subject, and / or (iii) tumor cells in a tumor sample (e.g., a tumor tissue sample) from a subject exhibit moderate membrane expression of 5T4, e.g., as measured by flow cytometry. For example, in some embodiments, the tumor cells exhibit higher cell membrane expression of 5T4 than a suitable low 5T4-expressing control, e.g., OVCAR-3, and / or lower cell membrane expression of 5T4 than a suitable high 5T4-expressing control, e.g., Caki-2.

[0022] In some embodiments, (i) tumor cells in a subject, (ii) tumor cells in a tumor in a subject, and / or (iii) tumor cells in a tumor sample (e.g., a tumor tissue sample) from a subject exhibit high membrane expression of 5T4, e.g., as measured by flow cytometry. For example, in some embodiments, the tumor cells exhibit higher or about the same level of membrane expression of 5T4 than a suitable high 5T4-expressing control, e.g., Caki-2, and / or higher than a suitable low 5T4-expressing control, e.g., OVCAR-3.

[0023] In some embodiments, (i) tumor cells in a subject, (ii) tumor cells in a tumor in a subject, and / or (iii) tumor cells in a tumor sample (e.g., a tumor tissue sample) from a subject exhibit lower, about the same, or higher levels of 5T4 membrane expression than a glioblastoma cell line, e.g., as measured by flow cytometry. In some embodiments, the glioblastoma cell line is U-138, U87, or GMS-10.

[0024] In certain embodiments of any of the foregoing methods, the 5T4 targeting agent is or includes an antibody, a bispecific T cell engager (BiTE), an immune cell (e.g., a T cell, e.g., a CAR-T cell), or a vaccine.

[0025] In some embodiments, the 5T4 targeting agent does not ablate or otherwise inhibit cancer stem cells. In some embodiments, the 5T4 targeting agent does not primarily mediate the treatment of glioblastoma in a subject by ablating or otherwise inhibiting cancer stem cells.

[0026] Various features and aspects of the invention are described in further detail below.

[0027] I. Definition Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For purposes of the present invention, the following terms are defined below.

[0028] As used herein, the terms "a" or "an" can mean one or more. For example, a statement such as "treatment with a superantigen and an immune cell" can mean treatment with one superantigen and one immune cell; with more than one superantigen and one immune cell; with one superantigen and more than one immune cell; or with more than one superantigen and more than one immune cell.

[0029] As used herein, unless otherwise indicated, the term "antibody" is understood to mean an intact antibody (e.g., an intact monoclonal antibody) or an antigen-binding fragment of an antibody, such as an optimized, engineered or chemically conjugated intact antibody or an antigen-binding fragment of an antibody (e.g., a phage display antibody, including a fully human antibody, a semi-synthetic antibody or a fully synthetic antibody). An example of an optimized antibody is an affinity matured antibody. Examples of engineered antibodies are Fc-optimized antibodies, antibodies engineered to reduce immunogenicity and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab') 2, Fv, single chain antibodies (e.g., scFv), minibodies and diabodies. Antibodies conjugated to a toxin moiety are examples of chemically conjugated antibodies.

[0030] As used herein, the terms "cancer" and "cancerous" are understood to mean the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, melanoma, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphocytic malignancies. More specific examples of cancer include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer, such as small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer, such as gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, brain cancer, retinoblastoma, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, as well as head and neck cancer, and gum or tongue cancer. Cancer includes cancers or cancerous cells, for example, cancer can include multiple individual cancers or cancerous cells, such as leukemia, or a tumor that includes multiple related cancers or cancerous cells.

[0031] As used herein, the term "cancer stem cell" or "CSC" refers to a subpopulation of cancer cells that has the characteristics normally associated with stem cells, such as self-renewal, the ability to differentiate into many cell types and generate many cancer cell types, an indefinite life span, and abbreviated cell cycle control.CSCs are tumorigenic and can form tumors from a small number of cells in animal tumor models.CSCs can persist in tumors as a separate subpopulation and can cause recurrence and metastasis by generating new tumors.

[0032] As used herein, the term "treatment-resistant" refers to a cancer that does not respond or no longer responds to treatment. In some embodiments, a treatment-resistant cancer can be resistant to treatment before or at the beginning of treatment. In other embodiments, a treatment-resistant cancer can become resistant during or after treatment. A treatment-resistant cancer is also referred to as a resistant cancer. As used herein, the term "recurrence" or "relapse" refers to the return of treatment-resistant cancer or signs and symptoms of treatment-resistant cancer after a positive response to a previous treatment (e.g., reduction in tumor burden, reduction in tumor volume, reduction in tumor metastasis, or changes in biomarkers that indicate a positive response to treatment).

[0033] As used herein, the term "immunogen" is a molecule that provokes (evokes, induces or causes) an immune response. This immune response may include antibody production, activation of specific cells, such as specific immunologically competent cells, or both. Immunogens can be derived from many types of substances, including, but not limited to, biologically derived molecules, such as proteins, subunits of proteins, killed or inactivated whole cells or lysates, synthetic molecules, and a variety of other agents, both biological and non-biological. It is understood that essentially any macromolecule, including naturally occurring macromolecules or those produced by recombinant DNA approaches, such as virtually any protein, can serve as an immunogen.

[0034] As used herein, the term "immunogenicity" refers to the ability of an immunogen to induce (cause, induce or cause) an immune response. Different molecules can have different degrees of immunogenicity, and it is known that a molecule with higher immunogenicity compared to another molecule can, for example, induce (cause, induce or cause) a higher immune response than an agent with lower immunogenicity.

[0035] As used herein, the term "antigen" refers to a molecule that is recognized by an antibody, a specific immunologically competent cell, or both. Antigens can be derived from many types of substances, including, but not limited to, biologically derived molecules, such as proteins, subunits of proteins, nucleic acids, lipids, killed or inactivated whole cells or lysates, synthetic molecules, and a variety of other agents, both biological and non-biological.

[0036] As used herein, the term "antigenicity" relates to the ability of an antigen to be recognized by antibodies, specific immunologically competent cells, or both.

[0037] As used herein, the term "epitope spreading" refers to the diversification of the epitope specificity of an immune response from an initial epitope-specific immune response directed against an antigen to other epitopes on the antigen (intramolecular spreading) or other antigens (intermolecular spreading). Epitope spreading allows the subject's immune system to determine additional target epitopes that are not initially recognized by the immune system in response to the original treatment protocol, while reducing the likelihood of escape variants in the tumor population, thereby affecting disease progression.

[0038] As used herein, the term "immune response" refers to a response to a stimulus by cells of the immune system, such as B cells, T cells (CD4+ or CD8+), regulatory T cells, antigen-presenting cells, dendritic cells, monocytes, macrophages, NKT cells, NK cells, basophils, eosinophils, or neutrophils. In some embodiments, the response is specific to a particular antigen ("antigen-specific response") and refers to a response by CD4+ T cells, CD8+ T cells, or B cells via antigen-specific receptors. In some embodiments, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can depend on the nature of the immune cells responding.

[0039] As used herein, the term "major histocompatibility complex" or "MHC" refers to a specific cluster of genes, many of which encode evolutionarily related cell surface proteins involved in antigen presentation and are key determinants of tissue compatibility. Class I MHC, or MHC-I, is primarily involved in the regulation of the CD8 + T lymphocytes (CD8 + Class II MHC or MHC-II functions primarily in antigen presentation to CD4 + T lymphocytes (CD4 + It functions in antigen presentation to T cells.

[0040] As used herein, the term "derived", e.g., "derived from", includes, but is not limited to, wild-type molecules derived from biological hosts such as bacteria, viruses, and eukaryotic cells and organisms, as well as modified molecules, e.g., molecules modified by chemical means or produced in recombinant expression systems.

[0041] As used herein, the term "seroreactive", "seroreaction" or "seroreactivity" is understood to mean the ability of an agent, such as a molecule, to react with antibodies in the serum of a mammal, such as, but not limited to, a human. This includes reactions with all types of antibodies, including antibodies specific for the molecule and non-specific antibodies that bind to the molecule, regardless of whether the antibodies inactivate or neutralize the agent. As is known in the art, different agents may have different seroreactivities with respect to each other, where an agent with lower seroreactivity than another will, for example, react with fewer antibodies and / or have a lower affinity and / or avidity for antibodies than an agent with higher seroreactivity. This may also include the ability of an agent to elicit an antibody immune response in an animal, such as a mammal, e.g., a human.

[0042] As used herein, the term "soluble T cell receptor" or "soluble TCR" is understood to mean a "soluble" T cell receptor that comprises a full-length (e.g., membrane-bound) receptor chain, except that the transmembrane region of the receptor chain is deleted or mutated such that the receptor does not insert into, traverse or otherwise associate with a membrane when expressed by a cell. A soluble T cell receptor may comprise only the extracellular domain of the wild-type receptor or an extracellular fragment of that domain (e.g., lacking the transmembrane and cytoplasmic domains).

[0043] As used herein, the term "superantigen" is understood to mean a class of molecules that stimulate a subset of T cells by binding to MHC class II molecules and the Vβ domain of the T cell receptor, thereby activating T cells that express a specific Vβ gene segment. The term includes wild-type, naturally occurring superantigens, such as those isolated from specific bacteria or expressed from their unmodified genes, and modified superantigens, where, for example, the DNA sequence encoding the superantigen has been modified, for example, by genetic engineering, to produce a fusion protein with a targeting moiety, and / or to modify a specific property of the superantigen, such as, but not limited to, its MHC class II binding (e.g., reducing affinity) and / or its seroreactivity, and / or its immunogenicity, and / or antigenicity (e.g., reducing its seroreactivity).The definitions are provided herein or in the following U.S. patents and applications: U.S. Patent Nos. 5,858,363, 6,197,299, 6,514,498, 6,713,284, 6,692,746, 6,632,640, 6,632,441, 6,447,777, 6,399,332, 6,340,461, and 6,338,845. , No. 6,251,385, No. 6,221,351, No. 6,180,097, No. 6,126,945, No. 6,042,837, No. 6,713,284, No. 6,632,6 No. 40, No. 6,632,441, No. 5,859,207, No. 5,728,388, No. 5,545,716, No. 5,519,114, No. 6,926,694, No. 7,12 Nos. 5,554, 7,226,595, 7,226,601, 7,094,603, 7,087,235, 6,835,818, 7,198,398, 6,774,218, 6,913,755, 6,969,616 and 6,713,284, U.S. Patent Application Nos. 2003 / 0157113, 2003 / 012414 2, 2002 / 0177551, 2002 / 0141981, 2002 / 0115190, 2002 / 0051765 and 2001 / 0046501, and PCT International Publication No. WO / 03 / 094846, as well as wild-type and modified superantigens and any immunologically reactive variants and / or fragments thereof.

[0044] As used herein, the term "targeting moiety" refers to any structure, molecule or moiety that can bind to a cell molecule, such as a cell surface molecule, preferably a disease-specific molecule, such as an antigen preferentially expressed on a cancer (or cancerous) cell. Exemplary targeting moieties include, but are not limited to, antibodies (including antigen-binding fragments thereof), soluble T-cell receptors, interleukins, hormones and growth factors.

[0045] As used herein, the term "tumor-targeted superantigen" or "TTS" or "cancer-targeted superantigen" is understood to mean a molecule comprising one or more superantigens covalently linked (either directly or indirectly) to one or more targeting moieties.

[0046] As used herein, the term "T cell receptor" is understood to mean a receptor specific to T cells, and includes the understanding of the term known in the art.The term also includes the receptor that comprises the disulfide-linked heterodimer of highly variable α or β chains that are expressed on cell membrane as a complex with the invariant CD3 chain, and the receptor that is made of variable γ and δ chains that are expressed on cell membrane as a complex with CD3 on a subset of T cells.

[0047] As used herein, the terms "therapeutically effective amount" and "effective amount" are understood to mean an amount of an active agent, such as a medicament active agent or pharmaceutical composition, that produces at least some effect in treating a disease or condition. The effective amount of the medicament active agent(s) used to implement the present invention for therapeutic treatment varies according to the mode of administration, the age, weight and general health of the subject. The effective amount may be administered in one or more administrations, applications or doses, and is not intended to be limited to a particular formulation or route of administration.

[0048] As used herein, the terms "subject" and "patient" refer to an organism that is treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably, humans.

[0049] As used herein, the terms "treat", "treating" and "treatment" are understood to mean the treatment of disease in a mammal, e.g., a human. The terms include (a) inhibiting disease, i.e., preventing the onset of disease; and (b) alleviating disease, i.e., causing regression of the disease state; and (c) curing disease. When used in the context of therapeutic treatment, the terms "prevent" or "block" are understood to completely prevent or inhibit, or not completely prevent or inhibit (e.g., partially prevent or inhibit) a given action, act, activity or event.

[0050] As used herein, the term "inhibiting cancer growth" is understood to mean measurably slowing, stopping or reversing the growth rate of cancer or cancerous cells in vitro or in vivo. Desirably, the growth rate is slowed by 20%, 30%, 50% or 70% or more as determined using an appropriate assay for determining cell growth rate. Typically, reversal of growth rate is achieved by initiating or promoting necrotic or apoptotic mechanisms of cell death in neoplastic cells, resulting in shrinkage of the neoplasm.

[0051] As used herein, the terms "variant", "variants", "modified", "altered", "mutated" and the like are understood to mean proteins or peptides and / or other agents and / or compounds that differ from a reference protein, peptide or other compound. Variants in this sense are described in more detail below and elsewhere. For example, the changes in the nucleic acid sequence of the variant may be silent, e.g., the changes may not alter the amino acids encoded by the nucleic acid sequence. If the changes are limited to this type of silent changes, the variant encodes a peptide having the same amino acid sequence as the reference peptide. The changes in the nucleic acid sequence of the variant may alter the amino acid sequence of the peptide encoded by the reference nucleic acid sequence. Such nucleic acid changes may result in substitutions, additions, deletions, fusions and / or truncations of amino acids in the protein or peptide encoded by the reference sequence, as described below. Generally, the differences in amino acid sequence are limited, so that the sequences of the reference and variant are similar overall and identical in many regions. A variant and a reference protein or peptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and / or truncations, which may be present in any combination. A variant may also be a fragment of a protein or peptide of the invention that differs from the reference protein or peptide sequence by being shorter than the reference sequence, for example by a terminal or internal deletion. Another variant of a protein or peptide of the invention also includes a protein or peptide that retains essentially the same function or activity as the reference protein or peptide.A variant may also be one in which (i) one or more amino acid residues are replaced by a conserved or non-conserved amino acid residue, where such replaced amino acid residue may or may not be one encoded by the genetic code, or (ii) one or more amino acid residues include a substituent group, or (iii) the mature protein or peptide is fused to another compound, such as a compound that increases the half-life of the protein or peptide (e.g., polyethylene glycol), or (iv) additional amino acids are fused to the mature protein or peptide, such as a leader sequence or secretory sequence or a sequence used for purification of the mature protein or peptide. Variants may be made by mutagenesis techniques and / or modification mechanisms, such as chemical modification, fusion, adjunct, including those applied to nucleic acids, amino acids, cells or organisms, and / or may be made by recombinant means.

[0052] As used herein, the term "sequential administration" and related terms refer to the administration of at least one agent (e.g., a superantigen conjugate) and at least one additional agent (e.g., immune cells), including staggered administration (i.e., staggered in time) and variations in dosage of these agents. This includes one agent being administered before, overlapping (partially or completely), or after the administration of another agent. The term "sequential administration" and related terms also include the administration of at least one superantigen, an immune cell, and one or more optional additional compounds, such as corticosteroids, immunomodulators, and other agents designed to reduce potential immune reactivity to the superantigen conjugate administered to the subject.

[0053] As used herein, the terms "systemic" and "systemically" are understood in the context of administration to mean administration of an agent such that the agent is exposed to at least one system associated with the entire body, including but not limited to the circulatory system, immune system, and lymphatic system, rather than only in a localized portion of the body, such as but not limited to, in a tumor. Thus, for example, a systemic treatment or a systemically administered agent is one in which at least one system associated with the entire body is exposed to the treatment or agent as opposed to, or different from, only the target tissue.

[0054] As used herein, the term "parenteral administration" includes any form of administration in which a compound is absorbed into a subject without absorption via the intestine. Exemplary parenteral administrations for use in the present invention include, but are not limited to, intramuscular, intravenous, intraperitoneal, or intraarticular administration.

[0055] When the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0056] In various places in this specification, values ​​are disclosed in groups or ranges. The description is specifically intended to include each and every individual subcombination of the members of such groups and ranges. For example, an integer in the range of 0 to 40 is specifically intended to disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 individually, and an integer in the range of 1 to 20 is specifically intended to disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 individually.

[0057] Throughout this description, when compositions are described as having, including, or comprising specific components, or when processes and methods are described as having, including, or comprising specific steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods according to the invention that consist essentially of or consist of the recited process steps.

[0058] In this application, when an element or component is said to be included in and / or selected from a list of described elements or components, it is to be understood that the element or component can be any one of the described elements or components, or the element or component can be selected from a group consisting of two or more of the described elements or components.

[0059] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether express or implied herein, can be combined in various ways without departing from the spirit and scope of the present invention. For example, when reference is made to a particular compound, the compound can be used in various aspects of the compositions of the present invention and / or in the methods of the present invention, unless otherwise understood from the context. That is, in this application, the embodiments are described and shown in a manner that allows for clear and concise application as described and illustrated, but it is intended and understood that the embodiments can be variously combined or separated without departing from the present teachings and invention(s). For example, it is understood that all features described and shown herein can be applicable to all aspects of the invention(s) described and shown herein.

[0060] The phrase "at least one of" should be understood to include each of the recited items following the phrase and various combinations of two or more of the recited items individually, unless otherwise understood from the context and application. The phrase "and / or" with respect to three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0061] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should generally be understood to be open-ended and open-ended, e.g., not excluding additional, unrecited elements or steps, unless specifically stated or understood from the context to the contrary.

[0062] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0063] The use of any and all examples or exemplary terms herein, such as "such as" or "including," is intended merely to better describe the invention and does not pose a limitation on the scope of the invention unless and until claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0064] II. 5T4 targeting agent As described above, the present invention is based in part on the discovery that glioblastoma tissue can express significant levels of 5T4 (particularly significant levels of membrane 5T4). As a result, 5T4 targeting agents can be used to target glioblastoma cells for therapy.

[0065] 5T4 is a 72 kDa oncofetal glycoprotein expressed on certain cancers. For example, immunohistochemical analysis shows that 5T4 expression is an indicator of poor prognosis in colorectal, ovarian, gastric and non-small cell lung cancers. The nucleotide and amino acid sequences of human 5T4 are available in GenBank under accession number Z29083. Sequence analysis identified 5T4 as a member of the leucine-rich repeat (LRR) family of proteins. The protein contains an extracellular domain that contains two leucine-rich repeat (LRR) regions with a short cytoplasmic tail of 44 amino acids and associated cysteine-containing flanking regions. All of the seven consensus NxS / TN glycosylation sites in the extracellular domain are glycosylated with a combination of complex glycans that include two high mannose chains and five sialylated, bi- to tetra-antennary complex chains with minor core fucosylation.

[0066] The term "5T4 targeting agent" as used herein refers to an agent that directs treatment to 5T4 antigen. Targeting agent can, for example, bind to 5T4 or alternatively, can, for example, mediate the production of additional molecules that bind to 5T4. For example, the term 5T4 targeting agent includes vaccines that mediate the production of immune response to 5T4 antigen in a subject.

[0067] In some embodiments, the targeting agent binds (e.g., directly binds) to 5T4. In such embodiments, the targeting agent may alternatively be referred to as a binding agent. The term "binding agent" as used herein refers to an agent that preferentially or specifically binds to an analyte of interest (e.g., 5T4). The terms "preferentially bind" or "specifically bind", when used in reference to a binding agent, refer to an agent that binds and / or associates with the particular target analyte to which it binds (i) more stably, (ii) more rapidly, (iii) with greater affinity, (iv) with greater duration, or (v) or a combination of any two or more of (i)-(iv), along with a molecule other than the target analyte. For example, a binding agent that specifically or preferentially binds to a target analyte is, for example, a binding domain that binds to the target analyte with greater affinity, avidity, more readily, and / or with greater duration than it binds to a different analyte. The binding agent may have an affinity for the analyte of about 100nM, 50nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM, 0.1nM, or 0.01nM or stronger, as measured by surface plasmon resonance. For example, the binding agent may have an affinity for the analyte in the range of about 0.01nM to about 100nM, about 0.1nM to about 100nM, or about 1nM to about 100nM. It is understood that a binding agent that preferentially binds to a first target analyte may or may not preferentially bind to a second target analyte. Thus, "preferential binding" does not necessarily require (although it may include) exclusive binding.

[0068] Exemplary binding agents include enzymes (e.g., those that bind substrates and inhibitors), antibodies (e.g., those that bind antigens), antigens (e.g., those that bind target antibodies), receptors (e.g., those that bind ligands), ligands (e.g., those that bind receptors), nucleic acid single-stranded polymers (e.g., those that bind nucleic acid molecules to form, for example, DNA-DNA, RNA-RNA or DNA-RNA duplexes), and synthetic molecules that bind target analytes. Natural, synthetic, semi-synthetic, and genetically modified macromolecules can be used as binding agents. Binding agents include biological binding agents, such as antibodies, aptamers, receptors, enzymes, or nucleic acids.

[0069] A. Antibodies Generally, antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains) and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are linked by interchain disulfide bonds. The immunoglobulin heavy chains are linked by interchain disulfide bonds. The light chains contain one variable region (V L ) and one constant region (C L The heavy chain consists of one variable region (V H ) and at least three constant regions (CH 1 , C.H. 2 and C.H. 3 The variable region determines the binding specificity of the antibody.

[0070] Each variable region contains three hypervariable regions, known as complementarity determining regions (CDRs), flanked by four relatively conserved regions, known as framework regions (FRs). The extent of the FRs and CDRs has been defined (Kabat, EA, et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, US Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. MOL. BIOL. 196:901-917). CDRs 1 , CDR 2 and CDR 3 The three CDRs, termed CDRs, contribute to the antibody binding specificity. Naturally occurring antibodies have been used as starting material for genetically engineered antibodies, such as chimeric and humanized antibodies.

[0071] As used herein, an antibody can include an intact antibody (e.g., an intact monoclonal antibody) or a fragment thereof, such as an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody) or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), e.g., an intact antibody, antigen-binding fragment or Fc fragment that has been modified, engineered or chemically conjugated. Examples of antigen-binding fragments include Fab, Fab', (Fab') 2 Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0072] The antibody molecule may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; in particular, from, for example, IgG1, IgG2, IgG3 and IgG4 (e.g., human) heavy chain constant regions. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, kappa or lambda (e.g., human) light chain constant regions. The constant region may be modified, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function and / or complement function). In one embodiment, the antibody has effector function and may fix complement. In another embodiment, the antibody does not recruit effector cells or does not fix complement. In another embodiment, the antibody has a reduced or no ability to bind to Fc receptors. For example, it may be an isotype or subtype, fragment or other variant that does not support binding to an Fc receptor, eg, it has the Fc receptor binding region mutagenized or deleted.

[0073] In certain embodiments, the antibody has a K of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or less for human 5T4 as measured using standard binding assays, such as surface plasmon resonance or biolayer interferometry. D Combine with.

[0074] The antibodies disclosed herein may be further optimized (e.g., affinity matured) to improve biochemical properties such as affinity and / or specificity, to improve biophysical properties such as aggregation, stability, precipitation and / or non-specific interactions, and / or to reduce immunogenicity. Affinity maturation procedures are within the ordinary skill in the art. For example, diversity may be introduced into the immunoglobulin heavy chain and / or immunoglobulin light chain by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis and / or site-directed mutagenesis.

[0075] In some embodiments, the isolated human antibody contains one or more somatic mutations. In these cases, the antibody can be modified relative to human germline sequences to optimize the antibody (i.e., a process called germline engineering).

[0076] Generally, an optimized antibody has at least the same or substantially the same affinity for an antigen as the non-optimized (or parent) antibody from which it is derived. Preferably, an optimized antibody has a higher affinity for an antigen when compared to the parent antibody.

[0077] In some embodiments, the antibody can be conjugated to an effector agent, such as a small molecule toxin or a radionuclide, using standard in vitro conjugation chemistry. If the effector agent is a polypeptide, the antibody can be chemically conjugated to the effector as a fusion protein or linked to the effector. The construction of fusion proteins is within the ordinary skill of the art.

[0078] The antibody can be conjugated to an effector moiety, such as a small molecule toxin or a radionuclide, using standard in vitro conjugation chemistry. If the effector moiety is a polypeptide, the antibody can be chemically conjugated to the effector as a fusion protein or linked to the effector. The construction of a fusion protein is within the ordinary skill of the art.

[0079] In some embodiments, the antibody may be a bispecific or multispecific antibody. A bispecific antibody is an antibody that has binding specificities for at least two different epitopes. An exemplary bispecific antibody may bind to two different epitopes of a single antigen. Other such antibodies may combine a binding site for a first antigen with a binding site for a second antigen. Alternatively, an antigen-specific arm may be combined with an arm that binds to a triggering molecule on a white blood cell, such as a T cell receptor molecule (e.g., CD3) or an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and Fc gamma RIII (CD16). Bispecific antibodies may also be used to localize cytotoxic agents to infected cells, for example, an antibody may have an antigen-binding arm and an arm that binds to a cytotoxic agent (e.g., saporin, anti-interferon alpha, vinca alkaloid, ricin A chain, methotrexate, or a radioisotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Methods for making bispecific antibodies are known in the art. One example of a bispecific antibody is a bispecific T cell engager (BiTE®). BiTEs are bispecific antibodies that direct the cytotoxic activity of T cells to targeted disease cells. BiTEs are typically fusion proteins that contain two single-chain variable fragments (scFv), one of which binds to T cells via the CD3 receptor and the other to infected cells via a specific molecule (e.g., 5T4). BiTEs form a link between T cells and target cells, allowing T cells to exert cytotoxic activity against target cells, for example, by producing perforin and granzymes independent of the presence of MHC I or costimulatory molecules.

[0080] Exemplary anti-5T4 antibodies include those described in U.S. Patent No. US7,531,648, U.S. Patent Publication Nos. US20060088522, US20100173382, US20160185859, US20160304617, US20180162937, US20190194346, and US20190374651, and International (PCT) Publication Nos. Examples of such antibodies include those described in WO9855607, WO2001036486, WO2003038098, WO2006031653, WO2007106744, WO2012131527, WO2013068874, WO2014137931, WO2016022939, WO2020076992, and WO2021048423. Additional anti-5T4 antibodies are commercially available, for example, from R&D Systems (MN, USA), Lifespan Biosciences, Inc (WA, USA), and Creative Biolabs (NY, USA).

[0081] In some embodiments, the anti-5T4 antibody comprises an anti-5T4 variable region sequence fused to the constant region sequence of the murine IgG1 / κ antibody C242, e.g., the anti-5T4 antibody comprises a chimeric heavy chain corresponding to residues 1-222 of SEQ ID NO: 7 and a chimeric 5T4 Fab light chain corresponding to residues 223-225 of SEQ ID NO: 7. The two protein chains are held together by non-covalent interactions between the Fab heavy and light chains.

[0082] B. Superantigen Conjugates 1. Superantigens Superantigens are bacterial, viral and human engineered proteins that can activate T lymphocytes, for example at picomolar concentrations. Superantigens can also activate a large subset of T lymphocytes (T cells). Superantigens can bind to major histocompatibility complex I (MHCI) without processing, and in particular to conserved regions outside the antigen-binding groove on MHC class II molecules (e.g., on monocytes), which avoids most of the polymorphism in conventional peptide-binding sites. Superantigens can also bind to the Vβ chain of the T cell receptor (TCR) rather than to the hypervariable loops of the T cell receptor. Examples of bacterial superantigens include, but are not limited to, Staphylococcal enterotoxins (SE), Streptococcus pyogenes exotoxins (SPE), Staphylococcus aureus toxic shock syndrome toxins (TSST-1), Streptococcal mitogenic exotoxins (SME), Streptococcal superantigens (SSA), Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin A (SEB), and Staphylococcal enterotoxin E (SEE).

[0083] Polynucleotide sequences encoding many superantigens have been isolated and cloned, and superantigens expressed from these or modified (again engineered) polynucleotide sequences have been used in anti-cancer therapy (see naptumomab estafenatox / ANYARA® below). The superantigens expressed by these polynucleotide sequences can be wild-type superantigens, modified superantigens, or wild-type or modified superantigens conjugated or fused to a targeting moiety. The superantigens can be administered to a mammal, such as a human, directly, e.g., by injection, or can be delivered, e.g., by exposing the patient's blood to the superantigen ex vivo, or by placing a gene encoding the superantigen in the mammal to be treated (e.g., via known gene therapy methods and vectors, e.g., cells that contain and are capable of expressing the gene) and expressing the gene in the mammal.

[0084] Examples of superantigens and their administration to mammals are found in the following United States patents and patent applications: U.S. Patent Nos. 5,858,363, 6,197,299, 6,514,498, 6,713,284, 6,692,746, 6,632,640, 6,632,441, 6,447,777, 6,399, and 6,450,772. ,332, No. 6,340,461, No. 6,338,845, No. 6,251,385, No. 6,221,351, No. 6,180,097, No. 6,126, No. 945, No. 6,042,837, No. 6,713,284, No. 6,632,640, No. 6,632,441, No. 5,859,207, No. 5,728,38 No. 8, No. 5,545,716, No. 5,519,114, No. 6,926,694, No. 7,125,554, No. 7,226,595, No. 7,226,601 No. 7,094,603, No. 7,087,235, No. 6,835,818, No. 7,198,398, No. 6,774,218, No. 6,913,755 Nos. 6,969,616 and 6,713,284, U.S. Patent Application Nos. 2003 / 0157113, 2003 / 0124142, 2002 / 0177551, 2002 / 0141981, 2002 / 0115190 and 2002 / 0051765, and PCT International Publication No. WO / 03 / 094846.

[0085] 2. Modified superantigens Within the scope of the present invention, superantigens can be engineered in a variety of ways, including modifications that retain or enhance the ability of the superantigen to stimulate T lymphocytes, and that may, for example, modify other aspects of the superantigen, such as, for example, its seroreactivity or immunogenicity. Modified superantigens include synthetic molecules that have superantigenic activity (i.e., the ability to activate subsets of T lymphocytes).

[0086] It is contemplated that various changes can be made to the polynucleotide sequence encoding the superantigen without appreciable loss of the bioavailability or activity of the superantigen, i.e., induction of T cell responses that result in tumor cell cytotoxicity.Furthermore, the affinity of the superantigen to MHC class II molecules can be reduced while minimizing the effect on the cytotoxicity of the superantigen.For example, this can help reduce the toxicity that would otherwise occur if the superantigen retains its wild-type ability to bind to MHC class II antigens (as in such cases, class II expressing cells, e.g., immune system cells, can also be affected by the response to the superantigen).

[0087] Techniques for modifying superantigens (e.g., polynucleotides and polypeptides), including making synthetic superantigens, are well known in the art and include, for example, PCR mutagenesis, alanine scanning mutagenesis and site-directed mutagenesis (see U.S. Pat. Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377 and 5,789,166).

[0088] In some embodiments, a superantigen may be modified such that its seroreactivity is reduced compared to a reference wild-type superantigen, but its ability to activate T cells is retained or enhanced compared to the wild-type. One technique for creating such modified superantigens includes substituting a specific amino acid in a specific region from one superantigen for another. This is possible because many superantigens, including but not limited to SEA, SEE and SED, share sequence homology in specific regions associated with specific functions (Marrack and Kappler (1990) SCIENCE 248(4959): 1066; see also FIG. 1, which shows the regions of homology between different wild-type and engineered superantigens). For example, in some embodiments of the present invention, a superantigen that has a desired T cell activation-inducing response but an undesirably high seroreactivity is modified such that the resulting superantigen retains its T cell activation ability but has a reduced seroreactivity.

[0089] It is known and understood by those skilled in the art that human serum usually contains antibodies of different titers against superantigens. For staphylococcal superantigens, for example, the relative titers are TSST-1>SEB>SEC-1>SE3>SEC2>SEA>SED>SEE. As a result, the seroreactivity of, for example, SEE (Staphylococcal Enterotoxin E) is lower than that of, for example, SEA (Staphylococcal Enterotoxin A). Based on this data, those skilled in the art may preferentially administer a low titer superantigen, for example SEE, instead of a high titer superantigen, for example SEB (Staphylococcal Enterotoxin B). However, as has also been discovered, different superantigens have different T cell activation properties relative to each other, and for wild-type superantigens, the best T cell activation superantigens often also have undesirably high seroreactivity.

[0090] These relative potencies sometimes correspond to potential problems with seroreactivity, such as problems with neutralizing antibodies. Thus, the use of low-potency superantigens, such as SEA or SEE, can be useful to reduce or avoid seroreactivity of parenterally administered superantigens. Low-potency superantigens have low seroreactivity, for example, as measured by typical anti-superantigen antibodies in the general population. In some instances, low-potency superantigens can also have low immunogenicity. Such low-potency superantigens can be modified to retain their low potency, as described herein.

[0091] Approaches to modify superantigens can be used to create superantigens with both desired T cell activation properties and reduced seroreactivity, and in some cases, reduced immunogenicity as well. Considering that certain regions of homology between superantigens are related to seroreactivity, it is possible to engineer recombinant superantigens with desired T cell activation and desired seroreactivity and / or immunogenicity. Furthermore, the protein sequences and immunological cross-reactivity of superantigens or staphylococcal enterotoxins are divided into two related groups. One group consists of SEA, SEE and SED. The second group is SPEA, SEC and SEB. Therefore, it is possible to select low titer superantigens to reduce or eliminate cross-reactivity with endogenous antibodies against high titer or staphylococcal enterotoxins.

[0092] Regions in superantigens which are believed to play a role in seroreactivity include, for example, region A, which includes amino acid residues 20, 21, 22, 23, 24, 25, 26, and 27; region B, which includes amino acid residues 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 49; region C, which includes amino acid residues 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84; region D, which includes amino acid residues 187, 188, 189, and 190; and region E, which includes amino acid residues 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, and 227 (see U.S. Pat. No. 7,125,554 and FIG. 1 herein). It is therefore contemplated that these regions may be mutated, for example using amino acid substitutions, to generate superantigens with altered seroreactivity.

[0093] The polypeptide or amino acid sequences for the above-mentioned superantigens can be obtained from any sequence databank, such as the Protein Databank and / or GenBank. Exemplary GenBank accession numbers include, but are not limited to, SEE: P12993; SEA: P013163; SEB: P01552; SEC1: P01553; SED: P20723; and SEH: AAA19777.

[0094] In certain embodiments of the invention, the wild-type SEE sequence (SEQ ID NO:1) or wild-type SEA sequence (SEQ ID NO:2) may be modified to substitute any of the amino acids in the identified regions A-E (see FIG. 1) with other amino acids. Such substitutions include, for example, K79, K81, K83 and D227 or K79, K81, K83, K84 and D227, or for example, K79E, K81E, K83S and D227S or K79E, K81E, K83S, K84S and D227A. In certain embodiments, the superantigen is SEA / E-120 (SEQ ID NO:3; see also U.S. Pat. No. 7,125,554), SEA D227A(SEQ ID NO:4; see also U.S. Pat. No. 7,226,601) or SEA / E-18 (SEQ ID NO:10; see also U.S. Pat. No. 7,125,554).

[0095] Biologically functional equivalents of polynucleotides encoding naturally occurring or referenced superantigens may include polynucleotides engineered to contain different sequences while retaining the ability to encode naturally occurring or referenced superantigens. This can be achieved due to the degeneracy of the genetic code, i.e., the presence of multiple codons that encode the same amino acid. In one example, it is possible to introduce a restriction enzyme recognition sequence into a polynucleotide without disrupting its ability to encode protein. Other polynucleotide sequences may encode superantigens that are different from the referenced superantigen but are functionally substantially equivalent to the referenced superantigen in at least one biological property or activity (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 98% biological property or activity, such as, but not limited to, the ability to induce a T cell response that causes tumor cell cytotoxicity).

[0096] In another example, a polynucleotide can be (can code for) a superantigen that may contain larger changes but is functionally equivalent to the reference superantigen. Certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of binding ability, such as the antigen-binding region of an antibody, a binding site on a substrate molecule, a receptor, or the like, for interacting with such structures. Furthermore, conservative amino acid substitutions may not disrupt the biological activity of a protein, and consequently structural changes often do not affect the ability of a protein to perform its designed function. Thus, it is contemplated that various changes may be made in the gene and protein sequences disclosed herein while still satisfying the goals of the invention.

[0097] Amino acid substitutions can be designed to take advantage of the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and / or type of amino acid side chain substituents reveals that arginine, lysine, and / or histidine are all positively charged residues; alanine, glycine, and / or serine are all similar in size; and / or phenylalanine, tryptophan, and / or tyrosine are all generally similar in shape. Therefore, based on these considerations, arginine, lysine, and / or histidine; alanine, glycine, and / or serine; and / or phenylalanine, tryptophan, and / or tyrosine are defined herein as biologically functional equivalents. It may also be possible to introduce non-naturally occurring amino acids. Approaches for making amino acid substitutions with other naturally occurring and non-naturally occurring amino acids are described in U.S. Pat. No. 7,763,253.

[0098] With respect to functional equivalents, it is understood that in the definition of "biologically functional equivalent" protein and / or polynucleotide, the concept that there is a limited number of changes that can be made in a given part of a molecule while retaining the molecule with an acceptable level of equivalent biological activity is implicit.Therefore, biologically functional equivalents are considered to be proteins (and polynucleotides) in which selected amino acids (or codons) can be substituted without substantially affecting biological function.Functional activities include induction of T cell response to produce tumor cell cytotoxicity.

[0099] It is also contemplated that modified superantigens may be generated by replacing homologous regions of various proteins via "domain swapping," which involves the generation of chimeric molecules using different, but in this case related, polypeptides. By comparing various superantigen proteins to identify functionally relevant regions of these molecules (see, for example, FIG. 1), it becomes possible to swap relevant domains of these molecules so that the criticality of these regions to superantigen function can be determined. These molecules may have additional value in that these "chimeras" may be distinguished from natural molecules while presumably providing the same function.

[0100] In certain embodiments, the superantigen comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of a reference superantigen selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, wherein the superantigen optionally retains at least 50%, 60%, 70% 80%, 90%, 95%, 98%, 99% or 100% of the biological activity or property of the reference superantigen.

[0101] In certain embodiments, the superantigen comprises an amino acid sequence encoded by a nucleic acid that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a nucleic acid encoding a superantigen selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, wherein the superantigen optionally retains at least 50%, 60%, 70% 80%, 90%, 95%, 98%, 99% or 100% of the biological activity or property of the reference superantigen.

[0102] Sequence identity can be determined in various ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm used by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES.25:3389-3402, incorporated by reference) is adjusted for searching sequence similarity. For a discussion of basic issues in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129, fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. Search parameters for histogram, description, alignment, expect (i.e., the statistical significance threshold for reporting matches to database sequences), cutoff, matrix and filter are at default settings. The default scoring matrix used by blastp, blastx, tblastn and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, fully incorporated by reference).Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). Equivalent Blastp parameter settings can be Q=9; R=2; wink=1; and gapw=32. Searches may also be performed using NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g.: -G, cost for open gap [integer]: default = 5 for nucleotides / 11 for proteins; -E, cost for extension gap [integer]: default = 2 for nucleotides / 1 for proteins; -q, penalty for nucleotide mismatch [integer]: default = -3; -r, reward for nucleotide match [integer]: default = 1; -e, expectation value [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, dropoff for blast extension in bits (X): default = 20 for blastn / 7 for others; -X, X dropoff value for gapped alignments (in bits): default = 15 for all programs but not applicable to blastn; and -Z, final X dropoff value for gapped alignments (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, for example, the Blosum62 matrix and a Gap Opening Penalty=10 and Gap Extension Penalty=0.1).Optimal comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty); equivalent settings for protein comparisons are GAP=8 and LEN=2.

[0103] 3. Targeted superantigens To increase specificity, the superantigen is preferably conjugated to a 5T4 targeting moiety to generate a targeted superantigen conjugate. The targeting moiety is a vehicle that can be used to bind the superantigen to cancerous cells, such as the surface of cancerous cells. The targeted superantigen conjugate should retain the ability to activate many T lymphocytes. For example, the targeted superantigen conjugate should activate many T cells and direct the T cells to tissues that contain the tumor-associated antigen bound to the targeting moiety. In such a situation, specific target cells are preferentially killed, leaving the rest of the body relatively unharmed. This type of treatment is desirable because non-specific anticancer agents, such as antiproliferative chemotherapy drugs, are non-specific and kill many cells that are not associated with the tumor being treated. For example, studies with targeted superantigen conjugates have shown that inflammation accompanied by infiltration of tumor tissue by cytotoxic T lymphocytes (CTLs) increases rapidly in response to the first injection of targeted superantigens (Dohlsten et al. (1995) PROC. NATL. ACAD. SCI. USA92:9791-9795). This inflammation accompanied by infiltration of CTLs into tumors is one of the major effectors of targeted superantigen antitumor therapy.

[0104] Tumor-targeted superantigens offer immunotherapy against cancer and are therapeutic fusion proteins that contain a targeting moiety conjugated to a superantigen (Dohlsten et al. (1991) PROC. NATL. ACAD. SCI. USA 88:9287-9291; Dohlsten et al. (1994) PROC. NATL. ACAD. SCI. USA 91:8945-8949).

[0105] The targeting moiety can be in principle any structure capable of binding to 5T4. The targeting moiety can be selected from an antibody, such as an antigen-binding fragment thereof, a soluble T-cell receptor, a growth factor, an interleukin (e.g., interleukin-2), a hormone, and the like.

[0106] In certain preferred embodiments, the targeting moiety is an antibody (e.g., a Fab, F(ab) 2 , Fv, single chain antibodies, etc. Exemplary anti-5T4 antibodies include those described in U.S. Patent No. US7,531,648, U.S. Patent Publication Nos. US20060088522, US20100173382, US20160185859, US20160304617, US20180162937, US20190194346, and US20190374651, and International (PCT) Publication Nos. Examples of such antibodies include those described in WO9855607, WO2001036486, WO2003038098, WO2006031653, WO2007106744, WO2012131527, WO2013068874, WO2014137931, WO2016022939, WO2020076992, and WO2021048423. Additional anti-5T4 antibodies are commercially available, for example, from R&D Systems (MN, USA), Lifespan Biosciences, Inc (WA, USA), and Creative Biolabs (NY, USA).

[0107] An example of a tumor-targeting superantigen that can be used in the present invention is 5T4Fab-SEA. D227A(SEQ ID NO: 6) and 5T4Fab-SEA / E-120 (SEQ ID NO: 7; see Figures 2 and 3).

[0108] In a preferred embodiment, the preferred conjugate is a superantigen conjugate known as naptumomab-estafenatox / ANYARA®, which is a fusion protein of a Fab fragment of an anti-5T4 antibody and the SEA / E-120 superantigen (see FIG. 2). Naptumomab-estafenatox / ANYARA® comprises two protein chains that cumulatively comprise an engineered Staphylococcal enterotoxin superantigen (SEA / E-120) and a targeting 5T4 Fab that contains modified 5T4 variable region sequences fused to the constant region sequences of the murine IgG1 / κ antibody C242. With reference to FIG. 3, the first protein chain comprises residues 1-458 of SEQ ID NO:7 (see also SEQ ID NO:8) and comprises a chimeric 5T4 Fab heavy chain corresponding to residues 1-222 of SEQ ID NO:7 covalently linked via a GGP tripeptide linker corresponding to residues 223-225 of SEQ ID NO:7 and a SEA / E-120 superantigen corresponding to residues 226-458 of SEQ ID NO:7. The second chain comprises residues 459-672 of SEQ ID NO:7 (see also SEQ ID NO:9) and comprises a chimeric 5T4 Fab light chain. The two protein chains are held together by non-covalent interactions between the Fab heavy and light chains. Residues 1-458 of SEQ ID NO:7 correspond to residues 1-458 of SEQ ID NO:8 and residues 459-672 of SEQ ID NO:7 correspond to residues 1-214 of SEQ ID NO:9. Naptumomab estafenatox / ANYARA® comprises the proteins of SEQ ID NOs:8 and 9 held together by non-covalent interactions between a Fab heavy chain and a Fab light chain. Naptumomab estafenatox / ANYARA® induces T cell-mediated killing of cancer cells at concentrations of approximately 10 pM, and the superantigen component of the conjugate has been engineered to have low binding to human antibodies and MHC class II.

[0109] It is contemplated that other antibody-based targeting moieties can be designed, modified, expressed and purified using techniques known in the art and described in more detail below.

[0110] Another type of targeting moiety comprises soluble T cell receptor (TCR).Some forms of soluble TCR can comprise either extracellular domain only or extracellular domain and cytoplasmic domain.Other modifications of TCR can be envisioned to produce soluble TCR with transmembrane domain deleted and / or TCR engineered to be not membrane-bound, as described in US Published Application Nos. US 2002 / 119149, US 2002 / 0142389, US 2003 / 0144474 and US 2003 / 0175212, and International Publication Nos. WO2003020763; WO9960120 and WO9960119.

[0111] The targeting moiety can be conjugated to the superantigen using either recombinant techniques or chemically linking the targeting moiety to the superantigen.

[0112] It is contemplated that genes encoding superantigens linked directly or indirectly (e.g., via amino acids including linkers) to a targeting moiety can be generated and expressed using conventional recombinant DNA techniques. For example, the amino terminus of the modified superantigen can be linked to the carboxy terminus of the targeting moiety, or vice versa. For antibodies or antibody fragments that can function as targeting moieties, either the light or heavy chain can be used to generate the fusion protein. For example, for a Fab fragment, the amino terminus of the modified superantigen can be linked to the first constant domain (CH) of the antibody heavy chain. 1) to the targeting moiety. In some instances, the modified superantigen may be linked to a Fab fragment by linking the VH and VL domains to the superantigen. Alternatively, a peptide linker may be used to link the superantigen and the targeting moiety together. When a linker is used, the linker preferably comprises hydrophilic amino acid residues such as Gln, Ser, Gly, Glu, Pro, His and Arg. A preferred linker is a peptide bridge consisting of 1-10 amino acid residues, more particularly 3-7 amino acid residues. An exemplary linker is the tripeptide -GlyGlyPro-. These approaches have been used successfully in the design and manufacture of naptumomab-estafenatox / ANYARA® superantigen conjugates.

[0113] It is also contemplated that superantigens can be linked to targeting moieties via chemical linkage. Chemical linkage of superantigens to targeting moieties can require a linker, for example, a peptide linker. The peptide linker is preferably hydrophilic and exhibits one or more reactive moieties selected from amide, thioether, disulfide, etc. (see U.S. Patent Nos. 5,858,363, 6,197,299 and 6,514,498). It is also contemplated that chemical linkage can use homo- or heterobifunctional cross-linking reagents. Chemical linkage of superantigens to targeting moieties often utilizes functional groups (e.g., primary amino or carboxy groups) that are present at multiple positions in a compound.

[0114] Examples of production systems for superantigens can be found, for example, in US Pat. No. 6,962,694. C. immune cells

[0115] Immune cells include, for example, lymphocytes, such as B cells and T cells, natural killer cells (NK cells), natural killer T cells (NKT cells), myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0116] In some embodiments, the immune cells are T cells, which may be, for example, cultured T cells, such as primary T cells or T cells from cultured T cell lines, such as Jurkat, SupTi, etc., or T cells obtained from a mammal, such as the subject to be treated. If obtained from a mammal, the T cells may be obtained from many sources, such as, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may also be enriched or purified. The T cells may be any type of T cell and may be at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells, such as Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc. The cells (eg, T cells) may comprise autologous cells from the subject being treated or, alternatively, allogeneic cells from a donor.

[0117] In some embodiments, T cells bind to 5T4 via a T cell receptor. The T cell receptor can be an endogenous or recombinant T cell receptor. The T cell receptor comprises two chains, termed α- and β-chains, that combine on the surface of the T cell to form a heterodimeric receptor that can recognize MHC-restricted antigens. Each of the α- and β-chains comprises two regions, a constant region and a variable region. The variable regions of each of the α- and β-chains contain CDRs that confer antigen-binding activity and binding specificity to the T cell receptor. 1 , CDR 2 and CDR 3 Each of these amino acids defines three loops called complementarity determining regions (CDRs), known as

[0118] In some embodiments, immune cells, such as T cells or NKT cells, bind to 5T4 via a chimeric antigen receptor (CAR), i.e., the T cells or NKT cells contain an exogenous nucleotide sequence encoding the CAR. As used herein, the term "chimeric antigen receptor" or "CAR" refers to any artificial receptor that includes an antigen-specific binding portion derived from an immune receptor and one or more signaling chains. The CAR may include a single-chain variable fragment (scFv) of an antibody specific for an antigen linked to a cytoplasmic domain of a T cell signaling molecule (e.g., a T cell co-stimulatory domain (e.g., from CD28, CD137, OX40, ICOS, or CD27) in tandem with a T cell induction domain (e.g., from CD3ζ)) and / or a cytoplasmic domain of a NK cell signaling molecule (e.g., DNAX-activating protein 12 (DAP12)) via a hinge and transmembrane region. T cells that express a chimeric antigen receptor are referred to as CAR T cells, NK cells that express a chimeric antigen receptor are referred to as CAR NK cells, and NKT cells that express a chimeric antigen receptor are referred to as CAR NKT cells.

[0119] Exemplary 5T4-targeted CAR T cells are described in U.S. Patent Publication Nos. US20200407461 and US20200010560 and International (PCT) Publication No. WO2016022939.

[0120] CAR T cells can be produced using methods known in the art. T cells can be obtained from several sources, such as peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, tumors and T cell lines. For example, T cells can be obtained by any of several techniques known to those skilled in the art, such as Ficoll. TMCells may be obtained from a unit of blood collected from a subject using separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically includes lymphocytes, e.g., T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. For example, the cells may be washed with phosphate buffered saline (PBS). After washing, the cells may be washed with, for example, Ca 2+ Non-containing or Mg 2+ The T cells may be resuspended in a variety of biocompatible buffers, such as free PBS, PlasmaLyte A, or other saline and / or buffer solutions. TM Peripheral blood lymphocytes can be isolated by lysing red blood cells and depleting monocytes by gradient centrifugation or countercurrent centrifugation elution. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 (Thermo Fisher Scientific), for a time sufficient for positive selection of desired T cells.

[0121] T cells can be engineered to express CAR by methods known in the art. In general, a polynucleotide vector encoding CAR is constructed, and the vector is transfected or transduced into a population of T cells. For example, a nucleotide sequence encoding CAR can be delivered to cells using a retroviral or lentiviral vector. Exemplary retroviral vectors include, but are not limited to, the vector backbone pMSGV1-CD8-28BBZ derived from pMSGV (mouse stem cell virus-based splice gag vector). For other exemplary lentiviral vectors, see, for example, Dull et al., (1998) J. VIROL. 72:8463-8471 and U.S. Patent Nos. 5,994,136, 6,682,907, 7,629,153, 8,329,462, 8,748,169, and 9,101,584. Retroviral transduction can be carried out using known techniques such as those of Johnson et al. (2009) BLOOD 114, 535-546. Surface expression of CAR on transduced T cells can be determined, for example, by flow cytometry. The nucleotide sequence encoding CAR can also be delivered to cells using in vitro transcribed mRNA.

[0122] T cells and / or T cells engineered to express a CAR can be activated and expanded generally using methods such as those described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. Generally, T cells are expanded by contact with an agent that stimulates a CD3 / TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. For example, a T cell population can be stimulated by contact with an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD2 antibody, or a protein kinase C activator (e.g., bryostatin) and / or a calcium ionophore.

[0123] Further methods for producing CAR T cells are described, for example, in Levine et al. (2016) MOL. THER. METHODS CLIN. DEV. 4:92-101.

[0124] Examples of systems for expression of CARs or for control of expression of CARs include "ON-Switch" CARs (Wu et al. (2015) SCIENCE 350: aab4077), combinatorial activation systems (Fedorov et al. (2014) CANCER JOURNAL 20:160-165;Kloss et al. (2013) NATURE BIOTECHNOLOGY 31: 71-75), doxycycline-inducible CARs (Sakemura et al. (2016) CANCER IMMUNOL. RES. 4:658-668), antibody-inducible CARs (Hill et al. (2018) NATURE CHEMICAL BIOLOGY 14:112-117), killing switches (Di Stasi et al. (2011) N. ENGL. J. MED. 365:1673-1683 (2011);Budde et al. (2013) PLoS ONE 8: e82742), the pause switch (Wei et al. (2012) NATURE 488: 384-388), the tunable receptor system (Ma et al. (2016) PROC. NATL. ACAD. SCI. USA 113: E450-458;Rodgers et al. (2016) PROC. NATL. ACAD. SCI. USA113: E459-468;Kudo et al. (2014) CANCER RES. 74: 93-103), and the proliferation switch (Chen et al. (2010) PROC. NATL. ACAD. SCI. USA 107, 8531-8536).

[0125] D. Expression Methods A protein of interest, such as an antibody, a superantigen conjugate, a chimeric antigen receptor and / or a T cell receptor subunit, can be expressed in a host cell of interest by incorporating a gene encoding the protein of interest into an appropriate expression vector.

[0126] Host cells can be genetically engineered, for example, by transformation or transfection techniques, to incorporate the nucleic acid sequence and express the superantigen. Introduction of the nucleic acid sequence into the host cell can be carried out by calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al. (1986) BASIC METHODS IN MOLECULAR BIOLOGY and Sambrook, et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0127] Representative examples of suitable host cells include bacterial cells, such as Streptococcus, Staphylococcus, Escherichia coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; mammalian cells, such as CHO, COS, HeLa, C127, 3T3, BHK, HEK-293 and Bowes melanoma cells.

[0128] When using recombinant DNA technology, protein of interest can be expressed using standard expression vectors and expression systems.The expression vector genetically engineered to contain the nucleic acid sequence encoding superantigen is introduced (e.g., transfected) into host cell to produce superantigen (see, for example, Dohlsten et al. (1994), Forsberg et al. (1997) J. BIOL. CHEM. 272:12430-12436, Erlandsson et al. (2003) J. MOL. BIOL. 333:893-905 and WO2003002143).

[0129] As used herein, "expression vector" refers to a vector that contains a recombinant polynucleotide that comprises an expression control sequence operably linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression can be supplemented by host cells or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), retrotransposons (e.g., piggyback, sleeping beauty) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that incorporate the recombinant polynucleotide of interest.

[0130] In some embodiments, the expression vector is a viral vector. The term "virus" is used herein to refer to obligate intracellular parasites that do not have protein synthesis or energy generation mechanisms. Exemplary viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Epstein-Barr virus (EBV) vectors, polyoma viral vectors (e.g., Simian vacuolating virus 40 (SV40) vectors), pox viral vectors and pseudotype viral vectors.

[0131] Viruses can be RNA viruses (having a genome made up of RNA) or DNA viruses (having a genome made up of DNA). In some embodiments, the viral vector is a DNA viral vector. Exemplary DNA viruses include parvoviruses (e.g., adeno-associated viruses), adenoviruses, asfarviruses, herpes viruses (e.g., herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), Epstein-Barr virus (EBV), cytomegalovirus (CMV)), papilloma viruses (e.g., HPV), polyoma viruses (e.g., simian vacuolating virus 40 (SV40)), and pox viruses (e.g., vaccinia virus, cowpox virus, smallpox virus, fowlpox virus, sheeppox virus, myxoma virus). In some embodiments, the viral vector is an RNA viral vector. Exemplary RNA viruses include bunyaviruses (e.g., hantaviruses), coronaviruses, flaviviruses (e.g., yellow fever virus, West Nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus, noroviruses (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)), and toroviruses.

[0132] In some embodiments, the expression vector comprises a control sequence or promoter operably linked to a nucleotide sequence encoding a protein of interest, such as a superantigen conjugate, a chimeric antigen receptor and / or a T cell receptor subunit. The term "operably linked" refers to the linkage of a polynucleotide element in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a gene when it affects the transcription of the gene. The nucleotide sequences that are operably linked are typically contiguous. However, some polynucleotide elements may be operably linked but not directly adjacent, and may even function in trans from different alleles or chromosomes, such that enhancers generally function when separated from the promoter by several kilobases, and intron sequences may be of variable length.

[0133] Exemplary promoters that can be used include, but are not limited to, retroviral LTR, SV40 promoter, human cytomegalovirus (CMV) promoter, U6 promoter or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters, including but not limited to histone, pol III and β-actin promoters).Other viral promoters that can be used include, but are not limited to, adenovirus promoter, TK promoter and B19 parvovirus promoter.

[0134] In some embodiments, the promoter is an inducible promoter. The use of an inducible promoter allows the expression of the polynucleotide sequence operably linked to be turned on or off as desired. In some embodiments, the promoter, such as the metallothionine promoter, glucocorticoid promoter, progesterone promoter and tetracycline promoter, is induced in the presence of exogenous molecules or activities. In some embodiments, the promoter, such as the IL-2 promoter, NFAT promoter, cell surface protein promoter (e.g., CD69 promoter or PD-1 promoter), cytokine promoter (e.g., TNF promoter), cellular activation promoter (e.g., CTLA4, OX40 or CD40L promoter) or cell surface adhesion protein promoter (e.g., VLA-1 promoter) is induced in the tumor microenvironment.

[0135] In some embodiments, the promoter mediates rapid, sustained expression measured over several days (e.g., the CD69 promoter). In some embodiments, the promoter mediates delayed, slow-inducible expression (e.g., the VLA1 promoter). In some embodiments, the promoter mediates rapid, transient expression (e.g., the TNF promoter, immediate early response gene promoters, and others).

[0136] For example, the selection of a promoter that is strong, weak, inducible, tissue-specific, developmental specific, has particular kinetics of activation (e.g., early and / or late activation), and / or has particular kinetics of expression of induced genes (e.g., short or long expression) is within the ordinary skill of one of ordinary skill in the art and will be apparent to one of ordinary skill in the art from the teachings contained herein.

[0137] In some embodiments, the protein of interest, such as an antibody or a superantigen conjugate, is purified prior to use, which can be achieved using a variety of purification protocols. The protein of interest can be purified using chromatography and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of pure peptides are ion exchange chromatography, size exclusion chromatography; affinity chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. The term "purified" as used herein is intended to refer to a composition that can be isolated from other components, where the macromolecule of interest (e.g., a protein) is purified to any degree compared to its original state. In general, the term "purified" refers to a macromolecule that has been subjected to fractionation to remove various other components, and the macromolecule substantially maintains its biological activity. The term "substantially purified" refers to a composition in which the macromolecule of interest forms a major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the content of the composition.

[0138] Various methods for quantifying the degree of purification of a protein are known to those skilled in the art, including, for example, determining the specific activity of an active fraction and assessing the amount of a given protein in a fraction by SDS-PAGE analysis, high performance liquid chromatography (HPLC), or any other fractionation technique.Various techniques suitable for use in protein purification include, for example, precipitation or heat denaturation with ammonium sulfate, PEG, antibodies, etc., followed by precipitation by centrifugation; chromatographic steps such as ion exchange, gel filtration, reverse phase, hydroxyapatite, affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques.It is contemplated that the order in which the various purification steps are performed may be altered, or certain steps may be omitted, resulting in a suitable method for the preparation of a substantially purified protein or peptide.

[0139] III. Pharmaceutical Compositions For therapeutic use, 5T4 targeting agent is preferably combined with pharmaceutically acceptable carrier.The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are within the scope of sound medical judgment and suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0140] The term "pharmaceutical acceptable carriers" as used herein refers to buffers, carriers and excipients suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic responses or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (such as oil / water or water / oil emulsions) and various types of wetting agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] . Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art.

[0141] In certain embodiments, pharmaceutical compositions may include formulation materials to modify, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids); bulking agents (such as phosphate buffers, ... agents) (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); colorants, flavors and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide; solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbates, triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see Remington's Pharmaceutical Sciences, 18th Edition, (Mack Publishing Company, 1990)).

[0142] In some embodiments, the pharmaceutical composition may comprise a nanoparticle, such as a polymeric nanoparticle, a liposome, or a micelle (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29).

[0143] In some embodiments, the pharmaceutical composition may comprise a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. The sustained-release preparation may comprise, for example, porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. The sustained-release matrix may comprise polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, poly(2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also comprise liposomes, which may be prepared by any of several methods known in the art.

[0144] The pharmaceutical composition comprising the 5T4 targeting agent disclosed herein may be provided in unit dosage form and may be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intramuscular, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. In some embodiments, the pharmaceutical composition comprising the 5T4 targeting agent disclosed herein is administered by IV infusion. Alternatively, the agent may be administered locally rather than systemically, for example by injection directly into the site of action of the agent(s), often in a depot or sustained release formulation. In some embodiments, the pharmaceutical composition comprising the 5T4 targeting agent disclosed herein is administered by intratumoral injection.

[0145] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose.

[0146] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be preserved against microorganisms. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), and suitable mixtures thereof.

[0147] Pharmaceutical preparations are preferably sterile.Sterilization can be achieved by any suitable method, for example, filtration through a sterile filtration membrane.If the composition is lyophilized, sterilization by filtration can be performed before or after lyophilization and reconstitution.

[0148] In some embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may contain from about 2% to about 75% by weight of the unit or from about 25% to about 60%, for example, any range that may be derived therein. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable. Such determinations are known and used by those skilled in the art.

[0149] The active agent is administered in an amount (or amounts) effective to reduce, reduce, inhibit or otherwise eliminate the growth or proliferation of cancer cells, induce apoptosis, inhibit the angiogenesis of cancer or tumors, inhibit metastasis, or induce cytotoxicity in cells. The effective amount of the active compound (or compounds) used to carry out the present invention for the therapeutic treatment of cancer varies according to the mode of administration, the age, weight and general health of the subject. These terms include synergistic situations where a single agent alone may act weakly or not at all, but when combined with another agent, for example, but not limited to, by sequential administration, two or more agents act to produce a synergistic result.

[0150] In general, a therapeutically effective amount of the active component is in the range of 0.1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, 1 mg / kg to 10 mg / kg. The amount administered depends on variables such as the type and extent of the disease or symptom being treated, the overall health of the patient, the in vivo efficacy of the antibody, the pharmaceutical formulation, and the route of administration. The starting dose may be increased beyond the upper level to rapidly achieve the desired blood or tissue level. Alternatively, the starting dose may be less than optimal, and the daily dose may be increased progressively during the course of treatment. Human doses may be optimized in conventional Phase I dose escalation studies designed to be performed, for example, from 0.5 mg / kg to 20 mg / kg. The frequency of administration may vary depending on factors such as the route of administration, the dosage, the serum half-life of the antibody, and the disease being treated. Exemplary dosing frequencies are once a day, once a week, and once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. In some embodiments, the 5T4 targeting agent is lyophilized and then reconstituted in buffered saline at the time of administration.

[0151] In some embodiments, when a 5T4 targeting agent is isolated, naturally occurring or genetically engineered immune cells, e.g., T cells, are administered at a dose of, e.g., 10 5 ~10 9 cells / kg, 105 ~10 8 cells / kg, 10 5 ~10 7 cells / kg, 10 5 ~10 6 cells / kg, 10 6 ~10 9 cells / kg, 10 6 ~10 8 cells / kg, 10 6 ~10 7 cells / kg, 10 7 ~10 9 cells / kg, 10 7 ~10 8 Cells / kg or 10 8 ~10 9 Cells / kg or 10 6 ~10 11 Total cells, 10 6 ~10 10 Total cells, 10 6 ~10 9 Total cells, 10 6 ~10 8 Total cells, 10 6 ~10 7 Total cells, 10 7 ~10 11 Total cells, 10 7 ~10 10 Total cells, 10 7 ~10 9 Total cells, 10 7 ~10 8 Total cells, 10 8 ~10 11 Total cells, 10 8 ~10 10 Total cells 10 8 ~10 9 Total cells, 10 9 ~10 11 Total cells, 10 9 ~10 10 Total cells or 10 10 ~10 11 The amount administered will depend on variables such as the type and severity of the disease or condition being treated, the overall health of the patient, the in vivo efficacy of the active agent, the pharmaceutical formulation and the route of administration. Progress can be monitored by periodic evaluation.

[0152] In certain non-limiting examples, doses of the 5T4 targeting agent can also include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 15 micrograms / kg / body weight, about 20 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight to about 1,000 mg / kg / body weight or more per administration, and any range that can be derivable therein. Non-limiting examples of ranges that may be derived from the numbers recited herein include ranges from about 5 mg / kg / body weight to about 100 mg / kg / body weight, from about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, from about 1 microgram / kg / body weight to about 100 milligrams / kg / body weight. Other exemplary dose ranges include about 1 microgram / kg / body weight to about 1,000 micrograms / kg / body weight, about 1 microgram / kg / body weight to about 100 micrograms / kg / body weight, about 1 microgram / kg / body weight to about 75 micrograms / kg / body weight, about 1 microgram / kg / body weight to about 50 micrograms / kg / body weight, about 1 microgram / kg / body weight to about 40 micrograms / kg / body weight, about 1 microgram / kg / body weight to about 30 micrograms / kg / body weight, about 1 microgram / kg / body weight to about 20 micrograms / kg / body weight, about 1 microgram / kg / body weight to about 15 micrograms / kg / body weight, about 1 microgram / kg / body weight to about 10 micrograms / kg / body weight, about 5 micrograms / kg / body weight to about 1,000 micrograms / kg / body weight, about 5 micrograms / kg / body weight to about 100 micrograms / kg / body weight, about 5 micrograms / kg / body weight to about 75 micrograms / kg / body weight, about 5 micrograms / kg / body weight to about 50 micrograms / kg / body weight, about 5 micrograms / kg / body weight to about 40 micrograms / kg / body weight, about 5 micrograms / kg / body weight to about 30 micrograms / kg / body weight, about 5 micrograms / kg / body weight to about 20 micrograms / kg / body weight, about 5 micrograms / kg / body weight to about 15 micrograms / kg / body weight, about 5 micrograms / kg / body weight to about 10 micrograms / kg / body weight, about 10 micrograms / kg / body weight to about 1,000 micrograms / kg / body weight, about 10 micrograms / kg / body weight to about 100 micrograms / kg / body weight, about 10 micrograms / kg / body weight to about 75 micrograms / kg / body weight, about 10 micrograms / kg / body weight to about 50 micrograms / kg / body weight gram / kg / body weight, about 10 micrograms / kg / body weight to about 40 micrograms / kg / body weight, about 10 micrograms / kg / body weight to about 30 micrograms / kg / body weight, about 10 micrograms / kg / body weight to about 20 micrograms / kg / body weight, about 10 micrograms / kg / body weight to about 15 micrograms / kg / body weight, about 15 micrograms / kg / body weight to about 1,000 micrograms / kg / body weight, about 15 micrograms / kg / body weight to about 100 micrograms / kg / body weight, about 15 micrograms / kg / body weight to about 75 micrograms / kg / body weight, about 15 micrograms / kg / body weight to about 50 micrograms / kg / body weight, about 15 micrograms / kg / body weight to about 40 micrograms / kg / body weight, about 15 micrograms / kg / body weight to about 30 micrograms / kg / body weight, about 15 micrograms / kg / body weight to about 20 micrograms / kg / body weight, about 20 micrograms / kg / body weight to about 1,Ranges such as about 20,000 micrograms / kg / body weight, about 20 micrograms / kg / body weight to about 100 micrograms / kg / body weight, about 20 micrograms / kg / body weight to about 75 micrograms / kg / body weight, about 20 micrograms / kg / body weight to about 50 micrograms / kg / body weight, about 20 micrograms / kg / body weight to about 40 micrograms / kg / body weight, about 20 micrograms / kg / body weight to about 30 micrograms / kg / body weight, etc., may be administered based on the numbers mentioned above.

[0153] In certain embodiments, for example, administration of a 5T4 targeting agent, the effective amount or dose of the 5T4 targeting agent administered is an amount in the range of 0.01 to 500 μg / kg of subject's body weight, for example, 0.1 to 500 μg / kg of subject's body weight, and for example, 1 to 100 μg / kg of subject's body weight.

[0154] The compositions described herein can be administered locally or systemically.Administration is generally parenteral administration.In a preferred embodiment, pharmaceutical compositions are administered subcutaneously, and in an even more preferred embodiment, intravenously.Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.

[0155] IV. Therapeutic uses The compositions and methods disclosed herein can be used to treat various forms of glioblastoma in a subject or to suppress the growth of glioblastoma in a subject.The present invention provides a method for treating glioblastoma in a subject.The method comprises administering to a subject an effective amount of the disclosed 5T4 targeting agent, either alone or in combination with another therapeutic agent for treating glioblastoma in a subject.For example, the disclosed 5T4 targeting agent can be administered to a subject to slow the growth rate of cancer cells, reduce the occurrence or number of metastases, reduce tumor size, inhibit tumor growth, reduce blood supply to tumors or cancer cells, promote immune response to cancer cells or tumors, prevent or suppress the progression of cancer, for example by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%. Alternatively, a 5T4 targeting agent can be administered to a subject to treat cancer, for example to increase the lifespan of a subject with cancer by, for example, 3 months, 6 months, 9 months, 12 months, 1 year, 5 years or 10 years.

[0156] It is contemplated that several glioblastomas, such as, but not limited to, proneural glioblastoma, neuroglial blastoma, classical glioblastoma, mesenchymal glioblastoma, primary glioblastoma, secondary glioblastoma, grade I glioblastoma, grade II glioblastoma, grade III glioblastoma, and / or grade IV glioblastoma, may be treated using the methods and compositions described herein.

[0157] It is contemplated that 5T4 expression can be measured in a sample, for example, a sample from a subject. 5T4 can be detected in tissue of interest, body fluid and / or cell sample from a subject using techniques known in the art. The body fluid sample can be, for example, blood, serum or plasma. The tissue sample can be, for example, tumor tissue. The cell sample can be, for example, cancer cell sample. It is understood that either tumor tissue or sample can be preserved or processed using techniques known in the art, for example, formalin-fixed paraffin-embedded section.

[0158] Preferably, patients to be treated have adequate bone marrow function (>2,000 / mm 3 Peripheral absolute granulocyte count and 100,000 / mm 3 platelet count <1.5 mg / dl), adequate liver function (bilirubin <1.5 mg / dl) and adequate renal function (creatinine <1.5 mg / dl).

[0159] Treatment regimen may vary and may often depend on tumor size, tumor location, disease progression and patient's health and age.Certain types of tumors may require more intensive treatment protocols, but at the same time, patients may not be able to tolerate more intensive treatment regimens.Clinicians are often best suited to make such decisions based on the physician's skill in the art and the known efficacy and toxicity (if any) of treatment formulations.

[0160] A typical course of treatment for a primary tumor or a post-resection tumor bed may include multiple administrations. A typical primary tumor treatment may include six administrations over a two-week period. The two-week regimen may be repeated one, two, three, four, five, six or more times. During the course of treatment, the need to complete the planned administrations may be reassessed.

[0161] Immunotherapy with superantigen conjugates often results in rapid (within hours) and potent polyclonal activation of T lymphocytes. A superantigen conjugate treatment cycle may include 4-5 daily intravenous superantigen conjugate drug injections. Such treatment cycles may be given, for example, at intervals of 4-6 weeks. Inflammation, accompanied by infiltration of CTLs into tumors, is one of the main effectors of antitumor therapeutic superantigens. After a short period of extensive activation and differentiation of CTLs, the T cell response declines rapidly (within 4-5 days) to return to baseline levels. Thus, the period of lymphocyte proliferation during which cytostatic drugs may interfere with superantigen treatment is short and well defined.

[0162] In some embodiments, a subject is administered a superantigen conjugate, such as a superantigen conjugate contemplated herein, daily for 2-6 consecutive days (e.g., 2, 3, 4, 5, or 6 consecutive days) every 2-12 weeks (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks). In some embodiments, a subject is administered a superantigen conjugate, such as a superantigen conjugate contemplated herein, daily for 4 consecutive days every 3-4 weeks (e.g., 3 or 4 weeks). In some embodiments, a subject is administered a superantigen conjugate, such as a superantigen conjugate contemplated herein, once every 2-8 weeks (e.g., once every 2, 3, 4, 5, 6, 7, or 8 weeks).

[0163] The methods and compositions described herein may be used alone or in combination with other therapeutic agents and / or modalities. The term "administered in combination," as used herein, is understood to mean that two (or more) different therapies are delivered to a subject during the course of the subject's affliction with a disorder, such that the effects of the therapies on the patient overlap at some point. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so there is an overlap in the administration period. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other begins. In some embodiments of either case, the treatments are more effective because of the combined administration. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, totally additive, or greater than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second is delivered.

[0164] In certain embodiments, the therapeutic regimens of the present invention may include contacting neoplastic or tumor cells with a first and a second treatment simultaneously. This may be accomplished by contacting the cells with a single composition or pharmaceutical formulation that includes both agents, or by contacting the cells simultaneously with two separate compositions or formulations, where one composition includes the first treatment and the other includes the second treatment.

[0165] Alternatively, the first treatment may precede or follow the second treatment by intervals ranging from minutes, days to weeks. In embodiments in which the first and second treatments are applied separately to the cells, it should be ensured that no significant time elapses between the times of delivery, so that the first and second treatments can still exert their advantageously combined effect on the cells. In such instances, it is contemplated that both modalities may be contacted with the cells within about 12 to 72 hours of each other. In some circumstances, it may be desirable to significantly extend the treatment time, where days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each administration.

[0166] Various combinations where the first treatment is "A" and the second treatment is "B" may be used: A / B / A, B / A / B, B / B / A, A / A / B, A / B / B, B / A / A, A / B / B / B, B / A / B / B, B / B / B / A, B / B / A / B, A / A / B / B, A / B / A / B, A / B / B / A, B / B / A / A, B / A / B / A, B / A / A / B, A / A / A / B, B / A / A / A, A / B / A / A and A / A / B / A.

[0167] In some embodiments, the methods or compositions described herein are administered in combination with one or more additional treatments, such as surgery, radiation therapy, or administration of another therapeutic preparation. In some embodiments, the additional treatment may include chemotherapy, such as a cytotoxic agent. In some embodiments, the additional treatment may include targeted treatment, such as a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor. In some embodiments, the additional treatment may include an anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative compound, such as a steroid, a biological immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodilator, a statin, an anti-inflammatory agent (e.g., methotrexate), or an NSAID. In some embodiments, the additional treatment may include a compound designed to reduce the possible immune reactivity of the subject to the administered 5T4 targeting agent. For example, the immune reactivity to the administered 5T4 targeting agent may be reduced by co-administration with, for example, an anti-CD20 antibody and / or an anti-CD19 antibody, which reduces the production of anti-drug antibodies in the subject. In some embodiments, the additional therapy may include a combination of therapeutic agents from different classes.

[0168] In certain embodiments, the methods or compositions described herein are administered in combination with an immune enhancing agent.

[0169] In certain embodiments, exemplary immune enhancing agents are those that (a) stimulate activation of T cell signaling; (b) suppress T cell inhibitory signaling between cancerous cells and T cells; (c) suppress inhibitory signaling that leads to expansion, activation and / or activity of T cells via a non-human IgG1 mediated immune response pathway, e.g., a human IgG4 immunoglobulin mediated pathway. 、 (d) a combination of (a) and (b); (e) a combination of (a) and (c); (f) a combination of (b) and (c); and (g) a combination of (a), (b) and (c).

[0170] In some embodiments, the immune enhancing agent is a checkpoint pathway inhibitor.For example, the checkpoint inhibitor can be selected from PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, adenosine A2A receptor antagonist, B7-H3 antagonist, B7-H4 antagonist, BTLA antagonist, KIR antagonist, LAG3 antagonist, TIM-3 antagonist, VISTA antagonist or TIGIT antagonist.

[0171] PD-1 is a receptor present on the surface of T cells that functions as an immune system checkpoint that inhibits or otherwise regulates T cell activity at the appropriate time to prevent overly active immune responses. However, cancer cells can exploit this checkpoint by expressing ligands such as PD-L1, PD-L2, etc., that interact with PD-1 on the surface of T cells to block or regulate T cell activity. Using this approach, cancers can evade T cell-mediated immune responses.

[0172] In the CTLA-4 pathway, the interaction of CTLA-4 on T cells with its ligands (e.g., also known as CD80, B7-1, and CD86) on the surface of antigen-presenting cells (not cancer cells) results in T cell inhibition. As a result, the ligands (e.g., CD80 or CD86) that inhibit T cell activation or activity are provided by antigen-presenting cells (important cell types in the immune system) rather than by cancer cells. Although both CTLA-4 and PD-1 binding have similar negative effects on T cells, the timing of downregulation by these two immune checkpoints, causative signaling mechanisms, and anatomical location of immune inhibition are different (Buchbinder et al. (2016) AMERICAN JOURNAL OF CLINICAL ONCOLOGY. 39(1)). Unlike CTLA-4, which is restricted to the early inductive phase of T cell activation, PD-1 functions much later during the effector phase (Keir et al. (2008) ANNU. REV IMMUNOL., 26:677-704). CTLA-4 and PD-1 represent two T cell inhibitory receptors with independent and non-overlapping mechanisms of action.

[0173] In some embodiments, the immune enhancer prevents (fully or partially) the antigen expressed by the cancerous cell from suppressing T cell inhibitory signaling between the cancerous cell and the T cell. In one embodiment, such an immune enhancer is a checkpoint inhibitor, such as a PD-1 system inhibitor. Examples of such an immune enhancer include, for example, anti-PD-1 antibody, anti-PD-L1 antibody, and anti-PD-L2 antibody.

[0174] In some embodiments, the 5T4 targeting agent is administered in conjunction with a PD-1 axis inhibitor, which may include (i) a PD-1 inhibitor, i.e., a molecule (e.g., an antibody or small molecule) that binds to PD-1 on a T cell and prevents binding of a PD-1 ligand expressed by the cancer cell of interest, and / or (ii) a PD-L inhibitor, e.g., a PD-L1 or PD-L2 inhibitor, i.e., a molecule (e.g., an antibody or small molecule) that binds to a PD-1 ligand (e.g., PD-L1 or PD-L2) and prevents the PD-1 ligand from binding to its cognate PD-1 on a T cell.

[0175] In certain embodiments, the 5T4 targeting agent is administered in conjunction with a CTLA-4 inhibitor, such as an anti-CTLA-4 antibody. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815 and 8,883,984, International (PCT) Publication Nos. WO98 / 42752, WO00 / 37504 and WO01 / 14424, and European Patent No. EP 1212422. B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0176] In some embodiments, the immune enhancer prevents (fully or partially) the antigens expressed by cancerous cells from suppressing the expansion, activation and / or activity of T cells, for example, via the human IgG4 (non-human IgG1)-mediated immune response pathway, rather than via the ADCC pathway. In some embodiments, it is contemplated that the immune response enhanced by the immune enhancer may include some ADCC activity, but the main component(s) of the immune response do not include ADCC activity. In contrast, some of the antibodies currently used in immunotherapy, such as ipilimumab (anti-CTLA-4 IgG1 monoclonal antibody), can kill targeted cells via ADCC through signaling via their Fc domains by Fc receptors on effector cells. Like many other therapeutic antibodies, ipilimumab is designed as a human IgG1 immunoglobulin, and although ipilimumab inhibits the interaction between CTLA-4 and CD80 or CD86, its mechanism of action is believed to involve ADCC depletion of tumor-infiltrating regulatory T cells that express high levels of cell surface CTLA-4. (Mahoney et al. (2015) NATURE REVIEWS, DRUG DISCOVERY 14: 561-584). Given that CTLA-4 is highly expressed on a subset of T cells (regulatory T cells) that function to negatively regulate T cell activation, when anti-CTLA-4 IgG1 antibodies are administered, the number of regulatory T cells is reduced via ADCC.

[0177] In some embodiments, it is desirable to use an immune enhancer whose mode of action is primarily to inhibit inhibitory signals between cancer cells and T cells without significantly depleting the T cell population (e.g., expanding the T cell population). To achieve this, it is desirable to use an antibody having or based on the human IgG4 isotype, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody. The human IgG4 isotype is preferred under certain circumstances, as it elicits little or no ADCC activity compared to the human IgG1 isotype (Mahoney et al. (2015) supra). Thus, in some embodiments, the immune enhancer, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, has or is based on the human IgG4 isotype. In some embodiments, the immune enhancer is an antibody that is not known to deplete Tregs, such as a non-CTLA-4 checkpoint-directed IgG4 antibody (e.g., an anti-PD-1 IgG4 inhibitor).

[0178] In some embodiments, the immune enhancer is an antibody having or based on the human IgG1 isotype or another isotype that elicits antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). In other embodiments, the immune enhancer is an antibody having or based on the human IgG4 isotype or another isotype that elicits little or no antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC).

[0179] Exemplary PD-1 system inhibitors are described in U.S. Patent Nos. 8,728,474, 8,952,136, and 9,073,994, and European Patent No. 1537878B1. Exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include nivolumab (OPDIVO®, Bristol-Myers Squibb), pembrolizumab (KEYTRUDA®, Merck), cemiplimab (LIBTAYO®, Regeneron / Sanofi), spartalizumab (PDR001), MEDI0680 (AMP-514), pidilizumab (CT-011), dostarlimab (TSR-042, WBP-285), sintilimab (IBI308), toripalimab (JS 001), camrelizumab (SHR1210), pimivalimab (JTX-4014), tislelizumab (BGB-A317), prolgolimab, INCMGA00012 (MGA012), and AMP-514. Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149.Exemplary anti-PD-L1 antibodies include avelumab (BAVENCIO®, EMD Serono / Pfizer), atezolizumab (TECENTRIQ®, Genentech) and durvalumab (IMFINZI®, Mediimmune / AstraZeneca), CS1001 (CStone Therapeutics), tagitanlimab (KL-A167), cosibelimab (CK-301; Checkpoint Therapeutics), TQB2450, envafolimab (KN035), SHR-1316, STI-A1014, BGB-A333, MSB2311, HLX-20, and BMS-936559 (Bristol-Myers Squibb).

[0180] In some embodiments, a subject is administered a PD-1 system inhibitor, e.g., an anti-PD-1 antibody, e.g., an anti-PD-1 antibody contemplated herein, every 1-5 weeks (e.g., every 1, 2, 3, 4, or 5 weeks). In some embodiments, a subject is administered a PD-1 system inhibitor, e.g., an anti-PD-1 antibody, e.g., an anti-PD-1 antibody contemplated herein, every 2-4 weeks (e.g., every 2, 3, or 4 weeks).

[0181] PD-1 inhibitors can be designed, expressed and purified using techniques known to those of skill in the art, for example, as described herein above. Anti-PD-1 antibodies can be designed, expressed, purified, formulated and administered as described in U.S. Patent Nos. 8,728,474, 8,952,136 and 9,073,994.

[0182] Other immune enhancing agents (e.g., antibodies and various small molecules) may target signaling pathways including one or more of the following ligands: B7-H3 (found especially in prostate, renal cell, non-small cell lung, pancreatic, stomach, ovarian, colorectal cells); B7-H4 (found especially in breast, renal cell, ovarian, pancreatic, melanoma cells); HHLA2 (found especially in breast, lung, thyroid, melanoma, pancreatic, ovarian, liver, bladder, colon, prostate, kidney cells); galectins (found especially in non-small cell lung, colorectal, and stomach cells); CD30 (found especially in Hodgkin's lymphoma, large cell lymphoma cells); CD70 (found especially in non-Hodgkin's lymphoma, renal cells); ICOSL (found especially in glioblastoma, melanoma cells); CD155 (found especially in kidney, prostate, pancreatic glioblastoma cells); and TIM3. Similarly, other potential immune enhancers that may be used include, for example, 4-1BB (CD137) agonists (e.g., fully human IgG4 anti-CD137 antibody urelumab / BMS-663513), LAG3 inhibitors (e.g., humanized IgG4 anti-LAG3 antibody LAG525, Novartis); IDO inhibitors (e.g., small molecule INCB024360, Incyte Corporation), TGFβ inhibitors (e.g., small molecule galunisertib, Eli Lilly), and other receptors or ligands found on T cells and / or tumor cells. In some embodiments, immune enhancers (e.g., antibodies and various small molecules) that target signaling pathways that include one or more of the aforementioned ligands are amenable to pharmaceutical intervention based on agonist / antagonist interactions but not via ADCC.

[0183] It is further envisioned that the present invention may be used in combination with surgical intervention. In the case of surgical intervention, the present invention may be used prior to surgery, for example to resect inoperable tumor subjects. Alternatively, the present invention may be used during and / or after surgery to treat residual or metastatic disease. For example, the resected tumor bed may be injected or perfused with a formulation comprising a 5T4 targeting agent. Perfusion may be continued after resection, for example by leaving an implanted catheter at the site of surgery. Periodic post-operative treatments are also envisioned. Any combination of the treatment of the present invention with surgery is within the scope of the present invention.

[0184] Continuous administration may also be applied where appropriate, for example, when a tumor is resected and the tumor bed is treated to eliminate residual microscopic disease. Delivery via syringe or cautery is preferred. Such continuous perfusion may be performed for a period of time from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 weeks, or longer, after the start of treatment. In general, the dose of therapeutic composition via continuous perfusion is equivalent to that given by single or multiple injections, adjusted over the time period during which perfusion is performed. It is further contemplated that limb perfusion may be used to administer the therapeutic composition of the present invention, particularly in the treatment of melanoma and sarcoma.

[0185] Exemplary cytotoxic agents that may be administered in combination with the methods or compositions described herein include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinating agents, cytotoxic agents, anti-inflammatory agents, anti-inflammatory drugs, anti-inflammatory agents ... agents), inhibitors of nucleic acid synthesis, histone deacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethylenimines, alkylsulfonates, triazenes, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating agents, agents that may interfere with signal transduction pathways, agents that promote apoptosis and radiation, or antibody molecule conjugates that bind to surface proteins to deliver toxic agents.In one embodiment, cytotoxic agents that may be administered with the methods or compositions described herein include platinum-based agents (e.g., cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacitidine, vorinostat, ixabepilone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicine, anthracyclines (e.g., doxorubicin or epirubicin). The anticancer drugs are daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansinoids. EXAMPLES

[0186] Working Example The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0187] Example 1 This example describes the expression analysis of 5T4 in human glioblastoma (GBM) tumor tissue.

[0188] Human GBM tissue samples were obtained from commercial sources and analyzed by immunohistochemistry (IHC) using rabbit anti-human 5T4 antibody (clone EPR5529; Abcam, ab134162). Stained samples were scored for 5T4 abundance by a trained pathologist. Abundance was defined as the percentage of tumor cores with at least 0.5% tumor cells with membrane 5T4 expression. Thirty samples were analyzed. Unexpectedly, 87% (26 / 30) of the analyzed GBM tissue samples showed tumor membrane positivity. Of the 26 positive cases, 67% (21 / 30) had tumor cells with up to 3% membrane 5T4 expression and 17% (5 / 30) had tumor cells with higher than 10% membrane 5T4 expression. Representative IHC images are shown in Figure 4.

[0189] Example 2 This example describes the analysis of 5T4 membrane expression in human glioblastoma (GBM) cell lines U-138, U-87 and GMS-10. A human renal cell carcinoma (RCC) cell line (Caki-2) known to express high levels of 5T4 and a human ovarian cancer cell line (OVCAR-3) known to express low levels of 5T4 were used as controls. 5T4 membrane expression was determined by flow cytometry.

[0190] Cell lines were analyzed using flow cytometry with Alexa Fluor 647-conjugated mouse IgG1 anti-human 5T4 monoclonal antibody (clone 524744; R&D systems, FAB49751R). Alexa Fluor 647-conjugated mouse IgG1 isotype control antibody was used as a negative control for staining. Cell lines were washed with PBS, labeled with viability dyes (Zombie Violet, BioLegend 77477 or Zombie NIR, Biolegend 423105) and then incubated with the indicated mAbs in flow cytometry buffer (PBS, 0.5% BSA, 2mM EDTA, 0.05% NaN3) for 30 min at room temperature. Cells were then washed with flow cytometry buffer and acquired on a MACSQuant flow cytometer (Miltenyi Biotec). Dead cells were excluded from the analysis. Data analysis was performed using FlowJo 10 software. As seen in Figure 5, all three GBM cell lines, U-138, U-87 and GMS-10, were positive for 5T4 expression. The fold change (FC) of 5T4 mAb staining relative to isotype control for the U-138, U-87 and GMS-10 cell lines was 5, 8 and 11, respectively, which was higher than expression on the OVCAR-3 cell line (FC=3) but lower than expression on the Caki-2 cell line (FC=19).

[0191] Example 3 This example describes an in vitro study testing the anti-cancer efficacy of T cells in combination with the 5T4-targeting superantigen naptumomab-estafenatox (NAP; ANYARA®) against human glioblastoma (GBM) cell lines U-138, U-87, and GMS-10.

[0192] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. PBMCs were incubated with 10 ng / ml Staphylococcal Enterotoxin A (SEA) for 4 days. T cells were then isolated and incubated with IL-2 for an additional day. 8x10 per well 3U-138, U-87 or GMS-10 cells were incubated with T cells for 1 h in 96-well plates. The effector:target ratio (T cells:GBM cells) was 10:1. After 1 h of incubation, NAP was added to the wells at different concentrations (ranging from 0 to 10 μg / ml) and the plates were incubated for another 4 to 48 h. At the end of the treatment, the culture supernatant containing the suspended T cells and tumor cells was removed and the attached tumor cells were washed once with culture medium. The viability of the remaining GBM cells was tested by CCK8 kit (Cell Counting Kit-8, Sigma Aldrich) according to the manufacturer's protocol. The viability of the control group was normalized to 100%. Cancer cell viability (%) = (OD value of treated group / OD value of control group) x 100. Data were analyzed using Graph Pad Prism software.

[0193] Dose-response curves for three GBM cell lines are shown: U-138 (Figure 6A), U-87 (Figure 6B) and GMS10 (Figure 6C). The combination of T cells with NAP reduced the viability of U-138, U-87 and GMS-10 cancer cells, with IC50 of NAP being 0.5ng / ml, 0.3ng / ml and 0.95ng / ml, respectively (4-13pM). These data indicate that NAP is highly potent against all three 5T4-expressing GBM cell lines, including those with relatively low levels of 5T4 expression.

[0194] Example 4 This example describes an in vitro study testing whether the anti-cancer effect of T cells in combination with 5T4-targeted superantigen NAP on the GMS-10 human glioblastoma (GBM) cell line is mediated by the 5T4 antigen.

[0195] PBMCs were isolated from healthy donors. PBMCs were incubated with 10 ng / ml Staphylococcal Enterotoxin A (SEA) for 4 days. T cells were then isolated and incubated with IL-2 for an additional day. 2x10 per well 4Carboxyfluorescein succinimidyl ester (CFSE)-labeled GMS-10 cells were incubated for 24 h in 96-well plates. To block the 5T4 antigen, these cells were incubated with 0–50 μg / ml of anti-5T4 Fab, 5T4FabV18. After 24 h of incubation, GMS-10 cells were washed and preactivated T cells were added to the wells at a 10:1 effector:target (T cells:GMS-10 cells) ratio. After 1 h of incubation, different concentrations (0–0.005 μg / ml) of NAP were added to the wells and the plates were incubated for another 48 h. At the end of the treatment, the culture supernatant containing the suspended T cells and tumor cells was removed. The attached tumor cells were detached, washed once with PBS and stained for dead cells. The viability of the remaining GMS-10 cells was determined by FACS. The viability of CFSE-gated cells in the control group was normalized to 100%. Cancer cell viability (%) = (treated cell count / control cell count) x 100.

[0196] The results of this study are shown in Figure 7. As shown, blocking 5T4 antigen with anti-5T4 Fab reduced the anti-cancer effect of NAP in a dose-dependent manner. These data indicate that NAP activity against GMS-10 GBM cell line is mediated by 5T4 antigen expressed on the surface of GMS-10 cells.

[0197] Incorporation by Reference The entire disclosures of each of the patent and scientific literature referenced herein are incorporated by reference for all purposes.

[0198] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

[0199] Sequence Listing [Table 1-1] [Table 1-2]

Claims

1. 13. A 5T4 targeting agent for use in reducing tumor volume in a subject having glioblastoma.

2. A 5T4 targeting agent for use in killing tumor cells in a subject with glioblastoma.

3. A 5T4 targeting agent for use in the treatment of glioblastoma in a subject in need thereof.

4. 4. The 5T4 targeting agent of any one of claims 1 to 3, wherein at least 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% of (i) tumor cells in a subject, (ii) tumor cells in a tumor in a subject, and / or (iii) tumor cells in a tumor sample (e.g. a tumor tissue sample) from the subject exhibit cell membrane expression of 5T4 as measured by immunohistochemistry.

5. 4. The 5T4 targeting agent of any one of claims 1 to 3, wherein (i) tumor cells in a subject, (ii) tumor cells in a tumor in a subject, and / or (iii) 0.5% to 10% (e.g. 0.5% to 3%, 3% to 5% or 5% to 10%) of tumor cells in a tumor sample (e.g. a tumor tissue sample) from the subject exhibit cell membrane expression of 5T4 as measured by immunohistochemistry.

6. The 5T4 targeting agent of any one of claims 1 to 3, wherein the 5T4 targeting agent comprises an antibody or an antigen-binding fragment thereof, a bispecific T cell engager (BiTE), an immune cell, or a vaccine.

7. The 5T4 targeting agent of any one of claims 1 to 3, wherein the 5T4 targeting agent is a superantigen conjugate comprising a superantigen covalently bound to an anti-5T4 antibody or antigen-binding fragment thereof.

8. 8. The 5T4 targeting agent of claim 7, wherein the superantigen comprises Staphylococcal enterotoxin A or an immunologically reactive variant and / or fragment thereof.

9. The 5T4 targeting agent of claim 7, wherein the superantigen comprises the amino acid sequence of SEQ ID NO:3 or an immunologically reactive variant and / or fragment thereof.

10. The 5T4 targeting agent of claim 7 , wherein the anti-5T4 antibody or antigen-binding fragment thereof comprises a Fab fragment that binds to the 5T4 cancer antigen.

11. The 5T4 targeting agent of claim 7, wherein the anti-5T4 antibody or antigen-binding fragment thereof comprises a heavy chain comprising amino acid residues 1 to 222 of SEQ ID NO:8 and a light chain comprising amino acid residues 1 to 214 of SEQ ID NO:

9.

12. The 5T4 targeting agent of claim 7 , wherein the superantigen conjugate comprises a first protein chain comprising SEQ ID NO:8 and a second protein chain comprising SEQ ID NO:

9.

13. The 5T4 targeting agent of claim 6 , wherein the 5T4 targeting agent is an immune cell.

14. The 5T4 targeting agent of claim 13, wherein the immune cell is a chimeric antigen receptor (CAR)-expressing immune cell.

15. 14. The 5T4 targeting agent of claim 13, wherein the immune cell is a T cell or a natural killer (NK) cell.

16. 16. The 5T4 targeting agent of claim 15, wherein the immune cell is a T cell.

17. A 5T4 targeting agent described in any of claims 1 to 3, wherein the 5T4 targeting agent is for use in combination with an immune enhancing substance, and optionally, the immune enhancing substance and the 5T4 targeting agent are administered to the subject in separate compositions.

18. The 5T4 targeting agent of claim 17, wherein the immune enhancing agent is a CTLA-4 or PD-1 inhibitor.

19. The 5T4 targeting agent of claim 18, wherein the PD-1 system inhibitor is a PD-1 or PD-L1 inhibitor.

20. 20. The 5T4 targeting agent of claim 19, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

21. 21. The 5T4 targeting agent of claim 20, wherein the anti-PD-1 antibody is selected from nivolumab pembrolizumab, cemiplimab, spartalizumab, MEDI0680, pidilizumab, dostallimab, sintilimab, toripalimab, camrelizumab, pimivalimab, tislelizumab, and prorugolimab.

22. 20. The 5T4 targeting agent of claim 19, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody.

23. 23. The 5T4 targeting agent of claim 22, wherein the anti-PD-L1 antibody is selected from atezolizumab, avelumab, durvalumab, CS1001, tagitanlimab, cosibelimab, TQB2450, embafolimab, SHR-1316, STI-A1014, BGB-A333, MSB2311, HLX-20, and BMS-936559.

24. The 5T4 targeting agent of any one of claims 1 to 3, wherein the subject is a human subject.

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