Orthogonal GPC3 chimeric antigen receptor T cell

Abstract

The present invention provides engineered T cells that express (a) a chimeric antigen receptor in which the antigen binding domain of the CAR binds to human GPC3 ("GPC-CAR"); and (b) an orthogonal receptor. The invention also provides methods of making and using such engineered T cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 317,935, filed on March 8, 2022, which is incorporated herein by reference for all purposes. Background Art

[0002] Glypican-3 (GPC3) is a member of the glypican family of heparan surface proteoglycans. GPC3 is also known as 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), comprising a 24 amino acid signal peptide (corresponding to amino acids 1-24 of SEQ ID NO: 1) sequence and a C-terminal sequence corresponding to amino acids 555-580 of SEQ ID NO: 1, and both the signal peptide sequence and the C-terminal sequence are removed in the mature form of the protein. Two heparan sulfates are connected at amino acids 495 and 508. hGPC3 contains a furin cleavage site between amino acids R359 and S359 (numbered according to SEQ ID NO: 1), providing an N-terminal 40 kDa fragment having a sequence corresponding to amino acids 25-358 of SEQ ID NO: 1 and a C-terminal 30 kDa fragment having a sequence corresponding to amino acids 359-554 of SEQ ID NO: 1 (SEQ ID NO: 2). When expressed on the cell surface after furin cleavage, the 40 kDa and 30-kDa fragments h are linked by three disulfide bonds. Hydrolysis of these disulfide bonds releases the 40 kDa subunit, which is referred to as "soluble GPC3" or "sGPC3", which can be detected in serum, and the level of GPC3 is considered to be a diagnostic marker for estimating the level of GPC3 expression in a subject and 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] GPC3 is rarely expressed in normal tissues, and due to its high expression in HCC, several studies have identified GPC3 as a cancer-specific target, especially a liver cancer-specific target. Baumhoer D, et al. (2008) Am J Clin Pathol 129(6):899–906. In addition, GPC3 is rarely expressed in other normal tissues in adults, making it suitable for targeted therapy as a tumor antigen. Li et al. (2018) Trends in cancer 4:741–54; Ho and Kim (2011) European journal of cancer 47:333–8. Although initially associated with liver cancer (particularly HCC), a variety of tumors 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), melanoma (Nakatsura and Nishimura (2005) BioDrugs: clinical immunotherapeutics, biopharmaceuticals and gene therapy 19:71–7), and ovarian clear cell carcinoma (Umezu (2010) Journal of clinical pathology 63:962–6). Therefore, GPC3 is not only a specific biomarker and prognostic factor for HCC, but also a potential target for the treatment of a variety of tumors. Given the correlation between a variety of tumors and GPC3, there are many GPC-3CARs. T cell therapies are under development, including multiple clinical trials (see, e.g., ClinicalTrials.gov trial numbers NCT02395250, NCT02723942, NCT03146234, NCT0295188, NCT03084380, and NCT03884751). These trials are evaluating GPC3 CAR-T therapy alone or in combination with other anticancer agents, such as checkpoint inhibitors.

[0004] Currently, the only CAR-T cell drugs approved for use by regulatory agencies are those that target cells expressing CD19 and are used to treat hematological malignancies. There are currently no CAR-T cell therapies approved for the treatment of solid tumors. In recent years, there has been significant progress in the development of CAR-T cells, with a variety of technology combinations producing what are known as first-, second-, third-, and fourth-generation CAR-T cells. Although clinical experience with CAR-T cells for the treatment of hematological malignancies has shown significant initial success, the rate of disease recurrence has been quite high over time. The "persistence" of CAR-T cells is a specific obstacle faced by existing technologies. It is well known that adoptively transferred human immune cells lose their activity relatively quickly after administration. Therefore, typical approaches to address this rapid loss of function are to: (a) administer excessively high doses of the cell therapy to maximize exposure of the cell therapy to the tumor before the cells lose their effectiveness, and / or (b) systemically administer high-dose IL2 (HD-hIL2) therapy to try to support the efficacy of the adoptively transferred cells. Both approaches have significant toxicity.

[0005] The effects of high doses of hIL2, such as those used to support adoptive cell therapy regimens, have been shown to result in significant toxicity in human subjects. The most common side effects observed from administration of HD-hIL2 in conjunction with adoptive cell transfer (ACT) include chills, high fever, hypotension, oliguria, and edema due to systemic inflammation and capillary leak syndrome, as well as reports of autoimmune phenomena such as vitiligo or uveitis. HD-hIL2 monotherapy may also induce generalized capillary leak syndrome, which can lead to death. Toxicity associated with HD-hIL2 requires expert management and is therefore typically applied in a hospital setting, often requiring admission to an intensive care unit. Dutcher, et al. (2014) J Immunother Cancer 2(1):26. This limits the use of HD-IL2 therapy to mostly young, very healthy patients with normal cardiopulmonary function.

[0006] In addition, high doses of engineered cell therapies are associated with life-threatening cytokine release syndrome (CRS). Currently available products show all grades of CRS in most treated subjects, and grade 3 or higher CRS in a large proportion of patients. Significant neurotoxicity has also been observed in most patients. However, lower doses of cell therapy agents are associated with a significant decrease in clinical outcomes. In addition, many patients who initially respond well to cell therapy relapse, primarily due to the lack of durability of cell therapy products. Currently, approximately 60% of patients treated with existing CD-19 CAR-T cell therapy drugs have been reported to relapse. Byrne M, et al. (2019) Biology of Blood and Marrow Transplantation 25(11):344-251.

[0007] Sockolosky, et al. (Science (2018) 359: 1037–1042) and Garcia, et al. (U.S. Patent Application Publication US2018 / 0228841A1 published on August 16, 2018) describe orthogonal IL2 / CD122 ligand / receptor systems to promote selective stimulation of cells engineered to express orthogonal receptors (particularly orthogonal CD122). In short, it has been specially modified to bind to variant IL2 molecules and activated by variant IL2 molecules. Engineered T cells expressing orthogonal CD122 are contacted with corresponding orthogonal ligands ("orthogonal IL2") homologous to this orthogonal CD122 to promote the specific activation of engineered T cells expressing orthogonal CD122. In particular, this orthogonal IL2 receptor ligand complex provides a mixed population of cells, particularly a mixed population of T cells engineered to express orthogonal receptors. This patent application incorporates the disclosures of WO 2019 / 104092 and US2018-0228842 A1) in their entirety by reference. Summary of the invention

[0008] The present disclosure provides GPC3 CAR-T cells using a selective regulatory system that exhibit efficacy in treating solid tumors, particularly solid tumors expressing GPC3 (GPC3+ tumors), are capable of selectively expanding GPC3 CAR-T cells in vivo, particularly in treated subjects, exhibit prolonged persistence, intratumoral infiltration of solid tumors, and the ability to treat relapse without the need to administer additional GPC3 CAR-T cells.

[0009] The present disclosure provides engineered T cells expressing (a) a chimeric antigen receptor, wherein the antigen binding domain of CAR binds to human GPC3 ("GPC-CAR"); and (b) an orthogonal receptor. The terms "chimeric antigen receptor T-cell" and "CAR-T cell" are used interchangeably and 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 orthogonal receptors ("orthogonal CAR-T cells" or "orthogonal CAR-T cells").

[0010] Also provided is a cell product substantially enriched for an orthogonal CAR-T cell population, the product being obtained by a process comprising the steps of: a) isolating a quantity of immune cells from a mammalian (eg, human) subject; (b) contacting the isolated amount of immune cells with a nucleic acid sequence encoding an orthogonal GPC3 CAR and an orthogonal hCD122 or a functional fragment thereof, under conditions such that the isolated immune cells take up the nucleic acid sequence; (c) contacting a certain amount of cells separated in step (b) ex vivo with a certain amount of orthogonal ligand, wherein the amount of the orthogonal ligand is sufficient to induce proliferation of the cells transduced by the contacting in step (b), and the contacting (c) is carried out for a period of time so that the transduced cells account for at least 20% of the cell population.

[0011] The present invention provides a mammalian immune cell, comprising (a) a nucleic acid sequence encoding an orthogonal hCD122 receptor, which is operably connected to one or more expression control elements so that the mammalian immune cell expresses the orthogonal hCD122 receptor, and (b) a nucleic acid sequence encoding GPC3 CAR, which is operably connected to one or more expression control elements so that the mammalian immune cell expresses CAR. In some embodiments, the nucleic acid sequence encoding GPC3 CAR and the nucleic acid sequence encoding the orthogonal receptor are provided on separate vectors, and each nucleic acid sequence is operably connected to an expression control sequence that is operable in a mammalian immune cell.

[0012] In some embodiments, the nucleic acid sequence encoding the GPC3 CAR and the nucleic acid sequence encoding the orthogonal receptor are provided on a single vector. In some embodiments, the nucleic acid sequence is operably linked to the same expression control element. In some embodiments, the vector comprises two nucleic acid sequences separated by the IRES element of the 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.

[0013] In some embodiments, a method of treating or preventing a disease, disorder or condition in a mammalian subject in need of treatment or prevention is provided, the method comprising the steps of: (a) isolating a certain amount of immune cells from a subject; (b) contacting the isolated amount of isolated immune cells with a nucleic acid sequence, the contacting being performed under conditions such that the isolated immune cells take up the nucleic acid sequence, the nucleic acid sequence encoding a transmembrane receptor comprising an intracellular signaling domain operatively connected to an extracellular domain, the extracellular domain of the receptor comprising an ECD of an orthogonal hCD122 or a functional fragment thereof; (c) contacting the isolated population of cells of step (b) ex vivo with an amount of an orthogonal ligand sufficient to induce proliferation of cells transduced by the contacting of step (b), wherein the contacting (c) is performed for a period of time such that the transduced cells comprise at least 20% of the cell population; and (d) administering to a mammalian subject a therapeutically effective amount of cells from the cell population produced in step (c), in combination with a therapeutically effective dose of an orthogonal ligand.

[0014] In some embodiments, after step (a) and before step (b), the cell population is manipulated ex vivo to enrich the population for activated immune cells or antigen-experienced T cells.

[0015] In some embodiments, the present invention provides orthogonal GPC3 CAR T cells, which are recombinantly modified to express orthogonal receptors (orthogonal immune cells). In some embodiments, the orthogonal IL2 receptor is a variant human CD122, which comprises amino acid substitutions at positions 133 and 134 according to the numbering of wild-type human CD122 (SEQ ID NO: 4). In some embodiments, according to SEQ ID NO.1 numbering, the orthogonal IL2 receptor is a variant human CD122 with amino acid substitutions at positions 133 and 134, wherein the substitution at position 133 is selected from H133D, H133E and H133F, and the substitution at position Y134 is selected from Y143F, Y134 or Y134R. In some embodiments, the orthogonal IL2 receptor is a variant human CD122 with amino acid substitutions H133D and Y134F, which is numbered according to SEQ ID NO: 1 (SEQ ID NO: 2).

[0016] In some embodiments, the present invention provides a method for preparing an engineered immune cell product substantially enriched in orthogonal GPC3 CAR T cells, the method comprising the following steps: (a) isolating a mixed immune cell population from a subject; (b) transfecting a portion of the isolated immune cell population with a recombinant vector capable of affecting the expression of orthogonal GPC3 CAR T cells in the transfected cells; (c) culturing the mixed immune cell population in the presence of orthogonal IL2 so that cells expressing orthogonal receptors selectively proliferate, thereby enriching the cell population for cells expressing orthogonal receptors. 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.

[0017] In some embodiments, the present invention provides orthogonal IL2, which specifically and selectively binds to the extracellular domain (ECD) of the transmembrane polypeptide comprising the modified CD122 polypeptide (orthogonal CD122). The binding of orthogonal IL2 to orthogonal CD122 participates in the transduction pathway of intracellular signal transduction, resulting in the activation of the natural intracellular signal transduction mode associated with IL2 binding to medium affinity or high affinity IL2 receptors, but it shows selectivity for the cells expressing the engineered orthogonal CD122.

[0018] The present disclosure further provides a method for prolonging the activated form ("persistence") of orthogonal GPC3 CAR T cells in vivo in a mammalian subject by administering an effective amount of orthogonal IL2 to the subject.

[0019] The present disclosure also provides a method of specifically and selectively activating and / or inducing proliferation of orthogonal GPC3 CAR T cells 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.

[0020] The present disclosure also provides a method of treating a mammalian subject having a neoplastic disease by administering to the mammalian subject an effective amount of orthogonal GPC3 CAR T cells in combination with a therapeutically effective amount of orthogonal IL2, wherein the orthogonal receptor is expressed on the orthogonal CAR-T cells.

[0021] The present disclosure also provides a method of treating a mammalian subject having 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 an orthogonal GPC3CAR T cell in combination with a therapeutically effective amount of a cognate orthogonal ligand directed against a receptor expressed on the orthogonal T cell.

[0022] The present disclosure also provides a method of restoring the activity of a depleted therapeutically effective amount of an orthogonal IL2 in a subject by administering to the subject a therapeutically effective amount of an orthogonal IL2.

[0023] The present invention further provides a method for treating a mammalian subject suffering from a recurrence of a neoplastic disease, characterized in that a therapeutically effective amount of an orthogonal is administered previously, the method comprising the following steps: (i) administering to the subject an effective amount of an orthogonal IL2 sufficient to restore the activity of the previously administered orthogonal CAR-T cells; and optionally (ii) periodically administering to the subject an effective amount of a cognate orthogonal ligand of an orthogonal receptor expressed on the previously administered orthogonal CAR-T cells to maintain the activity of the orthogonal CAR-T cells for a sufficient period of time; (iii) assessing the subject for the presence of a neoplastic disease, and when there is no evidence of a neoplastic disease, discontinuing administration of the orthogonal ligand, or periodically continuing to administer the orthogonal ligand according to a maintenance dosing regimen sufficient to maintain a certain amount of orthogonal CAR-T cells sufficient for immune surveillance of tumor cells.

[0024] The present disclosure further provides a method for treating a mammalian subject with a relapsed or refractory neoplastic disease, characterized in that an orthogonal CAR-T cell product is previously administered in a treatment regimen, the method comprising the following steps: (i) administering to the subject an effective amount of a cognate orthogonal ligand directed against an orthogonal receptor expressed on a previously administered orthogonal CAR-T cell, sufficient to restore the activity of the previously administered orthogonal CAR-T cell; (ii) periodically administering to the subject an effective amount of a cognate orthogonal ligand directed against a receptor expressed on a previously administered orthogonal CAR-T cell, so that the activity of the orthogonal CAR-T cell is maintained for a time sufficient to induce a therapeutic remission; (iii) assessing the subject for the presence of a neoplastic disease, and when there is no evidence of a neoplastic disease, continuing to periodically administer the orthogonal ligand or ceasing administration of the orthogonal ligand according to a maintenance dosing regimen sufficient to maintain a certain amount of orthogonal CAR-T cells sufficient for immune surveillance of tumor cells.

[0025] In some embodiments, the GPC3 CAR is a polypeptide having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or 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. 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).

[0026] In one embodiment, the present disclosure provides an orthogonal GPC3 CAR T cell, wherein on the surface of the orthogonal GPC3 CAR T cell are expressed: (a) a GPC3 CAR having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 37; and (b) an orthogonal CD122 having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0027] In one embodiment, the present disclosure provides an orthogonal GPC3 CAR T cell, wherein: (a) a GPC3 CAR having an amino acid sequence of SEQ ID NO: 37; and (b) an orthogonal CD122 having an amino acid sequence of SEQ ID NO: 4 are expressed on the surface of the orthogonal GPC3 CAR T cell.

[0028] In one embodiment, the present disclosure provides a method for preparing orthogonal GPC3 CAR T cells, the method comprising the following steps: a) obtaining human peripheral blood mononuclear cell (PBMC) samples; b) contacting the PBMC sample with magnetic beads coated with CD3 and CD28 ligands to provide an activated PBMC population c) isolating CD8+ and CD4+ T cells from the activated PBMC population; d) contacting the separated CD8+ and CD4+ T cell populations with a recombinant lentiviral vector comprising an expression cassette consisting, from 5' to 3', of a nucleic acid sequence encoding: Promoters active in T cells, such as the EF1a promoter; Nucleic acid sequence encoding signal peptide ( A nucleic acid sequence encoding GPC3 CAR; T2A sequence (SEQ ID NO:41) Nucleic acid sequence encoding signal peptide Nucleic acid sequence encoding SEQ ID NO:4 orthogonal CD122 thereby transducing the separated CD8+ and CD4+ T cell population fractions with the lentiviral vector; and e) contacting the cell population obtained from step (d) with an orthogonal IL2. In a preferred practice of the above method, the orthogonal IL2 is selected from the group consisting of: (a) a human IL2 mutant protein containing amino acid substitutions E15S / H16Q / L19V / D20L / Q22K / M23A; an orthogonal IL2 of SEQ ID NO 9 (STK-007), or a pegylated variant thereof (e.g., STK-009).

[0029] In some embodiments, the present disclosure provides orthogonal GPC3 CAR T cells prepared by a method comprising the steps of: a) obtaining human peripheral blood mononuclear cell (PBMC) samples; b) contacting the PBMC sample with magnetic beads coated with CD3 and CD28 ligands to provide an activated PBMC population c) isolating CD8+ and CD4+ T cells from the activated PBMC population; d) contacting the separated CD8+ and CD4+ T cell populations with a recombinant lentiviral vector comprising an expression cassette consisting, from 5' to 3', of a nucleic acid sequence encoding: Promoters active in T cells, such as the EF1a promoter; Nucleic acid sequence encoding signal peptide ( A nucleic acid sequence encoding GPC3 CAR; T2A sequence (SEQ ID NO:41) Nucleic acid sequence encoding signal peptide Nucleic acid sequence encoding SEQ ID NO:4 orthogonal CD122 thereby transducing the separated CD8+ and CD4+ T cell population fractions with the lentiviral vector; e) contacting the cell population obtained from step (d) with an orthogonal IL2 selected from the group consisting of a human IL2 mutant protein comprising amino acid substitutions E15S / H16Q / L19V / D20L / Q22K / M23A; an orthogonal IL2 of SEQ ID NO 9 (STK-007), or a pegylated variant thereof (e.g., STK-009).

[0030] 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 treatment or prevention, the method comprising administering to the subject a therapeutically effective amount of an orthogonal GPC3 CAR T cell prepared substantially according to the foregoing method in combination with a therapeutically effective dose of an orthogonal IL. In some embodiments of the foregoing methods, the orthogonal GPC3 CAR T is administered at 4x10 5 In some embodiments of the foregoing methods, when the orthogonal IL2 is STK-009, for human subjects, the therapeutically effective amount of STK-009 in combination with the orthogonal GPC3 CAR T cells is about 1.5 mg to about 12 mg administered subcutaneously weekly.

[0031] 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 consisting of the following steps: (1) Subjects treated with lymphocyte depletion regimen (2) administering to the subject a therapeutically effective amount of an orthogonal GPC3 CAR T in combination with a therapeutically effective dose of an orthogonal IL2, wherein the orthogonal GPC3 CAR T is prepared essentially according to the aforementioned method.

[0032] In some embodiments, the lymphodepleting regimen comprises administering cyclophosphamide and fludarabine. In some embodiments, the lymphodepleting regimen comprises administering cyclophosphamide 300 mg / m 2 / day and fludarabine 30 mg / m 2 / day for three days. In some embodiments of the above method, after lymphocyte depletion, 1x10 5 Up to 5x10 5 Orthogonal GPC3 CAR T cells / kg are administered. In some embodiments of the foregoing methods, after lymphocyte depletion, 4x10 5The orthogonal GPC3 CAR T is administered at a dose of CAR T cells / kg. In some embodiments of the foregoing methods, when the orthogonal IL2 is STK-009, for human subjects, the therapeutically effective amount of STK-009 combined with the orthogonal GPC3 CAR T cells is about 1.5 mg to about 12 mg administered subcutaneously weekly.

[0033] In one embodiment of the invention, the invention provides a method for treating a subject who has relapsed (e.g., relapse of a neoplastic disease) after administration of an orthogonal GPC CAR T cell therapy, the method comprising administering to the subject a therapeutically effective amount of an orthogonal IL2 such that the orthogonal ligand induces activation and / or proliferation of orthogonal GPC3 CAR T cells in the subject. In the treatment of relapse, a therapeutically effective amount of STK-009 is about 1.5 mg to about 12 mg administered subcutaneously weekly. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be better understood through the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, as a rule, the various features of the drawings are not to scale. On the contrary, the sizes of the various features are arbitrarily enlarged or reduced for clear display. The accompanying drawings include the following drawings.

[0035] Figure 1 A cartoon diagram of the arrangement of the coding sequences of the various functional domains of the GPC3 CAR and the orthogonal CD122 components used to generate the vectors for DR625, DR626, and DR628 is provided.

[0036] Figure 2A -D provides a graphical representation of data from a study evaluating the in vitro cell line efficacy of orthogonal GPC3 CAR T cells in cell lines expressing different levels of GPC3. The vertical axis measures luminescence, which is a measured value of cell viability, and the horizontal axis represents the relative dose of the effector (orthogonal GPC3 CAR T cell test agent) relative to the amount of target cells. Component A represents the results in the PLC / PRF / 5 cell line without GPC3 expression or very low GPC3 expression, component B provides data obtained using the Huh-7 cell line with low GPC3 expression, and component C provides data obtained from the HepG2 cell line with high-level GPC3 expression. Component D provides data illustrating the comparative level of GPC3 expression in the cell line evaluated.

[0037] Figure 3A-E provides a diagram of the relevant data generated by the study, which comes from the study of the effects of the orientation of CAR and orthogonal CD122 coding sequences on expression vectors in a variety of tumor cell lines, which are highly expressed (sub-figure A, HepG2), medium / lowly expressed (sub-figure B, PLC / PRF / 5) and substantially not expressed (sub-figure C, Raji). Sub-figures D and E are flow cytometry assessments, which are used to assess the level of GPC3 expression in HepG2 and PLC / PRF / 5 cells, respectively.

[0038] Figure 4 Provides a graphic representation of the data generated by the study, which evaluates the response of DR625 and DR626 orthogonal GPC3 CAR T cells and non-transduced cells in response to wild-type human IL2 (WT IL-2) and orthogonal IL2 (STK-009). The vertical axis is the proliferation signal normalized to non-stimulated cells, and the horizontal axis is the concentration of the test agent (wt hIL2 or STK-009).

[0039] Figure 5A -B provides a graphic representation of the relevant data generated by HepG2 study A, regarding the in vivo evaluation of the DR625 and DR626 orthogonal GPC3 CAR T cells. In sub-figure A, the measured tumor volume is provided on the Y axis and the time course of the study is provided on the X axis. The legend describes the treatment group and the molecules administered. Sub-figure B provides additional data generated by HepG2 study A, regarding the weight changes (Y axis) of the animals over the study time course (X axis).

[0040] Figure 6 A series of spider graphs are provided for data generated for individual animals in each treatment group in HepG2 Study A. For each graph, tumor volume is provided on the Y-axis and the time course of the study is provided on the X-axis. Each line of the spider graph is for an individual animal in the study.

[0041] Figure 7 A series of spider graphs are provided for data generated for individual animals in each treatment group in HepG2 Study A. The percent change in body weight relative to the start of the experiment is provided on the (Y-axis) and the time course of the study is provided on the (X-axis). Each line of the spider graph is for an individual animal in the study.

[0042] Figure 8 A graphical representation of data is provided relating to the activation levels (y-axis) of specific T cell subsets (as indicated) in mice participating in HepG2 Study A in response to treatment with the DR625 and DR626 orthogonal GPC3CAR T cells in combination with the orthogonal IL2 ligand STK-009.

[0043] Fig.9A-C provides a graphical representation of the data generated in HepG2 Study B. In Panel A, tumor volume (y-axis) is provided as a function of study duration (x-axis). In Panel B, body weight as a percentage relative to the start of the study (y-axis) is provided as a function of study duration (x-axis). In Panel C, the number of hCD3+ cells in the blood of study animals (y-axis) is provided as a function of study duration (x-axis).

[0044] Fig.10 are photomicrographs of tumor sections obtained from treated animals from each treatment group of HepG2 Study B, stained with various fluorescent markers to highlight the presence of GPC3, CD3, and DNA in the tumors. The legend provides the shading associated with each fluorescent marker.

[0045] Fig.11 are photomicrographs of tumor sections obtained from treated animals from each treatment group of HepG2 Study B, stained with various fluorescent markers to highlight the presence of nuclei, CD8, granzyme B, and CD4 in the tumor. The legend provides the shading associated with each fluorescent marker.

[0046] Fig.12 A graphical representation of the data generated in Huh7 Study A is provided. In Panel A, tumor volume (y-axis) is provided as a function of study duration (x-axis). In Panel B, body weight as a percentage relative to the start of the study (y-axis) is provided as a function of study duration (x-axis).

[0047] Fig.13 A spider diagram of the data generated for each treatment group in Huh7 Study B is provided. Each animal is represented by a line in the graph. Tumor volume (y-axis) is provided as a function of study duration (x-axis).

[0048] Fig.14 A graphical representation of data generated in a replicate Huh7 study subcutaneous study is provided. Tumor volume (y-axis) is provided as a function of study duration (x-axis).

[0049] Fig.15 A graphical representation of data generated in the intraperitoneal Huh7 replicates described in the experimental section is provided. Tumor volume (y-axis) is provided as a function of study duration (x-axis).

[0050] Fig.16 Data are presented pertaining to HepG2 re-challenge study A. Tumor volume (y-axis) is presented as a function of study duration (x-axis).

[0051] Fig.17Provided is a graphical representation of data from HepG2 re-challenge study B. Tumor volume (y-axis) is presented as a function of study duration (x-axis). DETAILED DESCRIPTION

[0052] In order to make the present invention more easily understood, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be read in accordance with the knowledge of those skilled in the art.

[0053] Before describing the methods and compositions of the present invention, it is to be understood that the invention is not limited to the particular methods or compositions described, as they may, of course, vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments only and is not intended to be limiting.

[0054] When providing a numerical range, it should also be considered that each intermediate value between the upper and lower limits of the range is specifically disclosed, unless the context clearly indicates otherwise. The present invention also includes a smaller range between any set value or intermediate value in the set range and any other set value or intermediate value in the set range. Depending on any explicitly excluded limits in the set range, the range may independently include or exclude the upper and lower limits of these smaller ranges, and the present invention also includes each range where these smaller ranges do not include limits, include any one or two limits. When the set range includes one or two limits, the present invention also includes a range that excludes one or two of the limits.

[0055] Unless otherwise indicated, all scientific and technical terms used herein have the same common meaning as understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein may be used to implement or test the present invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0056] It should be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes one or more peptides and equivalents thereof, such as polypeptides, known to those skilled in the art, and so forth.

[0057] The publications discussed herein are provided solely 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 publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0058] The publications discussed herein are provided solely 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 publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0059] 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 pressure. Standard abbreviations are used, including the following: bp = base pair; kb = kilobase; pl = picoliter; s or sec = second; 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 = myometrium intramuscular; ip = intraperitoneal (intraperitoneal); SC or SQ = subcutaneous (subcutaneous); QD = once daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; 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.

[0060] It should be understood that in the present disclosure, amino acids are referred to according to single-letter or three-letter codes. For the convenience of the reader, single-letter and three-letter amino acid codes are provided in Table 1 below:

[0061] Standard methods of 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, Vol. 1-4, John Wiley and Sons, Inc., New York, NY, which describes cloning and site-directed mutagenesis of DNA in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)). The scientific literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1-2, John Wiley and Sons, Inc., NY).

[0062] The present disclosure provides a variety of IL2 mutant proteins and CD122 receptor mutant proteins. The following nomenclature is used herein to represent substitution, deletion or insertion. In this article, the residue can be specified by the one-letter or three-letter amino acid code of the naturally occurring amino acid found in the wild-type molecule, but followed by the IL2 amino acid position of the mature IL2 molecule, such as "Cys125" or "C125" refers to the cysteine ​​residue at the 125th position of the wild-type hIL2 molecule. For orthogonal IL2, it is specified herein that the substitution is followed by the amino acid position of IL2 by the one-letter amino acid code, followed by the one-letter amino acid code of the substitution. For example, the orthogonal IL2 with "K35A" modification refers to the lysine (K) residue at the 35th position of the wild-type IL2 sequence being replaced by an alanine (A) residue at this position. The deletion of an amino acid residue is referred to as "des", followed by an amino acid residue and its position in the mature form of wild-type human IL2 (SEQ ID NO: 8). For example, the term "des-Ala1" or "desA1" refers to the alanine deletion at the 1st position of the polypeptide of the wild-type IL2 sequence. Similarly, for amino acid substitutions in orthogonal CD122, amino acid substitutions are designated herein by the one-letter amino acid code for the naturally occurring amino acid, followed by its position number in the wild-type IL2 sequence, followed by the one-letter amino acid code for the substituted amino acid at that position. For example, a hCD122 mutant protein having a tyrosine residue substituted with phenylalanine at position 134 is abbreviated as "Y134F". definition

[0063] Unless otherwise indicated, the following terms are intended to have the following meanings. Other terms are defined throughout the specification.

[0064] Activation : The term "activation / activation" as used herein refers to a receptor or a receptor complex to reflect a biological effect produced directly and / or by participating in a multi-component signal transduction cascade, which is a response to the binding of a ligand, produced by the binding of an agonist ligand to a receptor. For example, it is said that the binding of an IL2 agonist (IL2 agonist ligand) to an IL2 receptor "activates / activates" the signal transduction of the receptor to produce one or more intracellular biological effects (e.g., phosphorylation of STAT5).

[0065] activeAs used herein, the term "activity" is for a molecule to describe the properties of a molecule relative to a test system (e.g., an assay) or a biological or chemical property (e.g., the degree to which the molecule binds to another molecule) or a physical property of a material or cell (e.g., a change in 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, the ability to regulate immunological activity (such as an inflammatory response). "Activity" is generally expressed as the level of biological activity of a test agent per unit, such as [catalytic activity] / [mg protein], [immunological activity] / [mg protein], international units (IU) of activity, [STAT5 phosphorylation] / [mg protein], [T-cell proliferation] / [mg protein], plaque forming units (pfu), etc. As used herein, the term "proliferative activity" refers to an activity that promotes cell proliferation and replication, including the dysregulated cell division observed in neoplastic diseases, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis, and angiogenesis.

[0066] Affinity As 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 expressed as K. d The dissociation constant (K) between a molecule and its target is a measure of binding kinetics. off ) and the association constant (K on ) ratio.

[0067] Agonists:As 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 or promote an increase in target activation. In some cases, an agonist is an activator of a receptor protein, which can regulate cell activation, enhance activation, make cells sensitive to the activation of a second agent, or raise one or more genes, proteins, ligands, receptors, biological pathways, which may cause cell proliferation or cause cell cycle arrest, or cause the expression of the pathway of cell death (such as by apoptosis). In some embodiments, an agonist is an agent that is bound to a receptor and changes receptor state, causing a biological response. The response simulates the effect of the endogenous activator of a receptor. The term "agonist" includes partial agonists, full agonists, and super agonists. When an agonist causes a substantially complete biological response (i.e., a response related to a naturally occurring ligand / receptor binding interaction) induced by the receptor under study, this agonist can be described as a "full agonist", or an agonist can also be described as a partial agonist. In contrast to agonists, antagonists can specifically bind to receptors, but do not cause the signal cascades that are usually initiated by the receptors, and can change the effects of agonists on the receptors. An inverse agonist refers to an agent that produces a pharmacological response that is opposite to the direction of an agonist. A "superagonist" is an agonist that is able to produce a maximum response greater than that of the endogenous agonist of the target receptor, and therefore has an activity greater than 100% of the natural ligand. Superagonists are usually synthetic molecules that, when evaluated at similar concentrations in comparative tests, exhibit a response 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% in the quantitative or qualitative parameters that can be evaluated in the naturally occurring form of the molecule. It should be noted that the biological effects associated with full agonists may differ in degree and / or kind from those of partial or superagonists.

[0068] Antagonists : As used herein, the term "antagonist" or "inhibitor" refers to a molecule that opposes one or more effects of an agonist. Antagonists can prevent, reduce, inhibit or neutralize the activity of an agonist, and antagonists can also prevent, inhibit or reduce the constitutive activity of a target (such as a target receptor) even in the absence of an identified agonist. Inhibitors are molecules that reduce, block, prevent, delay activation, inactivate, desensitize or downregulate, for example, a gene, protein, ligand, receptor, biological pathway (including an immune checkpoint pathway) or cell.

[0069] AntibodyAs used herein, the term "antibody" refers collectively to: (a) glycosylated and non-glycosylated immunoglobulins (including but not limited to mammalian immunoglobulin classes IgG1, IgG2, IgG3 and IgG4), which specifically bind to a target molecule, and (b) immunoglobulin derivatives, including but not limited to IgG(1-4)ΔC H 2. F(ab')2, Fab, ScFv, V H 、V L , tetra-antibodies, tri-antibodies, bi-antibodies, dsFv, F(ab')3, scFv-Fc and (scFv)2, which compete with the immunoglobulins from which they are derived for binding to the target molecule. The term antibody is not limited to immunoglobulins, antibodies, or human antibodies from any mammalian species (including mice, humans, horses, and camels). The term antibody includes so-called "heavy chain antibodies" or "VHH" or Typically obtained from immunization of Camelidae (including camels, llamas and alpacas) (see, e.g., Hamers-Casterman, et al. (1993) Nature 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" includes antibodies that can be isolated from natural sources or from animals after immunization with an antigen, as well as engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted, veneered or deimmunized (e.g., T cell epitopes removed) antibodies. The term "human antibody" includes antibodies obtained from humans, as well as antibodies obtained from transgenic mammals containing human immunoglobulin genes, such that, after antigen stimulation, the transgenic animal produces antibodies containing amino acid sequence characteristics of antibodies produced by humans. The term includes parent antibodies and derivatives thereof, such as affinity matured, veneered, CDR grafted, humanized, camelized (in the case of VHH), or binding molecules containing the binding domains (such as CDRs) of antibodies in non-immunoglobulin scaffolds. The term "antibody" should be interpreted as not being limited to any particular synthetic method, including naturally occurring antibodies that can be isolated from natural sources, and engineered antibody molecules prepared by "recombinant" means, including antibodies isolated from transgenic animals that transfer human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with nucleic acid constructs that cause antibody expression, and antibodies isolated from combinatorial antibody libraries (including phage display libraries). In one embodiment, an "antibody" is a mammalian immunoglobulin. In some embodiments, the antibody is a "full-length antibody" that 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, each light chain including a variable region and a constant region. In some embodiments, the term "full-length antibody" is used to refer to a conventional IgG immunoglobulin structure, including two light chains and two heavy chains, each light chain including a variable region and a constant region that provide binding and effector functions. The term antibody includes antibody conjugates, which include modifications to extend the duration of action, such as fusion proteins (e.g., Fc fusions) or coupling to polymers (e.g., polyethylene glycol), as described in more detail below.

[0070] CDR.As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptide immunoglobulin polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977); Kabat et al., (1991) US Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (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), where the definitions include overlaps or subsets of amino acid residues when compared to each other. However, the definition of CDRs referring to antibodies or grafted antibodies or variants thereof is intended to fall within the scope of the term defined or used herein. In the context of the present invention, the numbering of CDR positions is provided according to the Kabat numbering.

[0071] Circulating tumor cells : The term "circulating tumor cell (CTC)" as used herein refers to tumor cells shed from a tumor mass (eg, a tumor) into the peripheral circulation.

[0072] Quite : The term "comparable" as used herein is used to describe the difference degree of two measurement results of evaluable quantitative or qualitative parameters. For example, when the first measurement result of an evaluable quantitative parameter (IL2 activity level determined by CTLL-2 proliferation or phosphorylation-STAT5 test) and the second measurement result of the evaluable parameter do not deviate from the scope that will be considered by those skilled in the art to produce statistically significant differences between the two results, the two measurements will be considered as "comparable". In some cases, if a measurement result deviates from another measurement result and is less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 7%, or less than 5%, or less than 4%, or less than 3%, or less than 2% or less than 1%, the measurement result can be considered as "comparable". In a specific embodiment, if a measurement result deviates from a reference standard and is less than 15%, or less than 10%, or less than 5%, the measurement result is comparable to the reference standard.

[0073] Derived from / derived from: As used herein, the term "derived from / derived from" in the context of an amino acid sequence (e.g., a polypeptide or polynucleotide sequence comprising an amino acid sequence "derived from / derived from" an IL2 polypeptide) is intended to indicate that the polypeptide or nucleic acid has a sequence based on a reference polypeptide or nucleic acid (e.g., a naturally occurring IL2 polypeptide or a nucleic acid encoding IL2), and is not intended to be limited to the source or method of making the protein or nucleic acid. For example, the term "derived from / derived from" includes homologues or variants of a reference amino acid or DNA sequence.

[0074] Effective concentration (EC) As used herein, the term "effective concentration" or its abbreviation "EC" is used interchangeably to indicate that the concentration of an agent (e.g., orthogonal hIL2) is an amount 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 the 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 the activating test agent. When the abbreviation EC is provided with a subscript (e.g., EC 40 ,EC 50 The subscript refers to the concentration at which the biological effect is observed. 最大 For example, in a test system, a concentration of a test agent that is sufficient to cause a reduction in a measurable biological parameter by 30% of the maximum level of the measurable biological parameter in response to the test agent is referred to as the "ECD" of the test agent for that biological parameter. 30 Similarly, the term “EC 100 " is used to denote the effective concentration of an agent that produces a maximal (100%) response of a measurable parameter to that agent. Similarly, the term EC 50 (which is commonly used in the field of pharmacokinetics) 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 maximum possible amount of a test agent that can be dissolved in a standard volume of a specific solvent (e.g., water) under conditions of standard temperature and pressure. In pharmacokinetics, the saturation concentration of a drug is often used to refer to the concentration of the drug that is sufficient to occupy all available receptors, while EC 50 is the concentration of drug that gives a half-maximal effect.

[0075] EnrichedAs used herein, the term "enriched" refers to non-natural manipulation of a sample so that the substance of interest (e.g., a molecule or cell) is present: (a) at a higher concentration (e.g., at least 3 times higher, or at least 5 times higher, or at least 10 times higher, or at least 50 times higher, or at least 100 times higher, or at least 1000 times higher) than the substance in the starting sample (e.g., a biological sample, for example, in which the molecule occurs naturally or after administration); or (b) at a higher concentration than in the environment in which the molecule was produced (e.g., in recombinantly modified bacteria or mammalian cells). In some embodiments, the term "enriched" is used herein to refer to a cell population comprising cells expressing an orthogonal receptor that is contacted with a cognate ligand in an amount sufficient to elicit a response in those cells expressing the orthogonal receptor, wherein the response is proliferation, such that the concentration of cells expressing the orthogonal receptor in the population is higher (e.g., at least 3-fold higher, or at least 5-fold higher, or at least 10-fold higher, or at least 50-fold higher, or at least 100-fold higher, or at least 1000-fold higher) after contacting the cell population with the cognate ligand.

[0076] Extracellular domain As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the cell surface protein portion (e.g., cell surface receptor) outside the cytoplasmic membrane. ECD can include the entire extracellular portion of a transmembrane protein, a cell surface or membrane-associated protein, a secreted protein, or a cell surface targeted protein.

[0077] hCD122 As used herein, the term "hCD122" refers to naturally occurring human CD122 polypeptides, including naturally occurring variants thereof. The amino acid sequence of naturally occurring mature hCD122 is provided as SEQ ID NO:4. Human CD122 (hCD122) is expressed as a preprotein of 551 amino acids, and the first 26 amino acids include a signal sequence, which is translated and cleaved in a mature protein of 525 amino acids. Amino acids 27-240 (amino acids 1-214 of mature proteins) correspond to extracellular domains, amino acids 241-265 (amino acids 225-239 of mature proteins) correspond to transmembrane domains, and amino acids 266-551 (amino acids 240-525 of mature proteins) correspond to intracellular domains. As used herein, the term CD122 includes naturally occurring variants of CD122 proteins, including CD122 variants containing S57F and D365E substitutions (according to the numbering of mature hCD122 proteins). hCD122 is cited as entry P14784 in the UniProtKB database. The nucleic acid and protein sequences of human CD122 can be found in Genbank accession numbers NM_000878 and NP_000869, respectively.

[0078] Identity: For polypeptides or DNA sequences, the term "identity" as used herein refers to the subunit sequence identity between two molecules. When a subunit position in two molecules is occupied by the same monomer subunit (i.e., the same amino acid residue or nucleotide), then the two molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the amount of the same position. Usually, the sequences are aligned to obtain the highest order match. If necessary, the identity can be calculated using published techniques and widely used 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). Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wisconsin 53705). Suitable algorithms for determining percent sequence identity and percent sequence similarity using their default parameters are the BLAST and BLAST 2.0 algorithms, respectively, described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402. Software for performing BLAST analysis is publicly available from the website of the National Center for Biotechnology Information (NCBI). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits are used as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence until the cumulative alignment score is improved. For nucleotide sequences, the cumulative score is calculated using the parameters M (a reward score for a pair of matching residues; always >0) and N (a penalty score for mismatched residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hit in each direction is terminated when the following occurs: the cumulative alignment score decreases by X from its maximum achieved value; the cumulative score becomes zero or below zero due to the accumulation of one or more negatively scored 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 comparison. The default values ​​used by the BLASTN program (for nucleotide sequences) are as follows: word length (W) 28, expectation (E) 10, M=1, N=-2, and comparison of both chains. For amino acid sequences, the default values ​​used by the BLASTP program are: word length (W) 3, expectation (E) 10, BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0079] IL2: As used herein, the term "interleukin-2" or "IL2" refers to an IL2 polypeptide having IL2 activity. In some embodiments, IL2 refers to mature wild-type human IL2. Mature wild-type human IL2 (hIL2) is a 133 amino acid polypeptide (minus the 20 N-terminal amino acids of the signal peptide of the preprotein), as described by Fujita, et al., PNAS USA, 80, 7437-7441 (1983). As used herein, the numbering of the residues of IL2 variants is based on the wild-type mature human IL2 sequence UniProt IDP60568, excluding the same signal peptide as SEQ ID NO: 8.

[0080] IL2 activity : The term "IL2 activity" refers to one or more biological effects on cells that respond to contact of cells with an effective amount of IL2 polypeptide. As mentioned above, IL2 is a pleiotropic cytokine that can produce one or more biological 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, enhances the growth of B cells, and promotes the proliferation and expansion of NK cells. An example of measuring IL2 activity is the use of CTLL-2 mouse cytotoxic T cells in a cell proliferation assay, see Gearing, AJH and CBBird (1987) in Lymphokines and Interferons, A Practical Approach. Clemens, MJ et al. (eds.): IRL Press. 295. The specific activity of recombinant human IL2 (rhIL2) is about 2.1x 10 4 IU / μg, which is calibrated against the recombinant human IL2 WHO international standard (NIBSC code: 86 / 500).

[0081] In an amount sufficient to produce a response:As used herein, the phrase "in an amount sufficient to produce a response / reaction / mitigation" is used to refer to the amount of the test agent sufficient to provide a detectable change in the level of the measured index before and after the test agent is applied to the test system (such as a baseline level). In some embodiments, the test system is a cell, tissue, or organism. In some embodiments, the test system is an in vitro test system, such as a fluorescence test. In some embodiments, the test system involves measuring the change in the parameter level of cells, tissues, or organisms reflecting biological functions before and after the test agent is applied to cells, tissues, or organisms. In some embodiments, an indicator (such as the concentration of phosphorylated STAT5) reflects the biological function (such as activation of IL2 receptors) of the cells evaluated in the test in response to the administration of a certain amount of test agents (such as IL2). In some embodiments, the test system involves measuring the change in the level of parameters (such as luminescence) of cells, tissues, or organisms (such as mice injected with luminescent tumor cells) reflecting biological conditions (such as the presence of tumors) before and after one or more test agents (such as CAR-T cells expressing orthogonal CD122 in combination with orthogonal IL2) are applied to cells, tissues, or organisms (such as mice). In some embodiments, the indicator (e.g., the concentration of phosphorylated STAT5) reflects the biological function (e.g., activation of IL2 receptor) of the cell (e.g., T cell) evaluated in the test in response to the administration of a certain amount of the test agent (e.g., IL2). An "amount sufficient to produce a response" can be an amount sufficient to be therapeutically effective, but an "amount sufficient to produce a response" may be more or less than a therapeutically effective amount.

[0082] Inhibitors : The term "inhibitor" as used herein is a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes or downregulates, for example, a gene, protein, ligand, receptor or cell. Inhibitors can also be defined as molecules that reduce, block or inactivate the constitutive activity of a cell or organism.

[0083] Separate : As used herein, the term "isolated" refers to a polypeptide of interest, if naturally occurring, in an environment different from that in which it may naturally occur. "Isolated" means that the polypeptide is included in a sample that is substantially enriched for the polypeptide of interest and / or the polypeptide of interest is partially or substantially purified. If the polypeptide does not occur naturally, "isolated" means that the polypeptide has been separated from the environment in which it was prepared by synthetic or recombinant means.

[0084] Kabat Number:The term "Kabat numbering" as used herein is a term recognized in the art and refers to a system for numbering 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, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the purposes of this disclosure, the positioning of CDRs in antibody variable regions follows Kabat numbering or simply "Kabat."

[0085] Ligand :As used herein, the term "ligand" refers to a molecule that specifically binds to a receptor and causes a change in the receptor, which changes the activity of the receptor or reacts in a cell expressing the receptor. In one embodiment, the term "ligand" refers to a molecule or a 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 mimics of cytokines and peptide mimics of antibodies. The complex of a ligand and a receptor is referred to as a "ligand-receptor complex". A ligand can include a domain of a polyprotein or fusion protein (e.g., an antibody-targeting ligand fusion protein).

[0086] Transfer : As used herein, the term "metastasis" describes the spread of cancer cells from a primary tumor to surrounding tissues and to distant organs.

[0087] adjust :As used herein, the terms "regulate", "modulate", etc. refer to the ability of a test agent to cause a response in a system (including a biological system or a biochemical pathway), which can be positive or negative, and can be direct or indirect. The term modulator includes agonists (including partial agonists, full agonists, and super agonists) and antagonists.

[0088] N-terminal : As used herein in the context of polypeptide structures, "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the extreme amino terminus and carboxyl terminus of a polypeptide, respectively, while the terms "N-terminal" and "C-terminal" refer to the relative positions toward the N-terminus and C-terminus, respectively, in the amino acid sequence of a polypeptide, and may include the N-terminal and C-terminal residues, respectively. "Near the N-terminus" or "Near the C-terminus" refers to the position of a first amino acid residue relative to a second amino acid residue, wherein the first and second amino acid residues are covalently bound to provide a continuous amino acid sequence.

[0089] Neoplastic diseases : As used herein, the term "neoplastic disease" refers to a disorder or condition caused by excessive cell proliferation or uncontrolled (or dysregulated) cell replication in a subject. The term neoplastic disease refers to a condition caused by the presence of a tumor in a subject. Tumors can be classified as: (1) benign (2) pre-malignant (or "precancerous"); and (3) malignant (or "cancerous"). The term "neoplastic disease" includes tumor-related diseases, disorders and conditions, and refers to conditions directly or indirectly related to a neoplastic disease, including, for example, angiogenesis and precancerous conditions, such as dysplasia.

[0090] Nucleic Acids : The terms "nucleic acid", "nucleic acid molecule", "polynucleotide" and the like are used interchangeably herein and refer to a polymeric form of nucleotides of any length, i.e., 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.

[0091] According to IL2 numbering : As used herein, the term "numbering according to IL2" refers to determining the position of a particular amino acid according to the position where the amino acid is normally found in the sequence of mature wild-type IL2 (SEQ ID NO: 8). For example, for hIL2, "R81" refers to the arginine at the 81st (numbering from the N-terminus) amino acid present in the sequence of mature wild-type hIL2. It should be noted that the amino acid sequences of IL2 molecules of different mammalian species have different numbers and sequences of amino acids. Therefore, when referring to residues according to this convention, it is helpful to identify the IL2 substance in question.

[0092] According to CD122 numbering : As used herein, the term "numbering according to CD122" refers to determining the position of a specific amino acid according to the position of the amino acid that is usually present in the sequence of a mature wild-type CD122 molecule. In one embodiment, the CD122 molecule is a mature wild-type human CD122 (SEQ ID NO.4). For example, for human CD122, H133 refers to the histidine at the 133rd (numbering from the N-terminus) amino acid of the sequence of mature wild-type hCD122.

[0093] Operationally connected: The term "operably linked" refers to the relationship between nucleic acid sequences encoding different functions when combined into a single nucleic acid sequence, which, when introduced into a cell, provides a nucleic acid capable of achieving transcription and / or translation of a specific nucleic acid sequence in the cell. For example, if it is expressed as a preprotein involved in the secretion of a polypeptide, the DNA of the signal sequence is operably linked to the DNA of the polypeptide; if it affects the transcription of the sequence, a promoter or enhancer is operably linked to the coding sequence; or if it is positioned to promote translation, a ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the DNA sequence being linked is continuous, and in the case of a secretory leader, is continuous and in the reading segment. However, some genetic elements, such as enhancers, do not need to be continuous with the sequence on which they act.

[0094] 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 produce a mutant or variant polypeptide. The parent polypeptide can be a wild-type (or native) polypeptide. The parent polypeptide can refer to the polypeptide itself or a composition comprising the parent polypeptide (e.g., glycosylated, pegylated, a fusion protein comprising the parent polypeptide).

[0095] Peptides : The terms "polypeptide", "peptide" and "protein" as used herein are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include genetically encoded or non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified polypeptide backbones. These 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 N-terminal methionine residues; fusion proteins with immunological tag proteins; fusion proteins of immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments), and the like.

[0096] Prevention / Prevention : The terms "prevention", "prevention" and the like as used herein refer to a course of action initiated before a disease, disorder, condition or its symptoms occur in a subject, so as to temporarily or permanently prevent, alleviate, inhibit or reduce the risk (e.g., as determined by the absence of clinical symptoms) of a subject suffering from a certain disease, disorder, condition or similar disease, or to postpone its onset, generally in the case where the subject is susceptible to a certain disease, disorder or condition due to genetic, empirical or environmental factors. In some cases, the terms "prevention" and "prevention" are also used to refer to slowing down the development of a disease, disorder or condition from its current state to a more harmful state.

[0097] Receptors: The term "receptor" as used herein refers to a polypeptide having a domain that specifically binds to a ligand, the binding of which results in a change in at least one biological property of the polypeptide. In some embodiments, the receptor is a "soluble" receptor that is not associated with the 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-associated domain that is used 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) connected by a membrane spanning domain commonly referred to as a transmembrane domain (TM). Ligand binding to the receptor results in a conformational change in the receptor, thereby producing a measurable biological effect. In some cases, if the receptor is a transmembrane polypeptide including ECD, TM and ICD, ligand binding to ECD causes a measurable intracellular biological effect mediated by one or more domains of ICD, in response to ligand binding to ECD. In some embodiments, the receptor is a component of a multicomponent complex to promote intracellular signaling. In some embodiments, the receptor is a transmembrane single-chain polypeptide including ECD, TM and ICD domains, wherein ECD, TM and ICD domains are derived from the same or 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).

[0098] Reorganization :As used herein, the term "recombinant" is used as an adjective to refer to a method for modifying a polypeptide, nucleic acid or cell using recombinant DNA technology. Recombinant protein is a protein produced using recombinant DNA technology, and a method for producing protein is usually represented by lowercase "r" (e.g., rhIL2). Similarly, if a cell is modified, by using recombinant DNA technology to include (e.g., transfection, transduction, infection) exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids, etc.), the cell is referred to as a "recombinant cell". The techniques and protocols used for recombinant DNA technology are well known in the art, for example, can be found in Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual" (Molecular Cloning: A Laboratory Manual) (2nd Edition, Cold Spring Harbor Laboratory Press, Plainview, New York) and other standard molecular biology experimental manuals.

[0099] Reaction / Response / Mitigation: The term "reaction / response / mitigation", for example, the reaction of cells, tissues, organs or organisms, including quantitative or qualitative changes in evaluable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy consumption rate (rate of energy consumption), differentiation level or state), wherein the change is related to activation, stimulation or processing, or internal mechanisms (such as genetic programming). In some cases, the terms "activation", "stimulation" and the like refer to cell activation regulated by internal mechanisms and external or environmental factors; and the terms "inhibition", "downregulation" and the like refer to the opposite effect. Examples of such standard protocols for assessing proliferation of CD3-activated primary human T-cells include bioluminescent assays that produce a luminescent signal that is proportional to the amount of ATP present, which in turn is proportional to the amount of cells present in culture, as described in Crouch, et al. (1993) "The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity" J. Immunol. Methods 160:81-8, or standardized commercially available assay systems, such as 2.0 Cell viability test or 3D cell viability kit, which is commercially available from Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711, catalog numbers G9241 and G9681, and is performed substantially in accordance with the instructions provided by the manufacturer, respectively. In some embodiments, the activation level of T cells in response to the administration of the test agent can be determined by the flow cytometry method, such as by STAT5 phosphorylation level determination, according to methods well known in the art. STAT5 phosphorylation can be measured using flow cytometry techniques, such as Horta, et al., supra, Garcia, et al., supra, as described, or using commercially available kits, such as phospho-STAT5 (Tyr694) kit (commercially available from Perkin-Elmer / cisbio, Waltham, Massachusetts, part number 64AT5PEG), substantially according to the instructions of the manufacturer. When the abbreviation EC ACT When used with a subscript, indicates the concentration of the test agent sufficient to produce an indication of the percentage of maximal STAT5 phosphorylation in T cells in response to application of the test agent as measured according to the test protocol. As an illustration, the abbreviation EC 30 PROCan be used for an orthogonal hIL2 to indicate the concentration that correlates with 30% of the maximal level of STAT5 phosphorylation in T cells in response to this orthogonal hIL2 as measured using the Phospho-STAT5 (Tyr694) kit.

[0100] In some cases, standardized, recognized bioactive measurements have been established for molecules. For example, for hIL2 potency, the standard method for evaluating hIL2 potency in international units (IU) is measured in the mouse cytotoxic T cell line CTLL-2 according to Wadhwa, et al. (2013) "The 2nd International standard for Interleukin-2 (IL2) Report of a collaborative study" Journal of Immunological Methods 397: 1–7, a more fully described standardized procedure. It should be noted in the context of the present invention that mouse IL2 receptor function is different from human IL2 receptor, particularly for signal transduction (e.g., STAT5 phosphorylation) that requires all components of the trimeric receptor complex to provide intracellular signal transduction. 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.

[0101] Selectivity:As used herein, the term "selectivity" is used to refer to the characteristic that a certain agent preferentially binds to and / or activates this particular cell type. In some embodiments, the present invention provides IL2 variants (orthogonal IL2), which selectively bind to engineered CD122 ECD polypeptides, thereby, in response to the binding of such orthogonal IL2 to a receptor comprising such homologous CD122 ECD polypeptides, the cells expressing the receptor comprising such CD122 ECD polypeptides are activated. In some embodiments, the present invention provides orthogonal hIL2, which is selective, i.e., this orthogonal IL2 shows preferential activation of immune cells expressing hoCD122 receptors. Selectivity is usually assessed by activity measurement in a characterization test of the activity induced by ligand / receptor binding reaction.

[0102] Significantly reduced binding: As used herein, the term "shows significantly reduced binding" is used to refer to the binding affinity of a variant of a ligand (e.g., orthogonal CD122) to a modified form of a receptor (e.g., orthogonal CD122) relative to the binding of a variant ligand to its naturally occurring form of a receptor. In some embodiments, if the binding of an orthogonal ligand to the natural form of a receptor is less than 20% of a naturally occurring ligand, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5%, then relative to the natural form of the ligand, the ligand (e.g., orthogonal IL2) shows significantly reduced binding. Similarly, if the natural form of the ligand is less than 20% of the affinity of the naturally occurring receptor to the orthogonal form of the receptor, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5%, then relative to the natural form of the ligand, the orthogonal receptor shows significantly reduced binding.

[0103] Specific binding: As used herein, the term "specific binding" refers to the degree of affinity / affinity of one molecule to bind to another molecule. In the context of a binding pair (such as a ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pair), when the first molecule of the binding pair is not bound to other components present in the sample in a significant amount, it is referred to as the first molecule of the binding pair specifically binding to the second molecule of the binding pair. When the affinity of the first molecule of the binding pair to the second molecule is at least 2 times greater, or at least 5 times greater, or at least 10 times greater, or at least 20 times greater, or at least 100 times greater than the affinity of the first molecule to other components present in the sample, the first molecule of the binding pair is referred to as specifically binding to the second molecule of the binding pair. In a specific embodiment in which the first molecule in the binding pair is an antibody, if the equilibrium dissociation constant between the antibody and the second molecule of the binding pair is less than about 10 -6 M, or less than about 10 -8 M, or less than about 10-10 M, or less than about 10 -11 M, or less than about 10 -10 M, less than about 10 -12 M, as determined, for example, by Scatchard analysis, then the antibody specifically binds to a second molecule (e.g., a protein, antigen, ligand, or receptor) in the binding pair (Munsen, et al. (1980) Analyt. Biochem. 107: 220-239). In one embodiment, wherein the ligand is orthogonal IL2 and the receptor comprises an orthogonal CD122 ECD, if the equilibrium dissociation constant of the orthogonal IL2 / orthogonal CD122 ECD is greater than about 10 -5 M, or less than about 10 -6 M, or less than about 10 -7 M, or less than about 10 -8 M, or less than about 10 -9 M, or less than about 10 -10 M, or less than about 10 -11 M, then orthogonal IL2 specific binding. Specific binding can be evaluated using techniques known in the art, including but not limited to competitive ELISA, radioligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting programs, and competitive binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and surface plasmon resonance assays (see, e.g., Drescher et al., Methods Mol Biol 493:323-343 (2009), using instruments commercially available from GE Healthcare Bio-Sciences, such as Biacore 8+, Biacore S200, Biacore T200 (GE Healthcare Bio-Sciences, Address: 100 Results Way, Marlborough MA 01752)); liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR) and immunoprecipitation); and solid phase ligand binding assays (e.g., multiwell plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filtration assays).

[0104] Basically pureAs used herein, the term "substantially pure" means that a component (e.g., a polypeptide) accounts for greater than about 50% of the total content of the composition, and generally accounts for greater than about 60% of the total content of the polypeptide. More generally, "substantially pure" refers to a composition in which at least 75%, at least 85%, at least 90% or more of the total composition is a component of interest. In some cases, the polypeptide will account for greater than about 90%, or greater than about 95% of the total content of the composition.

[0105] T-Cells: As used herein, the term "T-cell" or "T cell" is used in its conventional sense and refers to a lymphocyte differentiated in the thymus that possesses specific cell surface antigen receptors, including some that control the initiation or suppression of cell-mediated and humoral immunity and other cells 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, such as 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. Treg, induced Treg; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor infiltrating lymphocytes (TIL) and engineered variants of such T cells, including but not limited to CAR-T cells, recombinant modified TIL and TCR engineered cells.

[0106] Transmembrane domain: The term "transmembrane domain" or "TM" refers to the domain of a transmembrane polypeptide (such as a transmembrane polypeptide such as CD122 or CD132 or CAR), and when the transmembrane polypeptide is associated with a cell membrane, the domain is embedded in the cell membrane and is connected to the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide with a peptide bond (peptidyl linkage). The transmembrane domain can be homologous (naturally associated) or heterologous (not naturally associated) to one or both of the extracellular and / or intracellular domains. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with the ECD domain of the homologous receptor of the derived orthogonal receptor. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with the ICD domain of the homologous receptor of the derived orthogonal receptor. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with the proliferation signaling domain. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with different proteins. Alternatively, the transmembrane domain of the orthogonal receptor can be an artificial amino acid sequence that spans the plasma membrane. In some embodiments, the transmembrane domain of the orthogonal receptor is a transmembrane domain that is typically associated with the ICD of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, where the receptor is a chimeric receptor comprising an intracellular domain derived from a first parent receptor and a second extracellular domain derived from a second, different parent receptor, the transmembrane domain of the chimeric receptor is a transmembrane domain that is typically associated with the ICD or ECD of the parent receptor from which the chimeric receptor is derived.

[0107] Treatment / management : The terms "treatment", "therapy", "treatment" and the like refer to a course of action (e.g., administration of IL2, CAR-T cells or a pharmaceutical composition comprising the same) or similar situations initiated after diagnosis, observation of a disease, disorder or condition or its symptoms of a subject, in order to temporarily or permanently eliminate, reduce, inhibit, alleviate or improve at least one underlying cause of the disease, disorder or condition that afflicts the subject, or at least one symptom associated with the disease, disorder or condition. Treatment includes a course of action taken on a subject with a disease, wherein the course of action results in the subject's disease being inhibited (e.g., preventing the development of a disease, disorder or condition or improving one or more symptoms associated therewith).

[0108] Treg cells or regulatory T cells The term "regulatory T cells" or "Treg cells" as used herein refers to CD4 + A type of T cell that is able to suppress the response of other T cells, including but not limited to effector T cells (Teff). Treg cells are characterized by the expression of CD4, the alpha 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 + T cells.

[0109] Variants : The terms "protein variant" or "variant protein" or "variant polypeptide" are used interchangeably herein and 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 a modified form of a WT polypeptide. The term variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or a nucleic acid sequence encoding it. In some embodiments, the variant polypeptide comprises about 1 to about 10 amino acid modifications relative to the parent polypeptide, or about 1 to about 5 amino acid modifications relative to the parent, or about 1 to about 3 amino acid modifications relative to the parent, or 1 to 2 amino acid modifications relative to the parent, or a single amino acid modification relative to the parent. A variant may have at least about 99% identity, or at least about 98% identity, or at least about 97% identity, or at least about 95% identity, or at least about 90% identity with the parent polypeptide from which it is derived.

[0110] wild type: "Wild type" or "WT" or "native" herein refers to naturally occurring amino acid sequences or nucleotide sequences, including allelic variations. WT proteins, polypeptides, antibodies, immunoglobulins, IgG, etc. have amino acid sequences or nucleotide sequences that have not been artificially modified. DETAILED DESCRIPTION Overview

[0111] The present disclosure provides engineered T cells expressing (a) a chimeric antigen receptor, wherein the antigen binding domain of CAR binds to human GPC3 ("GPC-CAR"); and (b) an orthogonal receptor. The terms "chimeric antigen receptor T-cell" and "CAR-T cell" are used interchangeably and 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 orthogonal receptors ("orthogonal CAR-T cells" or "orthogonal CAR-T cells").

[0112] The present invention relates to compositions and methods related to the combination of orthogonal GPC3 CAR T cells and orthogonal IL2 ligands. The use of orthogonal GPC3 CAR T cells in combination with orthogonal IL2 can bring multiple benefits

[0113] In one embodiment, the present disclosure provides compositions and methods that provide for selective expansion of adoptively transferred human orthogonal GPC3 CAR-T cell populations in human subjects following administration of orthogonal IL2 to the subject.

[0114] In another embodiment, the present disclosure provides compositions and methods capable of selectively expanding adoptively transferred human orthogonal GPC3 CAR-T cells in vivo without significant off-target systemic activation of other immune cells.

[0115] In another embodiment, the present disclosure provides compositions and methods for supporting persistence of orthogonal GPC3CAR-T cells of adoptively transferred human immune cells without the significant toxicity associated with alternative support agents such as wt hIL2.

[0116] In another embodiment, the present disclosure provides compositions and methods for achieving in vivo therapeutic efficacy of a cell therapy product comprising orthogonal GPC3 CAR-T cells using an initial dose of a cell therapy agent previously reported to be ineffective and significantly lower than the dose of current similar cell therapy products in treating a neoplastic disease in a mammalian subject;

[0117] In another embodiment, the present disclosure provides compositions and methods that are capable of maintaining therapeutic levels of orthogonal GPC3 CAR-T cells at therapeutically effective levels in a subject for a longer period of time by periodically administering orthogonal IL2 to the subject.

[0118] In another embodiment, the present disclosure provides compositions and methods that are capable of treating the recurrence of a neoplastic condition in a subject previously treated with orthogonal GPC3 CAR-T cells by restoring the effectiveness of previously administered orthogonal cells through the administration of orthogonal IL2, without the need to administer additional orthogonal GPC3 CAR-T cells.

[0119] In another embodiment, the present disclosure provides compositions and methods that obviate the need for prior immune clearance of a subject prior to administration of a cell product comprising orthogonal GPC3 CAR-T cells.

[0120] In another embodiment, the present disclosure provides compositions and methods related to pharmaceutical formulations of orthogonal GPC3 CAR-T cells and orthogonal IL2.

[0121] In another embodiment, a method of treating a subject having a GPC3+ tumor is provided by administering a therapeutically effective amount of orthogonal GPC3 CAR-T cells in combination with a pharmaceutically acceptable orthogonal IL2 formulation. experiment

[0122] To demonstrate the efficacy of the compositions and methods within the scope of the present disclosure, a series of experiments were performed to evaluate the anti-GPC3+ tumor efficacy of orthogonal GPC3 CAR T cells in vitro and in vivo, as described in detail below, and the data are provided in the accompanying figures. A. Preparation of GPC3 CAR-T cells for research

[0123] Nucleic acid sequences encoding GPC3 CARS were synthesized using conventional DNA sequencing techniques. The amino acid sequences of GPC3CAR DR625, DR626 and DR628 are provided as SEQ ID NOs: 37, 38 and 39, respectively. DNA sequences encoding nucleic acids for constructing lentiviral vectors to produce GPC3 CAR DR625, DR626 and DR628 are provided as SEQ ID NOs: 42, 43 and 45, respectively.

[0124] The basis of the lentiviral vector used in this study for the construction of orthogonal GPC3 CAR T cells is a modified version of the LV200pLenti-EF1-MCS plasmid (available from Alstem, Inc., 2600 Hilltop Avenue, Richmond, California, USA, catalog number LV200). The vector was modified to insert an NheI restriction site into the MCS cloning site of the vector to facilitate the 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.

[0125] Human PBMC populations were obtained and CD4 / CD8 T cells were isolated from PBMC by flow cytometry.CD4 / CD8 T cells were stimulated with CD3.CD28 beads (Miltenyi) for 24 hours.

[0126] Healthy donor primary blood mononuclear cells (PBMCs) were isolated from leukopaks by Ficoll-Paque separation (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 antibodies (clone CD28.2, BD Biosciences).

[0127] 48 hours after stimulation, cells were transduced with lentivirus and maintained in complete OpTmizer T cell medium (Gibco) containing wild-type IL-2 (Miltenyi Biotech). 24-48 hours after transduction, cells were washed and maintained in medium containing WT IL-2, or replaced with medium containing STK-009. The medium was replaced every other day to expand the cells. Cell counts and viability were performed using Vi-Cell XR (Beckman Coulter). On days 7-14, cells were frozen with CryoStor CS10 cell freezing medium (STEMCELL).

[0128] Cells were transduced with lentivirus encoding a nucleic acid sequence encoding GPC3-CAR and an orthogonal CD122 coding sequence separated by a T2A sequence, which was added at an infectious ratio and cultured for one to three days, during which the cells were washed to remove excess lentivirus and resuspended in fresh culture medium.

[0129] The DNA sequences encoding the DR625, DR626 and DR628 constructs were inserted into the multiple cloning site (MCS) of the LV200pLenti-EF1-MCS plasmid (available from Alstem, Inc., 2600 Hilltop Avenue, Richmond, CA, USA, product number LV200 Alstem).

[0130] The orthogonal IL2 molecule used in these studies was a PEGylated hIL2 variant containing the amino acid deletion and substitution desAla1 / E15S-H16Q-L19V-D20L-Q22K-M23A, containing an N-terminal monoPEGylated 40 kDa branched (2x20 kD) PEG molecule and an aldehyde linker. The 40 kDa 2-arm branched PEG-aldehyde containing two 20 kDa linear PEG molecules was GL2-400AL3 (available from NOF America Corporation, 1 North Broadway, White Plains, NY 10601, USA.

[0131] Female NOD scidγ (NSG) mice aged 6 to 8 weeks were obtained from The Jackson Laboratory, 600 Main Street, Bar Harbor, ME USA 04609. Before the start of the study, the animals were weighed and given a clinical examination to ensure that they were in good health. Body weight was then tracked during the study.

[0132] Except as noted below, tumor cells were administered at a 50:50 ratio of tumor cells to phenol red-free Matrigel (Corning).

[0133] For the purpose of the following studies, GPC3 CAR T cells were identified by their expressed GPC3 CAR. B. In vitro evaluation of GPC3 negative; GPC3 low and GPC3 high cell lines

[0134] A series of initial experiments were performed to evaluate the role of different costimulatory domains in GPC3 CAR T cells. A series of experiments were performed to compare the in vitro activity of GPC3 CAR T cells containing CD28 (DR625) and 41BB (DR628) in various cell lines with different GPC3 expression levels and to evaluate the activity

[0135] The effect of the orientation of the co-stimulatory domain and CAR coding sequence on the vector was also evaluated. Figure 1 As shown, in DR625, the CD28 GPC3-CAR sequence is located 5' of the orthogonal CD122 sequence. However, in DR626, the orientation of the CD28 GPC3-CAR and orthogonal CD122 sequences is reversed, such that the orthogonal CD122 sequence is located 5' of the CD28 GPC3-CAR sequence.

[0136] A series of cellular experiments were performed to evaluate the cytotoxicity of orthogonal GPC3 CAR T cells in vitro and in vivo in cell lines with different GPC3 expression levels. The GPC low or negative hepatocellular carcinoma cell line PLC / PRF / 5 (ATCC Accession No. CRL-8024) was obtained.

[0137] The data presented in Figure 2 provide the results of this series of experiments. As shown in Figure 2, orthogonal GPC3 CAR T cells selectively kill GPC3 阳性 cells (Fig. 2 Panels B and C), but did not show significant specific cytotoxicity against GPC-negative (PLC / PRF / 5 cells, Panel A) cells.

[0138] The effect of the orientation of the co-stimulatory domain and CAR coding sequences on the expression vector was also evaluated. Figure 1As shown, in DR625, the CD28 GPC3-CAR sequence is located 5' of the orthogonal CD122 sequence. However, in DR626, the orientation of the CD28GPC3-CAR and orthogonal CD122 sequences is opposite, so that the orthogonal CD122 sequence is located 5' of the CD28GPC3-CAR sequence. The data is provided in Figure 3 of the accompanying drawings. As shown, both DR625 and DR626 orthogonal GPC3 CAR T cells exhibit selective cytotoxicity as a function of the expression level of GPC3 on target cells. The data show that the orientation of the GPC-CAR and orthogonal CD122 coding sequences on the vector has little or no effect on the cytotoxicity of the orthogonal GPC3CAR T cells, because DR625 and DR626 orthogonal GPC3CAR T cells behave similarly in this study.

[0139] To demonstrate that DR625 and DR626 orthogonal GPC3 CAR T cells selectively respond to exposure to orthogonal IL2, a dosing study was performed to compare DR625 and DR626 orthogonal GPC3 CAR T cells to non-transduced T cells. The results of this study are provided in the accompanying Figure Figure 4 Since all cells expressed the wild-type IL2 receptor, all cells responded to wild-type IL2. However, DR625 and DR626 orthogonal GPC3CAR T cells selectively expanded in the presence of the orthogonal IL2 ligand, while non-transduced cells did not respond to exposure to the orthogonal IL2. This data suggests that the orthogonal GPC3 CAR T cells selectively respond to contact with the orthogonal IL2 ligand. B. In vivo evaluation of orthogonal GPC3CAR T cells in tumors with high GPC3 expression (HepG2)

[0140] A series of experiments were performed using HepG2 cells to evaluate the performance of orthotopic GPC3 CAR-T cells in a solid epithelial tumor model of hepatocellular carcinoma xenografts. 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 with a poor prognosis. 1. HepG2 Study A (S2-21-001): Subcutaneous HepG2 Hepatocytes in NSG Mice Using DR625 and DR626 Cancer Model

[0141] HepG2 cells (Wistar Institute) were maintained in exponential growth phase before collection. Cells were collected by trypsinization and suspended in culture medium. Cell concentration and viability were determined using trypan blue (minimum 98% viability). The cell suspension was then adjusted to the concentration required for inoculation.

[0142] On day 0 of the study, approximately one million HepG2 cells in a volume of 100 μL (Matrigel + HepG2 suspension) were subcutaneously inoculated into approximately 10-week-old NSGs by a single injection. TM The injection area was monitored until a tumor was palpable. Tumors were measured using calipers and allowed to progress until the average size of the tumor before treatment was 50-150 mm 3 . Randomly group (sort) into treatment groups according to the table below.

[0143] On day 10 of the study, the CAR T cells shown in the table above were administered intravenously to mice. Additional treatments shown in the table above were administered subcutaneously every other day (qod). For treatment groups 3 and 5, STK-009 doses were interrupted due to weight loss, and a single dose of additional STK-009 was given to group 3 on day 40 of the study and to group 5 on day 33. Tumor measurements were recorded (daily) and mouse weights were recorded (3 times per week). When tumor size limit (maximum 2,000mm 3 ) when the animals were euthanized.

[0144] At the end of the treatment period, animals were sacrificed and necropsied. Tumors were excised, weighed and documented by digital imaging. Various tissues were collected from the sacrificed animals (immersion (Sigma Aldrich), snap frozen, nucleic acids isolated, and tissues prepared for histological analysis by performing a standard gross necropsy.

[0145] Data on tumor volume (efficacy) and body weight (toxicity) in this study are provided in Figure 5, panels A and B, respectively. Figure 6 Spider plots for each treatment group in this study are provided. Figure 7 Body weight spider plots are provided. Tumor volume spider plots demonstrate durable complete responses in mice treated with DR625 orthogonal GPC3 CAR T cells in combination with STK-009.

[0146] Although mice treated with STK-009 and DR625 and DR626 orthogonal GPC3-CAR T cells showed transient weight loss in this study, the animals did recover and the toxicity was reversible. Subsequent studies were conducted in which the STK-009 dosage regimen and dosage level were titrated to avoid toxicity while still maintaining anti-tumor efficacy. It should be noted that the dose of DR625 and DR626 orthogonal GPC3-CAR T cells is particularly high (e.g., 10 μg qod (given every other day)), and the efficacy at lower doses (e.g., 1 μg / week) has been confirmed.

[0147] During the course of this study, blood samples were collected from each treatment group weekly and CD8+T cells were selected by FACS. Cell types were distinguished by CCR7CD45RA status sorted by flow cytometry. The ability of four human CD8+T cell subsets, namely naive cells (CCR7+CD45RA+), central memory cells (TCM, CCR7+CD45RA-), effector memory cells (TEM, CCR7-CD45RA-) and CD45RA+ effector memory cells (TEMRA, CCR7-CD45RA+) to proliferate and differentiate in response to antigens or homeostatic cytokines was compared. 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 presented in the accompanying drawings Figure 8 In. Figure 8 As shown, STK-009 treatment induced a significant increase in the levels of GPC3_28z orthogonal CAR T cells in all memory subtypes. 2. HepG2 Study B (S2-21-009): Subcutaneous HepG2 Hepatocellular Carcinoma Model Using DR625

[0148] Another study was conducted to evaluate the effect of orthogonal GPC3 CAR T cells given in combination with orthogonal IL2 (STK-009), where STK-009 doses were provided less frequently but for a longer duration than in HepG2 Study A described above. This study was conducted essentially as described in HepG2 Study A, with the following modifications. 6 ) compared to lower cell doses (8.5x10 5) with a single dose of orthogonal GPC3 CAR T cells (DR625). In this HepG2 study B, a satellite arm was provided for additional immunohistochemistry analysis. Whole slide scanning and signal quantification were performed using the Akoya Vectra multispectral imaging system and the Akoya VectraInForm analysis software suite. 6 Compared to HepG2 cells, 5x10 6 HepG2G tumor cells. Treatment with orthogonal GPC3 CAR T cells began on day 7. Additional treatments were given every three days from day 7 to day 43. The table below summarizes the design of the study and the various treatment groups.

[0149] The results of this study are summarized as Fig.9A Efficacy (tumor volume) shown, Fig. 9B Toxicity (body weight) and Fig. 9C Activated T cells (CD3+ cells) shown in .

[0150] like Fig.9A As shown, the anti-tumor efficacy in response to the combination of orthogonal GPC3 CAR T cells (DR625) and orthogonal IL2 (STK-009) produced prolonged anti-tumor efficacy. It should be noted that in Group 3, tumors in animals treated with a combination of orthogonal GPC3 CAR T cells (DR625) and orthogonal IL2 (STK-009) did not grow throughout the study until Day 43, indicating that the efficacy of orthogonal GPC3 CAR T cells is durable when given in combination with orthogonal IL2.

[0151] like Fig. 9B As shown, in Group 3, no weight loss was observed in animals treated with orthogonal GPC3 CAR T cells (DR625) in combination with orthogonal IL2 (STK-009), indicating that orthogonal GPC3 CAR T cells are well tolerated in mammalian subjects when given in combination with orthogonal IL2.

[0152] like Fig. 9B As shown, the hCD3+ cells in the blood of Group 3 animals treated with a combination of orthogonal GPC3 CAR T cells (DR625) and orthogonal IL2 (STK-009) showed a specific increase, indicating that GPC3 CART cells expanded in vivo when given in combination with orthogonal IL2.

[0153] Immunohistochemical analysis was performed according to the previously described protocol. Fig.10Results from this analysis are provided, showing that intratumoral apoptosis occurred on day 29 of the study (22 days after the start of treatment with orthogonal GPC3 CAR T cells (DR625) in combination with orthogonal IL2 (STK-009). In this analysis, GPC3 was stained green, CD3 was stained red, and DNA was stained blue. This data shows that orthogonal GPC3 CAR T cells, when given in combination with orthogonal IL2, can cause intratumoral apoptosis of tumor cells in solid tumors.

[0154] Additional immunohistochemical analysis was performed and the results are shown in the accompanying Figures Fig.11 Here, the results of multicolor staining are shown, evaluating the presence of cell nuclei (blue), CD8+ cells (red), granzyme B (green), and CD4 cells (yellow). This data demonstrates that orthogonal GPC3 CAR T cells, when given in combination with orthogonal IL2, result in intratumoral activation of orthogonal GPC3 CAR T cells. B. In vivo evaluation of orthogonal GPC3 CAR T cells in tumors with low GPC3 expression

[0155] A series of studies were performed to evaluate the efficacy of orthogonal GPC3 CAR T cells (DR625) in combination with orthogonal IL2 (STK-009) in treating Huh7 tumors with lower GPC3 expression levels, compared to the above studies with HepG2 tumors with higher GPC3 expression levels. 1. Huh7 Study A (S2-21-017): DR625 in the Huh7 in vivo model

[0156] The Huh7 Study A study was conducted essentially according to the procedures described above in relation to the HepG2 Study A, with certain modifications as described below. 5 After 17 days, 2.5 x 10 Huh-7 cells failed to generate tumors. Therefore, a second subcutaneous implantation of 2.5 x 10 6 cells, triggering tumor growth. The treatment groups and treatments are summarized in the table below:

[0157] 2.5x10 were implanted subcutaneously 6 13 days after the initiation of the Huh7 cell line (study day 13), a single intravenous transfer of orthogonal GPC3CAR T cells (DR625) was performed. STK-009 was administered starting on study day 13 and continued until study day 43. The results of this study are shown in the accompanying figure Fig.12 .

[0158] like Fig.12As shown in the data summarized in , the combination of orthogonal GPC3 CAR T cells (DR625) and orthogonal IL2 (STK-009) failed to provide meaningful tumor control, while toxicity was relatively mild when assessed by tumor burden. Spider plots of individual animals ( Fig.13 ) indicating that some animals in Group 3 responded to treatment. Given the difficulty in obtaining initial tumor growth in this experiment, the study was repeated as described in Huh7 Study B below, with tumor cells given twice, intraperitoneally and subcutaneously. The above study was repeated with the following modifications: (a) Initial implants were 2x10 6 (a) Huh7 cells expressing luciferase were implanted to facilitate imaging; (b) Huh7 cells were implanted without Matrigel; (c) the dose of GPC3 orthogonal CAR T cells was increased to 5x10 6 orthogonal GPC3 CAR T cells; (d) STK-009 doses were given 3 times a week. The data obtained in this study are shown in the attached figure Fig.14 As shown, a significant fraction of mice treated with orthogonal GPC3 CAR T cells in combination with orthogonal IL2 survived. 2. Huh7 Research B S2-21-019: DR625 in the Intraperitoneal Huh-7 In Vivo Model

[0159] This Huh7 study B evaluated the efficacy of intraperitoneal administration of 1x10 6 The effect of Huh7 luciferase cells on NSG mice was as described above. The treatment groups and treatments for this study are summarized in the table below:

[0160] Tumor cell numbers were quantified twice weekly using an IVIS imager (Perkin Elmer). Mice were injected intraperitoneally with 100 ul of D-luciferin (15 mg / ml D-luciferin in PBS). Mice were placed under anesthesia by controlled low-flow isoflurane exposure (Kent Scientific Somnosuite). A region of interest (ROI) was drawn around each mouse and the total flux (photons / second) of luminescence was measured directly using Living Image software (Perkin Elmer). The results of this study are shown in the accompanying drawings Fig.15 .

[0161] Depend on Fig.15 The data shown show that higher CAR-T cell doses (5x10 6 Mice treated with CAR-T cells (2x10 6Mice treated with 50% (n=4) cells showed significant tumor control. Fig.14 The survival data shown in B indicate that the higher CAR-T cell dose (5x10 6 Mice treated with CAR-T cells (2x10 6 Mice treated with 100% CAR-T cells showed improved survival relative to controls (PBS), but less than that observed at higher CAR-T cell doses.

[0162] In summary, the above data on the treatment of Huh7 tumors indicate that orthogonal GPC3 CAR T cells given in combination with orthogonal IL2 are effective in treating tumors expressing low levels of GPC3. C. HepG2 research re-attack research

[0163] As mentioned previously, one of the most challenging aspects of CAR-T therapy is that the initial response to treatment is promising, with high response rates. However, over time, patients relapse, and the cancer recurs even with IL2 supportive therapy. In addition, the persistence of active CAR T cells in existing therapies is poor, and most of the activated CAR T cells administered become ineffective due to exhaustion or other mechanisms.

[0164] To evaluate the potential for treating GPC3+ tumor relapses, animals that were tumor-free at the completion of the above HepG2 study were re-challenged by administration of additional HepG2 tumor cells and treated with orthogonal IL2 alone without administration of orthogonal GPC3 CAR T cells. 1. HepG2 Rechallenge Study A: Rechallenge tumor-free mice from HepG2 Study A with additional tumors and STK-009 alone

[0165] In this study, surviving mice from the treatment group in the above HepG2 study A were re-challenged by administering an additional dose (2x10 6 ) of HepG2 tumor cells and again on day 80. The study design and treatment schedule are summarized in the table below. *Starting from the time of rechallenge implantation (Day 0)

[0166] The data from this experiment are shown in the attached figure Fig.16. From the data presented, it can be seen that the addition of STK-009 alone without any additional CAR-T cells resulted in tumor regression. This data suggests that in subjects who have relapsed and have recurrent tumors that were previously treated with orthogonal GPC3 CAR T cells, they can be successfully treated by administering orthogonal IL2 without the need for additional CAR T cell therapy. This data suggests that GPC3 CAR T cells previously administered in HepG2 Study A are able to be rejuvenated and retain anti-tumor efficacy by administering orthogonal IL2 (STK-009). 2. HepG2 Rechallenge Study B S2-21-012: Rechallenge tumor-free mice from Study HepG2 Study B S2-21-009 with additional tumors and STK-009 alone

[0167] A secondary rechallenge study was performed, the S2-21-012HepG2 rechallenge study was performed, using mice that survived HepG2 study B. In this study, 2.5E6 cells HepG2-luciferase cells were administered subcutaneously in Matrigel to the surviving mice from the previous study. The study design and treatment schedule are summarized in the table below. *STK-009 was administered starting from the day of tumor cell re-implantation (Day 0).

[0168] The data generated by this study are Fig.17 As shown. As can be seen from the data shown, the majority of mice previously cured with orthogonal GPC3CAR T cells were able to suppress tumor growth on rechallenge. Consistent with previous studies, this data suggests that in subjects with relapsed and recurrent tumors previously treated with orthogonal GPC3 CAR T cells, they can be successfully treated by administering orthogonal IL2 without the need for additional CAR T cell therapy. A. GPC3 Chimeric Antigen Receptor or GPC3 CAR:

[0169] The terms "chimeric antigen receptor" and "CAR" are used interchangeably and refer to a chimeric polypeptide comprising multiple functional domains, the sequence of which is arranged in sequence from the amino terminus to the carboxyl terminus: (a) an extracellular domain (ECD) comprising an antigen binding domain (ABD), optionally comprising a "hinge" domain; (b) a transmembrane domain (TM); and (c) one or more cytoplasmic signaling domains (CSD), wherein the above domains may be optionally connected by one or more spacer domains. CAR may further include a signal peptide sequence, which is usually removed during the presentation and post-translational processing of a cell surface CAR of a cell transformed with an expression vector comprising a nucleic acid sequence encoding CAR. CAR can be prepared according to principles well known in the art. See, e.g., Eshhaar 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 Med14(6):405-15; Brogdon, et al. (U.S. Patent No. 10.174,095, granted on January 8, 2019) Guedan, et al. (2019) Engineering and Design of Chimeric Antigen Receptors Molecular Therapy: Methods & Clinical Development Vol. 12: 145-156.

[0170] The present disclosure provides a GPC3 CAR, which is a CAR in which 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) ("anti-hGPC3sdAb") that selectively binds to hGPC3. In some embodiments, the anti-hGPC3sdAb of the present disclosure is a single domain antibody that selectively binds to mature human 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 human GPC3β subunit (SEQ ID NO: 2).

[0171] In some embodiments, the anti-GPC3 sdAb is a scFv (anti-hGPC3 scFv). ScFv is a polypeptide comprising the variable regions of the immunoglobulin heavy and light chains of an antibody 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; Ladner, U.S. Pat. No. 4,946,778, issued on August 7, 1990). In some embodiments, the anti-hGPC3 scFv selectively binds to mature human GPC3 (amino acids 25-580 of SEQ ID NO: 1). In some embodiments, the anti-hGPC3 scFv selectively binds to the human GPC3 β subunit (SEQ ID NO: 2). The preparation of anti-GPC3 antigen ScFv is first carried out by generating monoclonal antibodies against GPC3 or an antigenic fragment thereof. The generation of monoclonal antibodies and the isolation of hybridomas are well known techniques to those skilled in the art. See, for example, Monoclonal Antibodies: A Laboratory Manual, 2nd edition, Chapter 7 (E. Greenfield, ed. 2014, Cold Spring Harbor Press). The generation of anti-hGPC3 monoclonal antibodies is carried out by immunizing an immunocompetent species with hGPC3 polypeptide or an antigenic fragment thereof, including but not limited to mice, rabbits, horses, pigs, camels, llamas, dromedaries or sharks. 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 human Glypican-3 β subunit (SEQ ID NO: 2) or an antigenic fragment thereof. The immune response to the 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.

[0172] Antibodies generated by the immunization process can be optimized by techniques well known in the art (e.g., phage display and directed evolution) to select antibodies with specific desired characteristics. See, e.g., Barbas, et al. (1991) PNAS (USA) 88:7978-82; Ladner, et al. U.S. Pat. No. 5,223,409, issued June 29, 1993; Stemmer, W. (1994) Nature 370:389-91; Garrard U.S. Patent No. 5,821,047, issued October 13, 1998; Camps, et al. (2003) PNAS (USA) 100(17):9727-32; Dulbecco U.S. Patent No. 4,593,002, issued June 3, 1986; McCafferty U.S. Patent No. 6,806,079, issued October 19, 2004; McCafferty, U.S. Patent No. 7,635,666, issued December 22, 2009; McCafferty, U.S. Patent No. 7,662,557, issued February 16, 2010; McCafferty, U.S. Patent No. 7,723,271, issued May 25, 2010; and / or McCafferty U.S. Patent No. 7,732,377.

[0173] As an alternative to immunization with a GPC3 immunogen, an anti-GPC3 scFv can also be generated based on known anti-GPC3 antibody sequences. See, for example, The Protein Protocols Handbook, John M. Walker, ed. (2002) Humana Press, Section 150 "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. A variety of anti-GPC3 antibodies have been described in the literature. The CDRs of such antibodies can also be grafted onto scFv frameworks, particularly human or humanized scFv frameworks, to generate humanized anti-hGPC3 scFvs based on techniques known in the art. The following table summarizes examples of known anti-GPC3 antibodies and their CDRs (based on the Kabat numbering convention) that can be used to prepare anti-GPC3 scFvs:

[0174] Antibodies that selectively bind to GPC3 and GPC3 subunit B are known in the art, including but not limited to GC33 (Nakano, et al., U.S. Patent No. 7,919,086 and Nakano, et al. (2009) Biochem Biophys Res Comm 378(2):279-84), YP7, YP8, YP9, YP9.1, 32A9, which are described in Liu et al. (2020) J Translational Medicine 18, article number 295, published on August 3, 2020, and Chinese patent CN109021108B, authorized on June 25, 2019.

[0175] In one embodiment, the ABD of GPC3 CAR is a scFv comprising CDRs derived from GC33 antibody. In some embodiments, the ABD of GPC3 CAR is a scFv comprising heavy chain CDR1, 2 and 3 and light chain CDR 1, 2 and 3, wherein the heavy chain CDR1, 2 and 3 are independently at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 12, 13 and 115, 16, 17, and the light chain CDR 1, 2 and 3 are independently at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 12, 13 and 14, respectively. In some embodiments, the ABD of GPC3 is a scFv, wherein the heavy chain CDRs 1, 2, and 3 are identical to SEQ ID NOs: 12, 13, and 14, respectively, and wherein the light chain CDRs 1, 2, and 3 are identical to SEQ ID NOs: 15, 16, 17, respectively.

[0176] In some embodiments, the ABD of the GPC3 CAR is a scFv comprising a heavy chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences DYEMH (SEQ ID NO: 12), ALDPKTGDTAYSQKFKG (SEQ ID NO: 13), and FYSYTY (SEQ ID NO: 14), respectively, and a light chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences RSSQSLVHSNANTYLH (SEQ ID NO: 15), KVSNRFS (SEQ ID NO: 16), and SQNTHVPPT (SEQ ID NO: 17).

[0177] As described above, the heavy chain region and light chain region of scFv are connected by a polypeptide linker. In some embodiments, the linker between the heavy chain variable region and the light chain region is a polypeptide linker with 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 amino acids. In some embodiments, the linker between the heavy chain and light chain variable regions of scFv is a polypeptide of 15 amino acids. In some embodiments, the linker between the heavy chain and light chain variable regions of scFv is a polypeptide with an amino acid sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 18), referred to in the art as a Whitlow linker, as described in U.S. Patent No. 5,856,456.

[0178] In some embodiments, the linker is a polypeptide comprising primarily glycine (G) and serine (S) residues, referred to in the art as a "GS" linker. Glycine and glycine-serine polymers are relatively unstructured and can therefore be used as neutral tethers between components. 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, wherein m, n, and o are each independently selected from an integer of at least 1 to 20, e.g., 1-18, 216, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and other flexible linkers. An example of a GS linker that can be used to practice the present disclosure is a polypeptide having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 49), abbreviated herein as "G4Sx3."

[0179] In some embodiments, the ABD of the GPC3 CAR is a scFv comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences DYEMH (SEQ ID NO: 12), ALDPKTGDTAYSQKFKG (SEQ ID NO: 13), and FYSYTY (SEQ ID NO: 14), respectively, and the light chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences RSSQSLVHSNANTYLH (SEQ ID NO: 15), KVSNRFS (SEQ ID NO: 16), and SQNTHVPPT (SEQ ID NO: 17), wherein the heavy chain and light chain variable regions are connected by a polypeptide linker having the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 33).

[0180] In some embodiments, the ABD of GPC3 CAR is a scFv comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences DYEMH (SEQ ID NO: 12), ALDPKTGDTAYSQKFKG (SEQ ID NO: 13), and FYSYTY (SEQ ID NO: 14), respectively, and the light chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences RSSQSLVHSNANTYLH (SEQ ID NO: 15), KVSNRFS (SEQ ID NO: 16), and SQNTHVPPT (SEQ ID NO: 17), wherein the heavy chain and light chain variable regions are connected by a polypeptide linker having the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 18), and the scFv has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGSTSGSGKPGSG EGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID NO:11).

[0181] In some embodiments, the anti-GPC3 sdAb is a scFv comprising a heavy chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences DYEMH (SEQ ID NO: 12), ALDPKTGDTAYSQKFKG (SEQ ID NO: 13), and FYSYTY (SEQ ID NO: 14), respectively, and a light chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences RSSQSLVHSNANTYLH (SEQ ID NO: 15), KVSNRFS (SEQ ID NO: 16), and SQNTHVPPT (SEQ ID NO: 17), wherein the heavy and light chain variable regions are connected by a polypeptide linker having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 49). In some embodiments, the anti-GPC3 sdAb is a scFv comprising a heavy chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences DYEMH (SEQ ID NO: 12), ALDPKTGDTAYSQKFKG (SEQ ID NO: 19), and FYSYTY (SEQ ID NO: 14), respectively, and a light chain variable region having CDRs 1, 2, and 3 comprising the amino acid sequences RSSQSLVHSNANTYLH (SEQ ID NO: 15), KVSNRFS (SEQ ID NO: 16), and SQNTHVPPT (SEQ ID NO: 17), wherein the heavy chain and light chain variable regions are linked by a CDR having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 18). NO:49), wherein the scFv has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the following amino acid sequence: DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGGSGGGGS GGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID NO:10) Signal peptide (SP):

[0182] In order to promote the surface expression of GPC3 CAR, the nucleic acid sequence encoding GPC3 CAR can also encode a signal peptide, which promotes the surface expression of GPC3 CAR when expressed in mammalian cells and is cleaved from the polypeptide after translation. The signal peptide can be derived from a surface-expressed protein or a synthetic naturally occurring signal peptide. In one embodiment of the present invention, wherein the signal peptide is derived from a naturally occurring signal peptide of a surface-expressed protein, the signal peptide of GPC3 CAR is a signal peptide selected from the group consisting of human CD8a signal peptide, human serum albumin (HSA) signal peptide, prolactin albumin signal peptide, human IL2 signal peptide, human trypsinogen-2, human CD-5 signal peptide, human immunoglobulin kappa light chain signal peptide and its functional derivatives. The use of signal peptides in naturally occurring signal peptide sequences to increase the specific amino acid substitution of secretion efficiency 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. In an alternative scheme using a signal peptide derived from a naturally occurring signal peptide, the signal peptide can 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, GPC3CAR comprises a human CD8a signal peptide having an amino acid sequence of MALPVTALLLPLALLLHAARP (SEQ ID NO: 20).

[0183] 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). VHH is a heavy chain domain fragment of an antibody produced by camelid mammals (such as camels, llamas, dromedaries, alpacas, and llamas), and naturally lacks a light chain. A typical VHH has a molecular weight of about 12-15 kDa, which is much smaller than a traditional mammalian IgG class antibody (150-160 kDa) consisting of two heavy chains and two light chains. Similar to more traditional IgG class antibodies produced by other mammalian species, camel antibodies selectively target specific antigens. Anti-GPC3 camel antibodies, wherein anti-GPC3 VHH 1 is similarly produced by immunizing camelid animals, and immune-competent animals are immunized with hGPC3 polypeptides or antigenic fragments thereof. In some embodiments, the anti-GPC3 VHH binds to mature human GPC3 (amino acids 25-580 of SEQ ID NO: 1), which is derived from a camelid antibody obtained by immunizing a camelid 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 to human GPC3 β subunit (SEQ ID NO: 2), which is derived from a camelid antibody obtained by immunizing a camelid with a polypeptide sequence comprising amino acids 25-580 of SEQ ID NO: 1.

[0184] The GPC3 CAR of the present disclosure also provides a transmembrane domain, which connects the ABD (optionally including a polypeptide linker) to the intracellular domain (ICD) of the GPC3 CAR. The transmembrane domain is composed of any thermodynamically stable sequence in the eukaryotic cell membrane. The transmembrane domain used to construct the GPC3 CAR that can be used to practice the present invention includes about 20 amino acids that are conducive to the formation of an alpha helical secondary structure. The transmembrane domain can be derived from the transmembrane domain of a naturally occurring transmembrane protein. Alternatively, the transmembrane domain can be synthetic. When designing a synthetic transmembrane domain, it is preferred to use amino acids that are conducive to alpha-helical structure. Amino acids that are conducive to alpha-helical formation are well known in the art. See, for example, Pace, et al. (1998) Biophysical Journal 75: 422-427. In some embodiments, the CAR transmembrane domain may be derived from the transmembrane domain of a type I transmembrane protein, including but not limited to CD3ζ, CD4, CD8, CD28. In some embodiments, the transmembrane domain is the human CD28 transmembrane domain corresponding to amino acids 132-157 of the human CD28 precursor protein, numbered according to human CD28 precursor protein UniProt P10747 including the signal peptide.

[0185] The cytoplasmic domain of GPC3 CAR includes one or more intracellular signaling domains. In one embodiment, the intracellular signaling domain includes a cytoplasmic sequence of a T cell receptor (TCR) and a co-receptor that initiates signal transduction after antigen receptor engagement, as well as functional derivatives and subfragments thereof. Cytoplasmic signaling domains, such as those from T cell receptor ζ-chain signaling domains, are used as part of CAR to generate signals that stimulate T lymphocyte proliferation and effector function after chimeric receptors engage with target antigens. 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 known as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also known as ICOS), the p110α, β or δ catalytic subunit of PI3 kinase, the human CD3ζ-chain, the cytoplasmic domain of CD134 (also known as OX40 and TNFRSF4), FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (δ, Δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28.

[0186] In some embodiments, GPC3 CAR may also provide a costimulatory domain. The term "costimulatory domain" refers to the stimulatory domain of CAR, typically an intracellular domain (endodomain), which provides a secondary nonspecific activation mechanism through which a primary specific stimulus is propagated. The costimulatory domain refers to a portion of CAR that can enhance the proliferation, survival or development of memory cells. Examples of costimulation include antigen-nonspecific T cell costimulation after antigen-specific signaling through a T cell receptor, and antigen-nonspecific B cell costimulation after signaling through a B cell receptor. Costimulation, such as T cell costimulation, and the factors involved have been described in Chen and Flies. (2013) Nat Rev Immunol 13 (4): 227-42. In some embodiments of the invention, the CSD comprises one or more TNFR superfamily members, 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.

[0187] Those of ordinary skill will be aware of other costimulatory domains that can be used in conjunction with the teachings of the present disclosure.

[0188] 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 CD3ζ.

[0189] In one embodiment of the present invention, the ICD of GPC3-CAR comprises the cytoplasmic domain of 4-1BB and a portion of the intracellular signaling domain of CD3ζ.

[0190] In another embodiment, the ICD of GPC3-CAR comprises the signaling domain of CD3-ζ and the signaling domains of CD28 and CD137.

[0191] The GPC3 CAR of the present disclosure may optionally include one or more polypeptide spacers connecting the functional domains of CAR, in particular the connection between GPC3-ABD and the transmembrane domain of CAR. Although not an essential element of the CAR structure, it is generally believed that the inclusion of a spacer domain to promote antigen recognition of ABD is necessary. Moritz and Groner (1995) Gene Therapy 2 (8) 539-546. No specific amino acid sequence is required to achieve the function of the spacer, but the typical characteristic of the spacer is flexibility to allow the ABD to move freely to promote targeted antigen recognition. Similarly, it has been found that while retaining the function of CAR, the length of the spacer is also quite loose. Jensen and Riddell (2014) Immunol. Review 257 (1) 127-144. Sequences used as spacers in the construction of CARs useful in the practice of the present invention include, but are not limited to, the hinge region of IgG1, immunoglobulin 1 CH2-CH3 region, IgG4 hinge-CH2-CH3, IgG4 hinge-CH3 and IgG4 hinge. The hinge and transmembrane domain may be from the same molecule, such as the hinge and transmembrane domain of CD8-α. Imai, et al. (2004) Leukemia 18(4):676-684.

[0192] The present disclosure provides GPC3 CARs that can be used to prepare orthogonal GPC3 CAR T cells. In one embodiment, the GPC3 CAR comprises an hCD8a signal peptide amino acid sequence (italics), a GC33 scFv with a Whitlow linker as an antigen binding domain (Whitlow linker underlined), a polypeptide sequence with a small sequence of the hCD28 extracellular domain, a transmembrane and co-stimulatory domain, and a human CD3ζ domain, and the GPC3 CAR has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the following amino acid sequence: MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKR GSTSGSGKPGSG EGSTKG QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0193] In one embodiment, the GPC3 CAR comprises an hCD8a signal peptide amino acid sequence (italics), a GC33 scFv with a G4Sx3 linker as an antigen binding domain (the G4Sx3 linker is underlined), a polypeptide sequence having a small sequence of the hCD28 extracellular domain, a transmembrane and co-stimulatory domain, and a human CD3ζ domain, and the GPC3 CAR has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the following amino acid sequence: MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKR GGGGSGGGGSGG GGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0194] In one embodiment, the GPC3 CAR comprises the amino acid sequence of the hCD8a signal peptide (italics), a GC33 scFv with a Whitlow linker as an antigen binding domain (Whitlow linker is underlined), an hCD8a hinge and transmembrane domain, a human 4-1BB co-stimulatory domain, and has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the following amino acid sequence: MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKR GSTSGSGKPGSG EGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG TCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0195] In one embodiment, the GPC3 CAR comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence:

[0196] In one embodiment, the GPC3 CAR comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence:

[0197] In one embodiment, the GPC3 CAR comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPT FGQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVY YCTRFYSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:39)

[0198] In one embodiment, the GPC3 CAR comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following amino acid sequence: B. Orthogonal CD122 receptor

[0199] The second component of orthogonal GPC3 CAR T cells is an orthogonal CD122 receptor. The terms "orthogonal CD122 receptor" or "orthogonal CD122" and "oCD122" and "hoRb" are used interchangeably, meaning that the extracellular domain thereof includes the extracellular domain of human CD122 (hCD122) and the intracellular domain is capable of signaling in mammalian immune cells Transmembrane polypeptides, which include one or more amino acid substitutions (numbered according to mature human CD122 polypeptides) at positions H133 and Y134. In some embodiments, the amino acid substitutions at positions H133 and Y134 are H133D and Y134F.

[0200] In some embodiments, orthogonal CD122 is a human CD122 mutant protein, which comprises amino acid substitutions at positions H133 and Y134 according to the numbering of mature human CD122 polypeptides. In some embodiments, orthogonal CD122 is a human CD122 mutant protein, which comprises amino acid substitutions at positions H133D and Y134F according to the numbering of mature human CD122 polypeptides. In some embodiments, the orthogonal IL2 receptor has the following amino acid sequence:

[0201] The term "orthogonal CD122 receptor" or "orthogonal CD122" includes chimeric orthogonal receptors in which the intracellular domain of the naturally occurring CD122 is replaced by a heterologous intracellular signaling domain. As described in PCT International Publication No. WO 2021 / 050752 by Garcia et al., the intracellular domain of the orthogonal receptor may include a signaling domain of the receptor other than the CD122 intracellular domain. Orthogonal receptors comprising intracellular sequences heterologous to the CD122 ECD are referred to as chimeric orthogonal receptors. In one embodiment, when the transmembrane receptor comprises an orthogonal CD122 ECD and a CD122 ICD, binding of an orthogonal IL2 to this receptor produces an intracellular signal characteristic of activation of the CD25 / CD122 / CD132 high affinity IL2 receptor with CD122 / CD132 medium affinity. In an alternative embodiment, for a chimeric orthogonal receptor, binding of an orthogonal IL2 to the ECD of the chimeric orthogonal receptor results in intracellular signaling that is unique to the intracellular domain. For example, when the chimeric orthogonal receptor comprises a hoCD122 ECD and the intracellular domain is derived from an IL9 receptor, the intracellular signal induced by binding of hoIL2 to the chimeric receptor results in intracellular signaling specific to the IL9 receptor. Examples of orthogonal IL2 molecules are described in: Garcia, et al. (U.S. Patent Application Publication US2018 / 0228842A1, published on August 16, 2018) and Garcia, et al. provide certain modified IL2 polypeptides. U.S. Patent No. 10,869,887, issued on December 22, 2020, all teachings of which are incorporated herein by reference.

[0202] In one embodiment, orthogonal CD122 is a human CD122 comprising amino acid modifications at positions 133 and 134 according to the numbering of the mature human CD122 (SEQ ID NO: 4) in a naturally occurring form. In some embodiments, orthogonal CD122 is a hCD122 molecule with amino acid substitutions H133D and Y134. In one embodiment, an orthogonal receptor is a modified human CD122 having an amino acid sequence (minus a signal peptide) having an amino acid sequence of the ECD of hCD122 with substitutions H133D and Y134F and a transmembrane (TM) and intracellular domain (ICD) of a wild-type hCD122 molecule, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the following amino acid sequence: (SEQ ID NO:6)

[0203] In some embodiments, the ICD of orthogonal CD122 contains one or more STAT3 binding motifs in addition to the natural STAT5 recognition motif. The additional STAT3 binding motifs enhance signal transduction and stabilize the IL2 response. After being phosphorylated at the conserved tyrosine residue at the C-terminus, STAT proteins act as transcriptional activators and then translocate to the nucleus, where they bind to DNA and activate transcription of the target gene. Hennighausen and Robinson (2008) Genes Dev. 2008; 22: 711–21. In the STAT family, seven STAT proteins have been identified: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, which have functions in a variety of pathways from innate and acquired immunity to cell proliferation, differentiation, and survival. Basham et al., (2008) Nucleic Acids Res. 2008 Jun; 36(11): 3802–3818. STAT binding motifs are often found in cytokine receptors, where they bind to cytokine ligands to activate tyrosine kinases of the Janus kinase (JAK) family, which phosphorylate certain tyrosine residues in the intracellular domain. The phosphorylated receptor recruits STAT to the STAT recognition motif on the receptor and the STAT becomes phosphorylated. The phosphorylated STAT dimerizes and translocates to the nucleus, where they initiate transcription of various genes. Hennighausen, supra.

[0204] In some embodiments, the modified orthogonal CD122 may include one, two, three or more additional STAT3 binding motifs. In some embodiments, the STAT3 recognition motif has an amino acid sequence 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. CAR-T cell expression vector

[0205] Preparation of orthogonal GPC CAR T cells useful in practicing the present invention is accomplished by transforming isolated T cells with an expression vector comprising: (a) a nucleic acid sequence encoding the above-described GPC CAR and (b) a nucleic acid sequence encoding an orthogonal receptor.

[0206] The expression vector used to express CAR in T cells can be a viral vector or a non-viral vector. The term "non-viral vector" refers to an autonomously replicating extrachromosomal circular DNA molecule that, unlike a normal genome, is not necessary for cell survival under non-selective conditions that enable expression of coding sequences in target cells. Plasmids are examples of non-viral vectors. In order to promote transfection of target cells, target cells can be directly exposed to non-viral vectors under conditions that are conducive to the uptake of non-viral vectors. Examples of conditions that promote the uptake of exogenous nucleic acids by mammalian cells are well known in the art, and include but are not limited to chemical means (e.g. Thermo-Fisher Scientific), high salt, magnetic field (electroporation).

[0207] In some embodiments exemplified herein, the expression vector is a viral vector. As used herein, the term viral vector is used with its conventional meaning to refer to any obligate intracellular parasite that does not have protein synthesis or energy production mechanisms, generally referring to any enveloped or non-enveloped animal virus that is commonly used to deliver exogenous transgenes to mammalian cells. The viral vector can be a replication-competent type (e.g., essentially wild type), a conditional replication type (recombinant engineering to replicate under certain conditions) or a replication-deficient type (basically unable to replicate in the absence of a cell line that can complement the missing function of the virus). The viral vector can have certain modifications to make it "selectively replicated", i.e., it preferentially replicates in certain cell types or phenotypic cell states, such as cancer. Useful viral vector systems in the practice of the present invention include, for example, naturally occurring or recombinant viral vector systems. Examples of viral vectors suitable for use in the practice of the present disclosure include recombinant modified enveloped 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 retroviruses (including but not limited to Rous sarcoma virus) and hepatitis B virus.

[0208] Typically, a gene of interest is inserted into such a vector to allow packaging of a gene construct, usually accompanied by a viral genomic sequence, and then infects a sensitive host cell, resulting in expression of a gene of interest (eg, a targeted antigen). In addition, the expression vector encoding GPC CAR may also be an mRNA vector. When transfection is performed using a viral vector system, a retrovirus or lentiviral expression vector is preferably used to transfect T cells, because these systems can enhance the efficiency of gene transfer to T cells, thereby reducing the time for culturing a large number of T cells for clinical applications. In particular, γ 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 because they have been shown to be integrated into resting T cells. June, et al. (2009) Nat Rev Immunol 9 (10): 704-716.

[0209] In addition to the targeting antigen, the expression vector can also encode one or more polypeptides. In the practice of the present invention, when expressing multiple polypeptides, each polypeptide can be operably linked to an expression control sequence (monocistronic), or multiple polypeptides can be encoded by a polycistronic construct, wherein multiple polypeptides are expressed under the control of an expression control sequence.

[0210] Instead of using multiple expression cassettes, the nucleic acid sequences encoding the CAR and IL-10 polypeptides can be encoded by a multicistronic construct, which includes the nucleic acid sequence GPC3 CAR and the orthogonal receptor polypeptide, using an internal ribosome entry site (IRES) element or foot-and-mouth disease virus protein 2A (FMVD2A) to promote co-expression in the target cell. In one embodiment of the present disclosure, the nucleic acid sequence that promotes co-expression includes the nucleic acid sequence: GGGAGTGGAGAGGGCCGCGGATCACTTCTCACATGCGGCGACGTAGAGGAAAATCCTGGCCCC (SEQ ID NO: 41) Also referred to herein as T2A.

[0211] In one embodiment, the expression vector encoding GPC3 CAR may optionally further encode one or more immunomodulators. Examples of immunomodulators 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 capable of regulating immune responses. The expressed cytokines may be directed to intracellular expression or expressed as extracellular presentation or secretion with a signal sequence. It is reported that IL-12 leads to enhanced anti-tumor efficacy (see Yeku, et al. Scientific Reports Vol. 7, Article No.: 10541 (2017) published online on September 5, 2017.

[0212] In one embodiment, the expression vector encoding GPC3 CAR may optionally provide an additional expression cassette comprising a nucleic acid sequence encoding a "rescue" gene. A "rescue gene" is a nucleic acid sequence whose expression in a cell makes the cell susceptible to killing by external factors, or causes a toxic condition in the cell, causing the cell to be killed. Providing a rescue gene can achieve selective cell killing of transduced cells. Therefore, when the construct is incorporated into the cells of a mammalian subject, the rescue gene provides additional safety precautions to prevent the influence of the unwanted spread or replication ability vector system of the transduced cell. In one embodiment, the rescue gene is a thymidine kinase (TK) gene (see, for example, Woo, et al. U.S. Patent No. 5,631,236, issued on May 20, 1997, and Freeman, et al. U.S. Patent No. 5,601,818, issued on February 11, 1997), wherein cells expressing the TK gene product are susceptible to selective killing by administration of gancyclovir.

[0213] The expression vector may also include one or more selectable marker genes. Preparation of orthogonal GPC3 T cells

[0214] The present disclosure provides a method for preparing orthogonal GPC3 CAR T cells, the method comprising the following steps: a. obtaining a population of T cells to be engineered; 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, wherein each nucleic acid sequence is operably linked to an expression control sequence operable 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 affect T cell proliferation. a. Obtaining the T cell population to be engineered

[0215] T cells that can be used to prepare orthogonal GPC3-CAR T cell populations include naive T cells, central memory T cells, effector memory T cells, or a combination thereof. T cells can be obtained from the mammal to be treated, or various T cell lines available in the art. T cells used for transformation are usually obtained from the mammal to be treated. T cells can be obtained from many sources of mammalian subjects, 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 drawing blood. In another embodiment, T cells are isolated from peripheral blood, and specific T cells (e.g., CD3 + 、CD28+ 、CD4 + 、CD8 + 、CD45RA + and CD45RO + T cells) can be isolated by selection techniques well known in the art, such as incubation with anti-CD3 / anti-CD28 coupled beads.

[0216] In some embodiments, the T cells used to prepare orthogonal GPC3-CAR T cells are modified to remove endogenous TCRRa and TCRb functions, which are referred to as allogeneic T cells, and orthogonal GPC3-CART 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 orthogonal GPC3-CAR T cells are obtained from a subject, and the orthogonal GPC3-CAR T cells derived from the subject are to be administered to the subject. In this case, the orthogonal GPC3-CAR T cells are referred to as autologous orthogonal GPC3-CAR T cells. The above-mentioned T cells for engineering are collected from a subject or donor, can be separated from a cell mixture by a technique for enriching the desired cells, or can be engineered and cultured without separation. b. Enrichment of one or more T cell subtypes;

[0217] In some embodiments, before transforming cells with nucleic acid sequences encoding GPC3-CAR and orthogonal receptors, the isolated T cell population is enriched with certain T cell subtypes. A variety of techniques are known in the art for enriching specific T cell subtypes for mixed T cell populations. Technologies providing precise separation include fluorescence-activated cell sorters. By using dyes associated with dead cells (such as propidium iodide), dead cells can be selected for cells. The isolated cells can be collected in any appropriate culture medium to maintain the viability of the cells, usually with a serum pad at the bottom of the collection tube. A variety of culture media are commercially available and can be used according to the properties of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's culture medium, etc., usually supplemented with fetal calf serum (FCS). The collected and optionally enriched cell populations can be used immediately for genetic modification, or can be frozen and stored at liquid nitrogen temperature and can be reused after thawing. Cells are usually stored in 10% DMSO, 50% FCS, 40% RPMI 1640 culture medium. Orthogonal IL2 can be used to selectively expand such orthogonal GPC3-CAR T cells in vitro or in vivo. c. Transformation of T cells with nucleic acid sequences encoding GPC3 CAR and orthogonal receptors:

[0218] Transform the selected T cell population with an expression vector encoding GPC3-CAR, basically according to the teachings below. d. Will include The mixed population of orthogonal GPC3 CAR T cells is contacted with one or more agents that affect T cell proliferation.

[0219] Following transformation, T cells may be activated and expanded, generally using methods such as those described in U.S. Patents 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. Typically, the T cells of the present invention are expanded by culturing cells, which are contacted with the surface of an agent that stimulates CD3 TCR complex-related signals (e.g., anti-CD3 antibodies) and an agent that stimulates T cell surface co-stimulatory molecules (e.g., anti-CD28 antibodies). Suitable conditions for T cell culture are well known in the art. Lin, et al. (2009) Cytotherapy 11(7):912-922 (Optimization and validation of arobust 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 (“Exvivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement”). During the expansion phase, the T cell population is maintained under conditions sufficient to support cell proliferation, e.g., an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0220] It will be clear to those skilled in the art that the aforementioned step of contacting the cell population with one or more vectors encoding GPC3-CAR and orthogonal ligands will not result in a 100% transduction efficiency, so the resulting cell population is still a mixed cell population containing orthogonal GPC3-CAR T cells and non-transformed T cells. Since it is desirable that the cell population for administration to the subject be as enriched in orthogonal GPC3-CAR T cells as possible, in some embodiments, during the expansion phase, the cell population is contacted with orthogonal IL2. Contacting a mixed cell population consisting of orthogonal GPC3-CAR T cells and non-transformed T cells with orthogonal IL2 results in selective activation and proliferation of orthogonal GPC3-CAR T cells, so that the resulting cell product is significantly enriched in orthogonal GPC3-CAR T cells.

[0221] In some embodiments, the orthogonal IL2 used to selectively expand GPC3-CAR T cells in a mixed cell population is a human IL2 mutein comprising a set of amino acid substitutions: E15S / H16Q / L19V / D20L / Q22K / M23A, which is numbered according to mature wild-type human IL2.

[0222] In some embodiments, the orthogonal IL2 used to selectively expand GPC3-CAR T cells in a mixed cell population is STK-007.

[0223] In some embodiments, the orthogonal IL2 used to selectively expand GPC3-CAR T cells in a mixed cell population is STK-009.

[0224] In an alternative method of transducing separated T cells with a recombinant vector encoding GPC3 CAR and orthogonal CD122 sequences, the nucleic acid sequence encoding signal peptide-GPC3 CAR-T2A-orthogonal CD122 polypeptides can be inserted into the genome of the T cell genome. In some embodiments, the present invention provides orthogonal GPC3 CAR T cells, wherein the nucleic acid sequence encoding GPC3 CAR and orthogonal CD122 is inserted into the genome of mammalian T cells. Homologous recombination has been shown to promote site-specific integration of large transgenics in T cell genomes (Schumann. et al. use Cas9 ribonucleoproteins to produce knock-in primary human T cells (Generation of knock-in primary human Tcells using Cas9 ribonucleoproteins.) Proc Natl Acad Sci USA. (2015) 112: 10437–42. doi: 10.1073 / pnas.1512503112). In this method, after cleavage of the target gene DNA using Cas9 RNP, the gene of interest is delivered to the cleavage site using adeno-associated virus (AAV). Site-specific transgene integration is achieved through HDR. Using the combined action of Cas9 / RNP and AAV donor vectors, the anti-CD19 CAR gene has been successfully integrated into the TRAC locus (Eyquem, et al. Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumor rejection, Nature. (2017) 543:113–7). Targeting the nucleic acid sequence encoding the signal peptide-GPC3CAR-T2A-orthogonal CD122 polypeptide to the TRAC locus not only results in uniform expression of the GPC3 CAR and orthogonal CD122 polypeptides, but also reportedly provides resistance to CAR T cell exhaustion. In addition, inserting the GPC3 CAR and orthogonal CD122 sequences into a defined location within the host cell genome avoids the risk of inducing oncogenesis and places the GPC3 CAR and orthogonal CD122 expression under the control of endogenous regulatory elements.

[0225] The genome insertion of the nucleic acid sequence encoding GPC3 CAR and orthogonal CD122 polypeptides can be realized by the sequence-specific endonuclease of the nucleic acid molecule of recognition and cutting specific "target" sequence. Endonucleases are generally classified according to the degree of specificity and sequence identity characteristics of the target sequence. When this endonuclease has a polynucleotide recognition site with a length greater than about 12 base pairs (bp) (more preferably 14-55bp), some endonucleases are referred to as "rare cutting" endonucleases. Rare cutting endonucleases can be used for inactivating genes at the locus or integrating transgenes by homologous recombination (HR), i.e., by inducing DNA double-strand breaks (DSBs) at the locus and inserting foreign DNA at the 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) generated by engineered zinc finger domain fusions (Porteus M and Carroll D., Gene targeting using zinc finger nucleases (2005) Nature Biotechnology 23 (3): 967-973, TALE-nuclease, Cas9 nuclease from CRISPR system as or modified restriction endonuclease to expand sequence specificity (Eisenschmidt, et al. 2005; 33 (22): 7039–7047). In some embodiments of the present invention, immune cells (e.g., CAR-T of orthogonal receptor ECD of expression 1) are modified to reduce alloreactivity by inactivating more than one component of T cell receptor (TCR). Such modification methods of T cells are described in Galetto et al. U.S. Patent Application Publication No. US2013 / 015884A1 published on November 28, 2013, and in Galetto et al. U.S. Patent No. 10,426,795B2 issued on October 21, 2019, a method of restoring TCRα-deficient T cells by expressing pTα to restore functional CD3 complex is described, and its teachings are incorporated herein by reference. Orthogonal IL2:

[0226] As used herein, the term "orthogonal IL2" or "oIL2" refers to an IL2 mutant protein derived from an IL2 parent polypeptide, wherein the orthogonal IL2 specifically binds to the extracellular domain of an orthogonal CD122 receptor, and exhibits a significantly reduced binding to the extracellular domain of wild-type CD122. In some embodiments, the orthogonal IL2 exhibits specific binding to a receptor comprising an orthogonal CD122ECD, and (2) contacting a cell expressing a transmembrane receptor comprising an ECD of an orthogonal CD122 polypeptide, the contact being implemented in an amount sufficient to cause a response, resulting in a signal characteristic of a signal produced by the intracellular domain (ICD) of the transmembrane receptor.

[0227] As described in PCT International Publication No. WO2021 / 050752 by Garcia et al., the intracellular domain of an orthogonal receptor may include a signaling domain of a receptor other than the CD122 intracellular domain. An orthogonal receptor comprising an intracellular sequence heterologous to the CD122 ECD is referred to as a chimeric orthogonal receptor. In one embodiment, when a transmembrane receptor comprises an orthogonal CD122 ECD and a CD122 ICD, an orthogonal IL2 binds to this receptor to produce an intracellular signal characteristic of the activation of the CD25 / CD122 / CD132 high affinity IL2 receptor of the CD122 / CD132 medium affinity IL2 receptor. In an alternative embodiment, for a chimeric orthogonal receptor, the binding of an orthogonal IL2 to the ECD of a chimeric orthogonal receptor results in intracellular signaling specific to the intracellular domain. For example, when a chimeric orthogonal receptor comprises a hoCD122 ECD and the intracellular domain is derived from an IL9 receptor, the intracellular signal induced by the binding of hoIL2 to the chimeric receptor results in intracellular signaling specific to the IL9 receptor. Examples of orthogonal IL2 molecules are described in: Garcia, et al. (U.S. Patent Application Publication No. US2018 / 0228842A1, published on August 16, 2018) and Garcia, et al. Certain modified IL2 polypeptides are provided in U.S. Patent No. 10,869,887, issued on December 22, 2020, the entire teachings of which are incorporated herein by reference.

[0228] In some embodiments, the amino acid sequence of the orthogonal hIL2 has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to any of the polypeptides having the following amino acid sequences: (AA1)–(AA2)-(AA3)-(AA4)-(AA5)-(AA6)-(AA7)-(AA8)-(AA9) i-T10-Q11-L12-(AA13)-(AA14)-(AA15)-(AA16)-L17-(AA18)-(AA19)-(AA20)-L21-(AA22)- (AA23)-I24-L25-N26-(AA27)-I28-N29-N30-Y31-K32-N33-P34-K35-L36-T37-(AA38)-(AA3 9)-L40-T41-(AA42)-K43-F44-Y45-M46-P47-K48-K49-A50-(AA51)-E52-L53-K54-(AA55)-L 56-Q57-C58-L59-E60-E61-E62-L63-K64-P65-L66-E67-E68-V69-L70-N71-L72-A73-(AA74)- S75-K76-N77-F78-H79-(AA80-(AA81)-P82-R83-D84-(AA85)-(AA86)-S87-N88-(AA89)-N90 -(AA91)-(AA92)-V93-L94-E95-L96-(AA97)-G98-S99-E100-T101-T102-F103-(AA104)-C105 -E106-Y107-A108-(AA109)-E110-T111-A112-(AA113)-I114-V115-E116-F117-L118-N119- R120-W121-I122-T123-F124-(AA125)-(AA126)-S127-I128-I129-(AA130)-T131-L132-T133 in: AA1 is A (wild type) or missing; AA2 is P (wild type) or missing; AA3 is T (wild type), C, A, G, Q, E, N, D, R, K, P, or deleted; AA4 is S (wild type) or missing; AA5 is S (wild type) or missing; AA6 is S (wild type) or missing; AA7 is T (wild type) or deleted; AA8 is K (wild type) or missing; AA9 is K (wild type) or deleted; AA13 was Q (wild type), W, or missing; AA14 was L (wild type), M, W, or missing; AA15 is E (wild type), K, D, T, A, S, Q, H, or deleted; AA16 is H (wild type), N or 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, SML, 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 missing; 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 T (wild type), I or missing AA55 is H (wild type) or Y; AA74 is Q (wild type), N, H, S; AA80 is L (wild type), F, or V; AA81 is R (wild type), I, D, Y, T, or missing AA85 is L (wild type) or V; AA86 is I (wild type) or V; AA88 is N (wild type), E or 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.

[0229] In one embodiment, the orthogonal IL2 is a Human orthogonal IL2 with E15S / H16Q / L19V / D20L / Q22K / M23A, numbering is based on mature human IL2 (SEQ ID NO: 7).

[0230] In one embodiment, the orthogonal IL2 is a desAla1 / E15S-H16Q-L19V-D20L-Q22K-M23A] human orthogonal IL2. In one embodiment, the orthogonal IL2 is a polypeptide comprising the following amino acid sequence: PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:9)

[0231] Also known as STK-007.

[0232] In some embodiments, the present invention provides human orthogonal IL2 to facilitate recombinant expression in bacterial cells by eliminating the unpaired cysteine ​​residue at position 125 and / or eliminating the N-terminal Met of the directly expressed IL2 polypeptide and eliminating the alanine at position 1 by post-translational processing by endogenous bacterial proteases. When an amino acid is missing, it is referred to as "des". In some embodiments, the cysteine ​​at position 125 is substituted with alanine or serine (C125A or C125S). This mutation is often used to avoid misfolding of the protein when recombinantly expressed in bacteria and isolated from inclusion bodies.

[0233] In some embodiments, human orthogonal IL2 comprises one or more mutations in the position of the hIL2 sequence, which or contacts hCD122 or changes the orientation of other positions of contacting CD122, resulting in orthogonal IL2 having an increased affinity for CD122. The IL2 residues identified as participating in IL2 binding to CD122 include L12, Q13, H16, L19, D20, M23, Q74, L80, R81, D84, L85, I86, S87, N88, I89V91, I92 and E95. In some embodiments, orthogonal IL2 comprises 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, the orthogonal IL2 comprises one or more of the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F. In some embodiments, the orthogonal IL2 comprises one or more of the following amino acid substitutions: N74Q, L80F, R81D, L85V, I86V, I89V, and I92F. In some embodiments, the orthogonal IL2 comprises one or more of the following amino acid substitutions: Q74N, L80V, R81T, L85V, I86V, and I92F. In some embodiments, the orthogonal IL2 comprises one or more of the following amino acid substitutions: Q74H, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the orthogonal IL2 comprises one or more of the following amino acid substitutions: Q74S, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the orthogonal IL2 comprises one or more of the following amino acid substitutions: Q74N, L80F, R81D, L85V, I86V and I92F. In some embodiments, the orthogonal IL2 comprises one or more of the following amino acid substitutions: Q74S, R81T, L85V and I92F. In some embodiments, the orthogonal IL2 comprises [L80F-R81D-L85V-I86V-I92F]. In some embodiments, the orthogonal IL2 comprises the substitution L85V identified as increasing the affinity of IL2 for CD122. Modifications that regulate CD25 affinity

[0234] In some embodiments, the orthogonal IL2 comprises one or more mutations in a position of the IL2 sequence that either contacts CD25 or changes the orientation of other positions that contact CD25, resulting in a reduced affinity of the orthogonal IL2 for CD25. The mutations can be located in or near a region known to be very close to CD25 based on a published crystal structure (Wang, et al. Science 310: 1159 2005). It is believed that the IL2 residues that contact CD25 include K35, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, V69, L72, and Y107. In some embodiments, the orthogonal IL2 of the present invention 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 (Ast et al. U.S. Patent Application Publication 2012 / 0244112A1, published on September 27, 2012; U.S. Patent No. 9266938B2, issued on February 23, 2016). Specific combinations of substitutions have been identified to reduce binding to CD25. In some embodiments, the orthogonal IL2 of the invention comprises one or more of the following groups of substitutions: [R38A-F42A-Y45A-E62A], 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). Modifications that regulate CD132 affinity

[0235] In some embodiments of the invention, the orthogonal IL2 comprises one or more mutations at positions in the IL2 sequence that either contact CD132 or change the orientation of other positions that contact CD132, resulting in altered binding to CD132. Exemplary orthogonal IL2 comprises one or more mutations at positions in the IL2 sequence that either contact CD132 or change the orientation of other positions that contact CD122, resulting in altered binding to CD132. It is believed that the IL2 residues that contact CD132 include Q11, L18, Q22, E110, N119, T123, Q126, S127, I129, S130, and T133. In some embodiments, IL2 comprises modifications at L18 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.

[0236] When directly recombinantly produced in a bacterial expression system, in the absence of a leader sequence, endogenous proteases result in deletion of the N-terminal Met-Ala1 residue to provide a "desAla1" orthogonal IL2. In some embodiments, the present invention provides a human orthogonal IL2, which is a hIL2 polypeptide, comprising one of the following groups of amino acid modifications:

[0237] The orthogonal IL2 of the present invention may include a modification that eliminates the O-glycosylation site at the Thr3 position to promote the production of non-glycosylated orthogonal IL2 when expressing the orthogonal IL2 in mammalian cells (e.g., CHO or HEK cells). Therefore, in certain embodiments, the orthogonal IL2 includes a modification that eliminates the O-glycosylation site at the residue 3 position of human IL-2 corresponding to IL-2. In some embodiments, the modification that eliminates the O-glycosylation site at the residue 3 position of human IL-2 corresponding to IL-2 is an amino acid modification. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P, which remove the glycosylation site at position 3 without eliminating biological activity (see U.S. Patent No. 5,116,943; Weiger et al., (1989) Eur. J. Biochem., 180: 295-300). In some embodiments, the orthogonal IL2 may comprise a deletion of the first two amino acids (desAla1-desPro2), as well as substitution of Thr3 glycosylation with a cysteine ​​residue to facilitate selective N-terminal modification, particularly PEGylation of the thiol group of cysteine ​​(see, e.g., Katre et al., U.S. Pat. No. 5,206,344, issued April 27, 1993).

[0238] The orthogonal IL2 disclosed herein may optionally further comprise a modification at position M104, in one embodiment replacing methionine 104 with an alanine residue (M104A) to provide a more resistant orthogonal IL2 to oxidation (see Koths, et al., US Pat. No. 4,752,585).

[0239] When directly recombinantly produced 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 provide a "desAla1" orthogonal IL2. The orthogonal IL2 may comprise a deletion of the first two amino acids (desAla1-desPro2), as well as substitution of Thr3 glycosylation with a cysteine ​​residue (T3C) to facilitate N-terminal modification, particularly PEGylation of the thiol group of cysteine ​​(see, e.g., Katre, et al., U.S. Pat. No. 5,206,344, issued April 27, 1993).

[0240] The orthogonal IL2 may further comprise elimination of the N-terminal amino acid at one or more of positions 1-9, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2.

[0241] In some embodiments of the invention, the orthogonal IL2 comprises amino acid substitutions to avoid vascular leakage syndrome, which is a substantial negative and dose-limiting side effect of using IL2 therapy in humans, without substantial loss of efficacy. See, Epstein, et al., U.S. Patent No. 7,514,073B2, issued April 7, 2009. Examples of such modifications included in the orthogonal IL2 of the invention include one or more of R38W, R38G, R39L, R39V, F42K, and H55Y.

[0242] In some embodiments, orthogonal IL2 can be affinity matured to enhance its activity to orthogonal CD122. "Affinity matured" polypeptide is a polypeptide having one or more changes on one or more residues, which results in an improvement in the affinity of the orthogonal polypeptide to the homologous orthogonal receptor compared to a parent polypeptide that does not have these one or more changes, or vice versa. Compared to the "parent" polypeptide, affinity maturation can be performed to increase the binding affinity of the orthogonal IL2 by at least about 10%, or at least about 50%, or at least about 100%, or at least about 150%, or 1-5 times. When a sufficient amount of molecules are used under suitable experimental conditions to be evaluated by ELISA and / or FACS analysis, the engineered orthogonal IL2 of the present invention activates its homologous orthogonal receptor, as described above, but significantly reduces the binding and activation of the wild-type IL2 receptor.

[0243] As described above, the compositions of the invention include orthogonal IL2 that has been modified to provide for extended lifespan in vivo and / or extended duration of action in a subject. Such modifications that provide for extended lifespan and / or duration of action include modifications to the primary sequence of the orthogonal IL2, coupling to a carrier molecule (e.g., albumin, acylation, PEGylation), and Fc fusions.

[0244] In some embodiments, orthogonal IL2 may comprise certain amino acid substitutions that result in an extended lifespan in vivo. For example, Dakshinamurthi, et al. (International Journal of Bioinformatics Research (2009) 1(2): 4-13) describe that one or more substitutions of V91R, K97E, and T113N in an IL2 polypeptide will produce an IL2 variant with enhanced stability and activity. In some embodiments, an orthogonal IL2 of the present invention comprises one, two, or all three of the V91R, K97E, and T113N modifications.

[0245] In some embodiments, the orthogonal IL2 is modified to provide certain properties (e.g., prolonged duration of action in a subject) to the orthogonal IL2, which can be achieved by coupling to a carrier molecule to provide the desired pharmacological properties, such as extended half-life. In some embodiments, the orthogonal IL2 can be covalently linked to the Fc domain of IgG, albumin, or other molecules to extend its half-life, for example, by pegylation, glycosylation, fatty acid acylation, etc., as known in the art.

[0246] In some embodiments, the orthogonal IL2 is expressed as a fusion protein with an albumin molecule (eg, human serum albumin), which is known in the art to facilitate prolonged exposure in vivo.

[0247] In one embodiment of the invention, human orthogonal IL2 is conjugated to albumin, referred to herein as an "orthogonal IL2 albumin fusion". The term "albumin", when used in the context of human orthogonal IL2 albumin fusion, includes albumins such as human serum albumin (HSA), cyno serum albumin, and bovine serum albumin (BSA). In some embodiments, the HSA comprises a C34S or K573P amino acid substitution relative to the wild-type HSA sequence. According to the present invention, albumin can be conjugated to human orthogonal IL2 at the carboxyl terminus, the amino terminus, the carboxyl and amino termini, and internally (see, e.g., USP 5,876,969 and USP 7,056,701). In the HSA-human orthogonal IL2 polypeptide conjugates contemplated by the present disclosure, various forms of albumin can be used, such as albumin secretion pre-sequence and variants thereof, fragments and variants thereof, and HSA variants. Such forms generally have one or more desired albumin activities. In other embodiments, the present invention relates to a fusion protein comprising a human orthogonal IL2 polypeptide fused directly or indirectly to albumin, an albumin fragment, an albumin variant, 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, direct fusion is achieved through a linker, such as a peptide linker or a modified form thereof, as discussed more fully below.

[0248] Alternatively, the human orthogonal IL2 albumin fusion comprises an orthogonal IL2 that is a fusion protein comprising an albumin binding domain (ABD) polypeptide sequence and an orthogonal IL2 polypeptide. As described above, a fusion protein comprising an albumin binding domain (ABD) polypeptide sequence and a human orthogonal IL2 polypeptide can be achieved, for example, by genetic manipulation, such that a nucleic acid encoding HSA or a fragment thereof is joined to a nucleic acid encoding one or more orthogonal IL2 sequences. In some embodiments, the albumin binding peptide comprises the amino acid sequence DICLPRWGCLW (SEQ ID NO: 50).

[0249] Orthogonal IL2 polypeptides can also be coupled to large, slowly metabolized macromolecules, such as proteins; polysaccharides, such as sepharose, agarose, cellulose or cellulose beads; polyamino acids, such as polyglutamic acid or polylysine; amino acid copolymers; inactivated viral particles; inactivated bacterial toxins, such as toxoids or leukotoxin molecules from diphtheria, tetanus, cholera; inactivated bacteria, dendritic cells, thyroglobulin; tetanus toxoid; diphtheria toxoid; polyamino acids such as poly (D-lysine: D-glutamic acid); rotavirus VP6 polypeptide; influenza virus hemaglutinin, influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B virus core protein and surface antigen. If desired, this coupling format can be used to generate antibodies against the polypeptides of the present disclosure.

[0250] In some embodiments, orthogonal IL2 is coupled to XTEN (by chemical means or in fusion protein form), which provides a prolonged duration similar to PEGylation, which can be produced in Escherichia coli in the form of recombinant fusion protein. Suitable XTEN polymers coupled to orthogonal IL2 of the present invention are provided in Podust, et al. (2016) "Extension of in vivo half-life of biologically active molecules by XTEN protein polymers" by XTEN protein polymers, J Controlled Release 240:52-66 and Haeckel et al. (2016) "XTEN as a biological alternative to PEGylation allows complete expression of a protease-activatable killer cell inhibitor (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. The XTEN polymer fusion proteins can incorporate a protease-sensitive cleavage site, such as a MMP-2 cleavage site, between the XTEN polypeptide and the orthogonal IL2.

[0251] Other candidate components and molecules for coupling include other components and molecules suitable for separation or purification. Specific non-limiting examples include binding molecules such as biotin (biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or molecules comprising solid supports (including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips and membranes).

[0252] In some embodiments, the IL-2 mutant protein can also be linked to other therapeutic agents, including therapeutic compounds, such as anti-inflammatory compounds or anti-tumor agents, therapeutic antibodies (such as Herceptin), immune checkpoint modulators, immune checkpoint inhibitors (such as anti-PD1 antibodies), cancer vaccines, as described elsewhere herein. Antimicrobial agents include aminoglycosides (including gentamicin), antiviral compounds such as rifampicin, 3′-azido-3′-deoxythymidine (AZT) and acyclovir, antifungal agents such as azoles, including fluconazole, macrolides (plyremacrolide), such as amphotericin B and candicidin, antiparasitic compounds such as antimonials, etc. Orthogonal IL2 can be coupled to other 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. Nonsteroidal anti-inflammatory drugs, such as indomethacin, salicylic acid acetate, ibuprofen, sulindac, piroxicam and naproxen, and anesthetics or analgesics are also included. Radioisotopes, such as those useful for imaging and treatment, are also included.

[0253] The orthogonal IL2 of the present disclosure can be chemically coupled to such carrier molecules using well-known chemical coupling methods. Bifunctional cross-linking agents such as isofunctional and heterofunctional cross-linking agents known in the art can be used for this purpose. The type of cross-linking agent used depends on the properties of the molecule to be coupled to the IL-2 mutant protein, which can be easily determined by those skilled in the art. Alternatively or in addition, the orthogonal IL2 and / or the molecule intended to be coupled thereto can be chemically derivatized so that the two are coupled in a separate reaction, which is also well known in the art.

[0254] In some embodiments, the orthogonal IL2 is coupled 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 (polyvinyl pyrrolidone, copolymers of ethylene glycol and propylene glycol, poly (oxyethylated polyols), polyolefinic alcohols, polysaccharides, poly-α-hydroxy acids, polyvinyl alcohol (PVA), polyphosphazenes, polyoxazolines (POZ), poly (N-acryloylmorpholine), or combinations thereof.

[0255] In some embodiments, the orthogonal IL2 is conjugated to one or more polyethylene glycol molecules or "pegylated." Although the method or site of PEG attachment to the orthogonal IL2 may vary, in certain embodiments pegylation does not alter or only minimally alters the activity of the orthogonal IL2.

[0256] In some embodiments, cysteine ​​can be substituted with threonine at position 3 (3TC) to facilitate N-terminal PEGylation using specific chemistries.

[0257] In some embodiments, selective PEGylation of orthogonal IL2 (e.g., by incorporating non-natural amino acids with side chains to facilitate selective PEG coupling chemistry, as described in Ptacin, et al. (PCT International Application No. PCT / US2018 / 045257, filed on August 3, 2018, and published as International Publication No. WO on February 7, 2019) 2019 / 028419A1 disclosed) can be used to generate orthogonal IL2 with reduced affinity for one or more subunits of the IL2 receptor complex (such as CD25, CD132). For example, a human orthogonal IL2 that incorporates a non-natural amino acid with a PEGylated specific portion at a sequence or residue of an IL2 identified as interacting with CD25 comprising amino acids 34-45, 61-72, and 105-109 generally provides an orthogonal IL2 with reduced binding to CD25. Similarly, a human orthogonal IL2 that incorporates a non-natural amino acid with a PEGylated specific portion at a sequence or residue of an IL2 identified as interacting with hCD132 comprising amino acids 18, 22, 109, 126, or 119-133 provides an orthogonal IL2 with reduced binding to hCD132.

[0258] In certain embodiments, the increase in half-life outweighs any decrease in biological activity. PEG suitable for coupling to polypeptide sequences is generally soluble in water at room temperature and has the general formula R(O-CH2-CH2) nOR, wherein R is hydrogen or a protecting group, such as an alkyl or alkanol group, wherein n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons. The PEG coupled to the polypeptide sequence can be linear or branched. The present disclosure contemplates branched PEG derivatives "star-PEG" and multi-arm PEG.

[0259] The molecular weight of the PEG used in the present invention is not limited to any specific range. The PEG component of PEG-orthogonal IL2 can have a molecular weight greater than about 5kDa, greater than about 10kDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa or greater than about 50kDa. In some embodiments, the molecular weight is about 5kDa to about 10kDa, about 5kDa to about 15kDa, about 5kDa to about 20kDa, about 10kDa to about 15kDa, about 10kDa to about 20kDa, about 10kDa to about 25kDa or about 10kDa to about 30kDa. The molecular weight of a linear or branched PEG molecule is from about 2,000 to about 80,000 Daltons, or about 2,000 to about 70,000 Daltons, or about 5,000 to about 50,000 Daltons, or about 10,000 to about 50,000 Daltons, or about 20,000 to about 50,000 Daltons, or about 30,000 to about 50,000 Daltons, or about 20,000 to about 40,000 Daltons, or about 30,000 to about 40,000 Daltons. In one embodiment of the invention, the PEG is a 40 kD branched PEG comprising two 20 kD arms.

[0260] The present invention also contemplates compositions of conjugates in which PEG has different n values, so that multiple PEGs are present in specific ratios. For example, some compositions contain mixtures of conjugates in which n=1, 2, 3, and 4. In some compositions, the percentage of conjugates with n=1 is 18-25%, the percentage of conjugates with n=2 is 50-66%, the percentage of conjugates with n=3 is 12-16%, and the percentage of conjugates with n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. Chromatography / chromatography can be used to resolve fractions of conjugates, and then identify fractions containing conjugates with, for example, a desired number of PEGs attached, purified from unmodified protein sequences and conjugates with other numbers of PEGs attached.

[0261] PEG suitable for coupling to polypeptide sequences is generally soluble in water at room temperature and has the general formula R(O-CH2-CH2) n OR, wherein R is hydrogen or a protecting group, such as an alkyl or alkanol group, wherein n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons.

[0262] Two widely used first generation activated monomethoxy PEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15: 100-114) and benzotriazole methyl ester PEG (BTC-PEG; see, e.g., Dolence, et al. U.S. Pat. No. 5,650,234), which react preferentially with lysine residues to form carbamate linkages, but are also known to react with histidine and tyrosine residues. PEG-aldehyde linkers are used to target a single site at the terminus of a polypeptide by reductive amination.

[0263] Pegylation often occurs at the α-amino group at the N-terminus of a polypeptide, the ε-amino group on the side chain of a lysine residue, and the imidazole group on the side chain of a histidine residue. Since most recombinant polypeptides have a single α group and several ε groups and imidazole groups, many positional isomers can be produced depending on the chemical nature of the linker. General pegylation strategies known in the art can be applied herein.

[0264] PEG can be conjugated to the orthogonal IL2 of the invention via a terminal reactive group ("spacer") that mediates the bond between the free amino or carboxyl groups of one or more polypeptide sequences and the polyethylene glycol. PEGs with spacers that can bind to free amino groups include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide.

[0265] In some embodiments, the PEGylation of orthogonal IL2 is promoted by incorporating non-natural amino acids with unique side chains to promote site-specific PEGylation. It is known in the art to incorporate non-natural amino acids into polypeptides to provide functional moieties to achieve site-specific PEGylation of such polypeptides. See, for example, Ptacin, et al., (PCT International Application No. PCT / US2018 / 045257, filed on August 3, 2018 and published on February 7, 2019, International Publication No. WO 2019 / 028419A1. In one embodiment, the orthogonal IL2 of the present invention incorporates non-natural amino acids at position D109 of orthogonal IL2. In one embodiment of the present invention, orthogonal IL2 is PEGylated at position 109 of orthogonal IL2, and the PEG molecular weight is about 20kD, or about 30kD, or about 40kD.

[0266] The PEG coupled to the polypeptide sequence can be linear or branched. The present disclosure contemplates branched PEG derivatives "star-PEG" and multi-arm PEG. Specific embodiments of PEG useful in the practice of the present invention include 10 kDa linear PEG-aldehyde (e.g., ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10 kDa linear PEG-NHS ester (e.g., ME-100CS, ME-100AS, ME-100GS, ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g. ME-200AL, NOF, 20 kDa linear PEG-NHS ester (e.g., ME-200CS, ME-200AS, ME-200GS, ME-200HS, NOF), 20 kDa 2-arm branched PEG-aldehyde, the 20 kDA PEG-aldehyde, which contains two 10 kDA linear PEG molecules (e.g., GL2-200AL3, NOF), 20 kDa 2-arm branched PEG-NHS ester, the 20 kDA PEG-NHS ester, which contains two 10 kDA linear PEG molecules (e.g., GL2-200TS, GL200GS2, NOF), 40 kDa 2-arm branched PEG-aldehyde, the 40 kDA PEG-aldehyde, which contains two 20 kDA linear PEG molecules (e.g., GL2-400AL3), 40 kDa 2-arm branched PEG-NHS ester, the 40 kDA PEG-NHS ester, which contains two 20 kDA linear PEG molecules (e.g., GL2-400AL3, GL2-400GS2, NOF), linear 30 kDa PEG-aldehyde (e.g., ME-300AL) and linear 30 kDa PEG-NHS ester.

[0267] As previously described, PEG can be attached to the orthogonal IL2 directly or through a linker molecule. Suitable linkers include "flexible linkers" which are generally long enough to allow some movement between the modified polypeptide sequence and the connected components and molecules. Linker molecules are generally about 6-50 atoms long. The linker molecule can also be, for example, an aryl acetylene, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof. Suitable linkers can be easily selected and can be of any suitable length, such as 1 amino acid (such as Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 or more than 50 amino acids. Examples of flexible linkers include glycine polymers (G) n , glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers and other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and can therefore be used as neutral tethers between components. Other 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 can therefore be used as neutral tethers between components. Multimers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) can be linked together to provide a flexible linker that can be used to couple heterologous amino acid sequences to polypeptides disclosed herein.

[0268] Furthermore, such linkers can be used to link orthogonal IL2 to other heterologous polypeptide components described herein, the heterologous amino acid sequence can be a signal sequence and / or a fusion partner, for example, albumin, Fc sequence, etc.

[0269] In one embodiment of the present invention, the orthogonal IL2 is human orthogonal IL2, and its structure is: [PEG]-[Linker] n -[hoIL2] Where n = 0 or 1, or [PEG]-[Linker] n -[desAla1-hIL2[E15S-H16Q-L19V-D20L-Q22K-M23A] Where n = 0 or 1, or

[0270] In another embodiment of the present invention, the orthogonal IL2 is human orthogonal IL2, the structure of which is: 40kD-PEG-(Linker) n-PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT Where n = 0 (absent) or 1 (present).

[0271] In one embodiment, the orthogonal IL2 is a polypeptide: 40kD-PEG-(Linker) n -PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT Wherein 40kD-PEG-linker (n=1) is a molecule having the following structure: Also referred to herein as STK-009.

[0272] In some embodiments, the orthogonal IL2 is provided as a fusion protein with a polypeptide sequence ("targeting domain") to facilitate selective binding to a specific cell type or tissue expressing a cell surface molecule that specifically binds to such targeting domain, optionally including a linker molecule of 1-40 (or 2-20, or 5-20, or 10-20) amino acids between the orthogonal IL2 sequence and the targeting domain sequence of the fusion protein.

[0273] In other embodiments, chimeric polypeptides comprising an orthogonal IL-2 and an antibody or antigen binding portion thereof can be produced. The antibody or antigen binding component of the chimeric protein can serve as a targeting moiety. For example, it can be used to localize the chimeric protein to a specific cell subset or target molecule. Methods for producing cytokine-antibody chimeric polypeptides are described in, for example, U.S. Patent No. 6,617,135.

[0274] In some embodiments, the targeting domain of the orthogonal IL2 fusion protein specifically binds to the cell surface molecules of tumor cells. In one embodiment, wherein the ECD of the CAR of the CAR-T cell specifically binds to GPC3, the orthogonal IL2 can be provided as a fusion protein with a GPC3 targeting portion. For example, in one embodiment, wherein the ECD of the CAR of the CAR-T cell is a scFv molecule that provides specific binding to GPC3, and the orthogonal IL2 is provided as a fusion protein with a GPC3 targeting portion (e.g., 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 a scFv selected from SEQ ID NO: 10 or SEQ ID NO: 11. Treatment

[0275] The present disclosure provides methods and compositions for treating a subject suffering from a neoplastic disease.

[0276] 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 a T cell population from a subject; (b) contacting the T cell population with a recombinant viral vector, the vector comprising a first nucleic acid sequence encoding GPC3 CAR and a second nucleic acid sequence encoding an orthogonal receptor, the first and second nucleic acid sequences being operably linked to expression control elements functional in mammalian T cells, the contacting causing a portion of the separated T cell population to be transformed by the viral vector, such that the transformed cells express GPC3 CAR and the orthogonal receptor; (c) administering to the subject a therapeutically effective amount of the T cells produced by step (b), and concurrently administering to the subject a therapeutically effective amount of an orthogonal IL2.

[0277] In one embodiment, the present invention provides a method of treating a subject having a neoplastic disease, the method comprising administering to the subject a therapeutically effective amount of an orthogonal GPC3 CAR T cell in combination with a therapeutically effective amount of an orthogonal IL2. In some embodiments, the antigen binding domain of the GPC3 CAR cell is a scFv. In some embodiments, the scFv is a GC33 scFv. In some embodiments, the scFv is SEQ ID NO: 10 or SEQ ID NO: 11.

[0278] In some embodiments, the intracellular domain of GPC3 CAR T comprises a CD28 co-stimulatory domain and a CD3-ζ domain. In some embodiments, the intracellular domain of GPC3 CAR T comprises a 41BB co-stimulatory domain and a CD3-ζ domain.

[0279] In some embodiments, the GPC3 CAR is a polypeptide having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or 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.

[0280] In some embodiments, the neoplastic disease is selected from the group consisting of hepatocellular carcinoma, hepatoblastoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and ovarian clear cell carcinoma.

[0281] In some embodiments, the present disclosure provides a method of treating a subject having a neoplastic disease characterized by the presence of a tumor expressing GPC3 ("GPC3+ tumor") in the subject.

[0282] As used herein, the term "subject" is used interchangeably with the terms "recipient," "individual," and "patient," and refers to any mammalian subject in need of diagnosis, treatment, or therapy. For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, and the like. In some embodiments, the mammal is a human.

[0283] As used herein, the term "suffering from" refers to a physician's judgment that a subject needs or will benefit from treatment based on available information (including, but not limited to, X-rays, CT-scans, routine laboratory diagnostic tests (e.g., blood cell counts, etc.), genomic data, protein expression data, immunohistochemistry) that is recognized in the art to identify a disease, disorder, or condition. The term suffering from is often used in conjunction with a specific disease state, such as "suffering from a neoplastic disease" means that a patient is diagnosed as carrying a tumor. As discussed above, the presence of GPC3+ tumors can be identified by the detectable presence of the 40 kDa subunit of GPC3 (referred to as "soluble GPC3" or "sGPC3") in serum, and the level of GPC3 is considered a diagnostic marker.

[0284] As used herein, the term "neoplastic disease" refers to a disorder or condition caused by excessive cell proliferation or uncontrolled (or dysregulated) cell replication in a subject. The term neoplastic disease refers to a condition caused by the presence of a tumor in a subject. Tumors can be classified as: (1) benign; (2) pre-malignant (or "precancerous"); and (3) malignant (or "cancerous"). The term "neoplastic disease" includes tumor-related diseases, disorders and conditions, and refers to conditions directly or indirectly related to a neoplastic disease, including, for example, angiogenesis and precancerous conditions.

[0285] The terms "administer," "dosing," and "applying" are used interchangeably herein and refer to the act of contacting a subject, including contacting a subject's cells, tissues, organs, or biological fluids in vitro, in vivo, or ex vivo with an agent (e.g., an ortholog, an IL2 ortholog, an engineered cell expressing an orthogonal receptor, an engineered cell expressing an orthogonal IL2 receptor (e.g., a CAR-T cell expressing an orthogonal IL2 receptor), a chemotherapeutic agent, an antibody) or a pharmaceutical formulation comprising one or more of the foregoing. Administration of the agent can be achieved by any of a variety of methods recognized in the art, including, but not limited to, topical administration, intravascular injection (including intravenous or intraarterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal, transmucosal, iontophoretic delivery, intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhalation (e.g., respiratory inhaler, including dry powder inhaler), intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intracerebroventricular injection (ICVI), etc. The term "administering" includes contact of the agent with a cell, tissue, or organ, as well as contact of the agent with a liquid, wherein the liquid is in contact with the cell, tissue, or organ.

[0286] The term "therapeutically effective amount" as used herein refers to the administration of an agent to a subject, either alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a series of doses, in an amount that is capable of producing any detectable, positive effect on any symptom, aspect or feature of a disease, disorder or condition. The therapeutically effective amount can be determined by measuring relevant physiological effects and can be adjusted in conjunction with the dosing regimen and diagnostic analysis of the subject's condition, etc. The evaluation parameters used to determine the therapeutically effective amount of an agent are determined by a physician using recognized diagnostic criteria, including but not limited to indicators such as age, weight, sex, general health, ECOG score, observable physiological parameters, blood concentrations, blood pressure, electrocardiograms, computed tomography scans, X-rays, etc. Alternatively or in addition, other parameters commonly assessed in clinical settings can also be monitored to determine whether a therapeutically effective amount of the agent has been administered to the subject, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect or feature of a disease, disorder or condition, biomarkers (such as inflammatory cytokines, IFN-γ, granzymes, etc.), reduction in serum tumor markers, improvement in response evaluation criteria in solid tumors (RECIST), improvement in immune-related response criteria (irRC), prolonged survival, prolonged progression-free survival, prolonged time to progression, prolonged time to treatment failure, prolonged event-free survival, prolonged time to next treatment, improvement in objective response rate, improvement in duration of response, reduction in tumor burden, complete remission, partial remission, stable disease, etc., and clinicians in the field can assess the improvement of the subject's response to the administration of the agent based on these assessments. As used herein, the terms "complete remission (CR)", "partial remission (PR)", "stable disease (SD)" and "progressive disease (PD)" related to target lesions, "complete remission (CR)", "incomplete remission / stable disease (SD)" and "progressive disease (PD)" related to non-target lesions, should be understood as defined in RECIST criteria. As used herein, the terms "immune-related complete remission (irCR)", "immune-related partial remission (irPR)", "immune-related progressive disease (irPD)" and "immune-related stable disease (irSD)" are defined according to immune-related remission criteria (irRC).As used herein, the term "immune-related response criteria (irRC)" refers to a system for evaluating the 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. During the course of treatment for a subject, the therapeutically effective amount can be adjusted based on the dosing regimen and / or the assessment of the subject's condition and changes in the aforementioned factors. In one embodiment, the therapeutically effective amount is the amount of an agent that does not cause irreversible serious adverse events when used alone or in combination with another agent during administration to a mammalian subject.

[0287] As used herein, the term "in combination with" when referring to the administration of multiple agents to a subject refers to the administration of a first agent to a subject and at least one other (i.e., a second, third, fourth, fifth, etc.) agent. For purposes of the present invention, an 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, when the second agent is administered, the biological effect resulting from the administration of the first agent persists in the subject, such that the therapeutic effects of the first agent and the second agent are superimposed. For example, an engineered orthogonal cell therapeutic is typically administered infrequently (usually only once), while an IL2 ortholog is administered periodically while the orthogonal cell agent persists in the subject. An engineered orthogonal cell therapeutic provides a therapeutic effect over a longer period of time (weeks or months), and the administration of a second agent (e.g., an IL2 ortholog) provides its therapeutic effect while the therapeutic effect of the first agent is still ongoing, so the second agent is considered to be administered in combination with the first agent, even though the administration of the first agent may be performed at a time point (e.g., days or weeks) far from the time of administration of the second agent. In one embodiment, if the first and second reagents are given simultaneously (within 30 minutes of each other), in the same period or in sequence, it is considered that a reagent is given in combination with the second reagent. In some embodiments, if the first and second reagents are given 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, the first reagent is considered to be given "in the same period" with the second reagent. The term "combined with..." should also be understood to be applicable to the situation where the first and second reagents are co-formulated in a single pharmaceutically acceptable preparation and the co-preparation is given to an object.

[0288] In some embodiments, orthogonal IL2 is administered in combination with orthogonal GPC3 CAR T cells, wherein the orthogonal IL2 and the orthogonal GPC3 CAR T cells are administered to the subject at the same time.

[0289] In some embodiments, orthogonal IL2 is administered in combination with orthogonal GPC3 CAR T cells, wherein the orthogonal IL2 is administered to the subject at one or more time points 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to administration of the orthogonal GPC3 CAR T cells to the subject.

[0290] In some embodiments, the orthogonal IL2 is administered in combination with the orthogonal GPC3 CAR T cells, wherein the orthogonal IL2 is administered to the subject at one or more time points 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 30, 60, 90, 120, 150, 180, 240 days after the orthogonal GPC3 CAR T cells are administered to the subject.

[0291] In some embodiments, the orthogonal IL2 is administered in combination with the orthogonal GPC3 CAR T cells, wherein the orthogonal IL2 is administered (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days before administering the orthogonal GPC3 CAR T cells to the subject; (b) concurrently with administering the orthogonal GPC3 CAR T cells to the subject, and (c) at one or more time points 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 30, 60, 90, 120, 150, 180, 240 days after administering the orthogonal GPC3 CAR T cells to the subject.

[0292] In some embodiments, the orthogonal IL2 is administered in combination with the orthogonal GPC3 CAR T cells, wherein the orthogonal IL2 is administered (a) concurrently with administration of the orthogonal GPC3 CAR T cells to the subject, and (b) at one or more time points 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 30, 60, 90, 120, 150, 180, 240 days after administration of the orthogonal GPC3 CAR T cells to the subject.

[0293] In certain embodiments, orthogonal cells and IL2 orthologs are further combined with supplementary anti-tumor agents. One or more supplements may be administered before, during, and / or after the GPC3 CAR T cell and orthogonal IL2 combination therapy. In some embodiments, the supplementary anti-tumor agent is selected from the group consisting of chemotherapeutic agents, small molecules, supplementary biologics (including but not limited to checkpoint inhibitors (including but not limited to anti-PD1 antibodies, such as Keytruda, Opdivo), anti-tumor antigen antibodies (Herceptin)) and / or physical methods (surgery, radiation, etc.).

[0294] In other embodiments, one or more supplements are administered simultaneously, e.g., two or more agents are administered at the same time or about the same time; the two or more agents may be present in two or more separate formulations, or combined into a single formulation (i.e., a co-formulation). Regardless of whether the agents are administered sequentially or simultaneously, for purposes of the present invention, they are considered to be administered in combination. Administration of GPC3 CAR T cells

[0295] Orthogonal GPC3 CAR T cells and orthogonal IL2 can be provided in a pharmaceutical composition suitable for therapeutic use (e.g., for human treatment). Therapeutic preparations comprising these cells can be frozen or prepared for administration (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)) with a physiologically acceptable carrier, excipient, or stabilizer in the form of an aqueous solution. The cells will be formulated, dosed, and administered in a manner consistent with good medical practice. Considerations in this regard include the specific condition being treated, the specific mammal being treated, the individual clinical condition of the patient, the cause of disease, the agent delivery site, the method of administration, the dosing schedule, and other factors known to medical personnel.

[0296] Cells can be administered by any suitable means, usually parenterally. Parenteral infusions include intramuscular, intravenous (bolus or slow infusion), intraarterial, intraperitoneal, intrathecal or subcutaneous administration. In typical practice, CAR T cells can be infused into a subject in a physiologically acceptable medium, usually intravascularly, although it can also be introduced into any other convenient site where the cells can find a suitable site for growth.

[0297] The therapeutically effective amount of the orthogonal GPC3 CAR T cells for use in the method of treating a subject having a GPC+ tumor is about 1 x 10 4 to 5x10 6In some embodiments, the dose of orthogonal GPC3 CAR T cells administered to a subject in need of treatment during a treatment course is about 1x10 5 or 2x10 5 or 3x10 5 or 4x10 5 or 5x10 5 or 6x10 5 or 7x10 5 or 8x10 5 or 9x10 5 or 1x10 6 , or 2x10 6 or 3x10 6 or 4x10 6 or 5x10 6 Orthogonal GPC3 CAR cells / kg body weight.

[0298] The course of treatment for orthogonal GPC3 CAR T cells can be a single dose or multiple doses over a period of time. Typically, orthogonal GPC3 CAR T cells are administered in a single dose in each course of treatment, while orthogonal IL2 is administered regularly in combination with orthogonal GPC3 CAR T cells. However, orthogonal GPC3 CAR T cells can be administered in two or more divided doses 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. In this divided dosing regimen, the number of orthogonal GPC3 CAR T cells can be the same in each administration or can be provided at different levels. The multi-day dosing regimen over a period of time can be provided by a skilled technician (such as a physician), monitoring the administration of cells, considering the subject's response to treatment, including adverse reactions to treatment and their regulation, as described above. Orthogonal IL2 gives:

[0299] The embodiments of the methods disclosed herein relate to administering a pharmaceutical preparation comprising a therapeutically effective amount of an orthogonal IL2 (and / or a nucleic acid encoding an orthogonal IL2) to a subject in need of treatment. Administration to a subject can be achieved by intravenous injection or by continuous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intraspinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes. Orthogonal IL2 can also be suitably administered by intratumoral, para-cancer, intralesional, intranodal or perilesional routes, or by lymphatic administration to exert local and systemic therapeutic effects.

[0300] When the orthogonal IL2 is STK-009, a therapeutically effective amount of STK-009 in combination with orthogonal GPC3 CAR T cells for human subjects is about 0.5 mg to about 20 mg, or about 1 mg to about 15 mg, or about 1.5 mg to about 12 mg administered subcutaneously weekly.

[0301] In some embodiments, the subject orthogonal IL2 (and / or nucleic acid encoding the orthogonal IL2) can be incorporated into a composition, including a pharmaceutical composition. Such compositions generally include a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. The pharmaceutical composition is prepared to be compatible with the route in which it is intended to be administered and is compatible with therapeutic use for administering the orthogonal IL2 to a subject in need of treatment or prevention.

[0302] The orthogonal IL2 of the present invention (or the nucleic acid encoding it) can be administered to a subject in a pharmaceutically acceptable dosage form. The preferred formulation depends on the intended mode of administration and therapeutic application. Typically, protein therapeutics are formulated for parenteral administration, and the methods disclosed herein relate to parenteral administration of orthogonal IL2. Examples of parenteral routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Parenteral formulations include solutions or suspensions for parenteral administration, which may include carriers and buffers. Pharmaceutical formulations suitable for parenteral administration include sterile aqueous solutions (when water-soluble) or dispersants and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersants. Parenteral formulations may be packaged in ampoules, disposable syringes, or multidose vials made of glass or plastic. In one embodiment, the formulation is provided in a pre-filled syringe for parenteral administration.

[0303] In some embodiments of the methods of the present invention, the orthogonal IL2 is administered to a subject in need of treatment in a formulation that provides extended release of the orthogonal IL2 agent. Examples of extended release formulations of injectable compositions can be achieved by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) in the composition. In one embodiment, the main orthogonal IL2 or nucleic acid is prepared with a carrier that will protect the mutant IL-2 polypeptide from rapid clearance by the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. The materials can also be purchased from Alza Corporation and Novartis Pharmaceuticals, Inc. Liposomal suspensions (liposomes containing cells targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared by methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. In one embodiment, an orthogonal IL2 formulation is provided according to the teachings of Fernandes and Taforo, US Pat. No. 4,604,377, issued Aug. 5, 1986, and Yasui, et al., US Pat. No. 4,645,830.

[0304] In addition to administering to a subject an orthogonal IL2 protein pharmaceutical formulation comprising an orthogonal IL2, an orthogonal IL2 may also be provided to a subject by administering to the subject a pharmaceutically acceptable formulation comprising a recombinant vector encoding a nucleic acid sequence of the orthogonal IL2 to achieve sustained exposure of the subject to the selective orthogonal IL2. Administration of the recombinant vector encoding the orthogonal IL2 provides prolonged delivery of the orthogonal IL2 to the subject and prolonged activation of corresponding cells engineered to express a cognate orthogonal receptor associated with the orthogonal IL2. In some embodiments of the methods of the invention, a nucleic acid encoding an orthogonal IL2 is administered to a subject by transfection or infection using methods 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, erratum in Am. J. Health Syst. Pharm. 53:325, 1996).

[0305] In some embodiments, the orthogonal IL2 can be administered to a subject in the form of a nucleic acid expression construct in a viral vector encoding the orthogonal IL2. The terms "viral vector" and "virus" are used interchangeably herein to refer to any obligate intracellular parasite without protein synthesis or energy production mechanisms. The viral genome can be an RNA or DNA of a proteinaceous coating structure containing a lipid membrane. The terms virus and viral vector are used interchangeably herein. Useful viruses in the practice of the present invention include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesviridiae, poxviridae or adenoviridae. The virus is modified by DNA recombination techniques to contain an exogenous transgene expressing (e.g., a nucleic acid sequence encoding an orthogonal IL2), and can be engineered to be replication-deficient, conditionally replicative or replication-competent. The term "replication-competent viral vector" refers to a viral vector capable of infecting, DNA replication, packaging and lysing infected cells. The term "conditional replication-competent viral vector" used herein refers to a replication-competent vector designed to achieve selective expression in a specific cell type. This conditional replication can be achieved by operably connecting tissue-specific, tumor-specific or cell-type-specific or other selectively induced regulatory control sequences to early genes (such as the E1 gene of an adenovirus vector). Infecting an object with a recombinant virus or a non-viral vector can provide long-term expression of orthogonal IL2 in an object, and provide continuous selective maintenance of orthogonal GPC3 CART cells expressing CD122 orthogonal receptors. In one embodiment, the nucleic acid sequence encoding the IL2 receptor in the viral vector system is under the control of an adjustable promoter, an inducible promoter, a tissue-specific or tumor-specific promoter or a time-regulated promoter. With lymphatic clearance

[0306] Lymphodepletion regimens are often used in combination with CAR T cell therapy, and agents and dosage ranges for administering lymphodepletion agents are well known in the art. In one embodiment of the practice of the foregoing method, the subject is treated with a lymphodepletion regimen comprising a combination of cyclophosphamide and fludarabine. In some embodiments, the lymphodepletion regimen comprises administering a combination of cyclophosphamide and fludarabine prior to administering the GPC3 CAR T cells for a period of 1, 2, 3, 4, or 5 days. In some embodiments, the dose of cyclophosphamide used in the lymphodepletion regimen prior to administering the GPC CAR T cells is about 100, 200, 300, 400, 500, 600 mg / m 2 / day for a period of 1, 2, 3, 4, or 5 days. In some embodiments, the dose of fludarabine used in the lymphodepletion regimen prior to administration of GPC CAR T cells is about 10, 20, 30, 40, 50, 60 mg / m 2 / day for a period of 1, 2, 3, 4, or 5 days. In one embodiment, the dose of cyclophosphamide is about 500 mg / m prior to administration of GPC3 CAR T cells. 2 / day to about 600 mg / m 2 / day, the dose of fludarabine is about 30 mg / m 2 / day for a period of three days. In one embodiment, the dose of cyclophosphamide is about 300 mg / m 2 / day to about 600 mg / m 2 / day, the dose of fludarabine is about 30 mg / m 2 / day for a three-day period. No lymph node clearance

[0307] As previously described, GPC3 CAR T cells are capable of expansion in mammalian subjects. Therefore, this enables significantly lower doses of GPC3 CAR T cells to be administered to patients, thereby reducing or eliminating the need for lymphodepletion prior to administration of GPC3 CAR T cells. Lymphodepletion significantly damages the subject, making it susceptible to environmental factors, and avoiding lymphodepletion regimens, combined with cell therapy, will have significant benefits for patients.

[0308] In one embodiment, the present disclosure provides a method of treating a subject having a neoplastic disease, disorder or condition, wherein the tumor is characterized by expression of GPC3 and is suitable for treatment with GPC3 CAR-T cell therapy by administering an orthogonal ligand expressing CAR-T in the absence of lymphodepletion. Sequence Listing

Claims

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

11.

2. The GPC3 CAR of claim 1, wherein the GPC3 CAR comprises a CD3ζ signaling domain and a co-stimulatory domain selected from the co-stimulatory domains of C28 or 4-1BB.

3. The GPC3 CAR of claim 1, wherein the GPC3 CAR has at least 90%, 95%, 99% or 100% sequence identity with a GPC3 CAR selected from 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, wherein the GPC3 CAR is 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 according to any one of claims 1 to 4. A recombinant vector comprising the nucleic acid sequence according to claim 5. The recombinant vector according to claim 6 , wherein the recombinant vector is a lentiviral vector or a retroviral vector.

8. The recombinant vector of claim 6 or 7, wherein the vector further comprises a nucleic acid sequence encoding an orthogonal CD122 receptor.

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

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

12. An orthogonal GPC3 CAR T cell expressing an orthogonal CD122 and a GPC3 CAR, wherein the orthogonal CD122 comprises an amino acid substitution at position 133 and / or 134, the positions being numbered according to wild-type hCD122.

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

4.

14. A method for preparing orthogonal GPC3 CAR T cells, the method comprising the following steps: a) obtaining human peripheral blood mononuclear cell (PBMC) samples; b) contacting the PBMC sample with magnetic beads coated with CD3 and CD28 ligands to provide an activated PBMC population c) isolating CD8+ and CD4+ T cells from the activated PBMC population; d) contacting the separated CD8+ and CD4+ T cell populations with a recombinant lentiviral vector comprising an expression cassette consisting, from 5' to 3', of a nucleic acid sequence encoding: Promoters active in T cells, such as the EF1a promoter; · Nucleic acid sequence encoding signal peptide ( A nucleic acid sequence encoding GPC3 CAR; T2A sequence (SEQ ID NO: 41) Nucleic acid sequence encoding signal peptide Nucleic acid sequence encoding orthogonal CD122 of SEQ ID NO: 4 thereby transducing the separated CD8+ and CD4+ T cell population fractions with the lentiviral vector; e) contacting the cell population obtained from step (d) with an orthogonal IL2 selected from the group consisting of a human IL2 mutant protein comprising amino acid substitutions E15S / H16Q / L19V / D20L / Q22K / M23A; an orthogonal IL2 of SEQ ID NO 9 (STK-007), or a pegylated variant thereof (e.g., STK-009).

15. Orthogonal GPC3 CAR T cells prepared according to claim 14.

16. A method of treating or preventing a neoplastic disease, disorder or condition in a mammalian subject in need of treatment or prevention, the method comprising administering to the subject a therapeutically effective amount of an orthogonal GPC3 CAR T cell as described in claim 15 in combination with a therapeutically effective dose of an orthogonal IL.

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

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

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

20. A method of treating a subject who has relapsed after receiving orthogonal GPC3 CAR therapy, the method comprising administering to the subject a therapeutically effective amount of orthogonal IL2 such that the orthogonal ligand induces activation and / or proliferation of the subject's orthogonal GPC3 CAR T cells.

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

22. A method of generating an orthogonal GPC3 CAR T cell product that is substantially enriched for an orthogonal GPC3 CAR T cell population, the method comprising the steps of: (a) isolating a cell population from a mammalian subject, wherein the cell population comprises T cells; (b) contacting the T cell population isolated in step (a) with a recombinant vector, the recombinant vector comprising a first nucleic acid sequence encoding GPC3CAR and a second nucleic acid encoding orthogonal CD122, the first and second nucleic acid sequences being operably linked to an operative expression control sequence of a mammalian T cell, wherein the first and second nucleic acid sequences are separated by an IRES or T2A nucleic acid sequence, such that the cells express GPC3CAR and orthogonal CD122 (orthogonal GPC3CAR T cells); (c) contacting the cell population isolated in step (b) with an orthogonal IL2 ex vivo in an amount sufficient to induce proliferation of the cells transduced by the contact in step (b), wherein the contact with the orthogonal IL2 is maintained for a period of time such that the proportion of cells expressing GPC3 CAR and orthogonal CD122 accounts for at least 10% of the population.

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