Orthogonal GPC3 chimeric antigen receptor T cells

Abstract

Provided are engineered T cells that express (a) a chimeric antigen receptor ("GPC-CAR"), where the antigen-binding domain of the CAR binds to human GPC3, and (b) an orthogonal receptor. Also provided are methods of making and using such engineered T cells. TIFF2025509288000067.tif39170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 317,935, filed March 8, 2022, which is incorporated by reference for all purposes. [Background technology]

[0002] 2. Background of the Invention Glypican-3 (GPC3) is a member of the glypican family of heparan surface proteoglycans. GPC3 is also called DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, and SGBS1. The human GPC3 (hGPC3) gene encodes a 580 amino acid precursor protein (SEQ ID NO:1) that contains a 24 amino acid signal peptide sequence (corresponding to amino acids 1-24 of SEQ ID NO:1) and a C-terminal sequence corresponding to amino acids 555-580 of SEQ ID NO:1, both of which are removed from the mature form of the protein. Two heparan sulfates are attached to amino acids 495 and 508. hGPC3 contains a furin cleavage site between amino acids R359 and S359 (numbering according to SEQ ID NO:1), which generates a 40 kDa N-terminal fragment having a sequence corresponding to amino acids 25 to 358 of SEQ ID NO:1 and a 30 kDa C-terminal fragment having a sequence corresponding to amino acids 359 to 554 of SEQ ID NO:1 (SEQ ID NO:2). When expressed on the cell surface after furin cleavage, the 40 kDa fragment and the 30 kDa fragment are linked via three disulfide bonds. Hydrolysis of these disulfide bonds releases a 40 kDa subunit that is detectable in serum and is called "soluble GPC3" or "sGPC3", and the level of GPC3 has been proposed as a diagnostic marker for estimating the expression level of GPC3 in a subject and as a marker for the presence of tumors expressing GPC3, such as hepatocellular carcinoma (HCC). GPC3 is attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor.

[0003] Since GPC3 is hardly expressed in normal tissues and highly expressed in HCC, GPC3 has been identified as a cancer-specific target, especially a liver cancer-specific target, by several studies. Baumhoer D, et al. (2008) Am J Clin Pathol 129(6):899-906 (Non-Patent Document 1). In addition, GPC3 is hardly expressed in other normal tissues in adults, and therefore is suitable for targeted therapy as a tumor antigen. Li et al (2018) Trends in cancer 4:741-54 (Non-Patent Document 2); Ho and Kim (2011) European journal of cancer 47:333-8 (Non-Patent Document 3). GPC3 was initially associated with liver cancer (particularly HCC), but various neoplasms characterized by GPC3 expression include, but are not limited to, hepatoblastoma (Zhou S, et al (2017) Scientific reports 7:45932), lung squamous cell carcinoma (Li et al (2016) Oncotarget 7:2496-507). ,, ovarian yolk sac tumor (Esheba et al. (2008) American journal of surgical pathology;32:600-7 (Non-Patent Document 6)), melanoma (Nakatsura and Nishimura (2005) BioDrugs: clinical immunotherapeutics, biopharmaceuticals and gene therapy 19:71-7 (Non-Patent Document 7)), ovarian clear cell carcinoma (Umezu (2010) Journal of clinical pathology 63:962-6 (Non-Patent Document 8)). Thus, GPC3 is not only a specific biomarker and prognostic factor for HCC, but also a potential target for the treatment of various tumors. Given the correlation with GPC3 in a wide range of tumors, a number of GPC-3 CARs have been reported. T cell therapies are under development and include multiple clinical trials (see, for example, the following trial numbers at ClinicalTrials.gov: NCT02395250, NCT02723942, NCT03146234, NCT0295188, NCT03084380, and NCT03884751). These trials evaluated the efficacy of GPC3 CAR-T therapy alone or in combination with other anticancer drugs (e.g., checkpoint inhibitors).

[0004] At present, the only CAR-T cell agents approved for use by regulatory agencies target cells expressing CD19 and are used in the treatment of hematological malignancies. There are no approved CAR-T cell therapies for the treatment of solid tumors. The development of CAR-T cells has shown significant progress in recent years, with the combination of various technologies resulting in what are referred to as first, second, third, and fourth generation CAR-T cells. Although the clinical track record of CAR-T cells for the treatment of hematological malignancies has shown initial notable success, over time the rate of disease recurrence is substantial. What is referred to as the "persistence" of CAR-T cells is a particular obstacle for existing technologies. It is well established that adoptively transferred human immune cells lose their activity relatively rapidly after administration. Therefore, typical strategies to address this rapid loss of function include: (a) administration of very high doses of cell therapy to maximize exposure of the cell therapy to the tumor before the cells lose efficacy, and / or (b) systemic administration of high-dose IL2 (HD-hIL2) therapy to attempt to support the efficacy of the adoptively transferred cells. Both of these approaches result in significant toxicity.

[0005] High-dose hIL2 is used, for example, to support adoptive cell therapy regimens, but the action of such high-dose hIL2 has been described to result in significant toxicity in human subjects. The most common side effects observed in the administration of HD-hIL2 in combination with adoptive cell transfer (ACT) include chills, high fever, hypotension, oliguria, and edema due to systemic inflammatory syndrome and capillary leak syndrome, and also include reports of autoimmune phenomena, such as vitiligo or uveitis. HD-hIL2 monotherapy can also induce systemic capillary leak syndrome, which can be fatal. The toxicity associated with HD-hIL2 requires expert management and is therefore typically applied in hospitals and often requires admission to the intensive care unit. Dutcher, et al. (2014) J Immunother Cancer 2(1): 26 (Non-Patent Document 9). For this reason, the use of HD-IL2 therapy is largely limited to younger, extremely healthy patients with normal cardiopulmonary function.

[0006] In addition, high doses of engineered cell therapy agents are associated with life-threatening cytokine release syndrome (CRS). Currently available products have demonstrated CRS of all grades in the majority of treated subjects, with a significant proportion of patients experiencing grade 3 or higher CRS. Significant neurotoxicity has also been observed in the majority of patients. However, low doses of cell therapy agents are associated with significantly reduced clinical outcomes. In addition, many patients who initially appeared to respond well to cell therapy experience relapse, mainly due to the lack of persistence of cell therapy products. Currently, it has been reported that approximately 60% of patients treated with existing CD-19 CAR-T cell therapy agents experience relapse. Byrne M, et al (2019) Biology of Blood and Marrow Transplantation 25(11):344-251 (Non-Patent Document 10).

[0007] Sockolosky et al. (Science (2018) 359: 1037-1042) and Garcia et al. (US Patent Application Publication No. 2018 / 0228841 A1, published August 16, 2018) describe an orthogonal IL2 / CD122 ligand / receptor system to facilitate selective stimulation of cells engineered to express orthogonal receptors, particularly orthogonal CD122. Briefly, the receptor is specifically modified to bind and be activated by what are called variant IL2 molecules. Contacting engineered T cells expressing orthogonal CD122 with the corresponding orthogonal ligand ("ortho-IL2") that is cognate to such orthogonal CD122 promotes specific activation of the engineered T cells expressing orthogonal CD122. In particular, orthogonal IL2 receptor-ligand complexes result in the selective expansion of cells engineered to express the orthogonal receptor in a mixed population of cells, particularly a mixed population of T cells. This patent application is incorporated by reference in its entirety in the disclosures of International Publication WO 2019 / 104092 (Patent Document 2) and U.S. Patent Application Publication No. 2018-0228842 A1 (Patent Document 3). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US Patent Application Publication No. 2018 / 0228841 A1 [Patent Document 2] International Publication No. 2019 / 104092 [Patent Document 3] US Patent Application Publication No. 2018-0228842 A1 [Non-patent literature]

[0009] [Non-Patent Document 1] Baumhoer D, et al. (2008) Am J Clin Pathol 129(6):899-906

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Summary of the Invention

[0010] The present disclosure provides GPC3 CAR-T cells that utilize a selective regulatory system that has demonstrated efficacy in treating solid tumors, particularly solid tumors that express GPC3 (GPC3+ tumors), and that allows for the selective expansion of GPC3 CAR-T cells in vivo, particularly in subjects undergoing treatment, and also demonstrates extended persistence, intratumoral infiltration into solid tumors, and the ability to treat relapse without additional administration of GPC3 CAR-T cells.

[0011] The present disclosure relates to engineered T cells that express (a) a chimeric antigen receptor ("GPC-CAR"), where the antigen-binding domain of the CAR binds to human GPC3, and (b) an orthogonal receptor. The terms "chimeric antigen receptor T cells" and "CAR-T cells" are used interchangeably to refer to T cells that have been recombinantly modified to express a chimeric antigen receptor. As used herein, CAR-T cells can be engineered to express an orthogonal receptor ("orthogonal CAR-T cells" or "orthoCAR-T cells").

[0012] Also provided is a cell product having a substantially enriched population of orthogonal CAR-T cells, the cell product being obtained by a process comprising the steps of: (a) isolating a quantity of immune cells from a mammalian subject (e.g., a human subject); (b) contacting the quantity of isolated immune cells with a nucleic acid sequence under conditions to allow uptake of the nucleic acid sequence by the isolated immune cells, wherein the nucleic acid sequence encodes an orthogonal GPC3 CAR and an orthogonal hCD122, or a functional fragment thereof; (c) contacting ex vivo a quantity of the isolated cells from step (b) with an orthogonal ligand in an amount sufficient to induce proliferation of the cells transduced by the contacting in step (b), wherein the contacting in (c) is applied for a period of time such that the transduced cells constitute at least 20% of the population of cells.

[0013] The present disclosure provides a mammalian immune cell comprising: (a) a nucleic acid sequence encoding an orthogonal hCD122 receptor, operably linked to one or more expression control elements such that the mammalian immune cell expresses the orthogonal hCD122 receptor, and (b) a nucleic acid sequence encoding a GPC3 CAR, operably linked to one or more expression control elements such that the mammalian immune cell expresses the CAR. In some embodiments, the nucleic acid sequence encoding the GPC3 CAR and the nucleic acid sequence encoding the orthogonal receptor are provided on separate vectors, each nucleic acid sequence operably linked to an expression control sequence that functions in the mammalian immune cell.

[0014] In some embodiments, the nucleic acid sequence encoding GPC3 CAR and the nucleic acid sequence encoding orthogonal receptor are provided on a single vector.In some embodiments, these nucleic acid sequences are operably linked to the same expression control element.In some embodiments, the vector comprises the two nucleic acid sequences separated by the IRES element of T2A coding sequence.In some embodiments, the vector is a viral vector.In some embodiments, the vector is a lentiviral vector or a retroviral vector.

[0015] In some embodiments, there is provided a method of treating or preventing a disease, disorder, or condition in a mammalian subject in need of such treatment or prevention, comprising: (a) isolating a quantity of immune cells from a subject; (b) contacting the quantity of isolated immune cells with a nucleic acid sequence under conditions to allow uptake of the nucleic acid sequence by the isolated immune cells, the nucleic acid sequence encoding a transmembrane receptor, the transmembrane receptor comprising an intracellular signaling domain operably linked to an extracellular domain, the extracellular domain of the receptor comprising an orthogonal ECD of hCD122 or a functional fragment thereof; (c) contacting ex vivo a quantity of the isolated cells from step (b) with an orthogonal ligand in an amount sufficient to induce proliferation of the cells transduced by the contacting in step (b), wherein said contacting in (c) is applied for a period of time such that the transduced cells constitute at least 20% of the cell population; and (d) administering a therapeutically effective amount of cells of the cell population generated from step (c) to a mammalian subject in combination with administration of a therapeutically effective dose of an orthogonal ligand. A method is provided, comprising:

[0016] In some embodiments, after step (a) and before step (b), the population of cells is manipulated ex vivo such that activated immune cells or antigen-exposed T cells are enriched in the population.

[0017] In some embodiments, the disclosure provides orthogonal GPC3 CAR T cells (orthogonal immune cells) that have been recombinantly modified to express an orthogonal receptor. In some embodiments, the orthogonal IL2 receptor is human variant CD122, comprising amino acid substitutions at positions 133 and 134, as numbered according to human wild-type CD122 (SEQ ID NO:4). In some embodiments, the orthogonal IL2 receptor is human variant CD122 with amino acid substitutions at positions 133 and 134, as numbered according to SEQ ID NO.1, wherein the substitution at position 133 is selected from the group consisting of H133D, H133E, and H133F, and the substitution at position Y134 is selected from the group consisting of Y143F, Y134, or Y134R. In some embodiments, the orthogonal IL2 receptor is human variant CD122 (SEQ ID NO:2), having amino acid substitutions H133D and Y134F, as numbered according to SEQ ID NO:1.

[0018] In some embodiments, the present disclosure provides a method for preparing an engineered immune cell product substantially enriched in orthogonal GPC3 CAR T cells, the method comprising: (a) isolating a mixed population of immune cells from a subject; (b) transfecting a portion (fraction) of the isolated immune cell population with a recombinant vector capable of causing expression of orthogonal GPC3 CAR T cells in the transfected cells; (c) culturing the cell population in the presence of orthogonal IL2, such that cells expressing an orthogonal receptor selectively grow and the cells expressing the orthogonal receptor are enriched in the mixed immune cell population. In some embodiments, the present disclosure provides a method for preparing a cell population enriched in orthogonal GPC3 CAR T cells. In some embodiments, the present disclosure provides a mammalian cell population enriched in orthogonal GPC3 CAR T cells.

[0019] In some embodiments, the present disclosure provides an orthogonal IL2 that specifically and selectively binds to the extracellular domain (ECD) of a transmembrane polypeptide comprised of a modified CD122 polypeptide (orthogonal CD122). The binding of orthogonal IL2 to orthogonal CD122 is involved in intracellular signal transduction pathways that result in activation of the natural intracellular signaling pattern associated with IL2 binding to either the medium or high affinity IL2 receptor, but is selective for engineered cells expressing orthogonal CD122.

[0020] The disclosure further provides a method of extending activated ("long-lasting") orthogonal GPC3 CAR T cells in vivo in a mammalian subject by administering to the subject an effective amount of orthogonal IL2.

[0021] The disclosure further provides a method of specifically and selectively activating and / or inducing the proliferation of orthogonal GPC3 CAR T cells in vivo in a mammalian subject by administering to the mammalian subject an effective amount of orthogonal GPC3 CAR T cells in combination with an effective amount of orthogonal IL2.

[0022] The disclosure further provides a method of treating a mammalian subject suffering from a neoplastic disease by administering to the mammalian subject an effective amount of orthogonal GPC3 CAR T cells in combination with administration of a therapeutically effective amount of orthogonal IL2, where the orthogonal receptor is expressed on the orthogonal CAR-T cells.

[0023] The disclosure further provides a method of treating a mammalian subject suffering from a neoplastic disease characterized by the presence of a solid tumor (e.g., HCC) by administering to the mammalian subject a therapeutically effective amount of orthogonal GPC3 CAR T cells in combination with administration of an effective amount of an orthogonal ligand that is cognate to a receptor expressed in the orthogonal GPC3 CAR T cells.

[0024] The present disclosure further provides a method of restoring a therapeutically effective amount of exhausted orthogonal IL2 activity in a subject by administering a therapeutically effective amount of orthogonal IL2 to the subject.

[0025] The disclosure further provides a method of treating a mammalian subject suffering from a flare-up of a neoplastic disease in a treatment regimen characterized by prior administration of a therapeutically effective amount of an orthogonal, comprising: (i) administering to the subject an effective amount of orthogonal IL2 sufficient to restore activity of the previously administered orthogonal CAR-T cells; and, optionally, (ii) periodically administering to the subject an effective amount of an orthogonal ligand that is cognate to an orthogonal receptor expressed on the previously administered orthogonal CAR-T cells to maintain activity of the orthogonal CAR-T cells for a sufficient period of time; (iii) evaluating the subject for the presence of neoplastic disease, and in the absence of signs of neoplastic disease, either discontinuing administration of the orthogonal ligand or continuing periodic administration of the orthogonal ligand according to a maintenance dosing protocol sufficient to maintain a sufficient amount of orthogonal CAR-T cells for immune surveillance of the neoplastic cells.

[0026] The disclosure further provides a method of treating a mammalian subject suffering from a relapsed or refractory neoplastic disease in a treatment regimen characterized by prior administration of an orthogonal CAR-T cell product, the method comprising: (i) administering to the subject an effective amount of an orthogonal ligand, the orthogonal ligand being cognate to an orthogonal receptor expressed on the previously administered orthogonal CAR-T cells, sufficient to restore activity of the previously administered orthogonal CAR-T cells; (ii) periodically administering to the subject an effective amount of an orthogonal ligand, the orthogonal ligand being cognate to a receptor expressed on the previously administered orthogonal CAR-T cells, to maintain activity of the orthogonal CAR-T cells for a period sufficient to elicit a therapeutic response; (iii) administering to the subject an effective amount of an orthogonal ligand, the orthogonal ligand being cognate to a receptor expressed on the previously administered orthogonal CAR-T cells, sufficient to restore activity of the previously administered orthogonal CAR-T cells; evaluating the subject for the presence of neoplastic disease, and in the absence of signs of neoplastic disease, either discontinuing administration of the orthogonal ligand or continuing regular administration of the orthogonal ligand according to a maintenance dosing protocol sufficient to maintain a sufficient amount of orthogonal CAR-T cells for immune surveillance of the neoplastic cells.

[0027] In some embodiments, the GPC3 CAR is a polypeptide having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99% sequence identity to any one of SEQ ID NOs: 34, 35, 36, 37, 38, 39, and 40. In another embodiment exemplified herein, the GPC3 CAR is selected from the group consisting of SEQ ID NO: 37 (also referred to herein as DR625), SEQ ID NO: 38 (also referred to herein as DR626), and SEQ ID NO: 39 (also referred to herein as DR628).

[0028] In one embodiment, the disclosure provides an ortho-GPC3 CAR T cell, expressing on the surface of the ortho-GPC3 CAR T cell: (a) a GPC3 CAR having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99% sequence identity to the amino acid sequence of SEQ ID NO:37; and (b) an ortho-CD122 having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4.

[0029] In one embodiment, the disclosure provides an orthoGPC3 CAR T cell, expressing on the surface of the orthoGPC3 CAR T cell: (a) a GPC3 CAR having the amino acid sequence of SEQ ID NO:37; and (b) an orthoCD122 having the amino acid sequence of SEQ ID NO:4.

[0030] In one embodiment, the disclosure provides a method of making an OrthoGPC3 CAR T cell, the method comprising the steps of: (a) obtaining a sample of human peripheral blood mononuclear cells (PBMCs); (b) contacting a sample of PBMCs with magnetic beads coated with a CD3 ligand and a CD28 ligand to provide an activated PBMC population; (c) isolating CD8+ T cells and CD4+ T cells from the activated PBMC cell population; (d) contacting the isolated population of CD8+ T cells and CD4+ T cells with a recombinant lentiviral vector such that a fraction of the isolated population of CD8+ T cells and CD4+ T cells are transduced with the lentiviral vector, the lentiviral vector being 5' to 3' · promoters active in T cells, such as the EF1a promoter; A nucleic acid sequence encoding a signal peptide ( a nucleic acid sequence encoding a GPC3 CAR; T2A sequence (SEQ ID NO:41) A nucleic acid sequence encoding a signal peptide The nucleic acid sequence encoding ortho-CD122 of SEQ ID NO:4 and comprising an expression cassette comprising a nucleic acid sequence encoding (e) contacting the cell population obtained from step (d) with ortho-IL2. In a preferred practice of the above method, the ortho-IL2 is selected from the group consisting of: (a) a human IL2 mutein comprising the amino acid substitutions E15S / H16Q / L19V / D20L / Q22K / M23A; ortho-IL2 of SEQ ID NO 9 (STK-007), or a PEGylated variant thereof (e.g. STK-009).

[0031] In some embodiments, the disclosure provides an Ortho-GPC3 CAR T cell, wherein the Ortho-GPC3 CAR T cell is prepared by a method comprising the steps of: (a) obtaining a sample of human peripheral blood mononuclear cells (PBMCs); (b) contacting a sample of PBMCs with magnetic beads coated with a CD3 ligand and a CD28 ligand to provide an activated PBMC population; (c) isolating CD8+ T cells and CD4+ T cells from the activated PBMC cell population; (d) contacting the isolated population of CD8+ T cells and CD4+ T cells with a recombinant lentiviral vector such that a fraction of the isolated population of CD8+ T cells and CD4+ T cells are transduced with the lentiviral vector, the lentiviral vector being 5' to 3' · promoters active in T cells, such as the EF1a promoter; A nucleic acid sequence encoding a signal peptide ( a nucleic acid sequence encoding a GPC3 CAR; T2A sequence (SEQ ID NO:41) A nucleic acid sequence encoding a signal peptide The nucleic acid sequence encoding ortho-CD122 of SEQ ID NO:4 comprising an expression cassette comprising a nucleic acid sequence encoding (e) contacting the cell population obtained from step (d) with ortho-IL2, wherein the ortho-IL2 is selected from the group consisting of a human IL2 mutein comprising the amino acid substitutions E15S / H16Q / L19V / D20L / Q22K / M23A; ortho-IL2 of SEQ ID NO 9 (STK-007), or a PEGylated variant thereof (e.g., STK-009).

[0032] In some embodiments, the present disclosure provides a method for treating or preventing a neoplastic disease, disorder, or condition in a mammalian subject in need of such treatment or prevention, comprising administering to the subject a therapeutically effective amount of ortho-GPC3 CAR T prepared substantially according to the above-described method in combination with a therapeutically effective dose of ortho-IL. In some embodiments of the above-described method, the ortho-GPC3 CAR T is administered at a concentration of 4×10 per kg. 5 In some embodiments of the above methods, when the ortho-IL2 is STK-009, the therapeutically effective amount of STK-009 for a human subject in combination with the ortho-GPC3 CAR T cells is about 1.5 mg to about 12 mg administered subcutaneously once a week.

[0033] In some embodiments, the present disclosure provides a method of treating or preventing a neoplastic disease, disorder, or condition in a mammalian subject in need of such treatment or prevention, the method comprising the steps of: (1) treating a subject with a lymphocyte depletion regimen; (2) administering to the subject a therapeutically effective amount of ortho-GPC3 CAR T prepared substantially according to the method described above in combination with a therapeutically effective dose of ortho-IL2.

[0034] In some embodiments, the lymphocyte depletion regimen comprises administration of cyclophosphamide and fludarabine. In some embodiments, the lymphocyte depletion regimen comprises administration of 300 mg / m for a period of 3 days. 2 / day cyclophosphamide, and 30 mg / m 2 In some embodiments of the above methods, after lymphodepletion, the orthogonal GPC3 CAR T is administered to the subject at a dose of 1 x 10 per kg. 5 ~5 x10 5 In some embodiments of the above methods, after lymphodepletion, the ortho GPC3 CAR T is administered at a dose of 4×10 per kg. 5 In some embodiments of the above methods, when the ortho-IL2 is STK-009, the therapeutically effective amount of STK-009 for a human subject in combination with the ortho-GPC3 CAR T cells is about 1.5 mg to about 12 mg administered subcutaneously once a week.

[0035] In one embodiment of the invention, the invention provides a method of treating a subject having a flare-up (e.g., having a recurrence of a neoplastic disease) following administration of an ortho-GPC CAR T cell therapy, the method comprising administering to the subject a therapeutically effective amount of an ortho-IL2 ligand such that the ortho-IL2 induces activation and / or proliferation of ortho-GPC3 CAR T cells in the subject. In treating a flare-up, the therapeutically effective amount of STK-009 is about 1.5 mg to about 12 mg administered subcutaneously once weekly. [Brief description of the drawings]

[0036] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which: It is emphasized that, according to common practice, the various features of the drawings are not to scale. Rather, the dimensions of the various features have been arbitrarily expanded or reduced for the sake of clarity. The drawings include the following figures:

[0037] [Figure 1] FIG. 1 provides a schematic diagram of the arrangement of the coding sequences for the various functional domains of the GPC3 CAR and the orthogonal CD122 components in the vectors used to generate DR625, DR626, and DR628.

[0038] [Figure 2A]2A-2D provide graphical representations of data obtained from studies to evaluate the in vitro efficacy of orthogonal GPC3 CAR T cell lines against cell lines expressing various levels of GPC3. The vertical axis is luminescence measurement, which is a measure of cell viability, and the horizontal axis represents the relative amount of effector (test agent orthogonal GPC3 CAR T cells) to the amount of target cells. Panel A represents results in PLC / PRF / 5 cell lines, which do not express or have very low expression of GPC3, Panel B provides data obtained with Huh-7 cell lines, which express low levels of GPC3, and Panel C provides data obtained with HepG2 cell lines, which express high levels of GPC3. Panel D provides data showing a comparison of GPC3 expression levels in the cell lines evaluated. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A.

[0039] [Figure 3A] Figures 3A-3E provide graphical representations of data from studies comparing the effect of placing CAR coding sequences and orthogonal CD122 coding sequences on expression vectors in a variety of tumor cell lines: a high expressing cell line (Panel A, HepG2), a medium / low expressing cell line (Panel B, PLC / PRF / 5), and an essentially non-expressing cell line (Panel C, Raji). Panels D and E are flow cytometric evaluations to assess the levels of GPC3 expression in HepG2 and PLC / PRF / 5 cells, respectively. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A.

[0040] [Figure 4] Figure 4 provides a graphical representation of data obtained from studies evaluating the response of DR625 and DR626 ortho GPC3 CAR T cells, as well as untransduced cells, in response to wild type human IL2 (WT IL-2) and in response to orthogonal IL2 (STK-009). The vertical axis is the proliferation signal normalized to unstimulated cells, and the horizontal axis is the concentration of the test agent, either wt hIL2 or STK-009.

[0041] [Diagram 5] Figures 5A-5B provide graphical representations of data obtained from HepG2 Study A related to the in vivo evaluation of the described DR625 and DR626 orthogonal GPC3 CAR T cells. In Panel A, tumor volume measurements are listed on the Y-axis and the time course of the study is listed on the X-axis. The legend in the figure describes the treatment group and the molecule administered. Panel B provides additional data obtained from HepG2 Study A related to the animal weight change (Y-axis) during the time course of the study (X-axis).

[0042] [Figure 6-1] Figure 6 provides a series of spider plots of data obtained from individual animals in each of the treatment groups of HepG2 Study A. For each graph, tumor volume is listed on the Y-axis and the time course of the study is listed on the X-axis. Each line of the spider plot corresponds to an individual animal in the study. [Figure 6-2] A continuation of Figure 6-1 is shown.

[0043] [Figure 7-1] Figure 7 provides a series of spider plots of data obtained from individual animals in each of the treatment groups of HepG2 Study A. The percent change in body weight compared to the start of the experiment is listed on (Y-axis) and the time course of the study is listed on (X-axis). Each line of the spider plot corresponds to an individual animal in the study. [Figure 7-2] A continuation of Figure 7-1 is shown.

[0044] [Figure 8-1] Figure 8 provides a graphical representation of data regarding the level of activation (y-axis) of specific subsets of T cells (as indicated) from mice enrolled in HepG2 Study A in response to treatment with the indicated DR625 and DR626 orthogonal GPC3 CAR T cells in combination with the orthogonal IL2 ligand, STK-009. [Figure 8-2] A continuation of Figure 8-1 is shown. [Figure 8-3] This is a continuation of Figure 8-2.

[0045] [Figure 9] 9A-9C provide graphical representations of data obtained in HepG2 Study B. In Panel A, tumor volume (y-axis) is plotted against study duration (x-axis). In Panel B, percent body weight compared to start of study (y-axis) is plotted against study duration (x-axis). In Panel C, blood hCD3+ cell counts (y-axis) in study animals are plotted against study duration (x-axis).

[0046] [Figure 10] Figure 10 shows photomicrographs of tumor sections from animals treated in each of the treatment groups of HepG2 study B, which are stained with various fluorescent markers to highlight the presence of GPC3, CD3, and DNA in tumors.The legend in the figure describes the shading associated with each fluorescent marker.

[0047] [Figure 11]Figure 11 shows photomicrographs of tumor sections from animals treated in each of the treatment groups of HepG2 Study B, stained with various fluorescent markers to highlight the presence of nucleid, CD8, granzyme B, and CD4 in the tumor. The legend in the figure describes the shading associated with each fluorescent marker.

[0048] [Figure 12] Figure 12 provides a graphical representation of the data obtained in Huh7 Study A. In Panel A, tumor volume (y-axis) is plotted against study duration (x-axis). In Panel B, percent body weight compared to start of study (y-axis) is plotted against study duration (x-axis).

[0049] [Figure 13] Figure 13 provides a representation of the data obtained in Huh7 Study B for each treatment group as a spider graph. Each animal is represented by a line in the graph. Tumor volume (y-axis) is plotted against study duration (x-axis).

[0050] [Figure 14] Figure 14 provides a graphical representation of data obtained in one subcutaneous replicate of the Huh7 study, plotting tumor volume (y-axis) against study duration (x-axis).

[0051] [Figure 15] Figure 15 provides a graphical representation of data obtained in the Huh7 intraperitoneal replicates described in the "Experimental Methods" section, plotting tumor volume (y-axis) against study duration (x-axis).

[0052] [Figure 16-1] Figure 16 provides data for re HepG2 rechallenge study A. Tumor volume (y-axis) is plotted against study duration (x-axis). [Figure 16-2] A continuation of Figure 16-1 is shown.

[0053] [Figure 17-1] Figure 17 provides a graphical representation of the data obtained from HepG2 rechallenge study B. Tumor volume (y-axis) is plotted against study duration (x-axis). [Figure 17-2] A continuation of Figure 17-1 is shown. [Figure 17-3] A continuation of Figure 17-2 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Detailed Description of the Invention In order to facilitate understanding of the present invention, certain terms and expressions are defined below and also throughout the specification. The definitions provided herein are not limiting and should be read in the light of the knowledge that would be appreciated by one of ordinary skill in the art.

[0055] Before describing the present methods and compositions, it is to be understood at the outset that this disclosure is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing certain embodiments only, and is not intended to be limiting.

[0056] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each narrower range between any specified value or any intervening value within a specified range and any other specified value or any other intervening value within that specified range is also encompassed within the invention. The upper and lower limits of these narrower ranges may be independently included or excluded from the specified range, and each range where either limit is included in the narrower range, where neither limit is included in the narrower range, or where both limits are included in the narrower range is also encompassed within the invention, and each such range is also subject to the exclusion of any particular limit in the specified range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.In the practice or testing of this invention, any method and material similar or equivalent to those described herein can be used, but some methods and materials that are likely and preferred are described herein.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material for which the publication is cited.

[0058] As used herein and in the appended claims, it should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, and a reference to a "peptide" includes one or more peptides, as well as one or more equivalents thereof known to those skilled in the art, such as polypeptides.

[0059] The publications discussed herein are provided merely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0060] The publications discussed herein are provided merely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0061] Unless otherwise indicated, the following abbreviations are used herein: parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (° C.), and pressure is at or near atmospheric. Standard abbreviations are used, including: bp = base pairs; kb = kilobase; pl = picoliter; s or sec = seconds; min = minute; h or hr = hour; AA or aa = amino acid; kb = kilobase; nt = nucleotide; pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromole; mM = millimole; M = mole; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = once daily; BID = twice daily; QW = weekly; QM = monthly; HPLC = high performance liquid chromatography; BW = body weight; U = units; ns = Not statistically significant; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Dulbecco's modified Eagle's medium; EDTA = ethylenediaminetetraacetic acid.

[0062] It will be understood that throughout this disclosure, references to amino acids will be made according to the one-letter or three-letter code. For the convenience of the reader, the one-letter and three-letter codes for amino acids are provided in Table 1 below: Table 1: Amino acid abbreviations TIFF2025509288000002.tif93128

[0063] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) "Molecular Cloning" 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel, et al. (2001) "Current Protocols in Molecular Biology" Vols. 1-4 John Wiley and Sons, Inc. New York, NY, which describe cloning in bacterial cells and mutagenesis of DNA (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)). The scientific literature describes techniques for purifying proteins, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modifications, post-translational modifications, production of fusion proteins, and glycosylation of proteins (see, e.g., Coligan, et al. (2000) "Current Protocols in Protein Science", Vol. 1-2 John Wiley and Sons, Inc., NY).

[0064] The present disclosure provides various IL2 muteins, and various CD122 receptor muteins. The following nomenclature is used herein to refer to substitutions, deletions, or insertions. Residues may be designated herein by the one-letter or three-letter amino acid code of the naturally occurring amino acid found in the wild-type molecule, followed by the position of the amino acid in IL2 in the mature IL2 molecule, e.g., "Cys125" or "C125" refers to the cysteine ​​residue at position 125 of the wild-type hIL2 molecule. With respect to ortho-IL2, substitutions are designated herein by the one-letter amino acid code, followed by the position of the amino acid in IL2, followed by the one-letter amino acid code after the substitution. For example, ortho-IL2 with the modification "K35A" refers to the replacement of the lysine (K) residue at position 35 of the wild-type IL2 sequence with an alanine (A) residue at this position. Deletions of an amino acid residue are designated as "des" followed by the amino acid residue and its position in the mature form of human wild-type IL2 (SEQ ID NO:8). For example, the term "des-Ala1" or "desA1" refers to a deletion of an alanine at position 1 of the polypeptide of the wild-type IL2 sequence. Similarly, with respect to amino acid substitutions in orthogonal CD122, amino acid substitutions are designated herein by the one-letter amino acid code of the naturally occurring amino acid, followed by the number of its position in the wild-type IL2 sequence, followed by the one-letter amino acid code of the substituted amino acid at that position. For example, if a hCD122 mutein has a substitution of a tyrosine residue at position 134 with a phenylalanine residue, the substitution is abbreviated as "Y134F".

[0065] definition Unless otherwise specified, the following terms are intended to have the meanings set forth below. Other terms are defined elsewhere throughout the specification.

[0066] ActivateAs used herein, the term "activate" is used in reference to a receptor or receptor complex that directly reflects a biological effect resulting from the binding of an agonist ligand to a receptor that is responsive to the binding of the ligand and / or that reflects the biological effect by participating in a multicomponent signal transduction cascade. For example, an IL2 agonist (an IL2 agonist ligand) is said to "activate" receptor signal transduction by binding to the IL2 receptor, resulting in one or more intracellular biological effects (e.g., phosphorylation of STAT5).

[0067] Activity As used herein, the term "activity" is used in relation to a molecule to describe the properties of the molecule in relation to a test system (e.g., an assay), or to describe a biological or chemical property (e.g., the degree to which the molecule binds to another molecule), or to describe a physical property of a substance or cell (e.g., alteration of cell membrane potential). Examples of such biological functions include, but are not limited to, the catalytic activity of a biological agent, the ability to stimulate intracellular signaling, gene expression, cell proliferation, and the ability to modulate immunological activity, such as an inflammatory response. "Activity" is typically expressed as the level of biological activity per unit of the test agent, such as catalytic activity / mg protein, immunological activity / mg protein, international units (IU) of activity, phosphorylation of STAT5 / mg protein, proliferation of T cells / mg protein, plaque forming units (pfu), etc. As used herein, the term "proliferative activity" refers to activity that promotes cell growth and replication, including dysregulation of cell division, such as that observed in neoplastic diseases, inflammatory diseases, fibrosis, metaplasia, cellular transformation, metastasis, and angiogenesis.

[0068] affinityAs used herein, the term "affinity" refers to the degree to which a first molecule (e.g., a ligand) specifically binds to a second molecule (e.g., a receptor), and is defined as the K d The binding kinetics is measured by the binding kinetics expressed as d is the dissociation constant (K off ) and the association constant between the molecule and its target (K on ) is the ratio.

[0069] AgonistAs used herein, the term "agonist" refers to a first agent that specifically binds to a second agent ("target") and interacts with the target to cause and promote increased activation of the target. In some examples, agonist is an activator of receptor protein that regulates cell activation, enhances activation, sensitizes cells to activation by a second agent, or upregulates the expression of one or more genes, proteins, ligands, receptors, biological pathways that can cause cell proliferation, or upregulates pathways that cause cell cycle arrest or cell death, such as by apoptosis. In some embodiments, agonist is an agent that binds to a receptor and changes the state of the receptor to produce a biological response. The response mimics the action of the activator that is endogenous to the receptor. The term "agonist" includes partial agonists, full agonists, and superagonists. If agonist causes substantially complete biological response induced by receptor under test (i.e., the response related to naturally occurring ligand / receptor binding interaction), such agonist can be described as "full agonist" or partial agonist. In contrast to agonist, antagonist can specifically bind to receptor, but can modify the action of agonist at the receptor without causing signal cascade, which is typically initiated by receptor. Inverse agonist is an agent that causes pharmaceutical response opposite to that of agonist. "Superagonist" is a type of agonist that can cause maximum response exceeding that of the agonist that is endogenous to the target receptor, and therefore has more than 100% activity of natural ligand.A superagonist is typically a synthetic molecule that, when evaluated at similar concentrations in comparable assays, exhibits a response in an assessable quantitative or qualitative parameter of greater than 110%, or greater than 120%, or greater than 130%, or greater than 140%, or greater than 150%, or greater than 160%, or greater than 170% of the naturally occurring form of the molecule. It should be noted that the biological effects of a full agonist may differ in magnitude and / or type from the biological effects of a partial agonist or superagonist.

[0070] Antagonist As used herein, the term "antagonist" or "inhibitor" refers to a molecule that blocks the action of an agonist.Antagonists prevent, reduce, inhibit or neutralize the activity of agonists, and antagonists can prevent, inhibit or reduce the constitutive activity of targets, such as target receptors, even in the absence of identified agonists.Inhibitors are molecules that reduce, block, prevent, delay activation, inactivate, desensitize or downregulate, for example, genes, proteins, ligands, receptors, biological pathways, including immune checkpoint pathways, or cells.

[0071] antibody As used herein, the term "antibody" collectively refers to: (a) glycosylated and non-glycosylated immunoglobulins (including, but not limited to, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4) that specifically bind to a target molecule, and (b) immunoglobulin derivatives that compete with the immunoglobulin from which they are derived for binding to a target molecule, such as IgG(1-4)deltaC. H 2, F(ab')2, Fab, ScFv, V H , V LImmunoglobulin derivatives, including but not limited to, F(ab'), F(ab')3, scFv-Fc, and (scFv)2. The term antibody is not limited to immunoglobulins derived from any mammalian species, but includes murine, human, equine, camelid, and human antibodies. The term "antibody" includes so-called "heavy chain antibodies" or "VHH" or "Nanobodies®", such as those typically obtained from immunization of camelids, including camels, llamas, and alpacas (see, for example, Hamers-Casterman, et al. (1993) Nature 1999, 144:1311-1321, 1999). 363:446-448). Antibodies with a given specificity may also be derived from non-mammalian sources, such as VHHs obtained from immunization of cartilaginous fish, including but not limited to sharks. The term "antibody" encompasses antibodies that can be isolated from natural sources or from animals following immunization with an antigen, as well as engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, veneered antibodies, or deimmunized antibodies (e.g., those in which T-cell epitopes have been removed). The term "human antibody" includes antibodies that are human, It also includes antibodies obtained from a transgenic mammal that contains human immunoglobulin genes and thereby produces antibodies that, upon stimulation with an antigen, contain amino acid sequences characteristic of antibodies produced by humans. The term antibody includes both the parent antibody and its derivatives, such as affinity matured antibodies, veneered antibodies, CDR-grafted antibodies, humanized antibodies, camelized antibodies (in the case of VHHs) or binding molecules that contain the binding domains (e.g. CDRs) of an antibody in a non-immunoglobulin scaffold.The term "antibody" should not be construed as being limited to any particular synthetic means, and includes naturally occurring antibodies that can be isolated from natural sources, as well as engineered antibody molecules prepared by "recombinant" means, including: antibodies isolated from hybridomas isolated from transgenic animals that are transgenic for human immunoglobulin genes, antibodies isolated from host cells transformed with a nucleic acid construct that causes the expression of the antibody, and antibodies isolated from combinatorial antibody libraries, including phage display libraries. In one embodiment, the "antibody" is a mammalian immunoglobulin. In some embodiments, the antibody is a "full-length antibody", which includes variable and constant domains that provide binding and effector functions. In most cases, a full-length antibody includes two light chains and two heavy chains, with each light chain including a variable region and a constant region. In some embodiments, the term "full-length antibody" is used to refer to the general IgG immunoglobulin structure, which comprises two light chains and two heavy chains, with each light chain comprising a variable region and a constant region, providing binding and effector functions. The term antibody includes antibody conjugates that contain modifications to extend the duration of action, such as fusion proteins (e.g., fusion with Fc), or conjugation with polymers (e.g., polyethylene glycol), as described in more detail below.

[0072] CDRAs used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found in both the heavy and light chain variable regions of an immunoglobulin polypeptide. CDRs are described in the following, including overlapping or subsets of amino acid residues when comparing the two: Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996). Whichever definition is applied to refer to the CDRs of an antibody or grafted antibody, or variants thereof, it is intended to be within the scope of the term as defined and used herein. In the context of this disclosure, the numbering of the CDR positions is described according to the Kabat numbering convention.

[0073] Circulating tumor cells As used herein, the term "circulating tumor cells (CTCs)" refers to tumor cells that break away from a tumor mass (e.g., a neoplasm) and enter the peripheral circulation.

[0074] ComparableAs used herein, the term "comparable" is used to describe the degree of difference between two measured values ​​of an assessable quantitative or qualitative parameter. For example, if a first measured value of an assessable quantitative parameter (e.g., the level of IL2 activity determined by CTLL-2 proliferation assay or phospho-STAT5 assay) and a second measured value of the assessable parameter do not deviate from the range that a person skilled in the art would recognize as being substantially statistically significant between the two results in the situation, the two measured values ​​can be considered "comparable". In some examples, if one measured value deviates from another measured value by less than 35%, alternatively less than 30%, alternatively less than 25%, alternatively less than 20%, alternatively less than 15%, alternatively less than 10%, alternatively less than 7%, alternatively less than 5%, alternatively less than 4%, alternatively less than 3%, alternatively less than 2%, alternatively less than 1%. In certain embodiments, a measurement is comparable to a reference standard if the measurement deviates from the reference standard by less than 15%, alternatively less than 10%, alternatively less than 5%.

[0075] Origin As used herein, the term "derived from" in the context of an amino acid sequence (e.g., a polypeptide that includes an amino acid sequence "derived from" an IL2 polypeptide or polynucleotide sequence) is intended to indicate that the polypeptide or nucleic acid has a sequence that is based on the sequence of a reference polypeptide or nucleic acid (e.g., a naturally occurring IL2 polypeptide or a nucleic acid encoding IL2), but is not intended to limit the source or the manner in which the protein or nucleic acid is made. By way of example, the term "derived from" includes homologs or variants of the reference amino acid sequence or DNA sequence.

[0076] Effective concentration (EC)As used herein, the term "effective concentration", or its abbreviation "EC", is used interchangeably to refer to a concentration of an agent (e.g., Ortho-IL2) sufficient to cause a change in a given parameter in a test system. The abbreviation "E" refers to the magnitude of a given biological effect observed in a test system when the test system is exposed to a test agent. The abbreviation "EC" is used when the magnitude of the response is expressed as a factor of the concentration ("C") of the test agent. In the context of a biological system, the term Emax refers to the maximum magnitude of a given biological effect observed in response to a saturating concentration of an activating test agent. When the abbreviation EC is written with a subscript (e.g., EC 40 , E.C. 50 etc.), the subscript refers to the percentage of the Emax of the biological effect observed at that concentration. For example, if a concentration of a test agent sufficient to cause induction of a measurable biological parameter in a test system is 30% of the maximum level of such measurable biological parameter in response to such test agent, this is the "EC 30 Similarly, the term "EC 100 The term EC is used to mean the effective concentration of an agent that elicits a maximal (100%) response of a measurable parameter in response to such an agent. 50 The term "saturation concentration", which is widely used in the field of pharmacodynamics, refers to the concentration of an agent sufficient to cause a half-maximal (50%) change in a measurable parameter. The term "saturation concentration" refers to the largest possible amount of a test agent that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacodynamics, the saturation concentration of a drug is typically used to mean a sufficient concentration of the drug such that the drug has occupied all of the available receptors, and is generally used to describe the EC 50 is the concentration of a drug that produces half its maximal effect.

[0077] Concentrated As used herein, the term "enriched" refers to a sample that has been subjected to a non-natural manipulation such that a species (e.g., a molecule or cell) of interest is present at a concentration that is: (a) greater than the concentration of the species in a starting sample, e.g., a biological sample (e.g., a sample in which the molecule naturally occurs or in which the molecule will be present after administration) (e.g., at least 3-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 50-fold greater, alternatively at least 100-fold greater, alternatively at least 1000-fold greater); or (b) greater than the environment in which the molecule was produced (e.g., in recombinantly modified bacterial or mammalian cells). In some embodiments, the term "enriched" is used herein with reference to a cell population comprising cells expressing an orthogonal receptor, after contacting the cell population with a sufficient amount of cognate ligand to elicit a response, which is proliferation, in those cells expressing the orthogonal receptor, such that the concentration of cells expressing the orthogonal receptor in the cell population is higher (e.g., at least 3-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 50-fold greater, alternatively at least 100-fold greater, alternatively at least 1000-fold greater) after contacting the cell population with the cognate ligand.

[0078] Extracellular domain As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is on the outside of the plasma membrane of a cell. The ECD may include all but the cytoplasmic portion of a transmembrane protein, a cell surface or membrane-bound protein, a secreted protein, or a cell surface targeting protein.

[0079] hCD122As used herein, the term "hCD122" refers to naturally occurring human CD122 polypeptide, including naturally occurring variants thereof. The amino acid sequence of naturally occurring mature hCD122 is provided in SEQ ID NO 4. Human CD122 (hCD122) is expressed as a 551 amino acid preprotein, but the first 26 amino acids, including the signal sequence, are post-translationally cleaved in the 525 amino acid mature protein. Amino acids 27-240 (amino acids 1-214 of the mature protein) correspond to the extracellular domain, amino acids 241-265 (amino acids 225-239 of the mature protein) correspond to the transmembrane domain, and amino acids 266-551 (amino acids 240-525 of the mature protein) correspond to the intracellular domain. As used herein, the term CD122 includes naturally occurring variants of CD122 protein, including CD122 variants that include S57F substitution and D365E substitution (numbering according to mature hCD122 protein).hCD122 is referred to as entry P14784 in the UniProtKB database.The nucleic acid sequence and protein sequence of human CD122 can be found under Genbank accession numbers NM_000878 and NP_000869, respectively.

[0080] identityThe term "identity" as used herein with respect to a polypeptide sequence or a DNA sequence refers to the identity of subunit sequences between two molecules. If the same monomer subunit (i.e., the same amino acid residue or the same nucleotide) occupies a certain subunit position in both molecules, then the two molecules are identical at that position. The similarity between two amino acid sequences or between two nucleotide sequences is a direct function of the number of identical positions. The sequences are usually aligned to obtain the maximum degree of match. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990), etc. Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, using its default parameters. Suitable algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410, and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively.Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm first involves identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequences, either match or fall within some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a matched residue pair; always > 0), and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when: the cumulative alignment score falls by a number X from the maximum value achieved; the cumulative score falls below 0 due to the accumulation of one or more negatively scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M = 1, N = -2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0081] IL2 As used herein, the term "interleukin-2" or "IL2" refers to an IL2 polypeptide having IL2 activity. In some embodiments, IL2 refers to human wild-type mature IL2. Human wild-type mature IL2 (hIL2) occurs as a 133 amino acid polypeptide (without the N-terminal 20 amino acids, which are the signal peptide of the preprotein), as described below: Fujita, et. al., PNAS USA, 80, 7437-7441 (1983). As used herein, the numbering of residues in IL2 variants is based on the human wild-type mature IL2 sequence with the signal peptide removed, UniProt ID P60568, which is identical to the sequence of SEQ ID NO: 8.

[0082] IL2 activityThe term "IL2 activity" refers to one or more biological effects on a cell in response to contacting the cell with an effective amount of an IL2 polypeptide. As mentioned above, IL2 is a multifunctional cytokine that exerts one or more effects on a variety of cell types. IL2 promotes the proliferation and expansion of activated T lymphocytes, induces the proliferation and activation of naive T cells, promotes the proliferation of B cells, and promotes the proliferation and expansion of NK cells. An example of IL2 activity can be measured in a cell proliferation assay using mouse cytotoxic T cells, CTLL-2, see Gearing, AJH and CB Bird (1987) "Lymphokines and Interferons, A Practical Approach" Clemens, MJ et al., eds. IRL Press. 295. The specific activity of recombinant human IL2 (rhIL2) is approximately 2.1×10 4 IU / μg, calibrated against recombinant human IL2 WHO International Standard (NIBSC code: 86 / 500).

[0083] An amount sufficient to elicit a responseAs used herein, the phrase "amount sufficient to elicit a response" refers to an amount of a test agent sufficient to produce a detectable change in the level of an indicator measured before application of the test agent to the test system (e.g., a baseline level) and the level of the indicator measured thereafter. In some embodiments, the test system is a cell, a tissue, or an organism. In some embodiments, the test system is an in vitro test system, such as, for example, a fluorescent assay. In some embodiments, the test system involves measuring a change in the level of a parameter of a cell, tissue, or organism that reflects a biological function before and after application of the test agent to the cell, tissue, or organism. In some embodiments, the indicator (e.g., the concentration of phosphorylated STAT5) reflects the biological function of the cell assessed in the assay (e.g., activation of the IL2 receptor) in response to administration of an amount of a test agent (e.g., IL2). In some embodiments, the test system involves measuring a change in the level of a parameter (e.g., luminescence) of a cell, tissue, or organism (e.g., a mouse injected with luminescent neoplastic cells) that reflects a biological state (e.g., the presence of a neoplasm) before and after application of one or more test agents (e.g., CAR-T cells expressing orthogonal CD122 in combination with orthogonal IL2) to the cell, tissue, or organism (e.g., a mouse). In some embodiments, the indicator (e.g., concentration of phosphorylated STAT5) reflects a biological function (e.g., activation of the IL2 receptor) of the cell (e.g., T cell) assessed in the assay in response to administration of an amount of the test agent (e.g., IL2). An "amount sufficient to elicit a response" can be sufficient as a therapeutically effective amount, while an "amount sufficient to elicit a response" can be greater or less than a therapeutically effective amount.

[0084] inhibitory factorAs used herein, the term "inhibitor" refers to a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes, or downregulates, for example, a gene, a protein, a ligand, a receptor, or a cell. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates the constitutive activity of a cell or organism.

[0085] Isolated As used herein, the term "isolated" is used in reference to a polypeptide of interest that is in an environment different from the environment in which it may naturally occur, if the polypeptide of interest is naturally occurring. "Isolated" is intended to include a polypeptide that is in a sample, in which the polypeptide of interest is substantially enriched, and / or in which the polypeptide of interest is partially or substantially purified. In the case of a non-naturally occurring polypeptide, "isolated" refers to a polypeptide that is separated from the environment in which it is produced, either by synthetic or recombinant means.

[0086] Kabat Numbering: The term "Kabat numbering" as used herein is understood in the art and refers to the numbering system of amino acid residues that are more variable (e.g., hypervariable) than other amino acid residues in the heavy and light chain regions of immunoglobulins (Kabat, et al., (1971) Ann. NY Acad. Sci. 190:382-93; Kabat, et al., (1991) "Sequences of Proteins of Immunological Interest" 5th ed. US Department of Health and Human Services, NIH Publication No. 91-3242). For the purposes of this disclosure, the positioning of CDRs in the variable regions of antibodies is according to Kabat numbering, or simply "Kabat".

[0087] Ligand As used herein, the term "ligand" refers to a molecule that specifically binds to a receptor and causes a change in the activity of the receptor or a response of a cell expressing the receptor. In one embodiment, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor. As used herein, the term "ligand" includes natural and synthetic ligands. Ligand also includes small molecules, peptide mimetics of cytokines, and peptide mimetics of antibodies. A complex of a ligand and a receptor is referred to as a "ligand-receptor complex." A ligand may constitute a domain of a polyprotein or a fusion protein (e.g., a fusion protein of a ligand targeting an antibody).

[0088] Metastasis As used herein, the term "metastasis" refers to the spread of cancerous cells from a primary tumor to surrounding tissues and to distant organs.

[0089] Adjust As used herein, the terms "modulate," "modulation," and the like refer to the ability of a test agent to cause a response in a system, including a biological system or a biochemical pathway, that is either positive or negative, or that is either direct or indirect. The term modulator includes both agonists (including partial agonists, full agonists, and superagonists) and antagonists.

[0090] N-terminusAs used herein in the context of a polypeptide structure, the terms "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the terminal amino and carboxyl groups, respectively, of a polypeptide, while the terms "N-terminal" and "C-terminal" refer to relative positions toward the N-terminus and C-terminus, respectively, of the amino acid sequence of a polypeptide and can include the N-terminal and C-terminal residues, respectively. "Immediately N-terminal" or "immediately C-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue when the first and second amino acid residues are covalently linked to form a contiguous amino acid sequence.

[0091] neoplastic disease As used herein, the term "neoplastic disease" refers to a disorder or condition in a subject that is caused by cell hyperproliferation or unregulated (or dysregulated) cell replication. The term neoplastic disease refers to a disorder caused by the presence of a neoplasm in a subject. Neoplasms can be classified as: (1) benign; (2) premalignant (or "precancerous"); and (3) malignant (or "cancerous"). The term "neoplastic disease" includes neoplasm-related diseases, neoplasm-related disorders, and neoplasm-related conditions that refer to conditions that are directly or indirectly related to neoplastic disease, and includes, for example, angiogenesis, and precancerous conditions, such as dysplasia.

[0092] nucleic acid The terms "nucleic acid," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein to refer to a polymeric form of any length of nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

[0093] Numbering according to IL2 The term "numbering according to IL2" as used herein refers to identifying a particular amino acid position with reference to the position of that amino acid normally present in the sequence of wild-type mature IL2 (SEQ ID NO: 8). For example, with respect to hIL2, "R81" refers to arginine, the 81st amino acid (counting from the N-terminus) present in the sequence of wild-type mature hIL2. It should be noted that the amino acid sequences of IL2 molecules of various mammalian species each have a different number of amino acids and differ in amino acid sequence. Thus, referring to residues according to this convention is helpful in identifying the molecular species of IL2 being referred to.

[0094] Numbering according to CD122 The term "numbering according to CD122" as used herein refers to identifying a particular amino acid position with reference to the position of that amino acid normally occurring in the sequence of the wild-type mature CD122 molecule. In one embodiment, the CD122 molecule is human wild-type mature CD122 (SEQ ID NO. 4). For example, with respect to human CD122, H133 refers to histidine, the amino acid at position 133 (counting from the N-terminus) of the sequence of wild-type mature hCD122.

[0095] Functionally linkedThe term "operably linked" is used herein to refer to the relationship between multiple nucleic acid sequences encoding different functions when combined into a single nucleic acid sequence, which provides a nucleic acid capable of causing the transcription and / or translation of a particular nucleic acid sequence in a cell when introduced into the cell. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. However, some genetic elements, such as enhancers, need not be contiguous with the sequence to effect their action.

[0096] Parent Polypeptide As used herein, the terms "parent polypeptide" or "parent protein" are used interchangeably to refer to a naturally occurring polypeptide that is subsequently modified to create a mutein or variant polypeptide. A parent polypeptide may be a wild-type (or naturally occurring) polypeptide. A parent polypeptide may refer to the polypeptide itself, or to a composition that includes the parent polypeptide (e.g., a glycosylated protein, a PEGylated protein, a fusion protein that includes the parent polypeptide).

[0097] PolypeptidesAs used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, may include chemically or biochemically modified or chemically or biochemically derivatized amino acids, and may include polypeptides with modified polypeptide backbones. The terms include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without an N-terminal methionine residue; fusion proteins with immunologically tagged proteins; fusion proteins of immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments), and the like.

[0098] Prevent As used herein, the terms "prevent", "preventing", "prevention", and the like, generally refer to a course of action initiated on a subject prior to the onset of a disease, disorder, condition, or symptoms thereof, in the context of a subject predisposed to having a particular disease, disorder, or condition due to genetic, experiential, or environmental factors, which prevents, suppresses, inhibits, or reduces the subject's risk of developing a disease, disorder, condition, or the like, either temporarily or permanently (e.g., as determined by the absence of clinical symptoms), or delays its onset. In some instances, the terms "prevent", "preventing", and "prevention" are also used to refer to delaying the progression of a disease, disorder, or condition from its current state to a more severe state.

[0099] Receptor As used herein, the term "receptor" refers to a polypeptide having a domain that specifically binds to a ligand, where binding of the ligand causes a change in at least one of the biological properties of the polypeptide. In some embodiments, the receptor is a "soluble" receptor that is not bound to a cell surface. The soluble form of hCD25 is an example of a soluble receptor that specifically binds to hIL2. In some embodiments, the receptor is a cell surface receptor that includes an extracellular domain (ECD) and a membrane-binding domain, where the membrane-binding domain acts to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide that includes an intracellular domain (ICD) and an extracellular domain (ECD), where both domains are linked by a domain that spans the membrane, typically referred to as the transmembrane domain (TM). Binding of a ligand to a receptor causes a change in the conformation of the receptor, causing a measurable biological effect. In some examples, when the receptor is a transmembrane polypeptide comprising an ECD, a TM and an ICD, the binding of a ligand to the ECD causes a measurable intracellular biological effect mediated by one or more domains of the ICD in response to the binding of the ligand to the ECD. In some embodiments, the receptor is one component of a multi-component complex that promotes intracellular signal transduction. In some embodiments, the receptor is a transmembrane single-chain polypeptide comprising an ECD, a TM and an ICD domain, wherein the ECD, the TM and the ICD domain are derived from the same naturally occurring receptor variant or from different naturally occurring receptor variants. In some embodiments, the receptor can be a hoCD122 receptor. In some embodiments, the receptor is a chimeric antigen receptor (CAR).

[0100] RecombinationAs used herein, the term "recombinant" is used as an adjective to refer to the manner in which a polypeptide, nucleic acid, or cell is modified using recombinant DNA technology. A recombinant protein is a protein produced using recombinant DNA technology, and is often abbreviated with a lowercase "r" to indicate the manner in which the protein is produced (e.g., rhIL2). Similarly, if a cell is modified using recombinant DNA technology by incorporating (e.g., by transfection, by transduction, by infection) a foreign nucleic acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vector, plasmid, cosmid, etc.), the cell is referred to as a "recombinant cell." Techniques and protocols for recombinant DNA technology are well known in the art and can be found, for example, in Sambrook, et al. (1989) "Molecular Cloning: A Laboratory Manual" 2nd ed. (Cold Spring Harbor Laboratory Press, Plainview, NY), and other standard laboratory manuals in molecular biology.

[0101] responseFor example, the term "response" of a cell, tissue, organ, or organism includes quantitative or qualitative changes in the following, where the changes are correlated with activation, stimulation, or treatment, or where the changes are correlated with internal mechanisms, e.g., genetic programming: assessable biochemical or physiological parameters, such as concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, level of gene expression, rate of gene expression, rate of energy consumption, level or state of differentiation. In some contexts, terms such as "activation," "stimulation," and the like refer to activation of cells as regulated by internal mechanisms and as regulated by external or environmental factors; whereas terms such as "inhibition," "downregulation," and the like refer to the opposite effect. Examples of such standard protocols for assessing proliferation of CD3-activated human primary T cells include Crouch, et al. (1993) "The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity" J. Immunol. Methods 160: Examples of such protocols include bioluminescent assays that produce a luminescent signal, such as those described in US Pat. No. 81-8, where the luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of cells present in culture, or standardized commercially available assays, such as the CellTiter-Glo® 2.0 Cell Viability Assay or the CellTiter-Glo® 3D Cell Viability Kit, available from Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711, catalog numbers G9241 and G9681, respectively, in substantial accordance with the instructions provided by the manufacturer.In some embodiments, the level of T cell activation in response to administration of a test agent can be determined by flow cytometry, as described by STAT5 phosphorylation levels, according to procedures well known in the art. STAT5 phosphorylation can be measured using flow cytometry techniques, as described in Horta et al., supra, Garcia et al., supra, or using commercially available kits, such as the phospho-STAT5(Tyr694) kit (Perkin-Elmer / cisbio, Waltham MA, part number 64AT5PEG), substantially following the manufacturer's instructions. EC. ACT When the abbreviation EC is used with a subscript, it is provided to indicate the concentration of test agent sufficient to produce the stated percentage of maximal STAT5 phosphorylation in T cells in response to test agent application as measured according to the test protocol. 30 PRO The abbreviation may be used to indicate the concentration associated with 30% of the maximal level of STAT5 phosphorylation in T cells in response to orthogonal hIL2 as measured using a phospho-STAT5(Tyr694) kit.

[0102] In some instances, there are established, standardized, and accepted measurements of biological activity for a molecule. For example, the standard method for evaluating the potency of hIL2 in International Units (IU) is to measure it in the mouse cytotoxic T cell line CTLL-2, according to a standardized procedure described in more detail in: Wadhwa, et al. (2013) "The 2nd International standard for Interleukin-2 (IL2) Report of a collaborative study" Journal of Immunological Methods 397:1-7. In the context of the present disclosure, it should be noted that the mouse IL2 receptor functions differently from the human IL2 receptor, particularly in that it requires all components of the trimeric receptor complex to trigger intracellular signal transduction (e.g., phosphorylation of STAT5). See, e.g., Horta, et al., (2019) "Human and murine IL2 receptors differentially respond to the human-IL2 component of immunocytokines" Oncoimmunology 8(6):e1238538-1, e1238538-15, and Nemoto, et al. (1995) "Differences in the interleukin-2 (IL2) receptor system in human and mouse: alpha chain is require for formation of the functional mouse IL2 receptor" European J Immunology 25(11)3001-5.

[0103] SelectiveAs used herein, the term "selective" is used to refer to the property of an agent to preferentially bind and / or activate a particular cell type. In some embodiments, the disclosure provides an IL2 variant (ortho-IL2) that selectively binds to an engineered CD122 ECD polypeptide such that cells expressing a receptor comprising such a CD122 ECD polypeptide are activated in response to binding of such ortho-IL2 to a receptor comprising such a cognate CD122 ECD polypeptide. In some embodiments, the disclosure provides an orthogonal hIL2 that is selective in that such ortho-IL2 exhibits preferential activation for immune cells expressing the hoCD122 receptor. Selectivity is typically assessed by activity measured in an assay that characterizes the activity induced in response to ligand / receptor binding.

[0104] Significant decrease in binding As used herein, the term "exhibits significantly reduced binding" is used in reference to the affinity of binding of a variant ligand (e.g., orthogonal IL2) to a modified form of a receptor (e.g., orthogonal CD122) compared to the binding of the variant ligand to a naturally occurring form of the receptor. In some embodiments, a ligand (e.g., orthogonal IL2) exhibits significantly reduced binding relative to a native ligand if the orthogonal ligand binds to the native receptor with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, alternatively less than about 0.5% of the affinity of the naturally occurring ligand. Similarly, an orthogonal receptor exhibits significantly reduced binding to the natural ligand if the natural ligand binds to the orthogonal receptor with an affinity that is less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, alternatively less than about 0.5% of that of the naturally occurring receptor.

[0105] Specific binding As used herein, the term "specifically bind" refers to the degree of affinity with which one molecule binds to another molecule. In the context of binding pairs (e.g., ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pairs), a first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair if the first molecule of the binding pair does not bind in significant amounts to other components present in a sample other than the second molecule. In the case of a first molecule of a binding pair, a first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair if the affinity of the first molecule for the second molecule is at least 2-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 20-fold greater, alternatively at least 100-fold greater than the affinity of the first molecule for other components present in the sample. In certain embodiments where the first molecule of the binding pair is an antibody, the equilibrium dissociation constant between the antibody and the second molecule of the binding pair is about 10, as determined, for example, by Scatchard analysis (Munsen, et al. (1980) Analyt. Biochem. 107:220-239). -6 Less than M or about 10 -8 Less than M or about 10 -10 Less than M or about 10 -11 Less than M or about 10 -10 Less than M, about 10 -12 In one embodiment, where the ligand is ortho-IL2 and the receptor comprises an orthogonal CD122 ECD, the equilibrium dissociation constant of ortho-IL2 / orthogonal CD122 ECD is about 10 -5 More than M or about 10 -6 Less than M or about 10 -7 Less than M or about 10 -8 Less than M or about 10 -9 Less than M or about 10 -10 Less than M or about 10 -11Ortho-IL2 specifically binds if the binding affinity is less than M. Specific binding may be assessed using techniques known in the art, including, but not limited to, competitive ELISA, radioligand binding assays (e.g., saturation binding, Scatchard plots, non-linear curve fit programs, and competitive binding assays); non-radioligand binding assays (e.g., fluorescence polarization (FP) assays, fluorescence resonance energy transfer (FRET) assays, and surface plasmon resonance assays (e.g., using instruments commercially available from GE Healthcare Bio-Sciences, e.g., Biacore 8+, Biacore S200, Biacore T200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA 01752) (see Drescher et al., Methods Mol Biol 493:323-343). (2009)); liquid-phase ligand-binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid-phase ligand-binding assays (e.g., multi-well plate assays, on-bead ligand-binding assays, on-column ligand-binding assays, and filter assays).

[0106] Virtually pure As used herein, the term "substantially pure" indicates that a component (e.g., a polypeptide) constitutes more than about 50% of the total content of the composition, and typically constitutes more than about 60% of the total polypeptide content. More typically, "substantially pure" refers to a composition in which at least 75%, at least 85%, at least 90%, or more of the total composition is the component of interest. In some instances, the polypeptide constitutes more than about 90% or more than about 95% of the total content of the composition.

[0107] T cellsAs used herein, the terms "T-cells" or "T cells" are used in their conventional sense to refer to lymphocytes that differentiate in the thymus and have specific antigen receptors on their cell surface, including some that control the initiation and suppression of cellular and humoral immunity, and others that lyse antigen-bearing cells. In some embodiments, T cells include, but are not limited to: naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, e.g., T H 1. T H 2. T H 9. T H 11. T H 22, T FH Regulatory T cells, such as T R 1. Tregs, inducible Tregs, etc.; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor infiltrating lymphocytes (TILs), and engineered variants of such T cells, including, but not limited to, CAR-T cells, recombinantly modified TILs, and TCR engineered cells.

[0108] Transmembrane domainThe term "transmembrane domain" or "TM" refers to a domain of a transmembrane polypeptide (e.g., a transmembrane polypeptide such as CD122 or CD132, or CAR) that is embedded in the cell membrane when the transmembrane polypeptide is bound to the cell membrane and is peptidyl linked to the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide. The transmembrane domain may be homologous (naturally associated with) or heterologous (not naturally associated with) to either or both the extracellular domain and / or the intracellular domain. In some embodiments, the transmembrane domain is a transmembrane domain that is naturally associated with the ECD domain of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, the transmembrane domain is a transmembrane domain that is naturally associated with the ICD domain of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, the transmembrane domain is the transmembrane domain that is naturally associated with the domain that transmits growth signals. In some embodiments, the transmembrane domain is the transmembrane domain that is naturally associated with another protein. Alternatively, the transmembrane domain of the orthogonal receptor may be an artificial amino acid sequence that spans the plasma membrane. In some embodiments, the transmembrane domain of the orthogonal receptor is the transmembrane domain that is normally associated with the ICD of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, when the receptor is a chimeric receptor that comprises an intracellular domain from a first parent receptor and a second extracellular domain from a second, different parent receptor, the transmembrane domain of the chimeric receptor is the transmembrane domain that is normally associated with either the ICD or ECD of the parent receptor from which the chimeric receptor is derived.

[0109] TreatThe terms "treat", "treating", "treatment" and the like refer to a course of action (e.g., administration of IL2, CAR-T cells, or pharmaceutical compositions containing same) initiated on a subject after a diagnosis, observation, etc. of a disease, disorder, condition, or symptoms thereof has been made in the subject, which course of action eliminates, reduces, inhibits, alleviates, or ameliorates, either temporarily or permanently, at least one of the underlying causes of the disease, disorder, or condition afflicting the subject, or at least one of the symptoms associated with the disease, disorder, or condition. Treatment includes a course of action taken on a subject suffering from a disease that results in the inhibition of the disease in the subject (e.g., halting the progression of the disease, disorder, or condition, or improving one or more symptoms associated therewith).

[0110] Treg cells or regulatory T cells The term "regulatory T cells" or "Treg cells" as used herein refers to CD4 T cells that are capable of suppressing the responses of other T cells. + "Treg" refers to a type of T cell, including but not limited to effector T cells (Teff). Treg cells are characterized by expression of CD4, the a subunit of the IL2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004). "Conventional CD4 + "T cells" refers to CD4 T cells other than regulatory T cells. + It means T cells.

[0111] variantThe terms "protein variant" or "variant protein" or "variant polypeptide" are used interchangeably herein to refer to a polypeptide that differs from a parent polypeptide due to at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified version of a WT polypeptide. The term variant polypeptide may refer to the polypeptide itself, to a composition comprising the polypeptide, or to a nucleic acid sequence encoding it. In some embodiments, the variant polypeptide contains about 1 to about 10 amino acid modifications compared to the parent polypeptide, or about 1 to about 5 amino acid modifications compared to the parent, or about 1 to about 3 amino acid modifications compared to the parent, or 1 to 2 amino acid modifications compared to the parent, or a single amino acid modification compared to the parent. A variant may be at least about 99% identical, or at least about 98% identical, or at least about 97% identical, or at least about 95% identical, or at least about 90% identical to the parent polypeptide from which it is derived.

[0112] Wild type By "wild-type" or "WT" or "native" herein is meant an amino acid or nucleotide sequence found in nature, including allelic variations. A WT protein, WT polypeptide, WT antibody, WT immunoglobulin, WT IgG, etc. has an amino acid or nucleotide sequence that has not been modified by the hand of man.

[0113] Detailed Description Overview The present disclosure relates to engineered T cells that express (a) a chimeric antigen receptor ("GPC-CAR"), where the antigen-binding domain of the CAR binds to human GPC3, and (b) an orthogonal receptor. The terms "chimeric antigen receptor T cells" and "CAR-T cells" are used interchangeably to refer to T cells that have been recombinantly modified to express a chimeric antigen receptor. As used herein, CAR-T cells can be engineered to express an orthogonal receptor ("orthogonal CAR-T cells" or "orthoCAR-T cells").

[0114] The present invention is directed to compositions and methods relating to ortho-GPC3 CAR T cells in combination with ortho-IL2 ligand. The use of ortho-GPC3 CAR T cells in combination with ortho-IL2 provides a variety of benefits.

[0115] In one embodiment, the disclosure provides compositions and methods that, when ortho-IL2 is administered to a human subject, provide for selective expansion of a population of adoptively transferred human ortho-GPC3 CAR-T cells in the subject.

[0116] In another embodiment, the present disclosure provides compositions and methods that allow the selective expansion of a population of adoptively transferred orthogonal human GPC3 CAR-T cells in vivo without significant systemic off-target activation of other immune cells.

[0117] In another aspect, the disclosure provides compositions and methods that support the persistence of adoptively transferred human immune cells, orthoGPC3 CAR-T cells, without the significant toxicity associated with another supportive agent, e.g., wt hIL2.

[0118] In another aspect, the present disclosure provides compositions and methods for achieving in vivo therapeutic efficacy of a cell therapy product comprising ortho-GPC3 CAR-T cells in the treatment of a neoplastic disease in a mammalian subject using an initial dose of the cell therapy agent at a dose previously reported to be ineffective and significantly below the current dosage of the similar cell therapy product;

[0119] In another aspect, the present disclosure provides compositions and methods that allow for the maintenance of therapeutic levels of ortho-GPC3 CAR-T cells in a subject at therapeutically effective levels over an extended period of time by periodically administering ortho-IL2 to the subject.

[0120] In another aspect, the present disclosure provides compositions and methods that allow for the treatment of recurrence of a neoplastic condition in a subject previously treated with ortho-GPC3 CAR-T cells by administering ortho-IL2 to restore the efficacy of the previously administered orthogonal cells without the need for additional administration of ortho-GPC3 CAR-T cells.

[0121] In another aspect, the present disclosure provides compositions and methods that do not require prior immunodepletion of the subject prior to administration of a cell product comprising orthoGPC3 CAR-T cells.

[0122] In another aspect, the present disclosure provides compositions and methods relating to pharmaceutical formulations of ortho-GPC3 CAR-T cells and ortho-IL2.

[0123] In another embodiment, a method of treating a subject suffering from a GPC3+ tumor by administering a therapeutically effective amount of ortho-GPC3 CAR-T cells in combination with a pharma- ceutically acceptable formulation of ortho-IL2. EXAMPLES

[0124] Experimental Method For the purpose of proving the efficacy of the compositions and methods encompassed within the scope of the present disclosure, a series of experiments were performed to evaluate the anti-GPC3+ tumor efficacy of ortho-GPC3 CAR T cells both in vitro and in vivo, as described in detail below, and the data are provided in the accompanying figures.

[0125] A. Preparation of GPC3 CAR-T cells for testing The nucleic acid sequence encoding GPC3 CAR was synthesized using conventional DNA sequencing techniques. The amino acid sequences of DR625, DR626, and DR628 GPC3 CAR are provided in SEQ ID NOs: 37, 38, and 39, respectively. The DNA sequences encoding the nucleic acids used in the construction of lentiviral vectors to generate DR625, DR626, and DR628 GPC3 CAR are provided in SEQ ID NOs: 42, 43, and 45, respectively.

[0126] The lentiviral vector platform used to construct the ortho GPC3 CAR T cells used in this study was a modified version of the LV200 pLenti-EF1-MCS plasmid (commercially available from Alstem, Inc., 2600 Hilltop Drive, Richmond CA USA, catalog number LV200). The vector was modified to insert an NheI restriction site into the MCS cloning site of the vector to facilitate insertion of the GPC3 CAR sequence, and a Kozak sequence was added downstream of the EcoRI site. The DNA sequence of the resulting vector is SEQ ID NO: 47.

[0127] Human PBMC populations were obtained and CD4 / CD8 T cells were isolated from the PBMCs by flow cytometry. CD4 / CD8 T cells were stimulated with CD3·CD28 beads (Miltenyi) for a period of 24 hours.

[0128] Primary blood mononuclear cells (PBMCs) from healthy donors were isolated from leukopaks by separation on Ficoll-Paque (Global Life Sciences Solutions). CD4 and CD8 T cells were stained with CD4 and CD8 microbeads and isolated using MACS magnetic separation columns (Miltenyi Biotec). Cells were stimulated with anti-CD3 (clone OKT3, Miltenyi Biotec) and anti-CD28 (clone CD28.2, BD Biosciences) antibodies.

[0129] Forty-eight hours after stimulation, cells were transduced with lentivirus and maintained in OpTmizer T cell complete medium (Gibco) containing wild-type IL-2 (Miltenyi Biotec). 24–48 hours after transduction, cells were washed and either maintained in WT IL-2-containing medium or switched to STK-009-containing medium. Cells were expanded with medium changes every other day. Cell counts and viability were confirmed using Vi-Cell XR (Beckman Coulter). Cells were frozen in cell freezing medium CryoStor CS10 (STEMCELL) on days 7–14.

[0130] Cells were transduced with a lentivirus encoding a nucleic acid sequence encoding the coding sequence of GPC3-CAR and ortho-CD122 separated by a T2A sequence, where the lentivirus was added at an infection rate and cultured for a period of 1-3 days, at which point the cells were washed to remove excess lentivirus and resuspended in fresh medium.

[0131] The DNA sequences encoding the DR625, DR626, and DR628 constructs were inserted into the multiple cloning site (MCS) of the LV200 pLenti-EF1-MCS plasmid (commercially available from Alstem, Inc., 2600 Hilltop Drive, Richmond CA USA, catalog number LV200 Alstem).

[0132] The ortho-IL2 molecule used in these studies was a PEGylated hIL2 variant that contained the following amino acid deletions and substitutions: desAla1 / E15S-H16Q-L19V-D20L-Q22K-M23A, and contained 40 kD branched (2 x 20 kD) PEG molecules with an aldehyde linker, mono-PEGylated at the N-terminus. The 40 kDa 2-arm branched PEG-aldehyde, containing two 20 kDa linear PEG molecules, was Sunbright® GL2-400AL3 (commercially available from NOF America Corporation, One North Broadway, White Plains, NY 10601 USA).

[0133] Female NOD scid gamma (NSG) mice, 6-8 weeks of age, were obtained from The Jackson Laboratory, 600 Main Street, Bar Harbor, ME USA 04609. Prior to the start of the study, animals were weighed and clinically examined to ensure they were in good health. Body weights were then tracked over the course of the study.

[0134] Except as noted below, tumor cells were administered at a 50:50 ratio of tumor cells to phenol red-free Matrigel (Corning).

[0135] For subsequent testing, GPC3 CAR T cells are identified by the GPC3 CAR that they express.

[0136] B. In vitro evaluation in GPC3-negative cell lines, GPC3 low expressing cell lines, and GPC3 high expressing cell lines A first series of experiments was performed to evaluate the effect of different costimulatory domains in GPC3 CAR T cells, and a series of experiments was performed to compare GPC3 CAR T cells containing CD28 (DR625) and GPC3 CAR T cells containing 41BB (DR628) in vitro in different cell lines with different expression levels of GPC3, as well as to evaluate activity.

[0137] The effect of the arrangement of the coding sequences of the costimulatory domain and the CAR in the vector was also evaluated. As shown in Figure 1, in DR625 the CD28 GPC3-CAR sequence is 5' to the ortho-CD122 sequence. In DR626, the arrangement of the CD28 GPC3-CAR sequence and the ortho-CD122 sequence is reversed, such that the ortho-CD122 sequence is 5' to the CD28 GPC3-CAR sequence.

[0138] To evaluate the cytotoxicity of ortho-GPC3 CAR T cells in cell lines with different expression levels of GPC3 both in vitro and in vivo, a series of experiments were performed on cells obtained from PLC / PRF / 5 (ATCC accession number CRL-8024), a hepatocellular carcinoma cell line that low-expresses or is GPC-negative.

[0139] The data set forth in Figure 2 provide the results of this series of experiments. As shown in Figure 2, orthoGPC3 CAR T cells inhibited GPC3 陽性 cells (Figure 2, Panels B and C), but not GPC 陰性 The antibody did not show any significant specific cytotoxicity against cells (PLC / PRF / 5 cells, panel A).

[0140] The effect of the arrangement of the coding sequence of the costimulatory domain and the coding sequence of the CAR in the expression vector was also evaluated. As shown in Figure 1, in DR625, the CD28 GPC3-CAR sequence is 5' to the ortho-CD122 sequence. Meanwhile, in DR626, the arrangement of the CD28 GPC3-CAR sequence and the ortho-CD122 sequence is reversed, so that the ortho-CD122 sequence is 5' to the CD28 GPC3-CAR sequence. This data is provided in Figure 3 of the attached drawings. As shown, both DR625 and DR626 ortho-GPC3 CAR T cells showed selective cytotoxicity depending on the expression level of GPC3 in target cells. In this test, DR625 and DR626 ortho-GPC3 CAR T cells showed similar performance, and the data indicates that the arrangement of the coding sequence of the GPC-CAR and the ortho-CD122 coding sequence in the vector has little effect on the cytotoxicity of ortho-GPC3 CAR T cells.

[0141] To prove that DR625 and DR626 ortho-GPC3 CAR T cells are selectively responsive to exposure to ortho-IL2, a dose study was performed to compare DR625 and DR626 ortho-GPC3 CAR T cells and non-transduced T cells. The results of this study are provided in Figure 4 of the attached drawings. All cells express wild-type IL2 receptors, so all cells were responsive to wild-type IL2. However, DR625 and DR626 ortho-GPC3 CAR T cells selectively expanded in the presence of ortho-IL2 ligand, while non-transduced cells were unresponsive to exposure to ortho-IL2. This data proves that ortho-GPC3 CAR T cells are selectively responsive to contact with ortho-IL2 ligand.

[0142] B. In vivo evaluation of ortho-GPC3 CAR T cells in tumors highly expressing GPC3 (HepG2) A series of experiments was conducted to evaluate the performance of ortho-GPC3 CAR-T cells in an epithelial solid tumor xenograft model of hepatocellular carcinoma using HepG2 cells. Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Despite recent advances in diagnosis and treatment, HCC is often diagnosed at an advanced stage and has a poor prognosis.

[0143] 1. HepG2 Study A (S2-21-001): Application of DR625 and DR626 to the subcutaneous HepG2 hepatocellular carcinoma model in NSG mice HepG2 cells (Wistar Institute) were maintained in exponential growth phase before harvesting. Cells were harvested by trypsinizing the cells and suspending them in medium. Cell concentration and viability were determined using trypan blue (minimum 98% viability). The cell suspension was then adjusted to the required concentration for inoculation.

[0144] On day 0 of the study, approximately 1 million HepG2 cells in a volume of 100 μL (Matrigel+HepG2 suspension) were inoculated subcutaneously into the hind limb of approximately 10-week-old NSG™ mice (Jackson Labs) by a single injection. The injected area was monitored until tumors were palpable. Calipers were used to measure tumors, and tumors were measured at 100 nm to 150 nm, with an average tumor size of 50-150 mm prior to treatment. 3 After randomization, they were placed into treatment groups according to the table below. TIFF2025509288000003.tif51145

[0145] On day 10 of the study, mice were administered intravenously with CAR T cells as described in the table above. Additional treatments as shown in the table above were administered subcutaneously every other day (qod). For treatment of groups 3 and 5, administration of STK-009 was discontinued due to weight loss, and an additional single dose of STK-009 was administered on day 40 of the study for group 3 and on day 33 of the study for group 5. Tumor measurements were recorded (daily) and mouse weights were also recorded (three times a week). Tumors were removed when tumor size reached its limit (up to a maximum of 2,000 mm3 ), and the animals were euthanized.

[0146] At the end of the treatment period, animals were sacrificed and necropsies were performed. Tumors were excised, weighed, and documented by digital imaging. Various tissues were harvested from sacrificed animals (submerged in RNAlater® (Sigma Aldrich), flash frozen, nucleic acid isolated, and tissues prepared for histological analysis by gross necropsy).

[0147] Data from this study for tumor volume (efficacy) and body weight (toxicity) are provided in Figure 5, panels A and B, respectively. Spider plots for each of the treatment groups in this study are provided in Figure 6. Spider plots of body weight are provided in Figure 7. Spider plots of tumor volume demonstrate durable complete responses in mice treated with DR625 orthoGPC3 CAR T cells in combination with STK-009.

[0148] In this study, mice treated with STK-009 and DR625 ortho-GPC3-CAR T cells, and mice treated with STK-009 and DR626 ortho-GPC3-CAR T cells showed transient weight loss, but the animals recovered and the toxicity was reversible.Then, the next study was performed, which titrated the administration schedule and dose level of STK-009 to continue to maintain antitumor efficacy while avoiding toxicity.It should be noted that the dose of DR625 and DR626 ortho-GPC3-CAR T cells was particularly high (e.g., 10 μg qod (every other day)), but efficacy was demonstrated at low doses (e.g., 1 μg / week).

[0149] During the study, blood was drawn weekly from each treatment group and CD8+ T cells were selected by FACS. Cell types were differentiated by CCR7 and CD45RA status sorted by flow cytometry. Four subsets of human CD8+ T cells, naive (CCR7+CD45RA+), central memory (TCM, CCR7+CD45RA-), effector memory (TEM, CCR7-CD45RA-), and CD45RA+ effector memory (TEMRA, CCR7-CD45RA+), were compared for their ability to proliferate or differentiate in response to antigen or homeostatic cytokines. Geinat, et al (2003) "Proliferation and differentiation potential of human CD8+ memory T-cell subsets in response to antigen or homeostatic cytokines", Blood;101(11):4260-6. Data are shown in Figure 8 of the accompanying drawings. As shown in Figure 8, treatment with STK-009 induces a significant increase in the levels of GPC3_28z ortho CAR T cells across all memory subtypes.

[0150] 2. HepG2 Study B (S2-21-009): Application of DR625 to a subcutaneous HepG2 hepatocellular carcinoma model Further studies were conducted to evaluate the effect of ortho-GPC3 CAR T cells in combination with administration of ortho-IL2 (STK-009), in which STK-009 was administered less frequently but for a longer period of time when compared to HepG2 Study A discussed above. This study was conducted substantially according to HepG2 Study A discussed above, but with the following modifications. In this HepG2 Study B, one type of ortho-GPC3 CAR T cell (DR625) was administered in combination with the above-mentioned HepG2 Study A (1×10 6 ) compared with a lower dose (8.5 × 10 5In HepG2 Study B, a satellite arm was provided for further immunohistochemical analysis. Whole slide scanning and signal quantification were performed using an Akoya Vectra multispectral imaging system and Akoya Vectra InForm analysis software suite. In the HepG2 Study A described above, 1 × 10 6 HepG2 cells were administered, compared with 5 × 10 6 HepG2G tumor cells were used and administered on day 0 of the HepG2 Study A study. Treatment with OrthoGPC3 CAR T cells was started on day 7. Additional treatments were administered every 3 days starting on day 7 until day 43. The study design and the various treatment arms are summarized in the table below. TIFF2025509288000004.tif51159

[0151] The results of this study are summarized in Figure 9A for efficacy (tumor volume), in Figure 9B for toxicity (body weight), and in Figure 9C for activated T cells (CD3+ cells).

[0152] As shown in Figure 9A, the antitumor effect in response to the combination of ortho-GPC3 CAR T cells (DR625) combined with ortho-IL2 (STK-009) results in a prolonged antitumor effect. It should be noted that in animals in group 3 treated with ortho-GPC3 CAR T cells (DR625) combined with ortho-IL2 (STK-009), tumors were unable to grow throughout the study period until day 43, proving the sustained efficacy of ortho-GPC3 CAR T cells when administered in combination with ortho-IL2.

[0153] As shown in Figure 9B, no weight loss was observed in animals in Group 3 treated with ortho-GPC3 CAR T cells (DR625) in combination with ortho-IL2 (STK-009), demonstrating that ortho-GPC3 CAR T cells are well tolerated when administered in combination with ortho-IL2 in mammalian subjects.

[0154] As shown in Figure 9B, animals in Group 3 treated with ortho GPC3 CAR T cells (DR625) in combination with ortho IL2 (STK-009) showed a specific increase in blood hCD3+ cells, indicating that there is in vivo expansion of GPC3 CAR T cells when administered in combination with ortho IL2.

[0155] Immunohistochemical analysis was performed according to the protocol described above, and FIG. 10 provides the results of this analysis demonstrating intratumoral apoptosis on day 29 of the study (22 days after the start of treatment with ortho GPC3 CAR T cells (DR625) in combination with ortho IL2 (STK-009). In this analysis, GPC3 was stained green, CD3 was stained red, and DNA was stained blue. This data demonstrates that ortho GPC3 CAR T cells, when administered in combination with ortho IL2, cause intratumoral apoptosis of tumor cells in solid tumors.

[0156] Further immunohistochemical analysis was performed and the results are shown in Figure 11 of the accompanying drawings. Figure 11 shows the results of multiplex staining, assessed for the presence of nuclei (blue), CD8+ cells (red), Granzyme B (green), and CD4 cells (yellow). This data demonstrates that Ortho-GPC3 CAR T cells, when administered in combination with Ortho-IL2, cause intratumoral activation of Ortho-GPC3 CAR T cells.

[0157] B. In vivo evaluation of ortho-GPC3 CAR T cells in tumors with low GPC3 expression A series of experiments were performed to evaluate the efficacy of orthoGPC3 CAR T cells (DR625) in combination with orthoIL2 (STK-009) in treating Huh7 tumors, which express GPC3 at lower levels compared to the above-mentioned studies in HepG2 tumors, which express GPC3 at relatively high levels.

[0158] 1. Huh7 Study A (S2-21-017): DR625 in the Huh7 in vivo model This study, Huh7 Study A, was carried out essentially according to the procedure described above for HepG2 Study A, with the following modifications: First, 7.5×10 5 Huh-7 cells were implanted subcutaneously, but failed to give rise to tumors after 17 days. Therefore, 2.5 × 10 6 A second subcutaneous implant of cells was performed in the opposite flank, which led to the growth of a tumor. Treatment groups and treatments are summarized in the table below: TIFF2025509288000005.tif44153

[0159] A single intravenous transfer of orthoGPC3 CAR T cells (DR625) was performed in 2.5 × 10 6 The study was performed 13 days after subcutaneous implantation of Huh7 cells (study day 13). STK-009 administration began on study day 13 and continued through study day 43. The results of this study are shown in Figure 12 of the accompanying drawings.

[0160] As shown in the data summarized in Figure 12, the combination of ortho-GPC3 CAR T cells (DR625) combined with ortho-IL2 (STK-009) did not result in significant tumor control, while the toxicity was relatively mild when assessed in terms of tumor burden. Spider plots for individual animals (Figure 13) show that some animals in group 3 responded to treatment. Given the difficulty in growing tumors initially in this experiment, this study was repeated in Huh7 study B, described below, with both intraperitoneal and subcutaneous administration of tumor cells. The above experiment was repeated with the following modifications: (a) initial transplants were 2 × 10 6 (b) subcutaneous implantation of Huh7 cells, which expressed luciferase to facilitate imaging; (b) Huh7 cells were implanted without Matrigel; (c) the dose of GPC3 orthoCAR T cells was 5×10 6 orthoGPC3 CAR T cells; (d) STK-009 was administered three times per week. Data from this study are shown in Figure 14 of the accompanying drawings. As shown, a significant proportion of mice treated with the combination of orthogGPC3 CAR T cells combined with orthoIL2 survived.

[0161] 2. Huh7 Study B S2-21-019: DR625 in an intraperitoneal Huh-7 in vivo model This Huh7 study B, as discussed above, was performed using 1 × 10 6 The study evaluated the effect of intraperitoneal administration of Huh7 luciferase cells. The treatment groups and treatments for this study are summarized in the table below: TIFF2025509288000006.tif59145

[0162] The number of tumor cells was quantified twice weekly using an IVIS imager (Perkin Elmer). Mice received an intraperitoneal injection of 100 ul D-luciferin (15 mg / ml D-luciferin in PBS). Mice were anesthetized by controlled low-flow isoflurane exposure (Somnosuite, Kent Scientific). Regions of interest (ROIs) were drawn around each individual mouse, and luminescence was measured using Living Image software (Perkin Elmer) by directly measuring the total flux (photons / sec). The results of this study are shown in Figure 15 of the accompanying drawings.

[0163] As shown in the data depicted in Figure 15, higher CAR-T cell doses (5 x 10 6Mice treated with 2×10 CAR-T cell doses demonstrated significant tumor regression. 6 Mice treated with 5×10 cells demonstrated significant tumor control. Survival data shown in FIG. 14B demonstrate that mice treated with the higher CAR-T cell dose (5×10 6 Improved survival was demonstrated in mice treated with a lower CAR-T cell dose (2 × 10 cells), with the majority of animals still alive at the end of the study. 6 Mice treated with 100 ng / mL CAR-T cell doses demonstrated improved survival compared to control (PBS), although this was lower than that observed with higher CAR-T cell doses.

[0164] Taken together, the above data regarding the treatment of Huh7 tumors demonstrates that orthoGPC3 CAR T cells administered in combination with orthoIL2 are effective in treating tumors expressing low levels of GPC3.

[0165] C. Reloading test of HepG2 test As discussed above, one of the most challenging aspects of CAR-T therapy is that the initial response to treatment is promising, with a very high response rate. However, patients eventually experience relapse, and cancer recurs even with IL2 supportive therapy. In addition, active CAR T cells in existing therapies have poor persistence, and most of the administered activated CAR T cells lose their effectiveness due to exhaustion or other mechanisms.

[0166] To evaluate the potential to treat relapse of GPC3+ tumors, animals that became tumor-free upon completion of the HepG2 study described above were rechallenged by administration of additional HepG2 tumor cells and treated with ortho-IL2 alone, without administration of ortho-GPC3 CAR T cells.

[0167] 1. HepG2 Rechallenge Study A: Tumor-free mice from the HepG2 Study A study were rechallenged with additional tumors and with STK-009 alone. In this study, surviving mice from the treatment group in HepG2 Study A discussed above received an additional dose (2×10 6 ) HepG2 tumor cells, which was then repeated on day 80. The study design and treatment schedule are summarized in the table below. TIFF2025509288000007.tif50157* Counting from the time of reloading implantation (day 0)

[0168] The data of this experiment is shown in Figure 16 of the attached drawings. As can be seen from the data shown, in the absence of additional CAR-T cells, additional STK-009 alone causes tumor regression. This data demonstrates that subjects who have previously been treated with ortho GPC3 CAR T cells and have relapsed and whose tumors have recurred can be successfully treated by administering ortho IL2 without additional CAR T cell therapy. This data suggests that the GPC3 CAR T cells previously administered in HepG2 study A could be reactivated by administering ortho IL2 (STK-009) and that the cells continued to retain anti-tumor effects.

[0169] 2. HepG2 Rechallenge Study B S2-21-012: Rechallenge of tumor-free mice from the HepG2 Study B S2-21-009 study with additional tumors and STK-009 alone A second rechallenge study, the S2-21-012 HepG2 rechallenge study, was conducted with surviving mice from HepG2 study B. In this study, surviving mice from the previous study received subcutaneous administration of HepG2-luciferase cells, 2.5E6 cells in Matrigel. The study design and treatment protocol are summarized in the table below. TIFF2025509288000008.tif54153* STK-009 was administered starting on the day of tumor cell reimplantation (day 0).

[0170] The data generated from this study is provided in Figure 17. As can be seen from the data shown, the majority of mice previously cured by ortho-GPC3 CAR T cells were able to suppress the growth of re-challenged tumors. Consistent with previous studies, this data demonstrates that subjects previously treated with ortho-GPC3 CAR T cells who have relapsed and whose tumors have recurred can be successfully treated by administering ortho-IL2 without additional CAR T cell therapy.

[0171] A. GPC3 Chimeric Antigen Receptor or GPC3 CAR: The terms "chimeric antigen receptor" and "CAR" are used interchangeably to refer to a chimeric polypeptide that includes multiple functional domains, which are arranged in sequence from the amino terminus to the carboxy terminus as follows, and may be optionally linked by one or more spacer domains: (a) an extracellular domain (ECD) including an antigen binding domain (ABD) and optionally including a "hinge" domain; (b) a transmembrane domain (TM); and (c) one or more cytoplasmic signaling domains (CSDs). CARs may also further include a signal peptide sequence, which is usually removed during post-translational processing and presentation of the CAR on the cell surface of cells transformed with an expression vector that includes a nucleic acid sequence encoding the CAR. CARs can be prepared according to principles well known in the art. See, e.g., Eshhar et al. (U.S. Pat. No. 7,741,465 B1 issued June 22, 2010); Sadelain, et al. (2013) Cancer Discovery 3(4):388-398; Campana and Imai (U.S. Pat. No. 8,399,645 issued March 19, 2013); Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS(USA) 86(24):10024-10028; Curran, et al. (2012) J Gene Med 14(6):405-15; Brogdon et al. (U.S. Pat. No. 10,174,095 issued January 8, 2019); Guedan, et al. (2019) "Engineering and Design of "Chimeric Antigen Receptors", Molecular Therapy: Methods & Clinical Development Vol. 12: 145-156.

[0172] The present disclosure provides a CAR, a GPC3 CAR, wherein the antigen binding domain (ABD) of the CAR specifically binds to GPC3. In some embodiments, the ABD of the GPC3 CAR of the present disclosure is a single domain antibody (sdAb) that selectively binds to hGPC3 ("anti-hGPC3sdAb"). In some embodiments, the anti-hGPC3sdAb of the present disclosure is a single domain antibody that selectively binds to human mature GPC3 (amino acids 25-580 of SEQ ID NO:1). In some embodiments, the anti-hGPC3sdAb of the present disclosure is a single domain antibody that selectively binds to the β subunit of human GPC3 (SEQ ID NO:2).

[0173] In some embodiments, the anti-GPC3 sdAb is an scFv (anti-hGPC3 scFv). An scFv is a polypeptide consisting of the variable regions of the heavy and light chains of an antibody immunoglobulin, covalently linked by a peptide linker (Bird, et al. (1988) Science 242:423-426; Huston, et al. (1988) PNAS (USA) 85:5879-5883; Sz Hu, et al. (1996) Cancer Research, 56, 3055-3061; U.S. Patent No. 4,946,778 issued August 7, 1990 to Ladner). In some embodiments, the anti-hGPC3 scFv selectively binds to human mature GPC3 (amino acids 25 to 580 of SEQ ID NO:1). In some embodiments, the anti-hGPC3 scFv selectively binds to the β subunit of human GPC3 (SEQ ID NO:2). Preparation of anti-GPC3 antigen ScFv begins by generating monoclonal antibodies against GPC3 or antigenic fragments thereof. The generation of monoclonal antibodies and the isolation of hybridomas are techniques well known to those skilled in the art. For example, see "Monoclonal Antibodies: A Laboratory Manual", 2nd Edition, Chapter 7 (E. Greenfield, ed. 2014 Cold Spring Harbor Press). The generation of anti-hGPC3 monoclonal antibodies begins by immunizing an immunocompetent species with hGPC3 polypeptide or an antigenic fragment thereof, including but not limited to: mouse, rabbit, horse, pig, camel, llama, dromedary, or shark. In some embodiments, the immunogen used to generate an immune response is human glypican-3 (SEQ ID NO:1) or an antigenic fragment thereof. In some embodiments, the immunogen is the β subunit of human glypican-3 (SEQ ID NO:2) or an antigenic fragment thereof.The immune response to an immunogen can be enhanced by co-administering the immunogen with adjuvants well known in the art, including, but not limited to, alum, aluminum salts, Freund's complete adjuvant (FCA), and SP-21.

[0174] The antibodies generated by the immunization process may be optimized by techniques well known in the art, such as phage display and directed evolution, to select antibodies with certain desired characteristics.See, for example, Barbas, et al. (1991) PNAS(USA) 88:7978-82; U.S. Patent No. 5,223,409 to Ladner et al., issued June 29, 1993; Stemmer, W. (1994) Nature 370:389-91; U.S. Patent No. 5,821,047 to Garrard, issued October 13, 1998; Camps, et al. (2003) PNAS(USA) 100(17): 9727-32; U.S. Patent No. 4,593,002 to Dulbecco, issued June 3, 1986; U.S. Patent No. 6,806,079 to McCafferty, issued October 19, 2004; U.S. Patent No. 7,635,666 to McCafferty, issued December 22, 2009; U.S. Patent No. 7,662,557 to McCafferty, issued February 16, 2010; U.S. Patent No. 7,723,271 to McCafferty, issued May 25, 2010; and / or U.S. Patent No. 7,732,377 to McCafferty.

[0175] Instead of immunization with GPC3 as an immunogen, anti-GPC3 scFv can be generated based on known anti-GPC3 antibody sequences. For example, see: "Bacterial Expression, Purification and Characterization of Single-Chain Antibodies", Kipriyanov, S.; Feng, et al. "Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma" (2013) PNAS (USA) 1110 (12): E1083-E1091, Chapter 150 of "The Protein Protocols Handbook", edited by John M. Walker (2002) Humana Press. Various anti-GPC3 antibodies are described in the literature. The CDRs of such antibodies can also be grafted into scFv frameworks, particularly human scFv frameworks or humanized scFv frameworks, based on techniques known in the art, to generate humanized anti-hGPC3 scFvs. Examples of known anti-GPC3 antibodies and their CDRs (based on the Kabat numbering convention) useful for preparing anti-GPC3 scFv are summarized in the table below:

[0176] Antibodies that selectively bind to GPC3 and subunit B of GPC3 are known in the art and include, but are not limited to, GC33 (U.S. Patent No. 7,919,086 to Nakano et al., and Nakano, et al (2009) Biochem Biophys Res Comm 378(2):279-84); YP7, YP8, YP9, YP9.1, 32A9 are described in: Liu, et al (2020) J Translational Medicine, 18, Article No. 295, published August 3, 2020; and Chinese Patent No. 109021108B, granted June 25, 2019.

[0177] In one embodiment, the ABD of the GPC3 CAR is an scFv that comprises the CDRs from the GC33 antibody. In some embodiments, the s ABD of the GPC3 CAR is an scFv that comprises heavy chain CDR1, CDR2, and CDR3 that independently have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 12, 13, and 115, 16, 17, respectively, and light chain CDR1, CDR2, and CDR3 that independently have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 12, 13, and 14, respectively. In some embodiments, the s ABD of GPC3 is an scFv, wherein the heavy chain CDR1, CDR2, and CDR3 are identical to SEQ ID NOs: 12, 13, and 14, respectively, and the light chain CDR1, CDR2, and CDR3 are identical to SEQ ID NOs: 15, 16, and 17, respectively.

[0178] In some embodiments, the ABD of the GPC3 CAR is an scFv, each of which has the amino acid sequence A heavy chain variable region having CDR1, CDR2, and CDR3 composed of TIFF2025509288000009.tif11145 and an amino acid sequence and a light chain variable region having CDR1, CDR2, and CDR3 composed of TIFF2025509288000010.tif11128.

[0179] As described above, the heavy and light chain regions of the scFv are linked via a polypeptide linker. In some embodiments, the linker between the heavy and light chain regions is a polypeptide linker having the following number of amino acids: 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, or 30. In some embodiments, the linker between the heavy and light chain variable regions of the scFv is a 15 amino acid polypeptide. In some embodiments, the linker between the heavy and light chain variable regions of the scFv is the amino acid sequence TIFF2025509288000011.tif4128, which is referred to in the art as a Whitlow linker, as described in U.S. Pat. No. 5,856,456.

[0180] In some embodiments, the linker is a polypeptide composed primarily of glycine (G) and serine (S) residues, referred to in the art as a "GS" linker. Glycine and glycine-serine polymers are relatively unstructured and therefore can act as neutral tethers between moieties. Examples of glycine polymers include: (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., (GmSo)n, (GSGGS)n, (GmSoGm)n, (GmSoGmSoGm)n, (GSGGSm)n, (GSGSmG)n, and (GGGSm)n, and combinations thereof, where m, n, and o are each independently selected from integers at least 1-20, e.g., from 1-18, 216, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), as well as other flexible linkers. One example of a GS linker useful in the practice of the present disclosure is the amino acid sequence TIFF2025509288000012.tif4128, which is abbreviated herein as "G4Sx3."

[0181] In some embodiments, the ABD of the GPC3 CAR is an scFv, each of which has the amino acid sequence A heavy chain variable region having CDR1, CDR2, and CDR3 composed of TIFF2025509288000013.tif17144 and an amino acid sequence and a light chain variable region having CDR1, CDR2, and CDR3 comprised of the amino acid sequence TIFF2025509288000015.tif4128.

[0182] In some embodiments, the ABD of the GPC3 CAR is an scFv, each of which has the amino acid sequence A heavy chain variable region having CDR1, CDR2, and CDR3 composed of TIFF2025509288000016.tif11145 and an amino acid sequence and a light chain variable region having CDR1, CDR2, and CDR3 comprised of the amino acid sequence and the scFv has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000019.tif37128

[0183] In some embodiments, the anti-GPC3 sdAb is an scFv, each of which has the amino acid sequence A heavy chain variable region having CDR1, CDR2, and CDR3 composed of TIFF2025509288000020.tif17144 and an amino acid sequence and a light chain variable region having CDR1, CDR2, and CDR3 comprised of the amino acid sequence In some embodiments, the anti-GPC3 sdAb is an scFv, each of which has the amino acid sequence A heavy chain variable region having CDR1, CDR2, and CDR3 composed of TIFF2025509288000023.tif11128 and an amino acid sequence and a light chain variable region having CDR1, CDR2, and CDR3 comprised of the amino acid sequences TIFF2025509288000025.tif4128, wherein the scFv has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000026.tif37128

[0184] Signal peptide (SP): In order to promote the expression of GPC3 CAR on the surface, the nucleic acid sequence encoding GPC3 CAR may further encode a signal peptide, which promotes the expression of GPC3 CAR on the surface when expressed in mammalian cells and is cleaved from the polypeptide after translation.The signal peptide may be derived from the naturally occurring signal peptide of the protein expressed on the surface or may be synthetic.In one embodiment of the present invention, when the signal peptide is derived from the naturally occurring signal peptide of the protein expressed on the surface, the signal peptide of GPC3 CAR is a signal peptide selected from the group consisting of: the signal peptide of human CD8a, the signal peptide of human serum albumin (HSA), the signal peptide of prolactin albumin, the signal peptide of human IL2, the signal peptide of human trypsinogen-2, the signal peptide of human CD-5, the signal peptide of human immunoglobulin kappa light chain, and functional derivatives thereof. Substitution of specific amino acids in naturally occurring signal peptide sequences to increase the efficiency of secretion using signal peptides is described in Stern, et al. (2007) Trends in Cell and Molecular Biology 2:1-17 and Kober, et al. (2013) Biotechnol Bioeng. 1110(4):1164-73. Instead of using a signal peptide derived from a naturally occurring source, the signal peptide may be a synthetic sequence prepared according to established principles. See, for example, Nielsen, et al. (1997) Protein Engineering 10(1):1-6; Bendtsen, et al (2004) J. Mol. Biol 340(4):783-795; Petersen, et al (2011) Nature Methods 8:785-796. In one embodiment exemplified herein, GPC3 CAR is synthesized using the amino acid sequence Contains the signal peptide of human CD8a, with TIFF2025509288000027.tif4128.

[0185] In some embodiments, the ABD of the GPC3 CAR is a VHH that specifically binds to GPC3 (SEQ ID NO:1) or a fragment thereof (e.g., SEQ ID NO:2). A VHH is a heavy chain domain fragment of an antibody produced by mammals of the Camelidae family (e.g., camel, llama, dromedary, alpaca, and guanaco), which naturally lacks light chains. A typical VHH has a molecular weight of approximately 12-15 kDa, which is much smaller than the typical IgG class antibody of mammals (150-160 kDa), which is composed of two heavy chains and two light chains. Like the more common IgG class antibodies produced in other mammalian species, camelid antibodies are selective for a specific antigen. The camelid anti-GPC3 antibodies from which anti-GPC3VHH 1 is derived are generated by immunization of camelids in the same manner as proceeding by immunization of immunocompetent animals with hGPC3 polypeptides or antigenic fragments thereof. In some embodiments, the anti-GPC3 VHH that binds human mature GPC3 (amino acids 25-580 of SEQ ID NO:1) is derived from camelid antibodies obtained by immunizing camelids with a polypeptide sequence comprising amino acids 25-580 of SEQ ID NO:1. In some embodiments, the anti-GPC3 VHH is a VHH (anti-GPC3-VHH) that specifically binds the β subunit of human GPC3 (SEQ ID NO:2), which is derived from camelid antibodies obtained by immunizing camelids with a polypeptide sequence comprising amino acids of the β subunit of GPC3 (SEQ ID NO:2).

[0186] The GPC3 CAR of the present disclosure further provides a transmembrane domain that connects the ABD (optionally including a polypeptide linker) of GPC3 CAR to the intracellular domain (ICD) of GPC3 CAR. The transmembrane domain is composed of any sequence that is thermodynamically stable in eukaryotic cell membrane. The transmembrane domain useful for constructing the GPC3 CAR useful in the practice of the present invention is composed of approximately 20 amino acids that tend to form with an α-helical secondary structure. The transmembrane domain may be derived from the transmembrane domain of a naturally occurring transmembrane protein. Alternatively, the transmembrane domain may be synthetic. When designing a synthetic transmembrane domain, amino acids that tend to form an α-helical structure are preferred. Amino acids that tend to form α-helices are well known in the art. See, for example, Pace, et al. (1998) Biophysical Journal 75:422-427. In some embodiments, the transmembrane domain of the CAR may be derived from the transmembrane domain of a type I transmembrane protein, including, but not limited to, CD3zeta, CD4, CD8, CD28. In some embodiments, the transmembrane domain is the transmembrane domain of human CD28, corresponding to amino acids 132-157 of the human CD28 precursor protein, where the precursor protein is numbered according to the human CD28 precursor protein, UniProt P10747, including the signal peptide.

[0187] The cytoplasmic domain of GPC3 CAR comprises one or more intracellular signal domains.In one embodiment, the intracellular signal domain comprises the cytoplasmic sequence of T cell receptor (TCR) and the cytoplasmic sequence of co-receptor, and their functional derivatives and subfragments, which initiate signal transduction after antigen binds to the receptor.Cytoplasmic signal transduction domains, such as those derived from the zeta chain of T cell receptor, are utilized as part of CAR to generate stimulatory signals for T lymphocyte proliferation and effector function after target antigen binds to chimeric receptor. Examples of cytoplasmic signaling domains include, but are not limited to, the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also referred to as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also referred to as ICOS), the catalytic subunits of PI3 kinase p110α, p110β, or p110δ, the human CD3 ζ chain, the cytoplasmic domain of CD134 (also referred to as OX40 and TNFRSF4), the gamma and beta chains of FcεR1, MB1 (Igα) chain, B29 (Igβ) chain, etc., the CD3 polypeptides (δ, Δ, and ε), the syk family of tyrosine kinases (Syk, ZAP 70, etc.), the src family of tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell signaling, such as CD2, CD5, and CD28.

[0188] In some embodiments, the GPC3 CAR may also provide a costimulatory domain. The term "costimulatory domain" refers to the stimulatory domain of the CAR, typically intracellular, that transmits a first specific stimulus to provide a second non-specific activation mechanism. The costimulatory domain refers to the portion of the CAR that enhances the proliferation, survival, or development of memory cells. Examples of costimulation include the costimulation of antigen-nonspecific T cells after antigen-specific signaling through the T cell receptor, and the costimulation of antigen-nonspecific B cells after signaling through the B cell receptor. Costimulation, e.g., T cell costimulation, and factors involved therein, are described in Chen & Flies. (2013) Nat Rev Immunol 13(4):227-42. In some embodiments of the disclosure, the CSD comprises one or more of the following: a member of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or a combination thereof.

[0189] One of skill in the art will recognize other costimulatory domains that can be used in combination with the teachings of the present disclosure.

[0190] In one embodiment of the present disclosure, the ICD of GPC3-CAR comprises the cytoplasmic domain of CD28 and a portion of the intracellular signaling domain of CD3 zeta.

[0191] In one embodiment of the invention, the ICD of GPC3-CAR comprises the cytoplasmic domain of 4-1BB and a portion of the intracellular signaling domain of CD3 zeta.

[0192] In another embodiment, the ICD of GPC3-CAR comprises the signaling domain of CD3-zeta, the signaling domain of CD28 and the signaling domain of CD137.

[0193] The GPC3 CAR of the present disclosure may optionally include one or more polypeptide spacers that link the functional domains of the CAR, particularly between the GPC3-ABD and the transmembrane domain of the CAR. Although not a required component of the CAR structure, the inclusion of a spacer domain is generally considered desirable to facilitate antigen recognition by the ABD. Moritz and Groner (1995) Gene Therapy 2(8) 539-546. Although there is no specific amino acid sequence required to achieve the function of a spacer, a typical property of a spacer is flexibility, allowing free movement of the ABD to facilitate recognition of the target antigen. Similarly, it has been found that there is substantial tolerance for the length of the spacer that retains CAR function. Jensen and Riddell (2014) Immunol. Review 257(1) 127-144. In constructing a CAR useful in the practice of the present invention, sequences useful as spacers include, but are not limited to, the hinge region of IgG1, the CH2-CH3 region of immunoglobulin 1, the hinge-CH2-CH3 of IgG4, the hinge-CH3 of IgG4, and the hinge of IgG4. The hinge and transmembrane domains may be derived from the same molecule, for example, the hinge and transmembrane domains of CD8-α. Imai, et al. (2004) Leukemia 18(4):676-684.

[0194] The present disclosure provides a GPC3 CAR that can be useful in preparing orthogonal GPC3 CAR T cells. In one embodiment, the GPC3 CAR comprises a signal peptide of hCD8a (italics), a GC33 scFv with a Whitlow linker (Whitlow linker is underlined) as an antigen-binding domain, a polypeptide sequence with a short sequence of the extracellular domain of hCD28, a transmembrane domain and a costimulatory domain, and the following amino acid sequence of a human CD3 ζ domain, and the GPC3 CAR is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000028.tif71128

[0195] In one embodiment, the GPC3 CAR comprises a signal peptide of hCD8a (in italics), a GC33 scFv with a G4Sx3 linker as an antigen binding domain (the G4Sx3 linker is underlined), a polypeptide sequence having a short sequence of the extracellular domain of hCD28, a transmembrane domain and a costimulatory domain, and the following amino acid sequence of a human CD3 zeta domain, wherein the GPC3 CAR has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000029.tif71128

[0196] In one embodiment, the GPC3 CAR comprises the signal peptide of hCD8a (in italics), GC33 scFv with a Whitlow linker as the antigen binding domain (Whitlow linker is underlined), the hinge and transmembrane domains of hCD8a, the costimulatory domain of human 4-1BB, and the following amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000030.tif64129

[0197] In one embodiment, the GPC3 CAR comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000031.tif151128

[0198] In one embodiment, the GPC3 CAR comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000032.tif151128

[0199] In one embodiment, the GPC3 CAR comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000033.tif71128

[0200] In one embodiment, the GPC3 CAR comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000034.tif151128

[0201] B. Ortho CD122 Receptor The second component of ortho-GPC3 CAR T cells is an ortho-CD122 receptor. The terms "ortho-CD122 receptor" or "ortho-CD122" and "oCD122" and "hoRb" are used interchangeably to refer to a transmembrane polypeptide, the extracellular domain of which comprises or comprises more than one amino acid substitution at positions H133 and Y134 as numbered according to the human mature CD122 polypeptide, and the intracellular domain is capable of signaling in mammalian immune cells. In some embodiments, the amino acid substitution at positions H133 and Y134 is H133D and Y134F.

[0202] In some embodiments, the ortho-CD122 is a human CD122 mutein comprising amino acid substitutions at positions H133 and Y134, as numbered according to the human mature CD122 polypeptide. In some embodiments, the ortho-CD122 is a human CD122 mutein comprising amino acid substitutions at positions H133D and Y134F, as numbered according to the human mature CD122 polypeptide. In some embodiments, the ortho-IL2 receptor has the amino acid sequence:

[0203] The term "ortho-CD122 receptor" or "ortho-CD122" includes chimeric orthogonal receptors in which the naturally occurring intracellular domain of CD122 is replaced with a heterologous intracellular signaling domain. As described in PCT Publication WO 2021 / 050752 to Garcia et al., the intracellular domain of an orthogonal receptor may include a signaling domain of a receptor other than the intracellular domain of CD122. An orthogonal receptor that includes an intracellular sequence heterologous to the CD122 ECD is referred to as a chimeric orthoreceptor. In one embodiment, when a transmembrane receptor includes an orthogonal CD122 ECD and CD122 ICD, binding of ortho-IL2 to such a receptor triggers an intracellular signal characteristic of CD25 / CD122 / CD132 high affinity activation of the CD122 / CD132 medium affinity IL2 receptor. In another embodiment, with respect to the chimeric orthoreceptor, binding of ortho-IL2 to the ECD of the chimeric orthoreceptor causes intracellular signaling characteristic of the intracellular domain. For example, if the chimeric orthoreceptor comprises a hoCD122 ECD and the intracellular domain is derived from the IL9 receptor, the intracellular signal induced by hoIL2 binding to the chimeric receptor causes intracellular signaling characteristic of the IL9 receptor. Examples of ortho-IL2 molecules have been described and some modified IL2 polypeptides are provided in: Garcia et al. (U.S. Patent Application Publication No. 2018 / 0228842 A1, published August 16, 2018, and U.S. Patent No. 10,869,887, issued December 22, 2020 to Garcia et al.

[0204] In one embodiment, the orthogonal CD122 is human CD122 comprising amino acid modifications at positions 133 and 134 as numbered according to the naturally occurring form of human mature CD122 (SEQ ID NO: 4). In some embodiments, the orthogonal CD122 is a hCD122 molecule comprising amino acid substitutions H133D and Y134. In one embodiment, the orthogonal receptor is a modified human CD122, which has the following amino acid sequence (without the signal peptide) of the ECD of hCD122 with substitutions H133D and Y134F, and the transmembrane (TM) domain and intracellular domain (ICD) of the wild-type hCD122 molecule, and has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: TIFF2025509288000035.tif77128

[0205] In some embodiments, the orthogonal ICD of CD122 contains one or more STAT3 binding motifs in addition to the natural STAT5 recognition motif. The additional STAT3 binding motifs promote signal transduction and also stabilize the response to IL2. STAT proteins, upon phosphorylation of a conserved tyrosine residue at the C-terminus and subsequent translocation to the nucleus, act as transcriptional activators, binding to DNA in the nucleus and activating the transcription of target genes. Hennighausen and Robinson (2008) Genes Dev. 2008; 22:711-21. Seven STAT proteins have been identified in the STAT family: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, and have functions in a variety of pathways, from the innate and adaptive immune systems to cell proliferation, differentiation, and survival. Basham et al., (2008) Nucleic Acids Res. 2008 Jun; 36(11): 3802-3818. STAT binding motifs are typically present in cytokine receptors, and binding of their corresponding cytokine ligands activates tyrosine kinases in the Janus kinase (JAK) family, which phosphorylate several tyrosine residues in the intracellular domain. The phosphorylated receptor recruits STATs to the STAT recognition motifs in the receptor, and the STATs become phosphorylated. The phosphorylated STATs dimerize and translocate to the nucleus, where they initiate the transcription of various genes. Hennighausen, supra.

[0206] In some embodiments, the modified orthogonal CD122 may contain one, two, three, or even more additional STAT3 binding motifs. In some embodiments, the STAT3 recognition motif has an amino acid sequence of YX1X2Q. In some embodiments, X1 is selected from the group consisting of L, R, F, M, and X2 is selected from the group consisting of R, K, H, and P. In some embodiments, the STAT3 recognition motif has an amino acid sequence selected from the group consisting of: YLRQ; YLKQ; YRHQ; YLRQ; YFKQ; YLPQ; YMPQ, and YDKPH.

[0207] Expression vectors for CAR-T cells The preparation of orthogonal GPC CAR T cells useful in the practice of the present invention is achieved by transforming isolated T cells with an expression vector comprising: (a) a nucleic acid sequence encoding the GPC CAR described above, and (b) a nucleic acid sequence encoding an orthogonal receptor.

[0208] The expression vector for expressing CAR in T cells may be a viral vector or a non-viral vector. The term "non-viral vector" refers to an autonomously replicating, extrachromosomal circular DNA molecule that is separate from the normal genome and is not essential for cell survival under non-selective conditions, and can cause the expression of a coding sequence in a target cell. A plasmid is an example of a non-viral vector. To facilitate transfection into a target cell, the target cell may be directly exposed to the non-viral vector, which may be under conditions that facilitate the uptake of the non-viral vector. Examples of conditions that facilitate the uptake of foreign nucleic acid by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g., Lipofectamine®, Thermo-Fisher Scientific), high salt concentration, and magnetic field (electroporation).

[0209] In some embodiments exemplified herein, the expression vector is a viral vector. As used herein, the term viral vector is used in its conventional sense to refer to any obligate intracellular parasite that does not have the machinery to synthesize proteins or generate energy, and generally refers to any enveloped or non-enveloped animal virus that is widely used to deliver foreign transgenes to mammalian cells. Viral vectors can be replication-competent (e.g., substantially wild type), conditionally replicating (engineered by recombinant to replicate under certain conditions), or replication-defective (substantially unable to replicate in the absence of a cell line that can complement the missing function of the virus). Viral vectors can have some modifications that allow the viral vector to "selectively replicate", i.e., to replicate preferentially in certain cell types or in certain phenotypic cell conditions, such as cancerous cells. The viral vector system useful in the practice of the present invention includes, for example, naturally occurring viral vector system or recombinant viral vector system.The examples of viral vectors useful in the practice of the present disclosure include recombinantly modified enveloped DNA and RNA viruses or non-enveloped DNA and RNA viruses, such as adenovirus, vaccinia virus, lentivirus, retrovirus, herpes virus, adeno-associated virus, human immunodeficiency virus, Sindbis virus, and retrovirus (including but not limited to Rous sarcoma virus), and hepatitis B virus.

[0210] Typically, a gene of interest is inserted into such a vector, typically with a viral genomic sequence, which allows packaging of the gene construct and subsequent infection of a susceptible host cell, resulting in expression of the gene of interest (e.g., a target antigen). In addition, the expression vector encoding the GPC CAR may also be an mRNA vector. When a viral vector system is used for transfection, retroviral or lentiviral expression vectors are preferred for transfecting T cells, because the vector system has a high efficiency of gene transfer into T cells, resulting in a reduction in the time to culture a significant amount of T cells for clinical application. In particular, gamma retroviruses are particularly preferred for genetic modification of clinical grade T cells and have been shown to have therapeutic effects. Pule, et al. (2008) Nature Medicine 14(11):1264-1270. Similarly, self-inactivating lentiviral vectors are also useful, as they have been shown to integrate into resting T cells. June, et al. (2009) Nat Rev Immunol 9(10):704-716.

[0211] An expression vector may encode one or more polypeptides in addition to the target antigen. In the practice of the present invention, when multiple polypeptides are expressed, each polypeptide may be operably linked to its own expression control sequence (monocistronic), or multiple polypeptides may be encoded by a polycistronic construct, in which multiple polypeptides are expressed under the control of a single expression control sequence.

[0212] Instead of using multiple expression cassettes, the nucleic acid sequence encoding the CAR polypeptide and the nucleic acid sequence encoding the IL-10 polypeptide may be encoded by a polycistronic construct, where the expression cassette comprising the nucleic acid sequence of the GPC3 CAR polypeptide and the nucleic acid sequence of the orthogonal receptor polypeptide utilizes an internal ribosome entry site (IRES) element, or the foot and mouth disease virus protein 2A (FMVD2A), to facilitate co-expression in the target cell. In one embodiment of the disclosure, the nucleic acid sequence facilitating co-expression comprises the following nucleic acid sequence: TIFF2025509288000036.tif11132, which is also referred to herein as T2A.

[0213] In one embodiment, the expression vector encoding GPC3 CAR may optionally further encode one or more immunological regulators. Examples of immunological regulators useful in the practice of the present invention include, but are not limited to, cytokines. Examples of such cytokines are interleukins, including, but not limited to, one or more of the following: IL-1, IL-2, IL-3, IL-4, Il-10, IL-12, TNF-α, interferon α, interferon α-2b, interferon-β, interferon-γ, GM-CSF, MIP1-α, MIP1-β, MIP3-α, TGF-β, and other suitable cytokines that can regulate immune response. The cytokines to be expressed may be induced to express in cells, or may be expressed with a signal sequence for extracellular presentation or secretion. IL-12 has been reported to induce enhanced antitumor effects (see Yeku, et al. Scientific Reports Vol. 7, Article No. 10541 (2017), online publication: September 5, 2017).

[0214] In one embodiment, the expression vector encoding the GPC3 CAR may optionally provide an additional expression cassette that includes a nucleic acid sequence encoding a "rescue" gene. A "rescue gene" is a nucleic acid sequence that, when expressed, renders a cell susceptible to killing by an external agent or causes a toxic condition in a cell that causes the cell to die. The inclusion of a rescue gene allows selective cell killing of transduced cells. Thus, the rescue gene provides an additional safety precaution to prevent undesired spread of transduced cells or to prevent the action of replicative vector systems when the construct is integrated into the cells of a mammalian subject. In one embodiment, the rescue gene is a thymidine kinase (TK) gene (see, e.g., U.S. Pat. No. 5,631,236, issued May 20, 1997 to Woo et al., and U.S. Pat. No. 5,601,818, issued February 11, 1997 to Freeman et al.), in which cells expressing the TK gene product are sensitive to selective killing by administration of ganciclovir.

[0215] The expression vector may also contain one or more selectable marker genes.

[0216] Preparation of Ortho-GPC3 T cells The present disclosure provides a method for preparing orthoGPC3 CAR T cells, comprising: a. obtaining a population of T cells for manipulation; b. optionally, enriching for one or more T cell subtypes; c. transforming a T cell with a first nucleic acid sequence encoding an orthogonal receptor and a second nucleic acid sequence encoding a GPC3-CAR, each nucleic acid sequence operably linked to an expression control sequence functional in a mammalian T cell such that the GPC3-CAR and the orthogonal receptor are expressed on the surface of the T cell; d. contacting the population of T cells from step (c) with one or more agents that cause proliferation of the T cells. The present invention provides a method comprising:

[0217] a. Obtaining a population of T cells for manipulation T cells useful in preparing a population of orthogonal GPC3-CAR T cells include naive T cells, central memory T cells, effector memory T cells, or a combination thereof. T cells may be obtained from the mammalian subject to be treated or may be any of the various T cell lines available in the art. T cells for transformation are typically obtained from the mammalian subject to be treated. T cells can be obtained from several sources from the mammalian subject, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, spleen tissue, and tumors. In one embodiment, T cells are obtained by apheresis. In another embodiment, T cells are isolated from peripheral blood and specific T cells (e.g., CD3 + T cells, CD28 + T cells, CD4 + T cells, CD8 + T cells, CD45RA + T cells, and CD45RO + T cells) can be isolated by selection techniques well known in the art, such as incubation with anti-CD3 / anti-CD28 conjugated beads.

[0218] In some embodiments, the T cells used to prepare the orthogonal GPC3-CAR T cells are modified to eliminate the function of endogenous TCRa and TCRb, which are referred to as allogeneic T cells, and the orthogonal GPC3-CAR T cells based on such allogeneic T cells are referred to as allogeneic GPC3-CAR T cells. In some embodiments, the T cells used as the basis of the ortho GPC3-CAR T cells are obtained from the subject to whom the orthogonal GPC3-CAR T cells derived from the T cells will be administered. In such examples, the orthogonal GPC3-CAR T cells are referred to as autologous orthogonal GPC3-CAR T cells. The T cells for manipulation as described above are taken from the subject or donor, and the T cells may be separated from the cell mixture by techniques that enrich for the desired cells, or may be manipulated and cultured without separation.

[0219] b. enriching for one or more T cell subtypes; In some embodiments, the isolated population of T cells is enriched for a certain T cell subtype before transforming the cells with the nucleic acid sequence encoding GPC3-CAR and orthogonal receptor. Various techniques are known in the art for enriching a certain T cell subtype in a mixed population of T cells. Techniques for accurate separation include fluorescence-activated cell sorting. Non-dead cells may be selected by utilizing a dye that binds to dead cells (e.g., propidium iodide). The separated cells may be harvested in any suitable medium, which maintains the viability of the cells and usually has a cushion of serum at the bottom of the harvesting tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., which are often supplemented with fetal calf serum (FCS). The harvested and optionally enriched cell population may be used immediately for genetic modification, or may be frozen and stored at liquid nitrogen temperature, which can be thawed and reused. Cells are generally maintained in 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Ortho-IL2 can be used to selectively expand such ortho-GPC3-CAR T cells in vitro or in vivo.

[0220] c. Transforming T cells with a nucleic acid sequence encoding a GPC3 CAR and a nucleic acid sequence encoding an orthogonal receptor: The selected population of T cells is transformed with an expression vector encoding GPC3-CAR substantially in accordance with the teachings hereinabove.

[0221] d. Contacting the population of T cells, comprising a mixed population of ortho-GPC3 CAR T cells, with one or more agents that cause proliferation of the T cells. Following transformation, the T cells can be activated and expanded, generally using, for example, the methods described in 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. 2006 / 0121005. Generally, the T cells of the invention are expanded by culturing the cells in contact with a surface that provides an agent that stimulates signals associated with the CD3 TCR complex (e.g., an anti-CD3 antibody) and an agent that stimulates costimulatory molecules on the surface of the T cells (e.g., an anti-CD28 antibody). Suitable conditions for culturing T cells are known in the art, such as Lin, et al. (2009) Cytotherapy 11(7):912-922 ("Optimization and validation of a robust human T-cell culture method for monitoring phenotypic and polyfunctional antigen-specific CD4 and CD8 T-cell responses"); Smith, et al. (2015) Clinical & Translational Immunology 4:e31, published online January 16, 2015 ("Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement"). During the expansion phase, the population of T cells is maintained under conditions sufficient to support cell proliferation, such as an appropriate temperature (e.g., 37 degrees) and atmosphere (e.g., air supplemented with 5% CO2).

[0222] It will be clear to those skilled in the art that the above-mentioned step of contacting a cell population with one or more vectors encoding GPC3-CAR and an orthogonal ligand does not result in 100% transduction efficiency in the cell population obtained therefrom, and therefore the resulting cell population remains a mixed cell population comprising ortho-GPC3-CAR T cells and non-transformed T cells. Since it is desirable for the cell population to be administered to a subject to be as enriched as possible for ortho-GPC3-CAR T cells, in some embodiments, the cell population is contacted with ortho-IL2 during the expansion phase. Contacting a mixed cell population consisting of ortho-GPC3-CAR T cells and non-transformed T cells with ortho-IL2 causes selective activation and proliferation of ortho-GPC3-CAR T cells, resulting in substantial enrichment of the presence of ortho-GPC3-CAR T cells in the resulting cell product.

[0223] In some embodiments, the ortho-IL2 used to selectively expand GPC3-CAR T cells in a mixed cell population is a human IL2 mutein that comprises the following set of amino acid substitutions, numbered according to human wild-type mature IL2: E15S / H16Q / L19V / D20L / Q22K / M23A.

[0224] In some embodiments, the ortho-IL2 used to selectively expand GPC3-CAR T cells in a mixed cell population is STK-007.

[0225] In some embodiments, the ortho-IL2 used to selectively expand GPC3-CAR T cells in a mixed cell population is STK-009.

[0226] Instead of transducing isolated T cells with a recombinant vector encoding the GPC3 CAR sequence and the ortho-CD122 sequence, a nucleic acid sequence encoding a signal peptide-GPC3CAR-T2A-ortho-CD122 polypeptide may be inserted into the genome of the T cell genome. In some embodiments, the present invention provides an ortho-GPC3 CAR T cell, in which a nucleic acid sequence encoding the GPC3 CAR and a nucleic acid sequence encoding the ortho-CD122 are inserted into the genome of a mammalian T cell. Homologous recombination has been shown to facilitate site-specific integration of large transgenes in the T cell genome (Schumann. et al. "Generation of knock-in primary human T cells using Cas9 ribonucleoproteins" Proc Natl Acad Sci USA. (2015) 112:10437-42. doi: 10.1073 / pnas.1512503112). In this approach, the DNA of the target gene is cleaved using Cas9 RNP, followed by delivery of the gene of interest to the cleavage site using adeno-associated virus (AAV). Site-specific integration of the transgene is achieved by HDR. Anti-CD19 CAR gene has been successfully integrated into the TRAC locus using the combined action of Cas9 / RNP and AAV donor vector (Eyquem, et al. "Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumour rejection" Nature. (2017) 543:113-7. It is reported that directing the nucleic acid sequence encoding the signal peptide-GPC3CAR-T2A-orthoCD122 polypeptide into the TRAC locus can not only result in uniform expression of the GPC3 CAR polypeptide and the orthoCD122 polypeptide, but also in resistance to exhaustion of CAR T cells.Furthermore, inserting the GPC3 CAR sequence and ortho-CD122 sequence into a defined location on the genome of the host cell avoids the risk of inducing carcinogenesis and places the expression of GPC3 CAR and ortho-CD122 under the control of endogenous regulatory elements.

[0227] Insertion of the nucleic acid sequences encoding the GPC3 CAR and ortho-CD122 polypeptides into the genome can be accomplished by sequence-specific endonucleases that recognize and cleave nucleic acid molecules at a particular "target" sequence. Endonucleases are often categorized with respect to the degree of specificity and sequence identity characteristic of their target sequence. Some endonucleases are referred to as "rare-cutting" endonucleases when such endonucleases have a polynucleotide recognition site greater than about 12 base pairs (bp) in length, more preferably 14-55 bp in length. Rare-cutting endonucleases can be used to inactivate genes at a locus, or rare-cutting endonucleases can be used to integrate transgenes by homologous recombination (HR), i.e., by inducing a double-stranded DNA break (DSB) at a locus and inserting foreign DNA at this locus by gene repair mechanisms. Examples of rare-cutting endonucleases include: homing endonucleases (Grizot, et al (2009) Nucleic Acids Research 37(16):5405-5419), chimeric zinc finger nucleases (ZFNs) resulting from the fusion of engineered zinc finger domains (Porteus M and Carroll D., "Gene targeting using zinc finger nucleases" (2005) Nature Biotechnology 23(3):967-973, TALE nucleases, Cas9 endonucleases from the CRISPR system, or restriction endonucleases engineered to extend sequence specificity (Eisenschmidt, et al. 2005; 33(22): 7039-7047). In some embodiments of the invention, immune cells (e.g., CAR-Ts expressing the orthogonal receptor ECD of Formula 1) are engineered to reduce alloreactivity by inactivating another component of the T cell receptor (TCR).Methods for such modification of T cells are described in U.S. Patent Application Publication No. 2013 / 015884 A1, issued Nov. 28, 2013, to Galetto et al., the teachings of which are incorporated herein by reference, and for TCRα-deficient T cells by expressing pTα, which results in restoration of functional CD3 complexes, are described in U.S. Patent No. 10,426,795 B2, issued Oct. 21, 2019, to Galetto et al., the teachings of which are incorporated herein by reference.

[0228] Ortho-IL2: As used herein, the term "ortho-IL2" or "oIL2" refers to an IL2 mutein derived from a parent polypeptide of IL2, where the ortho-IL2 specifically binds to the extracellular domain of the ortho-CD122 receptor and exhibits significantly reduced binding to the extracellular domain of wild-type CD122. In some embodiments, the ortho-IL2 exhibits specific binding to a receptor comprising an orthogonal CD122 ECD, and (2) contact with a cell expressing a transmembrane receptor comprising the ECD of the orthogonal CD122 polypeptide in an amount sufficient to elicit a response produces a signal characteristic of a signal emanating from the intracellular domain (ICD) of the transmembrane receptor.

[0229] As described in PCT Publication WO 2021 / 050752 to Garcia et al., the intracellular domain of an orthogonal receptor may include a signaling domain of a receptor other than the intracellular domain of CD122. An orthogonal receptor that includes an intracellular sequence heterologous to the CD122 ECD is referred to as a chimeric orthoreceptor. In one embodiment, when a transmembrane receptor includes an orthogonal CD122 ECD and CD122 ICD, ortho-IL2 binding to such a receptor triggers an intracellular signal characteristic of CD122 / CD132 medium affinity IL2 receptor activation, CD25 / CD122 / CD132 high affinity. In another embodiment, for a chimeric orthoreceptor, ortho-IL2 binding to the ECD of the chimeric orthoreceptor triggers an intracellular signal characteristic of the intracellular domain. For example, if a chimeric ortho-receptor comprises a hoCD122 ECD and the intracellular domain is derived from the IL9 receptor, the intracellular signal induced by hoIL2 binding to the chimeric receptor causes intracellular signaling characteristic of the IL9 receptor. Examples of ortho-IL2 molecules have been described and some modified IL2 polypeptides are provided in Garcia et al. (U.S. Patent Application Publication No. 2018 / 0228842 A1, published August 16, 2018, and U.S. Patent No. 10,869,887, issued December 22, 2020 to Garcia et al.

[0230] In some embodiments, the amino acid sequence of ortho-hIL2 has at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99% sequence identity to any one of the identity to a polypeptide having the following amino acid sequence: TIFF2025509288000037.tif71128 where: AA1 is A (wild type) or deleted; AA2 is P (wild type) or deleted; AA3 is T (wild type), C, A, G, Q, E, N, D, R, K, P, or deleted; AA4 is S (wild type) or deleted; AA5 is S (wild type) or deleted; AA6 is S (wild type) or deleted; AA7 is T (wild type) or deleted; AA8 is K (wild type) or deleted; AA9 is K (wild type) or deleted; AA13 is Q (wild type), W, or deleted; AA14 is L (wild type), M, W, or deleted; AA15 is E (wild type), K, D, T, A, S, Q, H, or deleted; AA16 is H (wild type), N, Q, or deleted; AA18 is L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D, or T; AA19 is L (wild type), A, V, I, or deleted; AA20 is D (wild type), T, S, M, L, or deleted; AA22 is Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, F, or deleted; AA23 is M (wild type), A, W, H, Y, F, Q, S, V, L, T, or deleted; AA27 is G (wild type), K, S, or deleted; AA38 is R (wild type), W, or G; AA39 is M (wild type), L, or V; AA42 is F (wild type) or K; AA51 is either T (wild type), I, or deleted. AA55 is H (wild type) or Y; AA74 is Q (wild type), N, H, or S; AA80 is L (wild type), F, or V; AA81 is R (wild type), I, D, Y, T, or deleted; AA85 is L (wild type) or V; AA86 is I (wild type) or V; AA88 is N (wild type), E, ​​Q, or deleted; AA89 is I (wild type) or V; AA91 is V (wild type), R, or K; AA92 is I (wild type) or F; AA97 is K (wild type) or Q; AA104 is M (wild type) or A; AA109 is D (wild type), C, or an unnatural amino acid with an activated side chain; AA113 is T (wild type) or N; AA125 is C (wild type), A, or S; AA126 is Q (wild type) or H, M, K, C, D, E, G, I, R, S, or T; and / or AA130 is S (wild type), T, or R.

[0231] In one embodiment, the ortho-IL2 is human ortho-IL2, which comprises the following set of amino acid substitutions as numbered according to human mature IL2 (SEQ ID NO:7): E15S / H16Q / L19V / D20L / Q22K / M23A.

[0232] In one embodiment, the ortho-IL2 is human ortho-IL2 comprising the amino acid substitution set: desAla1 / E15S-H16Q-L19V-D20L-Q22K-M23A. In one embodiment, the ortho-IL2 is a polypeptide comprising the following amino acid sequence: TIFF2025509288000038.tif24128

[0233] This is also called STK-007.

[0234] In some embodiments, the present disclosure provides human ortho-IL2 that is amenable to recombinant expression in bacterial cells by eliminating the unpaired cysteine ​​residue at position 125 and / or by post-translational processing by endogenous bacterial proteases to eliminate the Met at the N-terminus of the directly expressed IL2 polypeptide and the alanine at position 1. When an amino acid is missing, it is referred to as "des". In some embodiments, the cysteine ​​at position 125 is replaced with an alanine or serine (C125A or C125S). Such mutations are typically used to prevent protein misfolding when the protein is recombinantly expressed in bacteria and isolated from inclusion bodies.

[0235] In some embodiments, human ortho-IL2 contains one or more mutations at positions on the hIL2 sequence that either contact hCD122 or that change the configuration of other positions that contact CD122, resulting in ortho-IL2 with increased affinity for CD122. Residues of IL2 that have been identified to be involved in binding of IL2 to CD122 include L12, Q13, H16, L19, D20, M23, Q74, L80, R81, D84, L85, I86, S87, N88, I89, V91, I92, E95. In some embodiments, ortho-IL2 contains one or more of the following amino acid substitutions: Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I92F, or a combination thereof. In some embodiments, ortho-IL2 comprises one or more of the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F. In some embodiments, ortho-IL2 comprises one or more of the following amino acid substitutions: N74Q, L80F, R81D, L85V, I86V, I89V, and I92F. In some embodiments, ortho-IL2 comprises one or more of the following amino acid substitutions: Q74N, L80V, R81T, L85V, I86V, and I92F. In some embodiments, ortho-IL2 comprises one or more of the following amino acid substitutions: Q74H, L80F, R81D, L85V, I86V, and I92F. In some embodiments, ortho-IL2 comprises one or more of the following amino acid substitutions: Q74S, L80F, R81D, L85V, I86V, and I92F. In some embodiments, ortho-IL2 comprises one or more of the following amino acid substitutions: Q74N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, ortho-IL2 comprises one or more of the following amino acid substitutions: Q74S, R81T, L85V, and I92F. In some embodiments, ortho-IL2 comprises [L80F-R81D-L85V-I86V-I92F]. In some embodiments, ortho-IL2 comprises the substitution L85V, which has been identified to increase the affinity of IL2 for CD122.

[0236] Modifications that modulate affinity to CD25 In some embodiments, ortho-IL2 contains one or more mutations at positions on the IL2 sequence that either contact CD25 or change the configuration of other positions that contact CD25, resulting in reduced affinity for CD25. The mutations can be in or near regions known to be close to CD25 based on published crystal structures (Wang, et al Science 310:1159 2005). Residues of IL2 believed to contact CD25 include K35, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, V69, L72, and Y107. In some embodiments, the ortho-IL2 of the disclosure comprises one or more of the following point mutations: R38A, F41A, and F42A (Suave, et al (1991) PNAS(USA)88:4636-4640); P65L (Chen et al. Cell Death and Disease (2018) 9:989); F42A / G / S / T / Q / E / N / R / K, Y45A / G / S / T / Q / E / N / D / R / K / , and / or L72G / A / S / T / Q / E / N / D / R / K (U.S. Patent Application Publication No. 2012 / 0244112 A1, published September 27, 2012 to Ast et al.; U.S. Patent Application No. 9,266,938 B2, issued February 23, 2016). Certain substitution combinations have been identified as reducing binding to CD25. In some embodiments, the ortho-IL2 of the present disclosure comprises one or more of the following substitution sets: [R38A-F42A-Y45A-E62A] as described in Carmenate, et al (2013) J Immunol 190:6230-6238; [F42A-Y45A-L72G] (Roche, RG7461 (RO6874281); and / or [T41P-T51P] (Chang, et al (1995) Molecular Pharmacology 47:206-211).

[0237] Modifications that modulate affinity to CD132 In some embodiments of the invention, ortho-IL2 contains one or more mutations at positions on the IL2 sequence that either contact CD132 or change the configuration of other positions that contact CD132, resulting in altered binding to CD132. Exemplary ortho-IL2 contains one or more mutations at positions on the IL2 sequence that either contact CD132 or change the configuration of other positions that contact CD122, resulting in altered binding to CD132. Residues of IL2 believed to contact CD132 include Q11, L18, Q22, E110, N119, T123, Q126, S127, I129, S130, and T133. In some embodiments, IL2 comprises the following modifications: AA18 is L (wild type), or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D, or T; AA126 is Q (wild type), or H, M, K, C, D, E, G, I, R, S, or T; and / or AA22 is Q (wild type), or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, or F in L18.

[0238] When recombinantly produced directly in a bacterial expression system in the absence of a leader sequence, endogenous proteases result in the deletion of the N-terminal Met-Ala1 residues resulting in "desAla1" ortho-IL2. In some embodiments, the disclosure provides a hIL2 polypeptide, human ortho-IL2, that includes one of the following sets of amino acid modifications:

[0239] To facilitate the production of unglycosylated ortho-IL2 upon expression in mammalian cells, e.g., CHO or HEK cells, the ortho-IL2 of the present disclosure may contain a modification that eliminates the O-glycosylation site at Thr3. Thus, in certain embodiments, the ortho-IL2 contains a modification that eliminates the O-glycosylation site of IL-2 at a position corresponding to the 3rd residue of human IL-2. In one embodiment, the modification that eliminates the O-glycosylation site of IL-2 at a position corresponding to the 3rd residue of human IL-2 is an amino acid substitution. Exemplary amino acid substitutions include the following, which remove the glycosylation site at position 3 without abolishing biological activity: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P (see U.S. Pat. No. 5,116,943; Weiger et al., (1989) Eur. J. Biochem., 180:295-300). In some embodiments, ortho-IL2 may include a deletion of the first two amino acids (desAla1-desPro2) and a substitution of Thr3 with a cysteine ​​residue for glycosylation to facilitate selective N-terminal modifications, particularly to facilitate PEGylation of the sulfhydryl group of cysteine ​​(see, for example, U.S. Pat. No. 5,206,344, issued Apr. 27, 1993 to Katre et al.).

[0240] Ortho-IL2 may optionally further comprise a modification at position M104, which in one embodiment is a substitution of a methionine at position 104 with an alanine residue (M104A), resulting in ortho-IL2 that is more oxidation-resistant (see U.S. Patent No. 4,752,585 to Koths et al.).

[0241] When produced directly recombinantly in a bacterial expression system in the absence of a leader sequence, endogenous proteases result in the deletion of the N-terminal Met-Ala1 residues to yield "desAla1" ortho-IL2. To facilitate N-terminal modifications, particularly PEGylation of the cysteine ​​sulfhydryl group, ortho-IL2 may include a deletion of the first two amino acids (desAla1-desPro2) and a substitution of Thr3 for glycosylation with a cysteine ​​residue (T3C) (see, for example, U.S. Patent No. 5,206,344, issued April 27, 1993 to Katre et al.).

[0242] Ortho-IL2 may further comprise a deletion of an N-terminal amino acid at one or more of positions 1-9, or of positions 1-8, or of positions 1-7, or of positions 1-6, or of positions 1-5, or of positions 1-4, or of positions 1-3, or of positions 1-2.

[0243] In some embodiments of the present disclosure, ortho-IL2 includes amino acid substitutions to avoid vascular leak syndrome, a significant adverse and dose-limiting side effect of IL2 therapy in humans, without substantial loss of efficacy. See U.S. Patent No. 7,514,073 B2, issued April 7, 2009 to Epstein et al. Examples of such modifications included in ortho-IL2 of the present disclosure include one or more of R38W, R38G, R39L, R39V, F42K, and H55Y.

[0244] In some embodiments, ortho-IL2 may be affinity matured to enhance its activity with respect to orthogonal CD122. An "affinity matured" polypeptide is one having one or more alterations in one or more residues that result in an improvement in the affinity of the orthogonal polypeptide for its orthogonal cognate receptor, or the affinity of the orthogonal receptor for its orthogonal cognate polypeptide, compared to a parent polypeptide that does not have such alterations. Affinity maturation can be done to increase the binding affinity of ortho-IL2 by at least about 10%, alternatively at least about 50%, alternatively at least about 100%, alternatively at least about 150%, or 1-5 fold, compared to the "parent" polypeptide. As discussed above, the engineered ortho-IL2 of the invention activates its cognate orthogonal receptor when assessed by ELISA and / or FACS analysis with sufficient amounts of the molecule under suitable assay conditions, but has significantly reduced binding to and activation of wild-type IL2 receptor.

[0245] As discussed above, the compositions of the present disclosure include ortho-IL2 that has been modified to provide increased longevity in vivo and / or increased duration of action in a subject. Such modifications that provide increased longevity and / or increased duration of action include modifications of the primary sequence of ortho-IL2, conjugation to a carrier molecule (e.g., albumin, acylation, PEGylation), and fusion to Fc.

[0246] In some embodiments, ortho-IL2 may contain some amino acid substitutions that extend in vivo life span. For example, Dakshinamurthi et al. (International Journal of Bioinformatics Research (2009) 1(2):4-13) describe that one or more of the following substitutions in the IL2 polypeptide, V91R, K97E, and T113N, can result in an IL2 variant with enhanced stability and activity. In some embodiments, the ortho-IL2 of the present disclosure contains one, two, or all three of the modifications V91R, K97E, and T113N.

[0247] In some embodiments, ortho-IL2 is modified to provide it with some property (e.g., extended duration of action in a subject), which may be achieved by conjugation to a carrier molecule that provides a desirable pharmaceutical property, such as extended half-life. In some embodiments, ortho-IL2 may be covalently linked to the Fc domain of IgG, to albumin, or to other molecules to extend its half-life, for example, by PEGylation, by glycosylation, by fatty acid acylation, and the like known in the art.

[0248] In some embodiments, ortho-IL2 is expressed as a fusion protein with an albumin molecule (eg, human serum albumin), which is known in the art to promote extended exposure in vivo.

[0249] In one embodiment of the present invention, human ortho-IL2 is conjugated with albumin, which is referred to herein as "ortho-IL2 albumin fusion". The term "albumin" used in the context of human ortho-IL2 albumin fusion includes albumins such as, for example, human serum albumin (HSA), cynomolgus monkey (cyno) serum albumin, and bovine serum albumin (BSA). In some embodiments, HSA contains an amino acid substitution of C34S or K573P compared to the wild-type HSA sequence. In accordance with the present disclosure, albumin can be conjugated to human ortho-IL2 at the carboxyl terminus, at the amino terminus, at both the carboxyl terminus and amino terminus, and internally (see, for example, U.S. Patent Nos. 5,876,969 and 7,056,701). In the conjugate of HSA and human ortho-IL2 polypeptide contemplated by the present disclosure, various forms of albumin can be used, such as albumin secretory presequence and variants thereof, fragments and variants thereof, and HSA variants. Such forms generally have one or more desirable albumin activities. In further embodiments, the present disclosure relates to a fusion protein comprising a human ortho-IL2 polypeptide, which is directly or indirectly fused to albumin, albumin fragments, albumin variants, etc., wherein the fusion protein has a higher plasma stability than the unfused drug molecule and / or the fusion protein retains the therapeutic activity of the unfused drug molecule. In some embodiments, the indirect fusion is effected by a linker, such as a peptide linker or modified version thereof, which will be discussed in more detail below.

[0250] Alternatively, an albumin fusion of human ortho-IL2 includes ortho-IL2, which is a fusion protein comprising an albumin binding domain (ABD) polypeptide sequence and an ortho-IL2 polypeptide. As described above, a fusion protein comprising an albumin binding domain (ABD) polypeptide sequence and a human ortho-IL2 polypeptide can be created, for example, by genetic engineering, in which a nucleic acid encoding HSA, or a fragment thereof, is linked to a nucleic acid encoding one or more of the ortho-IL2 sequences. In some embodiments, the albumin binding peptide comprises the amino acid sequence Includes TIFF2025509288000039.tif4128.

[0251] Ortho-IL2 polypeptides may also be conjugated to large, slowly metabolized macromolecules, such as proteins; polysaccharides, such as sepharose, agarose, cellulose, or cellulose beads; polymeric amino acids, such as polyglutamic acid or polylysine; amino acid copolymers; inactivated virus particles; inactivated bacterial toxins, such as toxoids from diphtheria, tetanus, cholera, or leukotoxin molecules; inactivated bacteria, dendritic cells, thyroglobulin; tetanus toxoid; diphtheria toxoid; polyamino acids, such as poly(D-lysine:D-glutamic acid); VP6 polypeptides of rotavirus; influenza virus hemagglutinin, influenza virus nucleoprotein; keyhole limpet hemocyanin (KLH); and core and surface antigens of hepatitis B virus. Such conjugates can be used to generate antibodies against the polypeptides of the present disclosure, if desired.

[0252] In some embodiments, ortho-IL2 is conjugated with XTEN (either chemically or as a fusion protein) and may be made as a recombinant fusion protein in E. coli, where XTEN extends duration similar to PEGylation. Suitable XTEN polymers for use in combination with ortho-IL2 of the present disclosure are provided in: Podust, et al. (2016) "Extension of in vivo half-life of biologically active molecules by XTEN protein polymers", J Controlled Release 240:52-66, and Haeckel et al. (2016) "XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic" PLOS ONE | DOI:10.1371 / journal.pone.0157193, June 13, 2016. Fusion proteins with XTEN polymers may incorporate a protease-sensitive cleavage site between the XTEN polypeptide and ortho-IL2, for example, an MMP-2 cleavage site.

[0253] Further candidate moieties and molecules for conjugation include those suitable for isolation or purification. Certain non-limiting examples include binding molecules such as biotin (the specific binding pair of biotin and avidin), antibodies, receptors, ligands, lectins, or molecules including solid supports, including for example plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.

[0254] In some embodiments, the IL-2 muteins may also be linked to additional therapeutic agents, including, for example, therapeutic compounds as described elsewhere herein, such as anti-inflammatory or anti-neoplastic compounds, therapeutic antibodies (e.g., Herceptin), immune checkpoint modulators, immune checkpoint inhibitors (e.g., anti-PD1 antibodies), cancer vaccines. Antimicrobial agents include aminoglycosides, including gentamicin, antiviral compounds, such as rifampicin, 3'-azido-3'-deoxythymidine (AZT), and acylovir, antifungal agents, such as azoles, including fluconazole, plyre macrolides, such as amphotericin B, and candicidin, antiparasitic compounds, such as antimonials, and the like. Ortho-IL2 may be conjugated with additional cytokines such as CSF, GSF, GMCSF, TNF, erythropoietin, immunomodulators, or cytokines such as interferons or interleukins, neuropeptides, reproductive hormones such as HGH, FSH, or LH, thyroid hormones, neurotransmitters such as acetylcholine, hormone receptors such as estrogen receptors, etc. Nonsteroidal anti-inflammatory drugs are also included, such as indomethacin, salicylic acid acetate, ibuprofen, sulindac, piroxicam, and naproxen, and anesthetics or analgesics. Radioisotopes are also included, such as those useful for imaging and therapy.

[0255] Ortho-IL2 of the present disclosure may be chemically conjugated to such carrier molecules using well-known chemical conjugation methods. Bifunctional cross-linking reagents, such as homofunctional and heterofunctional cross-linking reagents, well known in the art, can be used for this purpose. The type of cross-linking reagent used will vary depending on the nature of the molecule to be linked to the IL-2 mutein, and such type can be easily identified by one of skill in the art. Alternatively or additionally, ortho-IL2 and / or the molecule intended to be conjugated thereto may be chemically derivatized to allow the conjugation of both in separate reactions, as is well known in the art.

[0256] In some embodiments, ortho-IL2 is conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of the present invention include: polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), polyolefinic alcohols, polysaccharides, poly-α-hydroxy acids, polyvinyl alcohols (PVA), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof.

[0257] In some embodiments, ortho-IL2 is conjugated with one or more polyethylene glycol molecules, or "PEGylated." The method or site of attachment of PEG to ortho-IL2 can vary, but in certain embodiments, PEGylation does not alter, or only minimally alters, the activity of ortho-IL2.

[0258] In some embodiments, the threonine at position 3 can be substituted with a cysteine ​​(3TC) to facilitate N-terminal PEGylation using specific chemistry.

[0259] In some embodiments, selective PEGylation of ortho-IL2 (e.g., by incorporating unnatural amino acids with side chains that facilitate selective PEG conjugation chemistry as described in Ptacin et al. (PCT International Application PCT / US2018 / 045257, filed August 3, 2018, and published February 7, 2019 as WO 2019 / 028419 A1)) can be used to generate ortho-IL2 with reduced affinity for one or more subunits of the IL2 receptor complex (e.g., CD25, CD132). For example, human ortho-IL2 with incorporated unnatural amino acids with specific PEGylatable moieties at sequences or residues in IL2 identified to interact with CD25, including amino acids 34-45, 61-72, and 105-109, typically results in ortho-IL2 with reduced binding to CD25. Similarly, human ortho-IL2 incorporating unnatural amino acids with specific PEGylatable moieties at sequences or residues in IL2 identified to interact with hCD132 (including amino acids 18, 22, 109, 126, or 119-133) results in ortho-IL2 with reduced binding to hCD132.

[0260] In certain embodiments, the increase in half-life is greater than any decrease in biological activity. PEG suitable for conjugation to polypeptide sequences is generally soluble in water at room temperature and has the general formula R(O-CH2-CH2) n The PEG has the formula OR, where R is hydrogen or a protecting group, such as an alkyl or alkanol group, and where n is an integer between 1 and 1000. When R is a protecting group, it typically has 1 to 8 carbons. The PEG conjugated to the polypeptide sequence may be linear or branched. Branched PEG derivatives, "star PEGs," and multi-arm PEGs are contemplated by the present disclosure.

[0261] The molecular weight of PEG used in the present disclosure is not limited to any particular range. The PEG component of PEG-ortho-IL2 can have a molecular mass of more than about 5 kDa, more than about 10 kDa, more than about 15 kDa, more than about 20 kDa, more than about 30 kDa, more than about 40 kDa, or more than about 50 kDa. In some embodiments, the molecular mass is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. The PEG molecule, which may be linear or branched, has a molecular weight of about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, alternatively about 30,000 to about 40,000 daltons. In one embodiment of the invention, the PEG is a 40 kD branched PEG containing two 20 kD arms.

[0262] The present disclosure also contemplates compositions of conjugates, where PEG has various n values ​​and thus the various PEGs are present in a specific ratio. For example, some compositions include a mixture of conjugates where n = 1, conjugates where n = 2, conjugates where n = 3, and conjugates where n = 4. In some compositions, the percentage of conjugates where n = 1 is 18-25%, the percentage of conjugates where n = 2 is 50-66%, the percentage of conjugates where n = 3 is 12-16%, and the percentage of conjugates where n = 4 is up to 5%. Such compositions can be made by reaction conditions and purification methods known in the art. Chromatography can be used to separate fractions of the conjugates, and fractions are then identified that contain, for example, conjugates with a desired number of PEGs attached, and that have been purified free of unmodified protein sequences or conjugates with other numbers of PEGs attached.

[0263] PEG suitable for conjugation to a polypeptide sequence is generally soluble in water at room temperature and has the general formula R(O-CH-CH) n It has OR, where R is hydrogen or a protecting group, such as an alkyl or alkanol group, and where n is an integer from 1 to 1000. When R is a protecting group, it usually has from 1 to 8 carbons.

[0264] Two widely used types of first generation activated monomethoxy PEG (mPEG) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., U.S. Pat. No. 5,650,234 to Dolence et al.), which react preferentially with lysine residues to form carbamate linkages, but are also known to react with histidine and tyrosine residues. The use of PEG-aldehyde linkers targets only a single site at the N-terminus of a polypeptide by reductive amination.

[0265] PEGylation occurs most frequently at the α-amino group at the N-terminus of polypeptide, at the ε-amino group at the side chain of lysine residue, and at the imidazole group at the side chain of histidine residue.Many recombinant polypeptides have one α-amino group, several ε-amino groups, and several imidazole groups, so that depending on the chemical nature of the linker, a large number of positional isomers can be produced.General PEGylation strategies known in the art can be applied here.

[0266] PEG can be attached to the ortho-IL2 of the present disclosure via a reactive end group ("spacer"), which mediates a bond between a free amino or carboxyl group of one or more polypeptide sequences and polyethylene glycol. PEGs having a spacer capable of binding to a free amino group include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide.

[0267] In some embodiments, PEGylation of ortho-IL2 is facilitated by incorporating unnatural amino acids bearing unique side chains that facilitate site-specific PEGylation. The incorporation of unnatural amino acids into such polypeptides to provide functional moieties for achieving site-specific PEGylation of the polypeptide is known in the art. See, for example, Ptacin et al. (PCT International Application PCT / US2018 / 045257 filed August 3, 2018 and published February 7, 2019 as WO 2019 / 028419 A1). In one embodiment, the ortho-IL2 of the present invention incorporates an unnatural amino acid at position D109 of ortho-IL2. In one embodiment of the present invention, the ortho-IL2 is PEGylated at position 109 of ortho-IL2 with a PEG molecule having a molecular weight of about 20 kD, alternatively about 30 kD, alternatively about 40 kD.

[0268] PEG conjugated to a polypeptide sequence may be linear or branched. Branched PEG derivatives, "star PEGs," and multi-arm PEGs are contemplated by the present disclosure. Specific embodiments of PEG useful in the practice of the present invention include the following: 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601, USA);NY 10601 USA), 10 kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF, 20 kDa linear PEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), 20 kDa 2-arm branched PEG-aldehyde containing two 10 kDa linear PEG molecules, 20 kDA PEG-aldehyde (e.g., Sunbright® GL2-200AL3, NOF), 20 kDa 2-arm branched PEG-NHS ester containing two 10 kDA linear PEG molecules, 20 kDA PEG-NHS ester (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), 40 kDa 2-arm branched PEG-aldehyde containing two 20 kDA linear PEG molecules, 40 kDA PEG-aldehyde (e.g., Sunbright® GL2-400AL3), 40 kDa 2-arm branched PEG-NHS ester containing two 20 kDA linear PEG molecules, 40 30 kDa PEG-NHS ester (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), 30 kDa linear PEG-aldehyde (e.g., Sunbright® ME-300AL), and 30 kDa linear PEG-NHS ester.

[0269] As discussed above, PEG may be attached directly to ortho-IL2 or may be attached via a linker molecule. Suitable linkers include "flexible linkers," which are generally of sufficient length to allow some degree of movement between the modified polypeptide sequence and the linked moiety or molecule. Linker molecules are generally about 6 to about 50 atoms in length. Linker molecules may also be, for example, aryl acetylenes, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable linkers can be readily selected and may be of any length, for example, a single amino acid (e.g., Gly) or the following number of amino acids: 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50. Examples of flexible linkers include glycine polymers (Gly). n , glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and therefore can act as neutral tethers between moieties. Further examples of flexible linkers include glycine polymers (G) n , glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers. Glycine and glycine-serine polymers are relatively unstructured and therefore can act as neutral tethers between moieties. Multimers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of these linker sequences can be linked together to provide flexible linkers that can be used to conjugate heterologous amino acid sequences to the polypeptides disclosed herein.

[0270] Furthermore, such linkers may be used to link ortho-IL2 with additional heterologous polypeptide components as described herein, where the heterologous amino acid sequences may be signal sequences and / or fusion partners, such as, for example, albumin, Fc sequences, etc.

[0271] In one embodiment of the present disclosure, ortho-IL2 is selected from the group consisting of: [PEG]-[Linker] n -[hoIL2] where n=0 or n=1, or [PEG]-[Linker] n -[desAla1-hIL2[E15S-H16Q-L19V-D20L-Q22K-M23A] where n=0 or n=1, or

[0272] In another embodiment of the present invention, ortho-IL2 is Structure of human ortho-IL2 in TIFF2025509288000040.tif17128, where n = 0 (absent) or n = 1 (present).

[0273] In one embodiment, the ortho-IL2 is selected from the group consisting of: TIFF2025509288000041.tif17128, wherein the 40 kD-PEG-linker (n = 1) is The molecule has the structure of TIFF2025509288000042.tif20128, which is also referred to herein as STK-009.

[0274] In some embodiments, to facilitate selective binding to a particular cell type or tissue expressing a cell surface molecule that specifically binds to a polypeptide sequence (the "targeting domain"), Ortho-IL2 is provided as a fusion protein with such a targeting domain, optionally incorporating a linker molecule of 1 to 40 amino acids (alternatively 2 to 20 amino acids, alternatively 5 to 20 amino acids, alternatively 10 to 20 amino acids) between the Ortho-IL2 sequence and the targeting domain sequence of the fusion protein.

[0275] In other embodiments, chimeric polypeptides can be made that include orthogonal IL-2 and an antibody or its antigen-binding portion. The antibody or the antigen-binding element of the chimeric protein can act as a targeting moiety. For example, this can be used to localize the chimeric protein to a certain subset of cells or to localize to a target molecule. Methods for making chimeric polypeptides of cytokines and antibodies are described, for example, in U.S. Patent No. 6,617,135.

[0276] In some embodiments, the targeting domain of the ortho-IL2 fusion protein specifically binds to a cell surface molecule of a tumor cell. In one embodiment, if the ECD of the CAR of the CAR-T cell specifically binds to GPC3, the ortho-IL2 can be provided as a fusion protein with a targeting moiety to GPC3. For example, in one embodiment, if the ECD of the CAR of the CAR-T cell is an scFv molecule that provides specific binding to GPC3, the ortho-IL2 is provided as a fusion protein with a targeting moiety to GPC3, for example, as a fusion protein with a single chain antibody (e.g., scFv or VHH) that specifically binds to GPC3. In some embodiments, the targeting domain is an scFv derived from GC33. In some embodiments, the targeting domain is an scFv selected from SEQ ID NO:10 or SEQ ID NO:11.

[0277] Treatment method The present disclosure provides methods and compositions for treating a subject suffering from a neoplastic disease.

[0278] In one embodiment, the present invention provides a method of treating or preventing a neoplastic disease, disorder, or condition in a mammalian subject in need of such treatment or prevention, the method comprising the steps of: (a) isolating T cells from a subject; (b) contacting the population of T cells with a recombinant viral vector, the vector comprising a first nucleic acid sequence encoding a GPC3 CAR and a second nucleic acid sequence encoding an orthogonal receptor, the first nucleic acid sequence and the second nucleic acid sequence being operably linked to expression control elements functional in a mammalian T cell, the contacting causing transformation of a portion (fraction) of the isolated population of T cells with the viral vector, whereby the transformed cells express the GPC3 CAR and the orthogonal receptor; (c) administering to the subject a therapeutically effective amount of the T cells obtained from step (b) in combination with administering to the subject a therapeutically effective amount of ortho-IL2.

[0279] In one embodiment, the present invention provides a method of treating a subject suffering from a neoplastic disease, comprising administering to the subject a therapeutically effective amount of ortho-GPC3 CAR T cells in combination with a therapeutically effective amount of ortho-IL2. In some embodiments, the antigen-binding domain of the GPC3 CAR cells is an scFv. In some embodiments, the scFv is GC33 scFv. In some embodiments, the scFv is SEQ ID NO: 10 or SEQ ID NO: 11.

[0280] In some embodiments, the intracellular domain of GPC3 CAR T comprises the costimulatory domain of CD28 and the CD3-zeta domain.In some embodiments, the intracellular domain of GPC3 CAR T comprises the costimulatory domain of 41BB and the CD3-zeta domain.

[0281] In some embodiments, the GPC3 CAR is a polypeptide having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99% sequence identity to any one of SEQ ID NOs: 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the GPC3 CAR is selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39, and 40.

[0282] In some embodiments, the neoplastic disease is selected from hepatocellular carcinoma, hepatoblastoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and ovarian clear cell carcinoma.

[0283] In some embodiments, the disclosure provides methods of treating a subject suffering from a neoplastic disease, wherein the neoplastic disease is characterized by the presence in the subject of a neoplasm that expresses GPC3 (a "GPC3+ neoplasm").

[0284] As used herein, the term "subject" is used interchangeably with the terms "recipient," "individual," and "patient" to refer to any mammalian subject for whom diagnosis, treatment, or therapy is sought. For purposes of treatment, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, athletic, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0285] As used herein, the term "suffering" refers to a decision made by a physician regarding a subject who needs treatment or may benefit from treatment, based on available information recognized in the art to identify a disease, disorder, or condition, including but not limited to: X-ray, CT scan, conventional diagnostic tests on specimens (e.g., blood count, etc.), genomic data, protein expression data, immunohistochemical analysis. The term "suffering" is typically used in conjunction with a certain disease state, for example, "suffering from a neoplastic disease" refers to a subject who has been diagnosed with a neoplasm. As discussed above, the presence of GPC3+ neoplasms can be identified by the presence of the 40 kDa subunit of GPC3, referred to as "soluble GPC3" or "sGPC3", which is detectable in serum, and the level of GPC3 has been proposed as a diagnostic marker.

[0286] As used herein, the term "neoplastic disease" refers to a disorder or condition in a subject that is caused by cell hyperproliferation or unregulated (or dysregulated) cell replication. The term neoplastic disease refers to a disorder caused by the presence of a neoplasm in a subject. Neoplasms can be classified as follows: (1) benign; (2) premalignant (or "precancerous"); and (3) malignant (or "cancerous"). The term "neoplastic disease" includes neoplasm-related diseases, neoplasm-related disorders, and neoplasm-related conditions, which refer to conditions that are directly or indirectly related to neoplastic disease, and includes, for example, angiogenesis and precancerous conditions.

[0287] The terms "administration" and "administering" are used interchangeably herein to refer to the act of contacting a subject in vitro, in vivo, or ex vivo with an agent (e.g., an ortholog, an IL2 ortholog, engineered cells expressing an orthogonal receptor, engineered cells expressing an orthogonal IL2 receptor (e.g., CAR-T cells expressing an orthogonal IL2 receptor), a chemotherapeutic agent, an antibody), or a pharmaceutical formulation containing one or more of the above, including contacting the same with cells, tissues, organs, or biological fluids of the subject. Administration of the agent may be accomplished by any of a variety of techniques recognized in the art, including, but not limited to, local administration, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal delivery, transmucosal delivery, iontophoretic delivery, intralymphatic injection, intragastric injection, intraprostatic injection, intravesical injection (e.g., bladder), inhalation (e.g., respiratory inhaler, including dry powder inhaler), intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), etc. The term "administration" includes contacting the agent with a cell, tissue, or organ, and also includes contacting the agent with a fluid that contacts the cell, tissue, or organ.

[0288] The term "therapeutically effective amount" is used herein in reference to the administration of an agent to a subject, either alone or as part of a pharmaceutical composition or treatment regimen, either as a single dose or as part of a series of doses, in an amount that can produce any detectable positive effect on any symptom, any aspect, or any feature of a disease, disorder, or condition when administered to a subject.Therapeutically effective amount can be determined by measuring the relevant physiological effect, and it can be adjusted in relation to the dosage regimen and in response to the diagnostic analysis of the subject's condition, etc.The parameters for evaluation to determine the therapeutically effective amount of an agent are determined by a physician using diagnostic criteria recognized in the art, and include, for example, but are not limited to, the following signs: age, weight, sex, general health, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, X-ray, etc.Alternatively or additionally, other parameters commonly evaluated in a clinical setting may be monitored to determine whether a therapeutically effective amount of an agent has been administered to a subject, such as, for example, body temperature, heart rate, normalization of blood chemistry analysis, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or feature of a disease, disorder, or condition, biomarker (e.g., inflammatory cytokines, IFN-γ, granzymes, etc.), reduction in serum tumor markers, improvement in Response Evaluation Criteria In Solid Tumors (RECIST), Immune-Related Response Criteria (IRC), or other parameters that may be useful in determining whether a therapeutically effective amount of an agent has been administered to a subject. Improvement in Clinical Trials and Clinical Evaluation Criteria (irRC), prolonged survival, prolonged progression-free survival, prolonged progression-free survival, prolonged time to treatment failure, prolonged event-free survival, prolonged time to next treatment, improved objective response rate, improved duration of response, reduction in tumor burden, complete response, partial response, stable disease, and similar parameters used by clinicians in the art to evaluate the improvement of a subject's condition in response to administration of a certain agent. As used herein, the terms "Complete Response (CR)", "Partial Response (PR)", "Stable Disease (SD)", and "Progressive Disease (PD)" for target lesions, and the terms "Complete Response (CR)", "Incomplete Response / Stable Disease (SD)", and Progressive Disease (PD)" for non-target lesions are understood as defined in the RECIST criteria.As used herein, the terms "immune-related complete response (irCR)", "immune-related partial response (irPR)", "immune-related progressive disease (irPD)" and "immune-related stable disease (irSD)" are defined according to the immune-related response criteria (irRC). As used herein, the term "immune-related response criteria (irRC)" refers to a system for evaluating response to immunotherapy, as described in Wolchok, et al. (2009) "Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-Related Response Criteria", Clinical Cancer Research 15(23): 7412-7420. The therapeutically effective amount may be adjusted during the treatment of a subject in relation to the dosing regimen and / or in relation to the evaluation of the subject's condition and fluctuations in the above-mentioned factors. In one embodiment, a therapeutically effective amount is an amount of an agent that, when used alone or in combination with another agent, does not cause serious adverse events that are irreversible during administration to a mammalian subject.

[0289] As used herein, the term "combination," when used in reference to administering a plurality of agents to a subject, refers to administering a first agent and at least one additional agent (i.e., a second, third, fourth, fifth, etc.) to a subject. For purposes of the present invention, a first agent (e.g., an IL2 ortholog) is considered to be administered in combination with a second agent (e.g., an engineered human immune cell) if the biological effect in the subject resulting from administration of the first agent persists at the time of administration of the second agent, such that the therapeutic effect of the first agent overlaps with the therapeutic effect of the second agent. For example, an engineered orthogonal cellular therapy agent may typically be administered infrequently (typically only a single dose), while an IL2 ortholog is administered periodically while the orthogonal cellular agent persists in the subject. If an engineered orthogonal cellular therapy provides a therapeutic effect over an extended period of time (weeks or months) and administration of a second agent (e.g., an IL2 ortholog) provides its therapeutic effect while the therapeutic effect of the first agent continues, the second agent is considered to be administered in combination with the first agent, even if the first agent was administered at a time point (e.g., days or weeks) prior to the time of administration of the second agent. In one embodiment, a first agent is considered to be administered in combination with a second agent if the first and second agents are administered simultaneously (within 30 minutes of each other), contemporaneously, or sequentially. In some embodiments, a first agent is considered to be administered "contemporaneously" with a second agent if the first and second agents are administered within about 24 hours of each other, preferably within about 12 hours of each other, preferably within about 6 hours of each other, preferably within about 2 hours of each other, or preferably within about 30 minutes of each other. It should also be understood that the term "combination" applies to the situation where a first agent and a second agent are combined in a single pharma- ceutically acceptable formulation and the combination is administered to a subject.

[0290] In some embodiments, ortho-IL2 is administered in combination with ortho-GPC3 CAR T cells, wherein the ortho-IL2 is administered to the subject contemporaneously with administration of the ortho-GPC3 CAR T cells to the subject.

[0291] In some embodiments, ortho-IL2 is administered in combination with ortho-GPC3 CAR T cells, wherein the ortho-IL2 is administered to the subject at one or more of the following timepoints: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days prior to administration of the ortho-GPC3 CAR T cells to the subject.

[0292] In some embodiments, ortho-IL2 is administered in combination with ortho-GPC3 CAR T cells, wherein the ortho-IL2 is administered to the subject at one or more of the following timepoints: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 20 days, 30 days, 60 days, 90 days, 120 days, 150 days, 180 days, 240 days after administration of the ortho-GPC3 CAR T cells to the subject.

[0293] In some embodiments, ortho-IL2 is administered in combination with ortho-GPC3 CAR T cells, where ortho-IL2 is administered (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to administration of the ortho-GPC3 CAR T cells to the subject; (b) contemporaneously with administration of the ortho-GPC3 CAR T cells to the subject; and (c) concurrently with administration of the ortho-GPC3 CAR T cells to the subject. Ortho-IL2 is administered at one or more of the following timepoints: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 20 days, 30 days, 60 days, 90 days, 120 days, 150 days, 180 days, 240 days after administration of the T cells to the subject.

[0294] In some embodiments, ortho-IL2 is administered in combination with ortho-GPC3 CAR T cells, wherein the ortho-IL2 is administered at one or more of the following timepoints: (a) contemporaneous with administration of the ortho-GPC3 CAR T cells to the subject; and (b) 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 20 days, 30 days, 60 days, 90 days, 120 days, 150 days, 180 days, 240 days after administration of the ortho-GPC3 CAR T cells to the subject.

[0295] In certain embodiments, the orthogonal cells and IL2 orthologs are further combined in combination with an additional anti-neoplastic agent. The additional agent may be administered before, during, and / or after the treatment period with GPC3 CAR T cells in combination with ortho-IL2. In some embodiments, the additional anti-neoplastic agent is selected from the group consisting of: chemotherapeutic agents, small molecules, additional biologics including but not limited to checkpoint inhibitors (e.g., anti-PD1 antibodies such as Keytruda, Opdivo), anti-tumor antigen antibodies (Herceptin), and / or physical procedures (surgery, radiation therapy, etc.).

[0296] In other embodiments, the additional agents may be administered simultaneously, e.g., two or more agents are administered simultaneously or near simultaneously; the two or more agents may be in two or more separate formulations or may be combined in a single formulation (i.e., a combination formulation). Whether the agents are administered sequentially or simultaneously, for purposes of this disclosure, the agents are considered to be administered in combination.

[0297] Administration of GPC3 CAR T cells Ortho-GPC3 CAR T cells and ortho-IL2 can be provided as pharmaceutical compositions suitable for therapeutic use, e.g., human treatment. Therapeutic formulations containing such cells can be frozen in the form of an aqueous solution, or can be prepared for administration in the form of an aqueous solution with physiologically acceptable carriers, excipients, or stabilizers ("Remington's Pharmaceutical Sciences" 16th Edition, Osol, A. Ed. (1980)). The cells can be formulated, dosed, and administered in a manner consistent with good clinical practice. Factors to be considered in this context include: the particular disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site to which the agent is delivered, the method of administration, the administration schedule, and other factors understood by the physician.

[0298] Cells can be administered by any suitable means, usually parenteral administration.Parenteral infusion includes intramuscular administration, intravenous administration (bolus infusion or slow infusion), intraarterial administration, intraperitoneal administration, intrathecal administration, or subcutaneous administration.In typical practice, CAR T cells are usually intravascularly infused into subjects in a physiologically acceptable medium, but the cells can also be introduced into any other convenient site where the cells can find a suitable site for proliferation.

[0299] A therapeutically effective amount of ortho-GPC3 CAR T cells for use in a method of treating a subject suffering from a GPC+ neoplasm is about 1×10 per kg of subject body weight per course of therapy. 4 ~Approx. 5×10 6 In some embodiments, the dose of Ortho-GPC3 CAR T cells administered to a subject in need of treatment in one course of therapy is approximately 1×10 per kg of body weight. 5 pcs or 2×10 5 pcs or 3×10 5 pcs or 4×10 5 pcs or 5×10 5pcs or 6×10 5 pcs or 7×10 5 pcs or 8×10 5 pcs or 9×10 5 pcs or 1×10 6 pcs or 2×10 6 pcs or 3×10 6 pcs or 4×10 6 pcs or 5×10 6 ortho GPC3 CAR cells.

[0300] A course of ortho-GPC3 CAR T cell therapy may be a single administration or multiple administrations during a period. Typically, ortho-GPC3 CAR T cells are administered once per course of therapy, and ortho-IL2 is administered periodically in combination with ortho-GPC3 CAR T cells. However, two or more separate administrations of ortho-GPC3 CAR T cells may be administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. The amount of ortho-GPC3 CAR T cells administered in such a separate administration protocol may be the same in each administration, or may be provided at different levels in each administration. Administration protocols over a period of multiple days may be provided by one of skill in the art (e.g., a physician) monitoring the administration of cells, taking into account the subject's response to the treatment, including adverse effects of the treatment and their modulation, as discussed above.

[0301] Ortho-IL2 Administration: Therapeutic method embodiments of the present disclosure involve administering to a subject in need of treatment a pharmaceutical formulation comprising a therapeutically effective amount of ortho-IL2 (and / or a nucleic acid encoding ortho-IL2). Administration to the subject may be accomplished as an intravenous bolus injection or by continuous intravenous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intra-cerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Ortho-IL2 is also suitably administered by intratumoral, peritumoral, intralesional, intranodal, or perilesional routes or into the lymph to provide local and systemic therapeutic effects.

[0302] When the ortho-IL2 is STK-009, a therapeutically effective amount of STK-009 for a human subject in combination with ortho-GPC3 CAR T cells is from about 0.5 mg to about 20 mg, alternatively from about 1 mg to about 15 mg, alternatively from about 1.5 mg to about 12 mg, administered subcutaneously once weekly.

[0303] In some embodiments, the subject ortho-IL2 (and / or nucleic acid encoding ortho-IL2) may be incorporated into a composition, including a pharmaceutical composition. Such compositions typically include a polypeptide or a nucleic acid molecule and a pharma- ceutically acceptable carrier. The pharmaceutical composition is formulated to be compatible with its intended route of administration, and is also compatible with therapeutic use, which is why ortho-IL2 is administered to a subject in need of treatment or prophylaxis.

[0304] The ortho-IL2 (or nucleic acid encoding it) of the present disclosure may be administered to a subject in a pharma- ceutically acceptable dosage form. Preferred formulations vary depending on the intended mode of administration and depending on the intended therapeutic application. Typically, protein therapeutics are formulated for parenteral administration, and the methods of the present disclosure involve parenteral administration of ortho-IL2. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Parenteral formulations include solutions or suspensions used for parenteral application, which may include vehicles, carriers, and buffers. Pharmaceutical formulations for parenteral administration include sterile aqueous solutions (if water-soluble) or sterile aqueous dispersions, and also sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. In one embodiment, the formulation is provided as a pre-filled syringe for parenteral administration.

[0305] In some embodiments of the disclosed method, ortho-IL2 may be administered to a subject in need of treatment as a formulation to provide sustained release of the ortho-IL2 agent. An example of a sustained release formulation of an injectable composition may be provided by including an agent that delays absorption, such as aluminum monostearate and gelatin, in the composition. In one embodiment, the subject ortho-IL2 or nucleic acid is prepared with a carrier that prevents the mutant IL-2 polypeptide from being rapidly eliminated from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations may be prepared using standard techniques. Such materials may also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes that target infected cells using monoclonal antibodies against viral antigens) may also be used as pharmaceutically acceptable carriers. These can be prepared according to techniques known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811. In one embodiment, an ortho-IL2 formulation is provided according to the teachings of U.S. Patent No. 4,604,377, issued Aug. 5, 1986 to Fernandes and Taforo, and U.S. Patent No. 4,645,830, issued to Yasui et al.

[0306] Instead of administering to a subject an ortho-IL2 protein pharmaceutical formulation containing ortho-IL2, ortho-IL2 may be provided to the subject by administering to the subject a pharma- ceutically acceptable formulation of a recombinant vector comprising a nucleic acid sequence encoding ortho-IL2, thereby achieving continuous exposure of the subject to selective ortho-IL2. Administration of the recombinant vector encoding ortho-IL2 results in extended ortho-IL2 delivery to the subject and extended activation of corresponding cells engineered to express the orthogonal cognate receptor that binds to such ortho-IL2. In some embodiments of the methods of the present disclosure, a nucleic acid encoding ortho-IL2 is administered to a subject by transfection or infection using techniques known in the art, including, but not limited to, those described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20: 1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151-160, 1996, with errata in Am. J. Health Syst. Pharm. 53:325, 1996).

[0307] In some embodiments, Ortho-IL2 may be administered to a subject in the form of a construct expressing a nucleic acid in a viral vector encoding Ortho-IL2. The terms "viral vector" and "virus" are used interchangeably herein to refer to any of the obligate intracellular parasites that have no machinery for synthesizing proteins or generating energy. The viral genome may be RNA or DNA, which is encapsulated in a protein coat structure in a lipid membrane. The terms virus and viral vector are used interchangeably herein. Viruses useful in the practice of the present invention include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from the families baculoviridiae, parvoviridiae, picornoviridiae, herpesviridiae, poxviridae, or adenoviridiae. Viruses may be modified by recombinant DNA technology to include expression of a foreign transgene (e.g., a nucleic acid sequence expressing ortho-IL2) and engineered to be replication-incompetent, conditionally replicating, or replication-competent. The term "replicative viral vector" refers to a viral vector that is capable of infection, DNA replication, packaging, and lysis of infected cells. The term "conditionally replicating viral vector" is used herein to refer to a replication-competent vector that is designed to achieve selective expression in a particular cell type. Such conditional replication may be achieved by functionally linking tissue-, tumor-, or cell-type-specific regulatory control sequences, or other selectively induced regulatory control sequences, to an early gene (e.g., the E1 gene of an adenoviral vector).Infection of a subject with a recombinant viral or non-viral vector can result in long-term expression of ortho-IL2 in the subject and can also result in the continued selective maintenance of ortho-GPC3 CAR T cells expressing the orthogonal CD122 receptor. In one embodiment, the nucleic acid sequence encoding the IL2 receptor in the viral vector system is under the control of a regulatable promoter, an inducible promoter, a tissue-specific or tumor-specific promoter, or a temporally regulating promoter.

[0308] With lymphocyte depletion Lymphocyte depletion regimens are commonly utilized in combination with CAR T cell therapy, and the agents and dose ranges for administration of lymphocyte depleting agents are well known in the art. In one embodiment of practicing the above-mentioned method, the subject is treated with a lymphocyte depletion regimen, where the lymphocyte depletion regimen comprises cyclophosphamide in combination with fludarabine. In some embodiments, the lymphocyte depletion regimen involves administration of cyclophosphamide in combination with fludarabine for a period of 1, 2, 3, 4, or 5 days prior to administration of GPC3 CAR T cells. In some embodiments, the dose of cyclophosphamide used in the lymphocyte depletion regimen is about 100 mg / m for a period of 1, 2, 3, 4, or 5 days prior to administration of GPC CAR T cells. 2 / day to approximately 200 mg / m 2 / day to approximately 300 mg / m 2 / day to approximately 400 mg / m 2 / day to approximately 500 mg / m 2 / day to approximately 600 mg / m 2 / day. In some embodiments, the dose of fludarabine used in the lymphocyte depletion regimen is about 10 mg / m for a period of 1, 2, 3, 4, or 5 days prior to administration of the GPC CAR T cells. 2 / day to approximately 20 mg / m 2 / day to approximately 30 mg / m 2 / day to approximately 40 mg / m 2 / day to approximately 50 mg / m 2 / day to approximately 60 mg / m 2 / day. In one embodiment, the dose is from about 500 mg / m for a period of 3 days prior to administration of GPC3 CAR T cells. 2 / day ~ approx. 600 mg / m 2 / day dose of cyclophosphamide, and approximately 30 mg / m 2 Fludarabine at a dose of about 300 mg / m2 / day for a period of 3 days prior to administration of the GPC3 CAR T cells. 2 / day ~ approx. 600 mg / m 2 / day dose of cyclophosphamide, and approximately 30 mg / m 2 / day dose of fludarabine.

[0309] Without lymphocyte depletion As mentioned above, GPC3 CAR T cells can be expanded in vivo in mammalian subjects.This allows the GPC3 CAR T cells to be administered to patients at a significantly lower dose, and thus reduces or eliminates the need for lymphodepletion before administering GPC3 CAR T cells.Since lymphodepletion has a significant negative effect on subjects by making them vulnerable to environmental factors, avoiding lymphodepletion regimens combined with cell therapy can be a significant benefit to patients.

[0310] In one embodiment, the disclosure provides a method of treating a subject suffering from a neoplastic disease, disorder, or condition, wherein the neoplasm characterized by expression of GPC3 is amenable to treatment with GPC3 CAR-T cell therapy by administering a CAR-T expressing an orthogonal ligand, in the absence of lymphodepletion.

[0311] Sequence Listing TIFF2025509288000043.tif218153TIFF2025509288000044.tif226153TIFF2025509288000045.tif225153TIFF2025509288000046.tif225153TIFF2025509288000047.tif219153TIFF2025509288000048.tif225153TIFF2025509288000049.tif225153TIFF2025509288000050.tif222153TIFF2025509288000051.tif225153TIFF2025509288000052.tif225153TIFF2025509288000053.tif225153TIFF2025509288000054.tif225153TIFF2025509288000055.tif225153TIFF2025509288000056.tif225153TIFF2025509288000057.tif225153TIFF2025509288000058.tif225153TIFF2025509288000059.tif225153TIFF2025509288000060.tif225153TIFF2025509288000061.tif225153TIFF2025509288000062.tif225153TIFF2025509288000063.tif225153TIFF2025509288000064.tif225153TIFF2025509288000065.tif225153TIFF2025509288000066.tif122153

Claims

1. A GPC3 CAR comprising an antigen-binding domain that selectively binds to the beta subunit of human glypican-3 (SEQ ID NO:2), wherein the antigen-binding domain is an scFv having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:10 or SEQ ID NO:

11.

2. The GPC3 CAR of claim 1, comprising a CD3ζ signaling domain and a costimulatory domain selected from a costimulatory domain of C28 or a costimulatory domain of 4-1BB.

3. 2. The GPC3 CAR of claim 1, having at least 90%, 95%, 99%, or 100% sequence identity to a GPC3 CAR selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO:

40.

4. The GPC3 CAR of claim 1, selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO:

40.

5. A nucleic acid sequence encoding the GPC3 CAR of any one of claims 1 to 4.

6. A recombinant vector comprising the nucleic acid sequence of claim 5.

7. The recombinant vector of claim 6, which is a lentiviral vector or a retroviral vector.

8. 8. The recombinant vector of claim 6 or 7, further comprising a nucleic acid sequence encoding an ortho-CD122 receptor.

9. 9. The recombinant viral vector of claim 8, wherein the nucleic acid sequence encoding the GPC3 CAR and the nucleic acid sequence encoding the ortho-CD122 receptor are operably linked to at least one expression control sequence functional in a mammalian T cell.

10. The recombinant viral vector of claim 9, wherein the nucleic acid sequence encoding an orthogonal receptor, the nucleic acid sequence encoding the GPC3 CAR, and the nucleic acid sequence encoding the ortho CD122 receptor are operably linked to a single expression control sequence, and the nucleic acid sequence encoding the GPC3 CAR and the nucleic acid sequence encoding the ortho CD122 receptor are separated by an IRES sequence or a T2A sequence.

11. A recombinantly modified T cell comprising the recombinant viral vector of claim 10.

12. Ortho-GPC3 CAR T cells expressing ortho-CD122 and GPC3 CAR, wherein the ortho-CD122 comprises an amino acid substitution at position 133 and / or 134, as numbered according to wild-type hCD122.

13. The ortho GPC3 CAR T cell of claim 12, wherein (a) the GPC3 CAR comprises the amino acid sequence of SEQ ID NO: 37; and (b) the ortho CD122 has the amino acid sequence of SEQ ID NO:

4.

14. 1. A method for generating ortho-GPC3 CAR T cells, comprising: (a) obtaining a sample of human peripheral blood mononuclear cells (PBMCs); (b) contacting a sample of PBMCs with magnetic beads coated with a CD3 ligand and a CD28 ligand to provide an activated PBMC population; (c) isolating CD8+ T cells and CD4+ T cells from the activated PBMC cell population; (d) contacting the isolated population of CD8+ T cells and CD4+ T cells with a recombinant lentiviral vector such that a fraction of the isolated population of CD8+ T cells and CD4+ T cells are transduced with the lentiviral vector, the lentiviral vector being 5' to 3' - a promoter active in T cells, such as the EF1a promoter; - a nucleic acid sequence encoding a signal peptide; - a nucleic acid sequence encoding a GPC3 CAR; - T2A sequence (SEQ ID NO: 41); - a nucleic acid sequence encoding a signal peptide; A nucleic acid sequence encoding ortho-CD122 of SEQ ID NO:4 comprising an expression cassette comprising a nucleic acid sequence encoding (e) contacting the population of cells obtained from step (d) with ortho-IL2, wherein the ortho-IL2 is selected from the group consisting of a human IL2 mutein containing the amino acid substitutions E15S / H16Q / L19V / D20L / Q22K / M23A; ortho-IL2 of SEQ ID NO 9 (STK-007), or a PEGylated variant thereof (e.g., STK-009). A method comprising:

15. 15. OrthoGPC3 CAR T cells prepared according to claim 14.

16. 19. A method of treating or preventing a neoplastic disease, disorder, or condition in a mammalian subject in need thereof, comprising administering to the subject a therapeutically effective amount of ortho-GPC3 CAR T cells according to claim 15 in combination with a therapeutically effective dose of ortho-IL.

17. 15. The method of claim 14, wherein after step (a) and before step (b), the population of cells is enriched for CD8+ T cells or CD4+ T cells.

18. The method of claim 14, wherein the ortho-IL2 utilized ex vivo in step (b) is different from the orthogonal ligand used in vivo in step (c).

19. 15. The method of claim 14, wherein prior to step (d), the subject is treated with a lymphocyte depleting regimen.

20. 1. A method of treating relapse following ortho-GPC3 CAR therapy in a subject, comprising administering to the subject a therapeutically effective amount of ortho-IL2 such that an orthogonal ligand induces activation and / or proliferation of ortho-GPC3 CAR T cells in the subject.

21. 21. The method of claim 20, wherein the ortho-IL2 is STK-009.

22. 1. A method of making an ortho-GPC3 CAR T cell product having a substantially enriched population of ortho-GPC3 CAR T cells, comprising: (a) isolating a population of cells from a mammalian subject, said population of cells comprising T cells; (b) contacting the isolated population of T cells from step (a) with a recombinant vector comprising a first nucleic acid sequence encoding a GPC3 CAR and a second nucleic acid sequence encoding ortho-CD122, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to expression control sequences functional in a mammalian T cell, and the first nucleic acid sequence and the second nucleic acid sequence are separated by an IRES or T2A nucleic acid sequence, such that the cells express the GPC3 CAR and the ortho-CD122 (ortho-GPC3 CAR T cells); (c) contacting the isolated population of cells from step (b) ex vivo with ortho-IL2 in an amount sufficient to induce proliferation of the cells transduced by the contacting in step (b); maintaining contact with the ortho-IL2 for a period of time such that the fraction of cells expressing the GPC3 CAR and the ortho-CD122 constitutes at least 10% of the population. A method comprising:

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