IL2 orthologs and usage

Abstract

Methods and compositions useful in the practice of adoptive cell therapy are provided. [Solution] The present disclosure provides an orthogonal receptor. In some embodiments, the orthogonal receptor is orthogonal CD122. In some embodiments, the orthogonal receptor is orthogonal human CD122 (hCD122). In some embodiments, the orthogonal receptor is orthogonal CD122 that includes at least one STAT3 binding motif.

Description

[Technical Field]

[0001] Cross-reference to related patent applications This application claims the benefit of priority to each of U.S. Provisional Patent Application No. 62 / 961,200, filed January 14, 2020; U.S. Provisional Patent Application No. 63 / 015,476, filed April 24, 2020; and U.S. Provisional Patent Application No. 63 / 016,256, filed April 27, 2020, each of which is incorporated by reference for all purposes. [Background technology]

[0002] Background of the Invention Adoptive cell therapy has been reported as an effective treatment for diseases in human subjects. In some instances, cell therapy involves the administration of a cell product containing ex vivo expanded tumor-infiltrating lymphocytes (TILs) obtained from resected tumor tissue from a patient. See, for example, U.S. Patent No. 5,126,132A issued June 30, 1992 to Rosenberg, and Spiess, et al. (1987) J Natl Cancer Inst 79:1067-1075. Subjects suffering from metastatic melanoma treated with adoptive TIL therapy substantially according to this regimen have achieved objective tumor responses of around 50% in several Phase I / II clinical trials. See, for example, Rosenberg, et al. (2011) Clin Cancer Res 17:4550-4557 (Non-Patent Document 2); Andersen, et al. (2016) Clin Cancer Res 22:3734-3745 (Non-Patent Document 3); and Besser, et al. (2013) Clin Cancer Res 19:4792-4800 (Non-Patent Document 4).Building on the success of TIL therapy observed in melanoma patients, other researchers have investigated the efficacy of TIL therapy in cervical cancer (Stevanovic, et al (2015) J Clin Oncol 33:1543-1550 (Non-Patent Document 5)), renal cell carcinoma (Andersen, et al (2018) Cancer Immunol Res 6:222-235 (Non-Patent Document 6)), breast cancer (Lee, et al (2017) Oncotarget 8:113345-113359 (Non-Patent Document 7)), non-small cell lung cancer (Ben-Avi, et al (2018) Cancer Immunol Immunother 67:1221-1230 (Non-Patent Document 8)), and gastrointestinal cancer (Turcotte (2013) J Immunol 191:2217-2225 (Non-Patent Document 9) and Turcotte, et al (2014) Clin Cancer Res 20:331-343), cholangiocarcinoma (Tran, et al (2014) Science 344:641-645), pancreatic cancer (Hall, et al (2016) J Immunotherapy Cancer 4:61), head and neck cancer (Junker, et al (2011) Cytotherapy 13:822-834), and ovarian cancer (Fujita, et al (1995) Clin Cancer Res 1: 501-507).

[0003] A wide variety of engineered immune cells (e.g., T cells, NK cells, and TILs) have been developed for the immunotherapeutic treatment of human diseases. Human immune cells have been engineered for use in therapeutic applications such as the recognition and killing of cancer cells, intracellular pathogens, and cells involved in autoimmunity. The use of engineered cell therapy in cancer treatment is facilitated by the selective activation and expansion of engineered cells (such as T cells) that provide specific functions and are directed to selectively attack cancer cells. In some examples of adoptive immunotherapy, T cells are isolated from the subject's blood or tumor tissue, treated ex vivo, and reinfused into the subject. Therefore, compositions and methods that enable the selective activation of such targeted engineered cell populations are desirable.

[0004] The challenge in the clinical application of adoptive cell therapy is to maintain the viability of adoptively transferred cells so as to maintain and maximize their therapeutic efficacy after administration to a subject.The successful maintenance of the viability of adoptively transferred cells after administration to a subject facilitates the clinical response to such adoptive cell therapy.Although the cells administered to a subject in adoptive cell therapy regimen can be detected in the subject for several months or even years after administration, a significant proportion (typically the majority) of the administered cells become dormant, and these cells lose their therapeutic anti-tumor efficacy.The loss of activity of such adoptively transferred cells is often correlated with the loss of clinical efficacy, including the recurrence or relapse of neoplastic disease.

[0005] In clinical practice in human subjects, a commonly used means of supporting adoptively transferred cells (e.g., TIL therapy and engineered T cells such as CAR-T cells) after administration to a subject is the systemic administration of the pluripotent cytokine interleukin-2. Human interleukin-2 (IL2) is a 133-amino acid, four-alpha-helical bundle cytokine. IL2 is a member of the IL2 family of cytokines, which includes IL2, IL-4, IL-7, IL-9, IL-15, and IL21. The amino acid sequence of hIL2 (SEQ ID NO: 1) can be found in GenBank under the accession locator NP_000577.2. IL2 is produced by antigen-activated T cells and exerts a wide range of effects on the immune system, playing an important role in regulating both immune activation and suppression, and in homeostasis. IL2 promotes the proliferation and expansion of activated T lymphocytes, induces the proliferation and activation of naive T cells, enhances B cell growth, and promotes the proliferation and expansion of NK cells. Clinical experience has demonstrated that HD-IL2 treatment activates naive T cells and NK cells. Preclinical studies have implicated NK cells as the dominant mechanism of IL2-mediated acute toxicity. Assier, et al. (2004) J Immunol 172:7661-7668 (Non-Patent Document 15). Administration of HD-IL2 also stimulates the expansion of CD25+ regulatory T cells (Tregs), which mediate the activity of CD8+ T cells. The expansion of NK and Tregs has traditionally been thought to result in additional toxicities, such as CRS.

[0006] In current typical clinical practice of adoptive cell therapy using engineered tumor-infiltrating lymphocytes ("TILs") or CAR-T cells, administered simultaneously with or immediately after the infusion of TILs or CAR-T cells, subjects receive intravenous high-dose hIL2 (HD-hIL2) (720,000 IU / kg hIL2) every 8 hours as long as the subject tolerates the treatment. Administration of HD-hIL2 in conjunction with this adoptive cell therapy is believed to further enhance the viability and clinical efficacy of the engineered cell product. Anderson, et al. (2016) Clinical Cancer Research 22:3734-3745. A commonly used form of human IL2 (hIL2) is aldesleukin (Proleukin®), an hIL2 analogue in which the cysteine ​​at position 125 of the mature hIL2 molecule is replaced with serine (C125S).

[0007] However, systemic administration of hIL2 is associated with nonspecific stimulatory effects beyond the adoptively transferred cell population and significant toxicity in human subjects, especially at high doses. The effects of high-dose hIL2, such as those used in support of adoptive cell therapy regimens, have been reported to result in significant toxicity in human subjects. The most common side effects observed from the 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. Toxicity associated with HD-hIL2 requires specialized management; therefore, HD-hIL2 is typically used in hospitals, often requiring admission to the intensive care unit. Dutcher, et al. (2014) J Immunother Cancer 2(1): 26 (Non-Patent Document 16). HD-hIL2 treatment activates most lymphoid cells, including naive T cells and NK cells, which predominantly express intermediate-affinity receptors (CD122 / CD132), and CD25+ regulatory T cells (Tregs), which express high-affinity trimeric receptors (CD25 / CD122 / CD132). HD-hIL2 monotherapy can also induce systemic capillary leak syndrome, which can be fatal. This largely limits its use to young, extremely healthy patients with normal cardiac and pulmonary function. HD-IL2 therapy is typically administered in hospitals, often requiring admission to the intensive care unit. Nitric oxide synthase inhibitors have been proposed to improve the symptoms of VLS, but the common practice is to discontinue IL2 therapy if VLS is observed. Low-dose IL2 regimens have been tested in patients to alleviate VLS associated with HD-IL2 treatment. While low-dose IL2 treatment regimens partially alleviate VLS toxicity, this lower toxicity was achieved at the expense of suboptimal therapeutic outcomes in the treatment of neoplasms.

[0008] Furthermore, clinically approved forms of hIL2 (e.g., Proleukin®) have a relatively short in vivo lifespan (approximately a few hours), necessitating frequent dosing of IL2 to maintain sufficient exposure of engineered T cells to IL2, keeping the cells in an activated state. While modifications of IL2 to extend its in vivo lifespan have been described in the literature (e.g., PEGylation, U.S. Patent No. 5,206,344 to Katre et al., issued April 27, 1993; Meyers, et al. (1991) Clinical Pharmacology and Therapeutics 13(1):307-313), administration of such long-acting forms of wild-type hIL2 nonetheless presents toxicity concerns similar to the parent molecule, and prolonged exposure to such agents may exacerbate such toxicity. Thus, a significant challenge for cell-based therapies is to endow engineered cells with desired, tunable proliferative behavioral signals that are independent of modulation by endogenous signaling pathways, exhibit minimal cross-reactivity with non-target endogenous cells, and can be selectively controlled following administration of the engineered cell population to a subject.

[0009] Currently, prior to administration of adoptive cell therapy products, subjects are subjected to a lymphodepletion preparative regimen followed by interleukin-2 (IL-2) support. Because the dose of cells in cell therapy regimens is typically very high (typically 10 for current CD19 CAR T products), 6 ~10 8 Lymphocyte-depleting preparative regimens (single-dose administration of cells in the range of 100-150 cells) are thought to deplete Tregs, remove the cellular "sink," and provide "room" for adoptively transferred cells. However, lymphocyte depletion significantly weakens patients by making them vulnerable to environmental factors. Therefore, avoiding lymphocyte-depleting regimens in the context of cell therapy would greatly benefit patients.

[0010] A challenge with manufacturing cell therapy products is that such "living drugs" require strict control of their environment to maintain viability and functionality. Indeed, isolated cells, whether derived from a patient (autologous) or a single donor source (allogeneic), rapidly begin to lose function after removal from the target or controlled culture conditions. Successfully maintaining the viability of isolated cells while outside of the target or controlled culture conditions allows the isolated cells to regain functionality for reinsertion into the cell product manufacturing workflow or into the patient. During ex vivo preparation of cells for use in adoptive cell therapy, cells are often cultured in the presence of exogenous hIL2. Because the action of hIL2 is nonspecific to engineered cells in a mixed cell population containing both engineered and non-engineered immune cells, this exposure to IL2 leads not only to the expansion of desired therapeutically useful cells (e.g., CAR-T cells or antigen-experienced TILs) in the cell population, but also to the expansion of various undesired background cells from isolated tissue (e.g., neoplasm or blood) samples that do not provide clinical benefit, complicate the administration of the engineered cell product, and may contribute to toxicity. Therefore, current ex vivo expansion methods for the preparation of cells useful in autologous cell transfer result in cell products in which the proportion of desired therapeutically useful cells is compromised by undesired cells, resulting in suboptimal cell products. Because significant toxicity remains a significant challenge with current ACT protocols, methods that enable the preparation of cell products containing more homogeneous cell populations enriched for desired effective cells (e.g., CAR-T cells or antigen-experienced TILs) for use in ACT therapy are desirable.

[0011] In addition, current cell therapies are very expensive due to their complex nature and the management of associated toxicities, which often require patients to remain at or near a major medical facility for extended periods. Manufacturing large doses of cellular therapeutics is also complex and time-consuming. Much effort has been focused on reducing the time from the extraction of patient cells for autologous cell therapy to the reinfusion of their engineered version (the so-called "vein-to-vein" time), which currently stands at approximately three weeks. Allogeneic or "off-the-shelf" engineered T cells are currently under investigation to avoid delays in this therapy and to offset the costs associated with such personalized cell therapies. The lack of sustainability of current cell therapies also leads to the requirement for large doses of engineered cells, which further increases costs and results in additional vein-to-vein delays. Despite its proven therapeutic utility and promise, the cost of cell therapy places a significant burden on resource-limited healthcare systems, thus potentially limiting its wider availability to patients in need. The compositions and methods of the present disclosure address many of these challenges.

[0012] CD122 is a component of the medium- and high-affinity IL2 receptor complex. Sockolosky et al. (Science (2018) 359: 1037-1042) and Garcia et al. (U.S. Patent Application Publication US2018 / 0228841A1, published August 16, 2018) describe an orthogonal IL2 / CD122 ligand / receptor system to facilitate selective stimulation of cells engineered to express orthogonal receptors, particularly orthogonal CD122. This patent application incorporates by reference the entire disclosures of WO 2019 / 104092 and US 2018-0228842 A1. Contacting engineered T cells expressing orthogonal CD122 with the corresponding orthogonal ligand (an "IL2 ortholog") cognate to such orthogonal CD122 facilitates specific activation of such orthogonal CD122-expressing engineered T cells. In particular, this orthogonal IL2 receptor-ligand complex provides for selective expansion of cells engineered to express the orthogonal receptor in mixed cell populations, particularly mixed T cell populations. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Patent No. 5,126,132A [Patent Document 2] U.S. Patent No. 5,206,344 [Patent Document 3] U.S. Patent Application Publication US2018 / 0228841A1 [Patent Document 4] WO 2019 / 104092 [Patent Document 5] US 2018-0228842 A1 [Non-patent literature]

[0014] [Non-Patent Document 1] Spiess, et al (1987) J Natl Cancer Inst 79:1067-1075 [Non-licensed document 2] Rosenberg, et al. (2011) Clin Cancer Res 17:4550-4557 [Non-licensed document 3] Andersen, et al. (2016) Clin Cancer Res 22:3734-3745

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[0015] The present disclosure provides methods and compositions useful in the practice of adoptive cell therapy.

[0016] In some embodiments, the present disclosure provides an orthogonal receptor. In some embodiments, the orthogonal receptor is orthogonal CD122. In some embodiments, the orthogonal receptor is orthogonal human CD122 (hCD122). In some embodiments, the orthogonal receptor is orthogonal CD122 that includes at least one STAT3 binding motif.

[0017] In some aspects, the present disclosure provides a recombinant vector comprising a nucleic acid sequence encoding an orthogonal hCD122 receptor.

[0018] In some embodiments, the present disclosure provides a recombinant vector comprising a nucleic acid sequence encoding an orthogonal hCD122 receptor and a CAR.

[0019] In some aspects, the present disclosure provides a recombinant mammalian immune cell comprising a nucleic acid sequence encoding an orthogonal hCD122 receptor and a nucleic acid sequence encoding a CAR.

[0020] In some embodiments, the present disclosure provides orthologs that are cognate ligands for orthogonal receptors. In some embodiments, the orthologs are IL2 orthologs. In some embodiments, the IL2 orthologs are ligands for orthogonal CD122 receptors. In some embodiments, the IL2 orthologs are cognate ligands for a transmembrane receptor protein comprising the extracellular domain of the orthogonal hCD122 receptor. In some embodiments, the IL2 orthologs are cognate ligands for a transmembrane receptor protein comprising the extracellular domain of the orthogonal hCD122 receptor comprising amino acid substitutions at positions H133 and Y134. In some embodiments, the IL2 orthologs are ligands for orthogonal hCD122 comprising amino acid substitutions at positions H133 and Y134. In some embodiments, the IL2 orthologs are ligands for orthogonal hCD122 comprising amino acid substitutions H133D and Y134F.

[0021] In some aspects, the present disclosure provides a pharmaceutically acceptable formulation comprising a hIL2 ortholog of Formula 1.

[0022] In some aspects, the disclosure provides nucleic acid sequences encoding hIL2 orthologs of Formula 1.

[0023] In some aspects, the present disclosure provides a recombinant vector comprising a nucleic acid sequence encoding a hIL2 ortholog of Formula 1.

[0024] In some aspects, the present disclosure provides a pharmaceutically acceptable formulation of a recombinant vector comprising a nucleic acid sequence encoding a hIL2 ortholog of Formula 1.

[0025] In some aspects, the disclosure provides a recombinantly modified mammalian cell comprising a recombinant vector, wherein the vector comprises a nucleic acid sequence encoding a hIL2 ortholog of Formula 1.

[0026] In some aspects, the present disclosure provides orthogonal mammalian immune cells (orthogonal immune cells) that have been recombinantly modified to express an orthogonal receptor. In some aspects, in the orthogonal immune cells, the immune cells are T cells. In some aspects, the T cells are naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, e.g., T H 1. T H 2. T H 9. T H 11. T H 22, T FH ;regulatory T cells, e.g. R 1. Tregs, inducible Tregs; memory T cells, such as central memory T cells, effector memory T cells, NK cells, tumor infiltrating lymphocytes (TILs), and engineered variants of such T cells, including but not limited to CAR-T cells, recombinantly modified TILs, and TCR-engineered cells.

[0027] In some embodiments, the present disclosure provides orthogonal Treg cells. The present invention further provides methods for inducing immunosuppression in a subject by administering a therapeutically effective amount of orthogonal Tregs in combination with administration of an orthogonal ligand sufficient to cause proliferation and / or activation of orthogonal Tregs in the subject. In some embodiments, the orthogonal Tregs optionally provide a targeting domain (e.g., an engineered TCR or CAR). In some embodiments, the orthogonal Tregs are orthogonal CAR-Tregs (oCAR-Tregs). In some embodiments, the ABD of the CAR of the oCAR-Tregs specifically binds human factor VIII. In some embodiments, the ABD of the CAR of the oCAR-Tregs specifically binds to an alloantigen. In some embodiments, the ABD of the CAR of the oCAR-Tregs specifically binds to HLA-A2. In some embodiments, the oCAR-Tregs are CD19-oCAR-Tregs. Orthogonal Tregs are useful in treating diseases associated with inflammatory diseases.

[0028] In some embodiments, the present disclosure provides orthogonal NK cells. The present invention further provides methods of treating a neoplastic disease in a subject by administering a therapeutically effective amount of orthogonal NK (oNK) cells in combination with administration of an orthogonal ligand sufficient to cause the proliferation and / or activation of orthogonal Tregs in the subject. In some embodiments, the oNK cells optionally provide a targeting domain (e.g., an engineered TCR or CAR). In some embodiments, the orthogonal NK cells are orthogonal CAR-NK cells (oCAR-NK cells).

[0029] In some embodiments, the present disclosure provides mammalian immune cells recombinantly modified to express an orthogonal CD122 polypeptide. In some embodiments, the present disclosure provides mammalian cells recombinantly modified to express an orthogonal receptor comprising the extracellular domain of orthogonal hCD122. In some embodiments, the present disclosure provides mammalian cells recombinantly modified to express an orthogonal receptor comprising the extracellular domain of CD122 comprising amino acid substitutions at positions H133 and Y134. In some embodiments, the present disclosure provides mammalian cells recombinantly modified to express an orthogonal hCD122 comprising amino acid substitutions at positions H133 and Y134. In some embodiments, the present disclosure provides mammalian cells recombinantly modified to express an orthogonal hCD122 comprising amino acid substitutions H133D and Y134F.

[0030] In some aspects, the present disclosure provides methods of using an orthogonal receptor and its cognate orthogonal ligand (ortholog) to induce a signal in a mammalian cell expressing the orthogonal receptor. In some aspects, the present disclosure provides a method of eliciting a response in a mammalian cell engineered to express the orthogonal receptor, comprising contacting the engineered mammalian cell expressing the orthogonal receptor with an effective amount of the cognate orthogonal ligand (ortholog), wherein the response elicited by such contact is activation of the engineered cell, maintenance of the activated state of the engineered cell, and / or proliferation of the engineered cell. In some aspects, the present disclosure provides a method of eliciting a response in a mammalian cell engineered to express the orthogonal receptor, comprising contacting the engineered mammalian cell expressing the orthogonal receptor with an effective amount of the cognate orthogonal ligand (ortholog), wherein the response elicited by such contact is increased STAT5 phosphorylation and / or STAT3 phosphorylation in the engineered cell.

[0031] In some aspects, the present disclosure provides methods of eliciting a response in mammalian immune cells engineered to express an orthogonal receptor, comprising contacting the engineered mammalian cells expressing the orthogonal receptor with an effective amount of a cognate orthogonal ligand (ortholog), wherein the contacting is performed ex vivo (in vitro), and the response elicited by such contacting is activation of the engineered cells, maintenance of an activated state of the engineered cells, and / or induction of proliferation of the engineered cells.

[0032] In some aspects, the disclosure provides methods for preparing an engineered immune cell product substantially enriched in engineered cells, the method comprising: (a) isolating a mixed population of immune cells from a subject; (b) transfecting a portion of the isolated population of immune cells with a recombinant vector capable of achieving expression of an orthogonal receptor in the transfected cells; and (c) culturing the mixed immune cell population in the presence of an orthogonal ligand that specifically binds to the ECD of the orthogonal receptor, such that cells expressing the orthogonal receptor selectively grow, resulting in enrichment of the cell population for cells expressing the orthogonal receptor.

[0033] In some aspects, the present disclosure provides methods of eliciting a response in mammalian immune cells engineered to express an orthogonal receptor, comprising contacting the engineered mammalian cells expressing the orthogonal receptor with an effective amount of a cognate orthogonal ligand (ortholog), wherein the response elicited by such contact is activation of the engineered cells, maintenance of an activated state of the engineered cells, and / or proliferation of the engineered cells.

[0034] In some aspects, the disclosure provides a method of eliciting a response in a mammalian cell expressing an orthogonal receptor, comprising contacting the mammalian cell expressing the orthogonal receptor ex vivo (in vitro) with a cognate orthogonal ligand (ortholog) in an amount sufficient to elicit a response.

[0035] In some aspects, the disclosure provides a method of eliciting a response in a mammalian cell expressing an orthogonal receptor, the method comprising contacting the mammalian cell expressing the orthogonal receptor in vivo with a cognate orthologous ligand in an amount sufficient to elicit the response.

[0036] In some aspects, the present disclosure provides methods of use comprising using a first ortholog (i.e., cognate ligand) ex vivo and a second ortholog in vivo. In some aspects, the present disclosure provides methods of use comprising using a first ortholog ex vivo and a second ortholog in vivo, wherein the first and second orthologs are the same or different orthologs. In some aspects, the present disclosure provides methods of use of an ortholog to cause proliferation of mammalian cells expressing the orthogonal receptor.

[0037] In some embodiments, the present disclosure provides methods of using orthologs to cause activation of mammalian cells expressing an orthogonal receptor. In some embodiments, the present disclosure provides methods of using orthologs ex vivo and / or in vivo to cause proliferation of mammalian cells expressing an orthogonal receptor. In some embodiments, the present disclosure provides methods of using IL2 orthologs to cause proliferation of mammalian cells expressing orthogonal CD122.

[0038] In some embodiments, the present disclosure provides methods of using an IL2 ortholog to cause activation of mammalian cells that express an orthogonal CD 122. In some embodiments, the present disclosure provides methods of using an ortholog to cause activation of mammalian cells that have been recombinantly engineered to express an orthogonal receptor comprising the extracellular domain of CD 122 containing amino acid substitutions at positions H133 and Y134.

[0039] In some embodiments, the present disclosure provides methods of using orthologs to cause activation of mammalian cells recombinantly modified to express an orthogonal CD122 comprising amino acid substitutions at positions H133 and Y134. In some embodiments, the present disclosure provides methods of using orthologs to cause activation of mammalian cells recombinantly modified to express an orthogonal CD122 comprising amino acid substitutions H133D and Y134F.

[0040] In some aspects, the present disclosure provides methods for the preparation of a population of cells enriched for mammalian cells recombinantly modified to express an orthogonal receptor. In some aspects, the present disclosure provides a population of mammalian cells enriched for mammalian cells recombinantly modified to express an orthogonal receptor.

[0041] In some embodiments, the present disclosure provides the use of an ortholog that is a cognate ligand for an orthogonal receptor. In some embodiments, the ortholog is an ortholog of Formula 1. In some embodiments, the ortholog is STK-007. In some embodiments, the ortholog is STK-009.

[0042] In some aspects, the present disclosure provides IL2 orthologs that exhibit reduced affinity for the unengineered intermediate-affinity (CD122 / CD132) or high-affinity (CD25 / CD122 / CD132) IL2 receptor complex and are also useful for selectively biasing the activity of orthologous IL2 toward cells that constitutively express CD25 (e.g., Tregs) or cells that inducibly express CD25 (e.g., activated CD8+ T cells). IL2 orthologs that have significantly reduced affinity for the extracellular domain (ECD) of native wild-type CD122 but retain binding to the ECD of CD25 can also be used as competitive antagonists of wild-type IL2 by interfering with the formation of the high-affinity IL2 receptor complex and, therefore, can be used in the treatment of autoimmune diseases or graft-versus-host (GVH) disease.

[0043] In some embodiments, the present disclosure provides IL2 orthologs that specifically and selectively bind to the extracellular domain (ECD) of a transmembrane polypeptide comprising a modified CD122 polypeptide (orthogonal CD122). Binding of an IL2 ortholog to orthogonal CD122 participates in intracellular signaling transduction pathways, resulting in activation of the natural intracellular signaling pattern associated with IL2 binding to either a medium- or high-affinity IL2 receptor, but exhibits selectivity for engineered cells expressing orthogonal CD122. The IL2 ortholog can exhibit significantly reduced binding to the extracellular domain of wild-type CD122 compared to the binding of the IL2 orthogonal receptor, either alone or when CD122 is present in the form of an endogenous high- or medium-affinity IL2 receptor. In some embodiments, the affinity of the IL2 ortholog for the extracellular domain of orthogonal CD122 is equivalent to the affinity of wild-type IL2 for wild-type CD122. In some embodiments, the affinity of the IL2 ortholog for the extracellular domain of orthogonal CD122 is greater than the affinity of wild-type IL2 for the extracellular domain of wild-type CD122. In some embodiments, the affinity of the IL2 ortholog for the extracellular domain of orthogonal CD122 is less than the affinity of wild-type IL2 for the extracellular domain of wild-type CD122.

[0044] The present disclosure further provides methods for producing the IL2 orthologs of the present invention. In particular, the present disclosure provides recombinant expression vectors comprising a nucleic acid sequence encoding an IL2 ortholog operably linked to control elements to provide for expression of the nucleic acid sequence encoding the IL2 ortholog in a host cell.

[0045] The present invention further provides engineered mammalian cells that express orthogonal receptors. The present invention further provides pharmaceutically acceptable formulations of engineered mammalian cells that express orthogonal receptors.

[0046] The present disclosure further provides a method of making a pharmaceutically acceptable dosage form of an engineered cell therapy product, wherein the dosage form comprises a population of T cells, wherein the population of T cells is substantially enriched for one or more species of engineered T cells, and the engineered T cells express a receptor comprising an extracellular domain of a CD122 orthogonal polypeptide, the method comprising ex vivo culturing the population of T cells, comprising engineered T cells that express a receptor comprising an extracellular domain of a CD122 orthogonal polypeptide, in the presence of an IL2 ortholog of the invention, for a period of time sufficient to enrich the cell population for one or more such engineered T cells.

[0047] In some embodiments, a recombinant vector comprising a nucleic acid sequence encoding an IL2 ortholog described herein operably linked to regulatory elements to facilitate expression and secretion of the IL2 ortholog from mammalian cells is administered to a subject to provide in situ expression of the IL2 ortholog. In some embodiments, the recombinant vector is administered intratumorally to a subject suffering from cancer. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) or recombinant adenovirus (rAd), e.g., in some embodiments, a replication-deficient adenovirus derived from human adenovirus serotype 3 and / or 5. In some embodiments, the replication-deficient adenovirus has one or more modifications to the E1 region that disrupt the virus's ability to initiate the cell cycle and / or apoptotic pathway. Replication-deficient adenoviral vectors can optionally contain deletions in the E3 domain. In some embodiments, the adenovirus is a replication-competent adenovirus. In some embodiments, the adenovirus is a replication-competent recombinant virus that has been engineered to selectively replicate in neoplastic cells.

[0048] The present disclosure further provides methods for preparing a pharmaceutically acceptable dosage form of a cell therapy product comprising at least one (alternatively, two, three, four, or more) engineered T cells expressing a transmembrane receptor protein, wherein the extracellular domain of such transmembrane receptor protein comprises the extracellular domain of a CD122 orthogonal polypeptide, and wherein the fraction of engineered cells in the cell therapy product comprises at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90% of the total number of cells in the cell therapy product.

[0049] In some embodiments, a therapeutic method is provided that includes introducing a population of cells into a subject in need thereof, the cell population comprising engineered human immune cells comprising a nucleic acid sequence encoding a transmembrane polypeptide comprising an ECD of human orthogonal CD122, a transmembrane domain, and an intracellular signaling domain that provides an intracellular signal in response to binding of a ligand to the ECD of the transmembrane polypeptide, the nucleic acid sequence being operably linked to expression control elements to facilitate transcription, translation, and cell surface presentation of the transmembrane polypeptide. Such a cell population may comprise cells that have been modified ex vivo and are autologous or allogeneic with respect to the subject. In some embodiments, the therapeutic method includes the steps of: (1) contacting the cell population ex vivo with an amount of a cognate IL2 ortholog at a concentration and for a period of time sufficient to activate the engineered cells that contain a receptor comprising an extracellular domain that is the orthogonal CD122 ECD; and (2) administering the cell population to the subject; and (3) administering the cognate IL2 ortholog in vivo following administration of the engineered cells. In some embodiments, the transduced cell population is contacted in vivo with cognate orthogonal cytokines following administration of the engineered cells.

[0050] The present disclosure further provides a method for extending the in vivo active form ("persistence") of orthogonal engineered immune cells (e.g., orthogonal CAR-T cells) in a mammalian subject by administering to the subject an effective amount of an orthogonal ligand.

[0051] The present disclosure further provides methods for specifically and selectively activating and / or inducing in vivo expansion of orthogonal immune cells (e.g., orthogonal CAR-T cells) in a mammalian subject by administering to the mammalian subject an effective amount of orthogonal immune cells in conjunction with administration of an effective amount of an orthogonal ligand.

[0052] The present disclosure further provides a method of treating a mammalian subject suffering from a neoplastic disease by administering to the mammalian subject an effective amount of orthogonal CAR-T cells in combination with administration of an effective amount of a cognate orthogonal ligand for a receptor expressed on the orthogonal CAR-T cells.

[0053] The present disclosure further provides a method of treating a mammalian subject suffering from a diffuse (non-solid tumor) neoplastic disease by administering to the mammalian subject an effective amount of orthogonal CAR-T cells in conjunction with administration of an effective amount of a cognate orthogonal ligand for a receptor expressed on the orthogonal CAR-T cells.

[0054] The present disclosure further provides methods of treating a mammalian subject suffering from a neoplastic disease characterized by the presence of a solid tumor (e.g., lymphoma, prostate cancer, lung cancer, bladder cancer, HPV-associated cancers such as cervical cancer, neuroblastoma) by administering to the mammalian subject an effective amount of orthogonal T cells in conjunction with administration of an effective amount of a cognate orthogonal ligand for a receptor expressed on the orthogonal T cells.

[0055] The present disclosure further provides a method of restoring the activity of depleted orthogonal immune cells in a subject by administering to the subject an effective amount of an orthogonal ligand.

[0056] The disclosure further provides methods of treating a mammalian subject suffering from a recurrence of a neoplastic disease in a treatment regimen characterized by the administration of an orthogonal CAR-T cell product, comprising: (i) administering to the subject an effective amount of a cognate orthogonal ligand for a receptor expressed on the previously administered orthogonal CAR-T cells sufficient to restore the activity of the previously administered orthogonal CAR-T cells; (ii) periodically administering to the subject an effective amount of a cognate orthogonal ligand for a receptor expressed on the previously administered orthogonal CAR-T cells for a period sufficient to maintain the activity of the orthogonal CAR-T cells; and (iii) evaluating the subject for the presence and absence of neoplastic disease and discontinuing administration of the orthogonal ligand or continuing to periodically administer the orthogonal ligand according to a maintenance dosing protocol sufficient to maintain a quantity of orthogonal CAR-T cells sufficient for immune surveillance of the neoplastic cells.

[0057] The present disclosure further provides methods of preventing and / or treating relapse in the treatment of neoplastic diseases with CAR-T cell therapy by administering orthogonal CAR-T cells, followed by periodic administration of an effective amount of a cognate orthogonal ligand for a receptor expressed on the orthogonal CAR-T cells in a maintenance dosing protocol sufficient to maintain a quantity of orthogonal CAR-T cells sufficient for immune surveillance of neoplastic cells.

[0058] The disclosure further provides methods of treating a mammalian subject suffering from a neoplastic disease that has relapsed or is refractory to a treatment regimen characterized by the prior administration of an orthogonal CAR-T cell product, comprising: (i) administering to the subject an effective amount of a cognate orthogonal ligand for a receptor expressed on the previously administered orthogonal CAR-T cells sufficient to restore the activity of the previously administered orthogonal CAR-T cells; (ii) periodically administering to the subject an effective amount of a cognate orthogonal ligand for a receptor expressed on the previously administered orthogonal CAR-T cells for a period sufficient to maintain the activity of the orthogonal CAR-T cells to achieve a therapeutic response; and (iii) evaluating the subject for the presence and absence of neoplastic disease and discontinuing administration of the orthogonal ligand or continuing to periodically administer the orthogonal ligand according to a maintenance dosing protocol sufficient to maintain a quantity of orthogonal CAR-T cells sufficient for immune surveillance of the neoplastic cells.

[0059] The present disclosure further provides a method for treating a mammalian subject suffering from a neoplastic disease with orthogonal hCD122 cells and an orthogonal hIL2 ligand therefor, comprising the steps of: (i) administering to the subject a therapeutically effective dose of an orthogonal ligand for a receptor expressed on an orthogonal CAR-T within a period of 2 weeks to 1 day prior to administration of the orthogonal hCD122 cells (i.e., priming); (ii) administering to the subject an orthogonal hCD122 cell in combination with an orthogonal ligand, the orthogonal hCD122 cell being a cognate hIL2 ligand for the orthogonal CD122 receptor of the orthogonal hCD122 cells; and optionally (iii) evaluating the subject for the presence of neoplastic disease and absence of evidence of neoplastic disease and discontinuing administration of the orthogonal ligand or continuing to administer the orthogonal ligand thereafter at a dose sufficient to maintain a level of circulating orthogonal CAR-T cells sufficient to maintain immune surveillance of the neoplastic cells, and optionally, in the event of relapse, administering a therapeutically effective amount of an orthogonal ligand that is a cognate hIL2 ligand for the orthogonal CD122 receptor of the previously administered orthogonal hCD122 cells.

[0060] In another aspect, the present disclosure provides a method for treating chronic viral infections by administering orthogonal cells containing a targeting domain specific to an antigen expressed on the surface of virally infected cells. Examples of chronic viral infections that can be treated with the compositions and methods of the present disclosure include, but are not limited to, cytomegalovirus (CMV), HTLV1, herpes simplex virus type 2 (HSV-2), Epstein-Barr virus, human herpesvirus 6, human herpesvirus 7, hepatitis C virus (HCV), and human immunodeficiency viruses (HIV1 and HIV2).

[0061] In some embodiments, the engineered T cells are genomically modified to eliminate checkpoint expression (i.e., checkpoint knockout). Examples of such checkpoint knockout cells include PD1 knockout ("PD1KO") T cells. Strategies for generating PD1KO T cells are well known in the art. PD1KO T cells are well known in the art. McGowan et al. (121PD1 Disrupted Car-T Cells In The Treatment of Solid Tumors: Promises and Challenges) Biomedicine and Pharmacotherapy Biomedicine & Pharmacotherapy, Volume 121 (2020, available online 13 NOV 2019) 109625, https: / / doi.org / 10.1016 / j.biopha.2019.109625) provides an extensive review of engineered and isolated PD1KO T cells that have been investigated in various clinical applications (see Table 1).

[0062] As an alternative to completely eliminating PD1 function in orthogonal immune cells, in some embodiments, the orthogonal immune cells of the present disclosure provide a mechanism for downregulating PD1 activity in orthogonal immune cells. Expression of PDL1 on tumor cells and chronically virally infected cells allows such cells to evade immune surveillance by binding to PD1 expressed on human immune cells. While several groups have demonstrated that engineered T cells that knock out PD1 expression are effective, these cells are also promiscuous and prone to significant target toxicity, typically mediated by the self-tolerance mechanism of PD1 / PDL1 interaction, leading to significant toxicity. In some embodiments, downregulation is conditional on the response to signaling initiated by immune cell binding to its target, whether engineered cells express a specific targeting / activation domain such as a CAR (e.g., oCAR-T cells, oCAR NK cells, oTCR-engineered T cells) or engineered cells expressing an endogenous binding domain (e.g., ortho-TILs). Factors upregulated by immune cell binding can be used to selectively downregulate PD1 expression control sequences in orthogonal immune cells, resulting in downregulation of PD1 expression only when the orthogonal cells interact with their appropriate target. In some embodiments, domains can be engineered in the CAR ICD such that binding of the CAR to a target antigen results in intracellular signaling that suppresses PD1 expression after interaction of the targeting domain with the target, thereby enabling activated orthogonal immune cells to prevent downregulation, and potentially immune escape, caused by interaction of PD1 with cells expressing PDL1, such as chronically virally infected cells or tumor cells. In addition to those described above, other mechanisms for reducing PDL1 immunomodulation of engineered immune cells can be incorporated into the orthogonal immune cells of the present disclosure.See, for example, U.S. Patent Application Publication US2020 / 0407694A1 to Busser et al., published December 31, 2020, and U.S. Patent Application Publication US20180327470A1 to Li et al., published November 15, 2018.

[0063] As discussed above, a significant challenge with current cell therapy is the lack of a non-toxic means to support the growth of transfected cells after they have been administered to a patient.

[0064] The present disclosure provides a human orthogonal cell, which is a mammalian immune cell comprising a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more expression control elements such that the mammalian immune cell expresses the orthogonal hCD122 receptor.

[0065] The present disclosure provides a mammalian immune cell comprising: (a) a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more expression control elements such that the mammalian immune cell expresses the orthogonal hCD122 receptor; and (b) a nucleic acid sequence encoding a chimeric antigen receptor (CAR) operably linked to one or more expression control elements such that the mammalian immune cell expresses the CAR.

[0066] The present disclosure provides a recombinant expression vector comprising: (a) a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more expression control elements such that mammalian immune cells express the orthogonal hCD122 receptor; and (b) a nucleic acid sequence encoding a chimeric antigen receptor (CAR) operably linked to one or more expression control elements such that mammalian immune cells express the CAR.

[0067] The present disclosure provides methods of treating neoplastic disease by administering mammalian immune cells that contain a nucleic acid sequence encoding an orthogonal CD122 receptor.

[0068] The present disclosure provides methods of treating neoplastic disease in a human subject, comprising administering mammalian immune cells comprising a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more expression control elements such that the mammalian immune cells express the orthogonal hCD122 receptor, and administering an orthogonal ligand that binds to the ECD of the hCD122 receptor and results in intracellular signaling. In some embodiments, the ligand is administered prior to administration of the mammalian immune cells.

[0069] The present invention addresses a significant problem with current cell therapies, which is the lack of a non-toxic means to support the growth of transfected cells after they are administered to a patient.

[0070] The present disclosure provides a human orthogonal cell, which is a mammalian immune cell comprising a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more expression control elements such that the mammalian immune cell expresses the orthogonal hCD122 receptor.

[0071] The present disclosure provides a mammalian immune cell comprising: (a) a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more expression control elements such that the mammalian immune cell expresses the orthogonal hCD122 receptor; and (b) a nucleic acid sequence encoding a chimeric antigen receptor (CAR) operably linked to one or more expression control elements such that the mammalian immune cell expresses the CAR.

[0072] The present disclosure provides a recombinant expression vector comprising: (a) a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more expression control elements such that mammalian immune cells express the orthogonal hCD122 receptor; and (b) a nucleic acid sequence encoding a chimeric antigen receptor (CAR) operably linked to one or more expression control elements such that mammalian immune cells express the CAR.

[0073] The present disclosure provides methods of treating neoplastic disease by administering mammalian immune cells that contain a nucleic acid sequence encoding an orthogonal CD122 receptor.

[0074] The present disclosure provides methods of treating neoplastic disease in a human subject, comprising administering mammalian immune cells comprising a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more expression control elements such that the mammalian immune cells express the orthogonal hCD122 receptor, and administering an orthogonal ligand that binds to the ECD of the hCD122 receptor and results in intracellular signaling. In some embodiments, the ligand is administered prior to administration of the mammalian immune cells.

[0075] In some embodiments, there is provided a method of treating or preventing a disease, disorder, or condition in a mammalian subject in need thereof, comprising the steps of: (a) isolating a quantity of immune cells from said subject; (b) contacting the isolated amount of isolated immune cells with a nucleic acid sequence under conditions such that the nucleic acid sequence is taken up by the isolated immune cells, wherein the nucleic acid sequence encodes a transmembrane receptor, the transmembrane receptor comprising an intracellular signaling domain in functional communication with an extracellular domain, the extracellular domain of the receptor comprising an ECD of orthogonal hCD122 or a functional fragment thereof; (c) contacting the isolated quantity of cells from step (b) ex vivo with an orthogonal ligand in an amount sufficient to induce proliferation of the cells transduced by the contacting of step (b), wherein said contacting is applied for a period of time such that the transduced cells constitute at least 20% of the cells of the population; (d) administering to said mammalian subject a therapeutically effective amount of cells of the cell population produced from step (c) in combination with administration of a therapeutically effective dose of an orthogonal ligand.

[0076] In some embodiments, the population comprises one or more species of human immune cells selected from the group consisting of bone marrow cells, lymphocytes, peripheral blood mononuclear cells (PBMCs), tumor infiltrating lymphocytes (TILs), T cells, CD8+ T cells, CD25+CD8+ T cells, CAR-T cells, NK cells, CD4+ T cells, and Tregs.

[0077] In some embodiments, after step (a) but before step (b), the population of cells is manipulated ex vivo to enrich the population for activated immune cells or antigen-experienced T cells.

[0078] In some embodiments, the orthogonal hCD122 or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at positions 133 and / or 134, numbered according to wild-type hCD122.

[0079] In some embodiments, the contacting in step (b) further comprises incorporation of a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

[0080] In some embodiments, the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the receptor are provided on separate vectors, and each nucleic acid sequence is operably linked to an expression control sequence functional in a mammalian immune cell.

[0081] In some embodiments, the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the receptor are provided on a single vector.

[0082] In some embodiments, the nucleic acid sequences are operably linked to the same expression control elements.

[0083] In some embodiments, the vector comprises two nucleic acid sequences separated by an IRES element of the T2A coding sequence.

[0084] In some embodiments, the vector is a viral vector.

[0085] In some embodiments, the vector is a lentiviral or retroviral vector.

[0086] In some embodiments, the orthogonal ligand used ex vivo in step (b) is different from the orthogonal ligand used in vivo in step (c).

[0087] In some embodiments, prior to step (d), the subject is treated with a lymphodepleting regimen.

[0088] In some embodiments, the initial dose administered in step (d) is between 100,000 and 1,000,000 activated immune cells per kg of subject body weight.

[0089] In some embodiments, the orthogonal ligand is administered to the subject periodically over a period of at least two weeks to maintain a level of 100,000 to 1,000,000 activated immune cells per kg of the subject's body weight.

[0090] In some embodiments, the orthogonal ligand is administered to a point where there is no substantial evidence of residual tumor, at which time the dose of the orthogonal ligand is reduced to a level sufficient to maintain a low circulating level of approximately 10,000 to 100,000 orthogonal immune cells per kg of body weight over a period of at least 3 months of observation.

[0091] In some embodiments, the orthogonal ligand is administered to a point where there is no substantial evidence of residual tumor, at which time the dose of the orthogonal ligand is terminated.

[0092] In some embodiments, once a patient relapses from the initial course of immune cell therapy, the method further comprises administering to the relapsing patient a therapeutically effective amount of an orthogonal ligand that induces activation and / or proliferation of the previously administered orthogonal cells in the absence of an additional dose of orthogonal engineered cells, wherein the orthogonal ligand is administered to the subject for a period of time until remission of the relapsed tumor is observed.

[0093] In some embodiments, the disease, disorder or condition is a neoplastic disease.

[0094] In some embodiments, the disease, disorder or condition is a chronic viral disease.

[0095] In some embodiments, the disease, disorder or condition is an inflammatory disease.

[0096] Also provided is a cell product substantially enriched for a population of activated orthogonal immune cells obtained by a process comprising the steps of: (a) isolating a quantity of immune cells from a mammalian subject; (b) contacting the isolated amount of isolated immune cells with a nucleic acid sequence under conditions such that the nucleic acid sequence is taken up by the isolated immune cells, wherein the nucleic acid sequence encodes a transmembrane receptor, the transmembrane receptor comprising an intracellular signaling domain in functional communication with an extracellular domain, the extracellular domain of the receptor comprising an ECD of orthogonal hCD122 or a functional fragment thereof; (c) contacting the isolated quantity of cells from step (b) ex vivo with an orthogonal ligand in an amount sufficient to induce proliferation of the cells transduced by the contacting of step (b), wherein the contacting is applied for a period of time such that the transduced cells constitute at least 20% of the cells of the population.

[0097] In some embodiments, the cell product comprises one or more species of human immune cells selected from the group consisting of bone marrow cells, lymphocytes, peripheral blood mononuclear cells (PBMCs), tumor infiltrating lymphocytes (TILs), T cells, CD8+ T cells, CD25+CD8+ T cells, CAR-T cells, NK cells, CD4+ T cells, and Tregs.

[0098] In some embodiments, the cell product is further engineered to delete an endogenous TCR domain of said cell. [The present invention 1001] 1. A method for treating or preventing a disease, disorder or condition in a mammalian subject in need thereof, comprising: (a) isolating a quantity of immune cells from said subject; (b) contacting the isolated amount of isolated immune cells with a nucleic acid sequence under conditions such that the nucleic acid sequence is taken up by the isolated immune cells, wherein the nucleic acid sequence encodes a transmembrane receptor, the transmembrane receptor comprising an intracellular signaling domain in functional communication with an extracellular domain, the extracellular domain of the receptor comprising an ECD of orthogonal hCD122 or a functional fragment thereof; (c) contacting the isolated quantity of cells from step (b) ex vivo with an orthogonal ligand in an amount sufficient to induce proliferation of the cells transduced by the contacting of step (b), wherein said contacting is applied for a period of time such that the transduced cells constitute at least 20% of the cells of the population; (d) administering to said mammalian subject a therapeutically effective amount of cells of the cell population produced from step (c) in combination with administration of a therapeutically effective dose of an orthogonal ligand. [The present invention 1002] 1001. The method of claim 1001, wherein said population comprises one or more species of human immune cells selected from the group consisting of bone marrow cells, lymphocytes, peripheral blood mononuclear cells (PBMCs), tumor infiltrating lymphocytes (TILs), T cells, CD8+ T cells, CD25+CD8+ T cells, CAR-T cells, NK cells, CD4+ T cells, and Tregs. [The present invention 1003] The method of claim 1001, wherein after step (a) but before step (b), the population of cells is manipulated ex vivo to enrich the population for activated immune cells or antigen-experienced T cells. [The present invention 1004] 1001. The method of claim 1001, wherein the orthogonal hCD122 or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at positions 133 and / or 134, numbered according to wild-type hCD122. [The present invention 1005] The method of any of claims 1001 to 1004, wherein the contacting in step (b) further comprises incorporation of a nucleic acid sequence encoding a chimeric antigen receptor (CAR). [The present invention 1006] The method of claim 1005, wherein the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding said receptor are provided on separate vectors, and each nucleic acid sequence is operably linked to an expression control sequence functional in a mammalian immune cell. [The present invention 1007] 1005. The method of claim 1005, wherein the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding said receptor are provided on a single vector. [The present invention 1008] 1007. The method of claim 1007, wherein said nucleic acid sequences are operably linked to the same expression control element. [The present invention 1009] 1008. The method of claim 8, wherein the vector comprises said two nucleic acid sequences separated by an IRES element in the T2A coding sequence. [The present invention 1010] 1009. The method of claim 10, wherein the vector is a viral vector. [The present invention 1011] The method of claim 1010, wherein the vector is a lentiviral vector or a retroviral vector. [The present invention 1012] 1012. The method of any of claims 1001 to 1011, wherein the orthogonal ligand used ex vivo in step (b) is different from the orthogonal ligand used in vivo in step (c). [The present invention 1013] The method of any of claims 1001 to 1012, wherein prior to step (d), the subject is treated with a lymphodepleting regimen. [The present invention 1014] The method of any of claims 1001 to 1013, wherein the initial dose administered in step (d) is 100,000 to 1,000,000 activated immune cells per kg of subject body weight. [The present invention 1015] Any of the methods of inventions 1001 to 1014, wherein the orthogonal ligand is administered to the subject periodically over a period of at least two weeks to maintain a level of 100,000 to 1,000,000 activated immune cells per kg of the subject's body weight. [The present invention 1016] Any of the methods of inventions 1001-1015, wherein the orthogonal ligand is administered to a point where there is no substantial evidence of residual tumor, at which time the dose of the orthogonal ligand is reduced to a level sufficient to maintain a low circulating level of approximately 10,000-100,000 orthogonal immune cells per kg of body weight over a period of at least 3 months after observation. [The present invention 1017] The method of any of claims 1001 to 1015, wherein the orthogonal ligand is administered to a point where there is no substantial evidence of residual tumor, at which time the dose of the orthogonal ligand is terminated. [The present invention 1018] The method of claim 1017, further comprising administering to the relapsing patient, if the patient relapses from the initial course of immune cell therapy, a therapeutically effective amount of an orthogonal ligand that induces activation and / or proliferation of the previously administered orthogonal cells in the absence of an additional dose of orthogonal engineered cells, wherein the orthogonal ligand is administered to the subject over a period of time until remission of the relapsed tumor is observed. [The present invention 1019] The method of any of claims 1001 to 1018, wherein the disease, disorder or condition is a neoplastic disease. [The present invention 1020] The method of any of claims 1001 to 1019, wherein the disease, disorder or condition is a chronic viral disease. [The present invention 1021] The method of any of claims 1001 to 1019, wherein the disease, disorder or condition is an inflammatory disease. [The present invention 1022] 1. A cell product substantially enriched for a population of activated orthogonal immune cells, obtained by a process comprising the steps of: (a) isolating a quantity of immune cells from a mammalian subject; (b) contacting the isolated amount of isolated immune cells with a nucleic acid sequence under conditions such that the nucleic acid sequence is taken up by the isolated immune cells, wherein the nucleic acid sequence encodes a transmembrane receptor, the transmembrane receptor comprising an intracellular signaling domain in functional communication with an extracellular domain, the extracellular domain of the receptor comprising an ECD of orthogonal hCD122 or a functional fragment thereof; (c) contacting the isolated quantity of cells from step (b) ex vivo with an orthogonal ligand in an amount sufficient to induce proliferation of the cells transduced by the contacting of step (b), wherein the contacting is applied for a period of time such that the transduced cells constitute at least 20% of the cells of the population. [The present invention 1023] The cell product of the present invention 1022, comprising one or more species of human immune cells selected from the group consisting of bone marrow cells, lymphocytes, peripheral blood mononuclear cells (PBMCs), tumor infiltrating lymphocytes (TILs), T cells, CD8+ T cells, CD25+CD8+ T cells, CAR-T cells, NK cells, CD4+ T cells, and Tregs. [The present invention 1024] The composition of claim 1023, wherein the cell product is further engineered to delete an endogenous TCR domain of said cell. [Brief explanation of the drawings]

[0099] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, the dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures:

[0100] [Figure 1] Figure 1 of the accompanying figures provides Celltiterglo values ​​for NKL cells treated with 293T transfection supernatants from the experiment described in Example 6. Duplicate Celltiterglo values ​​are shown in parallel columns for NKL cells that received the indicated dilutions of each supernatant indicated in bold. [Figure 2] Figure 2 of the accompanying figures provides Celltiterglo values ​​for NKL hoRB cells treated with 293T transfection supernatants from the experiment described in Example 6. Duplicate Celltiterglo values ​​are shown in parallel columns for NKL hoRB cells that received the indicated dilutions of each supernatant indicated in bold. [Figure 3] Figures 3A-B provide the results of the disseminated in vivo RAJI tumor study, described in more detail in Example 7 below, demonstrating the effect of treatment with CAR-T orthogonal cells together with an orthogonal ligand compared to CAR-T or PBS alone. [Figure 4] Figures 4A-B provide the results of a rechallenge study, described in more detail in Example 8 below, demonstrating that administration of the orthogonal ligand alone (without the need to provide additional cells) can restore the anti-tumor activity of CAR T cells even in the absence of prolonged antigen or tumor-ligand exposure. [Figure 5] Figures 5A-B provide the results of rechallenge in a subcutaneous RAJI-luc lymphoma relapse mouse model, described in more detail in Example 8. The data demonstrate that administration of STK-009 alone can achieve CAR-T cell anti-tumor activity in animals that have relapsed from a prior course of treatment. [Figure 6]Figure 6 is a histogram of FACS analysis results from data described in more detail in Example 8, demonstrating that an orthogonal ligand (STK-009) can expand orthogonal CAR-T cells and preserve stem cell memory CAR-T cell populations. [Figure 7] Figures 7A-C provide caliper measurement data (relating to the efficacy of orthogonal CAR-T cells in combination with an orthogonal ligand in in vivo treatment of a solid tumor model) generated from the subcutaneous Raji solid tumor model, which is described in more detail in Example 11 below. [Figure 8] Figure 8 provides a graphical representation of physical measurements of tumor response data generated from the experiments described in Example 11 herein, demonstrating that administration of CD19 orthogonal CAR-T cells in combination with an orthogonal ligand (STK-009) is effective in treating solid tumors. [Figure 9] Figure 9 provides Kaplan-Meier survival plots for the subcutaneous solid tumor model study described in Example 8. The data provided demonstrate that administration of CD19 orthogonal CAR-T cells in combination with an orthogonal ligand (STK-009) is effective in treating solid tumors and confers a significant survival advantage. [Figure 10] FIG. 10 provides immunohistochemistry of tissues isolated from mice sacrificed after completion of the subcutaneous solid tumor model study described in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0101] Detailed Description Introduction In order that this disclosure may be more readily understood, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be read in light of the knowledge known to those of ordinary skill in the art.

[0102] Before the present methods and compositions are described, it is to be understood that this disclosure is not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0103] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the lower limit, unless the context clearly dictates otherwise, is also specifically disclosed. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range, and each range in which one, neither, or both of the upper and lower limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of the upper and lower limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Any method and material similar or equivalent to those described herein can be used to carry out or test the present invention, but some promising and preferred methods and materials are described herein.All publications mentioned herein are incorporated by reference in order to disclose and describe the method and / or material cited together with this publication.

[0105] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g., polypeptides, known to those skilled in the art, and so forth.

[0106] 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 publication 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.

[0107] 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 publication 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.

[0108] Unless otherwise indicated, the following abbreviations are used herein: parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric. Standard abbreviations are used, including: bp = base pairs; kb = kilobase; pl = picoliter; s or sec = seconds; min = minutes; h or hr = hours; 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 = micromolar; mM = millimolar. Molarity; M = molar concentration; kDa = kilodaltons; im = intramuscular (into the muscle); ip = intraperitoneal (into the peritoneal cavity); SC or SQ = subcutaneous (under the skin); QD = daily; BID = twice daily; QW = weekly; QM = 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.

[0109] It will be appreciated that throughout this disclosure, amino acids will be referred to according to their single-letter or three-letter code. For the convenience of the reader, the single-letter and three-letter amino acid codes are provided in Table 1 below:

[0110] (Table 1) Amino acid abbreviations TIFF2026041861000002.tif96128

[0111] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis 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, Vols. 1-2, John Wiley and Sons, Inc., NY).

[0112] B. Definition Unless otherwise indicated, the following terms are intended to have the meanings indicated below. Other terms are defined elsewhere throughout the specification.

[0113] ActivateAs used herein, the term "activate," with respect to a receptor or receptor complex, refers to a biological effect that results from the binding of an agonist ligand to the receptor in response to the binding of the ligand, either directly and / or through participation in a multicomponent signaling cascade. For example, the binding of an IL2 agonist (IL2 agonist ligand) to the IL2 receptor can be said to "activate" receptor signaling to produce one or more intracellular biological effects (e.g., phosphorylation of STAT5).

[0114] activity As used herein, the term "activity" is used to describe the properties of a molecule in a material or cellular test system (e.g., assay) or in terms of biological or chemical properties (e.g., the degree of binding of one molecule to another) or physical properties (e.g., alteration of cell membrane potential). Examples of such biological functions include, but are not limited to, the catalytic activity of a biopharmaceutical, intracellular signaling, gene expression, the ability to stimulate cell proliferation, and the ability to modulate immunological activities such as inflammatory responses. "Activity" is typically expressed as the level of biological activity per unit of the tested agent, for example, [catalytic activity] / [mg protein], [immunological activity] / [mg protein], International Units of activity (IU), [STAT5 phosphorylation] / [mg protein], [T-cell proliferation] / [mg protein], plaque-forming units (pfu), etc. As used herein, the term "proliferative activity" refers to the activity of promoting cell growth and replication, including uncontrolled cell division, as observed in neoplastic diseases, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis, and angiogenesis.

[0115] Administer / AdministerThe terms "administration" and "administering" are used interchangeably herein and refer to the act of contacting a subject (including a cell, tissue, organ, or biological fluid of a subject 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 may be achieved through any of a variety of art-recognized methods, including, but not limited to, topical administration, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal, transmucosal, iontophoretic delivery, intralymphatic injection, intragastric injection, intraprostatic injection, intravesical instillation (e.g., bladder), inhalation (e.g., respiratory inhaler, including dry powder inhaler), intraocular injection, intraabdominal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intracerebroventricular injection (ICVI), etc. The term "administration" includes contact of the agent with a cell, tissue, or organ as well as contact of the agent with a fluid (where the fluid is in contact with the cell, tissue, or organ).

[0116] Adverse events As used herein, the term "adverse event" refers to any undesirable experience associated with the use of a therapeutic or preventive agent in a subject. Adverse events do not necessarily have to be caused by the administration of a therapeutic or preventive agent (e.g., IL2 ortholog), and may arise from unrelated circumstances. Adverse events are typically classified as mild, moderate, or severe. As used herein, the classification of adverse events as used herein follows the Common Terminology Criteria for Adverse Events v5.0 (CTCAE), published by the US Department of Health and Human Services, the US National Institutes of Health, and the US National Cancer Institute on November 27, 2017.

[0117] affinityAs used herein, the term "affinity" refers to the degree of specific binding of a first molecule (e.g., a ligand) to a second molecule (e.g., a receptor), and is expressed as K d (the dissociation constant (K off ) and the binding constant (K on The binding kinetics is measured by the ratio of the binding activity of the ATP to the binding activity of the ATP.

[0118] AgonistsAs used herein, the term "agonist" refers to a first agent that specifically binds to a second agent ("target") and interacts with the target, causing or promoting increased activation of the target. In some examples, an agonist is an activator of a receptor protein that can modulate cell activation, enhance activation, sensitize cells to activation by a second agent, or upregulate the expression of one or more genes, proteins, ligands, receptors, biological pathways, resulting in pathways that lead to cell proliferation or cell death, such as by cell cycle arrest or apoptosis. In some embodiments, an agonist is an agent that binds to a receptor and changes the state of the receptor, resulting in a biological response. The response mimics the action of the receptor's endogenous activator. The term "agonist" includes partial agonists, full agonists, and superagonists. An agonist can be described as a "full agonist" if it induces substantially the full biological response (i.e., the response associated with the naturally occurring ligand / receptor binding interaction) induced by the receptor or partial agonist under study. In contrast to an agonist, an antagonist can specifically bind to a receptor but does not typically induce the signal cascade initiated by the receptor, and can alter the action of an agonist at that receptor. An inverse agonist is an agent that produces a pharmacological response opposite in direction to that of an agonist. A "superagonist" is a type of agonist that can generate a maximum response greater than that of the endogenous agonist at the target receptor, and thus has more than 100% activity of the natural ligand. A superagonist is typically a synthetic molecule that exhibits a response of greater than 110%, or greater than 120%, or greater than 130%, or greater than 140%, or greater than 150%, or greater than 160%, or greater than 170% of an appreciable quantitative or qualitative parameter of the naturally occurring form of the molecule when evaluated at similar concentrations in equivalent assays. It should be noted that the biological effects associated with a full agonist may differ in extent and / or type from the biological effects of a partial agonist or superagonist.

[0119] Antagonist As used herein, the term "antagonist" or "inhibitor" refers to a molecule that opposes the action of an agonist.An antagonist blocks, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also block, inhibit, or reduce the constitutive activity of a target, for example, a target receptor, even in the absence of a specified agonist.An inhibitor is, for example, a molecule that reduces, blocks, blocks, delays activation, inactivates, desensitizes, or downregulates a gene, protein, ligand, receptor, biological pathway, including an immune checkpoint pathway, or cell.

[0120] antibody As 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) that specifically bind to a target molecule, and (b) IgG(1-4) delta C that compete with the immunoglobulin from which it is derived for binding to the target molecule. H 2, F(ab')2, Fab, ScFv, V H , V LThe term "antibody" refers to immunoglobulin derivatives, including, but not limited to, tetrabodies, triabodies, diabodies, dsFv, F(ab')3, scFv-Fc, and (scFv)2. The term antibody is not limited to immunoglobulins derived from any particular mammalian species, and includes murine, hominid, equine, camelid, and human antibodies. The term antibody includes so-called "heavy chain antibodies" or "VHHs" or "Nanobodies®," such as those typically obtained from immunization of camelids (including camels, llamas, and alpacas) (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448). Antibodies of a given specificity can also be derived from non-mammalian sources, such as VHHs obtained from immunization of cartilaginous fish, including, but not limited to, sharks. The term "antibody" encompasses antibodies isolable from natural sources or from animals after immunization with an antigen, as well as monoclonal, bispecific, trispecific, chimeric, humanized, human, CDR-grafted, veneered, or deimmunized (e.g., to remove T-cell epitopes) antibodies. The term "human antibody" includes not only antibodies obtained from humans, but also antibodies obtained from transgenic mammals containing human immunoglobulin genes such that, upon stimulation with antigen, the transgenic animal produces antibodies containing amino acid sequences characteristic of antibodies produced by humans. The term antibody includes both the parent antibody and derivatives thereof, such as those that are affinity matured, veneered, CDR-grafted, humanized, camelized (in the case of VHHs), or molecules containing the binding domains (e.g., CDRs) of an antibody in a non-immunoglobulin scaffold.The term "antibody" should not be construed as limited to any particular synthetic means and includes naturally occurring antibodies isolatable from natural sources, as well as engineered antibody molecules prepared by "recombinant" means, including antibodies isolated from transgenic animals transgenic for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with nucleic acid constructs that result in the expression of antibodies, 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" comprising variable and constant domains that provide binding and effector function. In most cases, a full-length antibody comprises two light chains and two heavy chains, each light chain comprising a variable region and a constant region. In some embodiments, the term "full-length antibody" is used to refer to the traditional IgG immunoglobulin structure comprising two light chains and two heavy chains, each light chain comprising a variable region and a constant region that provide binding and effector function. The term antibody includes antibody conjugates that contain modifications that extend duration of action, such as conjugation to fusion proteins (e.g., Fc fusions) or polymers (e.g., polyethylene glycol), as described in more detail below.

[0121] biological samplesAs used herein, the term "biological sample" or "sample" refers to a sample obtained from (or derived from) a subject. By way of example, a biological sample includes a material selected from the group consisting of bodily fluids, blood, whole blood, plasma, serum, mucosal secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluid (e.g., vitreous humor, aqueous humor), lymphatic fluid, lymph node tissue, spleen tissue, bone marrow, and immunoglobulin-enriched fractions derived from one or more of these tissues. In some embodiments, the sample is obtained from a subject who has been exposed to a therapeutic treatment regimen, such as repeated exposure to one or more therapeutic agents, wherein the therapeutic agent comprises a pharmaceutical formulation of an IL2 ortholog. In other embodiments, the sample is obtained from a subject who has not recently been exposed to an IL2 ortholog, or from a subject prior to scheduled administration of an IL2 ortholog or a treatment regimen including an IL2 ortholog.

[0122] "CAR" or "Chimeric Antigen Receptor"As used herein, the terms "chimeric antigen receptor" and "CAR" are used interchangeably and refer to a chimeric polypeptide comprising multiple functional domains arranged in the following order from amino to carboxy terminus: (a) an extracellular domain (ECD) comprising an antigen-binding domain (ABD), optionally including a "hinge" domain; (b) a transmembrane domain (TD); and (c) one or more cytoplasmic signaling domains (CSDs), which may optionally be linked by one or more spacer domains. CARs may also further comprise a signal peptide sequence that is routinely removed during post-translational processing of the CAR and presentation on the cell surface of cells transformed with an expression vector comprising a nucleic acid sequence encoding the CAR. CARs may be prepared according to principles well known in the art. See, for example, Eshhaar et al. (U.S. Patent No. 7,741,465 B1, issued June 22, 2010); Sadelain, et al. (2013) Cancer Discovery 3 (4):388-398; Campana and Imai (U.S. Patent No. 8,399,645, issued March 19, 2013); Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS (USA) 86 (24):10024-10028; Curran, et al. (2012) J Gene Med 14 (6):405-15; Brogdon et al. (U.S. Patent No. 10,174,095, issued January 8, 2019); Guedan, et al. (2019) Engineering and Design of Chimeric Antigen Receptors Molecular See Therapy: Methods & Clinical Development Vol. 12: 145-156.In terms of nomenclature, by convention, CARs are designated according to the target of the CAR's antigen-binding domain (ABD), such that a "CD19 CAR" refers to a CAR whose ABD specifically binds to CD19, a "BCMA CAR" refers to a CAR whose ABD specifically binds to BCMA, and so on. In some embodiments, the ABD of the CAR is bivalent (or multivalent) so as to specifically bind to more than one target antigen, e.g., the CD19 and CD20 tumor antigens. In such instances, the CAR will generally be referred to as a "CD19 / CD20" CAR, in reference to the multivalent nature of its ABD.

[0123] CAR-T cellsAs used herein, the terms "chimeric antigen receptor T-cells" and "CAR-T cells" 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 a modified receptor comprising the extracellular domain of an orthogonal CD122 polypeptide (orthogonal CAR-T cells). In terms of nomenclature, CAR-T cells are conventionally designated according to the target of the antigen-binding domain of the CAR: "CD19 CAR-T cells" refer to CAR-T cells comprising a CAR whose ABD selectively binds to CD19, "BCMA CAR-T cells" refer to CAR-T cells comprising a CAR whose ABD selectively binds to BCMA, and so on. Examples of commercially available CD19 CAR-T cell products that can be modified to incorporate the orthogonal receptors of the present invention include axicabtagene ciloreucel (sold by Gilead Pharmaceuticals under the trade name Yescarta®) and tisagenlecleucel (sold by Novartis under the trade name Kymriah®). In some embodiments, the ABD of the CAR is bivalent (or multivalent) so as to specifically bind to more than one target antigen, e.g., the CD19 and CD20 tumor antigens. In such instances, CAR-T cells comprising such a CAR will generally be referred to as a "CD19 / CD20" CAR, referring to the multivalent nature of its ABD.

[0124] CD25 As used herein, the terms "CD25," "IL2 receptor alpha," "IL2Rα," "IL2Ra," and "p55" are used interchangeably and refer to a 55 kD polypeptide that is constitutively expressed in Treg cells and inducibly expressed on other T cells in response to activation. CD25 is also referred to in the literature as the "low affinity" IL2 receptor. hIL2 binds to hCD25 approximately 10 -8 K of M dThe nucleic acid and protein sequences of human CD25 can be found under Genbank accession numbers NM_000417 and NP_0004Q8, respectively. Human CD25 is expressed as a 272 amino acid preprotein containing a 21 amino acid signal sequence, which is post-translationally removed to yield a 251 amino acid mature protein. Amino acids 22-240 of the preprotein (amino acids 1-219 of the mature protein) correspond to the extracellular domain. Amino acids 241-259 of the preprotein (amino acids 220-238 of the mature protein) correspond to the transmembrane domain. Amino acids 260-272 of the preprotein (amino acids 239-251 of the mature protein) correspond to the intracellular domain. The amino acid sequence of the mature form of hCD25 (without the preprotein signal sequence) is as follows: TIFF2026041861000003.tif36132

[0125] CD122As used herein, the terms "CD122," "interleukin-2 receptor beta," "IL2Rb," "IL2Rβ," "IL15Rβ," and "p70-75" are used interchangeably and refer to the CD122 transmembrane protein. Human CD122 (hCD122) is a single-pass, type 1 transmembrane receptor expressed as a 551-amino acid preprotein, the first 26 of which contain a signal sequence that is post-translationally cleaved from the 525-amino acid mature protein. Amino acids 27-240 of the preprotein (amino acids 1-214 of the mature protein) correspond to the extracellular domain, amino acids 241-265 of the preprotein (amino acids 225-239 of the mature protein) correspond to the transmembrane domain, and amino acids 266-551 of the preprotein (amino acids 240-525 of the mature protein) correspond to the intracellular domain. As used herein, the term hCD122 includes naturally occurring variants of hCD122 protein, including S57F and D365E (residues numbered according to the mature hCD122 protein). hCD122 is referred to as entry P14784 in the UniProtKB database. The nucleic acid and protein sequences of human CD122 can be found under Genbank accession numbers NM_000878 and NP_000869, respectively. The amino acid sequence of the mature hCD122 protein without the signal sequence is as follows: TIFF2026041861000004.tif69132, and the extracellular domain (ECD) amino acid sequence of hCD122 is as follows: The file is TIFF2026041861000005.tif28132.

[0126] CD132As used herein, the terms "CD132," "IL2 receptor gamma," "IL2Rg," and "IL2Rγ" are used interchangeably and refer to the type 1 cytokine receptor shared by the receptor complexes for IL-4, IL-7, IL-9, IL-15, and IL21, referred to herein as the "shared" gamma chain. Human CD132 (hCD132) is expressed as a 369-amino acid preprotein containing a 22-amino acid N-terminal signal sequence. Amino acids 23-262 of the preprotein (amino acids 1-240 of the mature protein) correspond to the extracellular domain, amino acids 263-283 of the preprotein (amino acids 241-262 of the mature protein) correspond to the 21-amino acid transmembrane domain, and amino acids 284-369 of the preprotein (amino acids 262-347 of the mature protein) correspond to the intracellular domain. hCD132 is referred to as entry P31785 in the UniProtKB database. The nucleic acid and protein sequences of human CD132 can be found under Genbank accession numbers NM_000206 and NP_000197, respectively. The amino acid sequence of the mature hCD132 protein is as follows: TIFF2026041861000006.tif44133

[0127] CDR.As used herein, the term "CDR" or "complementarity-determining region" refers to the discontinuous antigen-binding site found in the variable region of both heavy and light chain immunoglobulin polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes overlapping or subsets of amino acid residues when compared with each other. In any event, application of either definition to refer to the CDRs of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. In the context of this disclosure, the numbering of CDR positions is provided according to the Kabat numbering convention.

[0128] Circulating tumor cells As used herein, the term "circulating tumor cells (CTCs)" refers to tumor cells that have shed from a tumor mass (e.g., a neoplasm) into the peripheral circulation.

[0129] equivalentAs used herein, the term "equivalent" is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, two measurements would be considered "equivalent" if a first measurement of an evaluable quantitative parameter (e.g., CTLL-2 proliferation or the level of IL2 activity as determined by a phospho-STAT5 assay) and a second measurement of the evaluable parameter do not deviate beyond a range that one skilled in the art would recognize as not producing a statistically significant difference in effect between the two results in that situation. In some examples, measurements can be considered "equivalent" if one measurement deviates from another measurement by less than 35%, alternatively less than 30%, alternatively less than 25%, alternatively less than 20%, alternatively less than 15%, alternatively less than 10%, alternatively less than 7%, alternatively less than 5%, alternatively less than 4%, alternatively less than 3%, alternatively less than 2%, or alternatively less than 1%. In certain embodiments, a measurement is equivalent to a reference standard if it deviates from the reference standard by less than 15%, alternatively less than 10%, or alternatively less than 5%.

[0130] Originates from As used herein, the term "derived from," in the context of amino acid sequence (e.g., a polypeptide comprising an amino acid sequence "derived from" an IL2 polypeptide or polynucleotide sequence), is meant to refer to a polypeptide or nucleic acid having a sequence based on that of a reference polypeptide or nucleic acid (e.g., a naturally occurring IL2 polypeptide or a nucleic acid encoding IL2), but is not meant to be limiting with respect to the source or manner in which the protein or nucleic acid is obtained. By way of example, the term "derived from" includes homologs or variants of the reference amino acid or DNA sequence.

[0131] Effective concentration (EC)As used herein, the term "effective concentration" or its abbreviation "EC" are used interchangeably and refer to the concentration of an agent (e.g., an IL2 ortholog) in an amount sufficient to produce 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 substance. The abbreviation "EC" is used when the magnitude of the response is expressed as a factor of the concentration ("C") of the test substance. In the context of a biological system, the term Emax refers to the maximum value of a given biological effect observed in response to a saturating concentration of an activating test substance. When the abbreviation EC is provided with a subscript (e.g., EC 40 , E.C. 50 (e.g., where the subscript refers to the percentage of Emax of the biological response observed at that concentration. For example, if a concentration of a test substance sufficient to result in the induction of a measurable biological parameter in a test system is 30% of the maximum level of that measurable biological parameter in response to that test substance, this is the "EC 30 Similarly, it is called "EC 100 The term "effective concentration" is used to describe the effective concentration of an agent that produces a maximal (100%) response of a measurable parameter in response to the agent. Similarly, the EC 50 The term "saturation concentration" (commonly used in the field of pharmacodynamics) refers to the concentration of an agent sufficient to produce a half-maximal (50%) change in a measurable parameter. The term "saturation concentration" refers to the maximum possible amount of test substance that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacodynamics, the saturation concentration of a drug is typically used to describe a concentration of drug sufficient such that all available receptors are occupied by the drug, and is referred to as the EC 50 is the drug concentration that gives half-maximal effect.

[0132] concentratedAs used herein, the term "enriched" refers to a sample that is non-naturally engineered such that a species of interest (e.g., a molecule or cell) is present at a concentration that is (a) greater (e.g., at least 3-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 50-fold greater, alternatively at least 100-fold greater, alternatively at least 1000-fold greater) than the concentration of the species in a starting sample, such as a biological sample (e.g., a sample in which the molecule naturally occurs or a sample in which the molecule occurs after administration); or (b) greater than the environment in which the molecule was made (e.g., as in recombinantly modified bacteria or mammalian cells). In some embodiments, the term "enriched" is used herein with respect to a population of cells comprising cells that express an orthogonal receptor when, after contacting the population of cells with a cognate ortholog in an amount sufficient to elicit a response in the cells that express the orthogonal receptor (the response being proliferation), the concentration of cells that express the orthogonal receptor in the population is greater (e.g., at least 3-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 50-fold greater, alternatively at least 100-fold greater, alternatively at least 1000-fold greater) after contacting the population of cells with the cognate ortholog.

[0133] Extracellular domain As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is outside the plasma membrane of a cell. An ECD can include the entire extracytoplasmic portion of a transmembrane protein, a cell surface or membrane-associated protein, a secreted protein, or a cell surface-targeted protein.

[0134] hCD122 As used herein, the term "hCD122" refers to the naturally occurring human CD122 polypeptide, including naturally occurring variants thereof. The amino acid sequence of naturally occurring mature hCD122 is provided as SEQ ID NO:2.

[0135] identityThe term "identity," when used herein with respect to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. If a subunit position in both molecules is occupied by the same monomer subunit (i.e., the same amino acid residue or nucleotide), the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. Generally, sequences are aligned to obtain the highest order match. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux, et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and 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, Wis. 53705) with its default parameters. Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).The algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet a certain positive threshold score, T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residues; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0136] IL2:As used herein, the term "interleukin-2" or "IL2" refers to an IL2 polypeptide that possesses IL2 activity. In some embodiments, IL2 refers to mature wild-type human IL2. Mature wild-type human IL2 (hIL2) occurs as a 133 amino acid polypeptide (less the N-terminal 20 amino acids of the signal peptide of the preprotein) as described in Fujita, et. al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of a naturally occurring variant of mature wild-type human IL2 (hIL2) is as follows: TIFF2026041861000007.tif23134. As used herein, the residue numbering of the IL2 variants is based on the IL2 sequence UniProt ID P60568, excluding the signal peptide, which is the same as that of SEQ ID NO:5.

[0137] IL2 activity The term "IL2 activity" refers to one or more biological effects on a cell in response to contacting the cell with an effective amount of an IL2 polypeptide. As previously mentioned, IL2 is a pleiotropic cytokine that exerts 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 IL2 activity can be measured in a cell proliferation assay using CTLL-2 murine cytotoxic T cells; see Gearing, AJH and CB Bird (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 approximately 2.1 x 10 4IU / μg, which is calibrated against the recombinant human IL2 WHO International Standard (NIBSC code: 86 / 500). In some embodiments, for example, if an IL2 orthogonal polypeptide ligand of interest exhibits (or is engineered to possess) reduced affinity for CD25, the level of IL2 activity can be assessed in human cells such as YT cells, which do not require CD25 to effect signaling through the IL2 receptor but can signal through the intermediate-affinity CD122 / CD132 receptor. In some embodiments, an orthogonal human IL2 ligand of the present disclosure may exhibit less than about 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the activity of WHO International Standard (NIBSC code: 86 / 500) wild-type mature human IL2 when assessed at equivalent concentrations in a comparable assay.

[0138] IL2 orthologsAs used herein, the term "IL2 ortholog" refers to a variant of IL2 derived from an IL2 parent polypeptide, wherein the IL2 ortholog specifically binds to an orthogonal CD122 ECD and exhibits significantly reduced binding to the extracellular domain of wild-type CD122. In some embodiments, the IL2 ortholog (1) exhibits specific binding to a receptor comprising an orthogonal CD122 ECD, and (2) when contacted with a cell expressing a transmembrane receptor comprising the ECD of the orthogonal CD122 polypeptide in an amount sufficient to elicit a response, produces a signal characteristic of that generated by the intracellular domain (ICD) of the transmembrane receptor. When the transmembrane receptor comprises an orthogonal CD122 ECD and a CD122 ICD, binding of the IL2 ortholog to such a receptor produces an intracellular signal characteristic of CD25 / CD122 / CD132 high-affinity activation of the CD122 / CD132 intermediate-affinity IL2 receptor. The IL2 ortholog (2) exhibits significantly reduced binding to wild-type hCD122. The term IL2 ortholog includes IL2 orthogonal variants and modified IL2 orthologs. In some embodiments, IL2 orthologs are derived from naturally occurring variants of human IL2, and such human IL2 orthologs may be referred to as "hoCD122" or "hoRb." Certain modified IL2 polypeptides are provided in Garcia et al. (U.S. Patent Application Publication US2018 / 0228842A1, published August 16, 2018). As used herein, the term IL2 ortholog includes modified hIL2 polypeptides described in Garcia et al., U.S. Patent Application Publication US2018 / 0228842A1, published August 16, 2018. In some embodiments, the affinity of an IL2 ortholog for the extracellular domain of orthogonal CD122 is equivalent to the affinity of wild-type IL2 for the ECD of wild-type CD122. In some embodiments, the affinity of the IL2 ortholog for the ECD of orthogonal CD122 is greater than the affinity of wild-type IL2 for the ECD of wild-type CD 122. In some embodiments, the affinity of the IL2 ortholog for the ECD of orthogonal CD122 is less than the affinity of wild-type IL2 for the ECD of wild-type CD122.

[0139] in sufficient quantity to produce a response As used herein, the phrase "in an amount sufficient to elicit a response" refers to an amount of test substance sufficient to provide a detectable change in the level of an indicator measured before (e.g., baseline level) and after application of the test substance to the test system. 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 assay. In some embodiments, the test system involves measuring a change in the level of a parameter of a cell, tissue, or organism that reflects a biological function before and after application of the test substance to the cell, tissue, or organism. In some embodiments, the indicator (e.g., concentration of phosphorylated STAT5) reflects the biological function of the cell being assessed in the assay (e.g., activation of the IL2 receptor) in response to administration of an amount of the test substance (e.g., IL2). In some embodiments, the test system involves measuring a change in the level of a parameter (e.g., luminescence) of a cell, tissue, or organism (e.g., a mouse) that reflects a biological state (e.g., the presence of a neoplasm) before and after application of one or more test substances (e.g., CAR-T cells expressing orthogonal CD122 in combination with an IL2 ortholog) to the cell, tissue, or organism (e.g., a mouse injected with luminescent neoplastic cells). In some embodiments, the indicator (e.g., the concentration of phosphorylated STAT5) reflects the biological function (e.g., activation of the IL2 receptor) of the cell (e.g., T cell) being assessed in the assay in response to administration of an amount of the test substance (e.g., IL2). An "amount sufficient to effect a response" can be sufficient to be a therapeutically effective amount, while "in an amount sufficient to elicit a response" can be more or less than a therapeutically effective amount.

[0140] Needs treatment The term "in need of treatment," as used herein, refers to a judgment made by a physician or other caregiver regarding a subject that the subject needs or will potentially benefit from treatment. This judgment is made based on a variety of factors within the physician's or caregiver's area of ​​expertise.

[0141] Needs prevention As used herein, the term "in need of prevention" refers to a judgment made by a physician or other caregiver regarding a subject that the subject needs or would potentially benefit from preventative care. This judgment is made based on a variety of factors within the physician's or caregiver's area of ​​expertise.

[0142] inhibitors As used herein, the term "inhibitor" refers to a molecule that reduces, blocks, prevents, delays the activation of, inactivates, desensitizes, or downregulates, for example, a gene, protein, ligand, receptor, or cell. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates the constitutive activity of a cell or organism.

[0143] isolated As used herein, the term "isolated" refers to a polypeptide of interest that is in an environment different from the environment in which it may naturally occur, if it occurs in nature. "Isolated" refers to a polypeptide that is substantially enriched in the polypeptide of interest and / or is in a sample in which the polypeptide of interest is partially or substantially purified. If the polypeptide does not occur in nature, "isolated" refers to the polypeptide being separated from the environment in which it was created, either synthetically or by recombinant means.

[0144] Orthogonal receptor intracellular domainAs used herein, the term "orthogonal receptor intracellular domain" or "ICD-OR" refers to the portion of a transmembrane orthogonal receptor that is located inside the plasma membrane of a cell that expresses the transmembrane orthogonal receptor. The ICD-OR may contain one or more "proliferation signaling domains" or "PSDs," which refer to protein domains that signal cells to enter mitosis and initiate cell proliferation. Examples include Janus kinases, including but not limited to JAK1, JAK2, JAK3, Tyk2, and Ptk-2; homologous members of the Janus kinase family from other mammalian or eukaryotic species; IL2 receptor β and / or γ chains; and other subunits from proteins of the cytokine receptor superfamily that can interact with proteins of the Janus kinase family to transduce signals, or portions, modifications, or combinations thereof. An example of a signal includes phosphorylation of one or more STAT molecules, including but not limited to one or more of STAT1, STAT3, STAT5a, and / or STAT5b.

[0145] "combined with"As used herein, the term "in combination with," when used in reference to administering multiple agents to a subject, refers to administering a first agent to a subject together with at least one additional (i.e., second, third, fourth, fifth, etc.) agent. For purposes of the present invention, one agent (e.g., an IL2 ortholog) is considered to be administered in combination with a second agent (e.g., engineered human immune cells) if the biological effect resulting from administration of the first agent persists in the subject such that, upon administration of the second agent, the therapeutic effect of the first agent overlaps with the therapeutic effect of the second agent. For example, an engineered orthogonal cellular therapy agent is typically administered infrequently (typically administered only once), while an IL2 ortholog is administered periodically while the orthogonal cellular agent persists in the subject. Because engineered orthogonal cellular therapy agents provide a therapeutic effect over an extended period of time (weeks or months), and administration of a second agent (e.g., an IL2 ortholog) provides its therapeutic effect while the therapeutic effect of the first agent is still ongoing, a second agent is considered to be administered in combination with a first agent even if the first agent is administered at a time significantly separated (e.g., days or weeks) from the time of administration of the second agent. In one embodiment, an agent is considered to be administered in combination with a second agent if the first and second agents are administered simultaneously (within 30 minutes of each other), during the same time period, or sequentially. In some embodiments, a first agent is considered to be administered "within the same time period" as a second agent if the first and second agents are administered within about 24 hours of each other, preferably within about 12 hours of each other, preferably within about 6 hours of each other, preferably within about 2 hours of each other, or preferably within about 30 minutes of each other. The term "in combination with" should also be understood to apply to situations where the first and second agents are combined into a single pharmaceutically acceptable formulation and the combination is administered to a subject. In certain embodiments, the orthogonal cells and IL2 ortholog are further combined with an additional adjunctive agent.The auxiliary agents may be administered or applied sequentially, e.g., where one agent is administered before one or more other agents. In other embodiments, the IL2 mutein and the auxiliary agents are administered simultaneously, e.g., where two or more agents are administered at the same or about the same time; the two or more agents may be present in two or more separate formulations or may be combined in a single formulation (i.e., a combination formulation). Whether the agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

[0146] High-affinity IL2 receptor As used herein, the term "high affinity IL2 receptor" refers to a trimeric receptor complex (also referred to as "IL2Rαβγ") that contains the CD25, CD122, and CD132 proteins. Wild-type hIL2 (SEQ ID NO: 5) binds approximately 10 -11 Holds Kd of M.

[0147] Intermediate affinity IL2 receptor As used herein, the term "intermediate affinity IL2 receptor" refers to the dimeric IL2 receptor complex (also referred to as "IL2Rβγ") comprising CD122 and CD132. The association of IL2 molecules with the intermediate affinity IL2 receptor expressed on the surface of mammalian immune cells results in IL2 signaling in the cells. Wild-type hIL2 (SEQ ID NO:5) binds approximately 10 to the intermediate affinity CD122 / CD132 (IL2Rβγ) receptor complex. -9 Holds Kd of M.

[0148] Kabat numbering:The term "Kabat numbering," as used herein, is recognized in the art and refers to a numbering system for 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, US Department of Health and Human Services, NIH Publication No. 91-3242). For purposes of this disclosure, the positioning of CDRs in the variable regions of antibodies is according to Kabat numbering or simply "Kabat."

[0149] Ligand As used herein, the term "ligand" refers to a molecule that specifically binds to a receptor and causes a change in the receptor that results in a change in the activity of the receptor or a response in a cell that expresses the receptor. In one aspect, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of the receptor. As used herein, the term "ligand" encompasses natural and synthetic ligands. "Ligand" also encompasses small molecules, peptide mimetics of cytokines, and peptide mimetics of antibodies. A complex of a ligand and a receptor is called a "ligand-receptor complex." A ligand can comprise one domain of a polyprotein or a fusion protein (e.g., an antibody-targeting ligand fusion protein).

[0150] metastasis As used herein, the term "metastasis" describes the spread of cancerous cells from a primary tumor to surrounding tissues and distant organs.

[0151] Modified IL2 orthologsAs used herein, the term "modified IL2 ortholog" is used to refer to an IL2 ortholog that has been modified by one or more modifications, such as pegylation, glycosylation (N-linked and O-linked), acylation, or polysialylation, or by conjugation (either chemically or as a fusion protein) to other polypeptide carrier molecules, including, but not limited to, albumin fusion polypeptides comprising serum albumin (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)), Fc-fusion proteins, targeting IL2 ortholog fusion proteins (e.g., scFv-IL2 ortholog fusion proteins, VHH-IL2 orthogonal polypeptide fusion proteins), and the like. Modified IL2 orthologs can be prepared to enhance one or more properties, for example, to modulate immunogenicity (conjugation or fusion to an immunogen), methods to increase water solubility, bioavailability, serum half-life, and / or therapeutic half-life; and / or to modulate biological activity. Certain modifications may be useful, for example, for generating antibodies for use in detection assays (e.g., epitope tags) or to facilitate protein purification (e.g., poly-His tags). Modified IL2 orthologs can be prepared to enhance one or more properties, for example, to modulate immunogenicity; methods to increase water solubility, bioavailability, serum half-life, and / or therapeutic half-life; and / or to modulate biological activity. Certain modifications may be useful, for example, for generating antibodies for use in detection assays (e.g., epitope tags) or to facilitate protein purification. In some embodiments, the modified IL2 ortholog has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO:5, excluding any modifications such as those encompassed by the modifications of Formula 1.

[0152] ModulateAs used herein, the terms "modulate," "modulation," and the like refer to the ability of a test substance to induce a response, either positively or negatively, directly or indirectly, in a biological system or a system containing a biochemical pathway. The term modulator includes both agonists (including partial agonists, full agonists, and superagonists) and antagonists.

[0153] Neoplastic disease As used herein, the term "neoplastic disease" refers to a disorder or condition in a subject resulting from cellular hyperproliferation or uncontrolled (or deregulated) cell replication. The term neoplastic disease refers to a disorder resulting from the presence of a neoplasm in a subject. Neoplasms can be classified as (1) benign, (2) pre-malignant (or "pre-cancerous"); and (3) malignant (or "cancerous"). The term "neoplastic disease" includes neoplasia-related diseases, disorders, and conditions that refer to conditions directly or indirectly related to neoplastic disease, and includes, for example, angiogenesis and dysplasia or pre-cancerous conditions such as smoldering multiple myeloma.

[0154] N-terminus As used herein in the context of a polypeptide's structure, the terms "N-terminus" (or "amino-terminus") and "C-terminus" (or "carboxy-terminus") refer to the extreme amino and carboxyl ends of a polypeptide, respectively, while the terms "N-terminal" and "C-terminal" refer to the relative position in the amino acid sequence of a polypeptide relative to the N-terminus and C-terminus, respectively, and can include residues at the N-terminus and C-terminus, respectively. "Immediately N-terminal" or "immediately C-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue, where the first and second amino acid residues are covalently linked to provide a contiguous amino acid sequence.

[0155] Neoplastic diseaseAs used herein, the term "neoplastic disease" refers to a disorder or condition in a subject resulting from cellular hyperproliferation or uncontrolled (or deregulated) cell replication. The term neoplastic disease refers to a disorder resulting from the presence of a neoplasm in a subject. Neoplasms can be classified as (1) benign, (2) pre-malignant (or "pre-cancerous"); and (3) malignant (or "cancerous"). The term "neoplastic disease" includes neoplasia-related diseases, disorders, and conditions that refer to conditions directly or indirectly related to neoplastic disease, and includes pre-cancerous conditions such as, for example, angiogenesis and dysplasia.

[0156] nucleic acid The terms "nucleic acid," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

[0157] Numbered according to IL2 The term "numbered according to IL2," as used herein, refers to identifying the position of a particular amino acid relative to the position at which that amino acid normally occurs in the sequence of mature, wild-type IL2. In some embodiments, the IL2 is hIL2 (SEQ ID NO:5). For example, with respect to hIL2, "R81" indicates that the 81st amino acid (numbered from the N-terminus) present in the sequence of mature, wild-type hIL2 is arginine. It should be noted that the amino acid sequences of IL2 molecules from different mammalian species have different amino acid numbers and arrangements. Therefore, referring to residues according to this convention helps identify the IL2 species in question.

[0158] Numbered according to CD122The term "numbered according to CD122," as used herein, refers to identifying the position of a particular amino acid relative to the position at which that amino acid normally occurs in the sequence of a mature, wild-type CD122 molecule. In one embodiment, the CD122 molecule is human CD122 (SEQ ID NO. 2). For example, with respect to human CD122, H133 indicates that the 133rd amino acid (numbered from the N-terminus) in the sequence of mature, wild-type hCD122 is a histidine.

[0159] Numbered according to the extracellular domain of CD122 The terms "numbered according to the extracellular domain of CD122" or "numbered according to CD122 ECD," as used herein, refer to identifying the position of a particular amino acid relative to the position at which that particular amino acid normally occurs in the extracellular domain (ECD) sequence of a mature, wild-type CD122 molecule. In one embodiment, the CD122 ECD molecule is human CD122 ECD (SEQ ID NO. 3). For example, with respect to human CD122 ECD, H133 indicates that the 133rd amino acid (numbered from the N-terminus) in the sequence of mature, wild-type hCD122 ECD is a histidine.

[0160] Functionally linkedThe term "operably linked," as used herein, refers to the relationship between nucleic acid sequences encoding different functions when they are combined into a single nucleic acid sequence, providing a nucleic acid that, when introduced into a cell, is capable of effecting transcription and / or translation of the particular nucleic acid sequence in the cell. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, certain genetic elements, such as enhancers, need not be contiguous with respect to the sequences to provide their effect.

[0161] Orthogonal cellsAs used herein, the term "orthogonal cell" refers to a mammalian cell that has been recombinantly modified to express an orthogonal receptor. In some embodiments, the orthogonal cell expresses an orthogonal CD122 (oCD122) polypeptide or a receptor comprising an orthogonal CD122 ECD. In some embodiments, the orthogonal cell is a modified human cell ("human orthogonal cell"). The orthogonal cell can be an immune cell, e.g., a human immune cell. A "human orthogonal immune cell" is a human immune cell that has been recombinantly modified to express human orthogonal CD122 (hoCD122) or a chimeric receptor comprising a human orthogonal CD122 ECD. In some embodiments, the immune cells to be engineered to express oCD122 (or a receptor comprising an oCD122 ECD) are selected from the group consisting of bone marrow cells, lymphocytes, peripheral blood mononuclear cells (PBMCs), tumor-infiltrating lymphocytes (TILs), T cells, CD8+ T cells, CD25+CD8+ T cells, CAR-T cells, NK cells, CD4+ T cells, and engineered versions thereof, including but not limited to Tregs, engineered TILs, engineered Tregs, and engineered NK cells. In some embodiments, the orthogonal cells are CAR-T derived from human immune cells recombinantly engineered to express human orthogonal CD122 ("hoCAR-T" cells). In some embodiments, the orthogonal cells are TILs isolated from a neoplasm of a human subject recombinantly engineered to express human orthogonal CD122 or a receptor comprising the oCD122 ECD ("hoTIL" cells). In some embodiments, the orthogonal cells are NK cells isolated from a human subject that have been recombinantly modified to express a receptor comprising human orthogonal CD122 or oCD122 ECD ("hoNK" cells). In some embodiments, the cells are human hematopoietic stem cells ("hoHSC" cells) that have been recombinantly modified to express a receptor comprising human orthogonal CD122 or oCD122 ECD.In some embodiments, the orthogonal cells are TCR-engineered cells ("hoTCR cells") derived from human immune cells that have been recombinantly modified to express a non-native T cell receptor that has been further modified to express a receptor comprising a human orthogonal CD122 or oCD122 ECD. As used herein, the term "orthogonal cells" refers to mammalian cells that have been recombinantly modified to express a receptor comprising an orthogonal CD122 or oCD122 ECD. In addition to the recombinant modifications required to express an orthogonal CD122 polypeptide or receptor comprising an oCD122 ECD, orthogonal cells may incorporate recombinant modifications, including the introduction of nucleic acid molecules encoding marker proteins operably linked to expression control sequences to facilitate expression in the orthogonal cells, including, but not limited to, marker proteins (proteins that confer antibiotic resistance, fluorescent proteins, or luminescent proteins); biologically active intracellular proteins, including, but not limited to, DNA- or RNA-binding proteins, transcription factors, including transcriptional repressors or derepressors, pro-apoptotic proteins, anti-apoptotic proteins, and intracellular regulatory proteins; and biologically active secreted proteins (typically extracellular proteins that include a signal peptide or secretory leader sequence to facilitate extracellular transport after expression in the orthogonal cells), such as growth factors, peptide hormones, cytokines, or chemokines, including biologically active therapeutic proteins such as antibodies. Additional recombinant modifications for orthogonal cells will be apparent to those skilled in the art. In some embodiments, the orthogonal CD122 receptor expressed on the orthogonal cells may comprise a human CD122 intracellular domain (ICD) that has been modified to provide one or more STAT3 binding motifs.

[0162] Orthogonal CD122As used herein, the terms "orthogonal CD122" or "CD122 orthogonal receptor" are used interchangeably herein and refer to a CD122 polypeptide variant that contains amino acid substitutions that result in specific binding to an IL2 ortholog that is the cognate ligand for the CD122 polypeptide variant, but does not specifically bind to naturally occurring forms of IL2.

[0163] Orthogonal Receptors As used herein, the term "orthogonal receptor" refers to a receptor variant, i.e., an orthogonal receptor containing modifications to its amino acid sequence such that it exhibits significantly reduced binding to its cognate ligand but exhibits specific binding to an orthogonal ligand that it is engineered to interact with. In some embodiments, an orthogonal receptor may contain an extracellular domain that exhibits significantly reduced binding to its cognate natural ligand, while the orthogonal ligand exhibits significantly reduced binding to the ECD of its cognate natural receptor. In some embodiments, the affinity of the orthogonal ligand for the cognate orthogonal receptor is comparable to that of the natural ligand for the native receptor, e.g., at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, or even higher, e.g., 2x, 3x, 4x, 5x, 10x, or more, of the affinity of the natural cytokine for the native receptor. An orthogonal receptor may be designated by the parent molecule from which it is derived (e.g., orthogonal CD122) or by the cognate ligand from which an orthogonal ligand for the orthogonal receptor is derived (e.g., orthogonal IL2 receptor).

[0164] OrthologsAs used herein, the terms "ortholog" and "orthogonal ligand," used interchangeably herein, refer to the ligand component of an orthogonal ligand / receptor pair and refer to a polypeptide that incorporates modifications into its primary structure that provide the polypeptide variant with (a) significantly reduced affinity for its native cognate receptor (i.e., the native receptor relative to the parent polypeptide from which the ortholog is derived); and (b) specific binding to an engineered orthogonal receptor that is a variant of the cognate receptor for the ortholog. Upon binding of the ortholog to the orthogonal receptor (which is expressed on the surface of cells modified by recombinant DNA technology to incorporate a nucleic acid sequence encoding the orthogonal receptor operably linked to regulatory elements to effect expression of the orthogonal receptor in the recombinantly modified cells), the activated orthogonal receptor initiates signal transduction that is transduced through native cellular elements to provide a biological activity that mimics the natural response of its cognate receptor but is specific to the recombinantly modified cell population expressing the orthogonal receptor. In some embodiments of the present invention, orthologs possess significant selectivity for orthogonal receptors compared to their cognate receptors, and optionally possess significantly reduced potency with respect to their cognate receptors. Selectivity is typically measured by the activity measured in an assay characteristic of the activity induced in response to ligand / receptor binding. In some embodiments, orthologs possess an EC50 that is at least 5-fold, alternatively at least 10-fold, alternatively at least 20-fold, alternatively at least 30-fold, alternatively at least 40-fold, alternatively at least 50-fold, alternatively at least 100-fold, alternatively at least 200-fold different when measured in the same assay.

[0165] Parent PolypeptideAs used herein, the terms "parent polypeptide" or "parent protein" are used interchangeably and refer to a naturally occurring polypeptide that is subsequently modified to create a variant polypeptide. A parent polypeptide can be a wild-type (or naturally occurring) polypeptide. A parent polypeptide can refer to the polypeptide itself or a composition comprising the parent polypeptide (e.g., a glycosylated, pegylated fusion protein comprising the parent polypeptide).

[0166] Partial agonist As used herein, the term "partial agonist" refers to a molecule that specifically binds to a given receptor and activates it, but only partially activates the receptor compared to a full agonist. A partial agonist can exhibit both agonist and antagonist activity. For example, when both a full agonist and a partial agonist are present, the partial agonist acts as a competitive antagonist by competing with the full agonist for receptor binding, resulting in a net decrease in receptor activation compared to when the receptor is contacted with the full agonist in the absence of the partial agonist. In clinical practice, partial agonists can be used to activate receptors in the presence of an inadequate amount of endogenous ligand to provide a desired submaximal response, or they can reduce overstimulation of the receptor in the presence of an excessive amount of endogenous ligand. The maximum response (E) generated by a partial agonist is max ) is referred to as its intrinsic activity and can be expressed on a percentage scale where a full agonist would produce a 100% response. An IL2 partial agonist of the present disclosure can have more than 10%, alternatively more than 20%, alternatively more than 30%, alternatively more than 40%, alternatively more than 50%, alternatively more than 60%, or alternatively more than 70% of the activity of WHO International Standard (NIBSC code: 86 / 500) wild-type mature human IL2 when evaluated at similar concentrations in an equivalent assay.

[0167] PEG-IL2 orthologsAs used herein, the term "PEG-IL2 ortholog" refers to an IL2 ortholog covalently linked to at least one polyethylene glycol (PEG) molecule, where the at least one PEG molecule is covalently attached to at least one amino acid residue of the IL2 ortholog. PEGylated polypeptides may also be referred to as mono-PEGylated, di-PEGylated, tri-PEGylated (etc.), each representing a PEG-IL2 ortholog containing one, two, three (or more) PEG moieties attached to the IL2 ortholog. In some embodiments, PEG may be covalently attached directly to the IL2 ortholog (e.g., via a lysine side chain, a sulfhydryl group of cysteine, or the N-terminal amine), or a linker may optionally be used between the PEG and the IL2 ortholog. In some embodiments, the PEG-IL2 ortholog comprises more than one PEG molecule, each attached to a different amino acid residue. In some embodiments, the PEG-IL2 ortholog is derived from SEQ ID NO:4 (mature human wild-type hIL2). Pegylated forms of IL2 and methodologies for PEGylation of IL2 polypeptides are well known in the art (see, e.g., U.S. Patent 4,931,544 to Katre et al., issued June 5, 1990; U.S. Patent 5,206,344 to Katre et al., issued April 27, 1993; and U.S. Patent No. 9,861,705 to Bossard et al., issued January 9, 2018). In some embodiments, IL2 muteins may be modified by the incorporation of unnatural amino acids bearing non-naturally occurring amino acid side chains to facilitate site-specific PEGylation, as described in U.S. Patent Application Publication US20170369871A1 to Ptacin et al., published December 28, 2017. In other embodiments, cysteine ​​residues may be incorporated at various positions within the IL2 molecule to facilitate site-specific PEGylation via cysteine ​​side chains, as described in PCT International Patent Application No. PCT / US2015 / 044462 to Greve et al., published February 18, 2016 as WO2016 / 025385.

[0168] PolypeptidesAs used herein, the terms "polypeptide," "peptide," and "protein," which are used interchangeably herein, refer to polymeric forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified polypeptide backbones. This term includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without an N-terminal methionine residue; fusion proteins with immunologically tagged proteins; fusion proteins of immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments), and the like.

[0169] Prevent As used herein, the terms "prevent," "preventing," "prevention," and the like, generally in the context of a subject who is prone to having a particular disease, disorder, or condition due to genetic, experiential, or environmental factors, refer to a course of action initiated with respect to a subject prior to the onset of the disease, disorder, condition, or symptoms thereof, to prevent, suppress, inhibit, or reduce, either temporarily or permanently, the subject's risk of developing the disease, disorder, condition, etc. (e.g., as determined by the absence of clinical symptoms) or delaying its onset. In certain instances, the terms "prevent," "preventing," and "prevention" are also used to refer to slowing the progression of a disease, disorder, or condition from its current state to a more severe state.

[0170] receptorAs used herein, the term "receptor" refers to a polypeptide having a domain that specifically binds to a ligand, and ligand binding alters at least one biological property of the polypeptide. In some embodiments, the receptor is a "soluble" receptor that is not associated with a cell surface. The soluble form of hCD25 is an example of a soluble receptor that specifically binds to hIL2. In some embodiments, the receptor is a cell surface receptor comprising an extracellular domain (ECD) and a membrane-associated domain that serves to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide comprising an intracellular domain (ICD) and an extracellular domain (ECD) connected by a transmembrane domain, typically referred to as a transmembrane domain (TM). Binding of a ligand to the receptor results in a conformational change in the receptor, resulting in a measurable biological effect. In some examples where the receptor is a transmembrane polypeptide comprising an ECD, a TM, and an ICD, binding of a ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to binding of the ligand to the ECD. In some embodiments, the receptor is a component of a multicomponent complex that facilitates intracellular signal transduction. For example, a ligand may bind to a cell surface receptor that is not involved in any intracellular signal transduction alone, but whose binding facilitates the formation of heteromultimeric or homomultimeric (homodimeric, homotrimeric, homotetrameric) complexes, including heterodimers (e.g., intermediate-affinity CD122 / CD132 IL2 receptor) and heterotrimers (e.g., high-affinity CD25 / CD122 / CD132 hIL2 receptor), which result in activation of an intracellular signal transduction cascade (e.g., the Jak / STAT pathway). In some embodiments, the receptor is a membrane-spanning single-chain polypeptide comprising ECD, TM, and ICD domains, the ECD, TM, and ICD domains being 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).

[0171] RecombinationAs used herein, the term "recombinant" is used as an adjective to refer to the process by which a polypeptide, nucleic acid, or cell has been modified using recombinant DNA technology. A recombinant protein is a protein produced using recombinant DNA technology and is often abbreviated with a lowercase "r" to indicate that the protein was produced by that process (e.g., rhIL2). Similarly, if a cell has been modified (e.g., transfected, transduced, infected) using recombinant DNA technology to incorporate an exogenous nucleic acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids, etc.), the cell is referred to as a "recombinant cell." Techniques and protocols for recombinant DNA technology are well known in the art; for example, they can be found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0172] responseFor example, the term "response" of a cell, tissue, organ, or organism encompasses quantitative or qualitative changes in assessable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy consumption rate, level or state of differentiation) that correlate with activation, stimulation, or treatment by internal mechanisms such as genetic programming. In certain contexts, terms such as "activation," "stimulation," and the like refer to cellular activation as regulated by internal mechanisms and by external or environmental factors; whereas terms such as "inhibition," "downregulation," and the like refer to the opposite effect. Examples of such standard protocols for assessing proliferation of CD3-activated primary human T-cells include bioluminescence assays that generate a luminescent signal proportional to the amount of ATP present, which is directly proportional to the number 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 that substantially follow the instructions provided by the manufacturer, such as the CellTiter-Glo® 2.0 Cell Viability Assay or CellTiter-Glo® 3D Cell Viability kits, commercially available from Promega Corporation (2800 Woods Hollow Road, Madison, WI 53711) under catalog numbers G9241 and G9681, respectively. In some embodiments, the level of T-cell activation in response to administration of a test substance can be determined by flow cytometry methods as described, such as determining the level of STAT5 phosphorylation according to methods well known in the art.STAT5 phosphorylation can be measured using flow cytometry techniques as described in Horta et al., supra, Garcia et al., supra, or commercially available kits substantially following the manufacturer's instructions, such as the Phospho-STAT5(Tyr694) kit (commercially available from Perkin-Elmer / cisbio, Waltham MA, Part Number 64AT5PEG). Abbreviations used with subscripts: EC. ACT In the case of , this is provided to indicate the concentration of test substance sufficient to produce the indicated percentage of STAT5 phosphorylation relative to maximum in T cells in response to application of the test substance, as measured according to the test protocol. 30 PRO can be used with respect to hIL2 orthologs to indicate the concentration associated with 30% of the maximum level of STAT5 phosphorylation in T cells in response to such hIL2 orthologs as measured with the Phospho-STAT5(Tyr694) kit.

[0173] In some instances, there are standardized and accepted metrics of biological activity established for molecules. For example, with respect to hIL2 potency, the standard methodology for assessing hIL2 potency in International Units (IU) is measured in the murine cytotoxic T cell line CTLL-2 according to standardized procedures, as described in detail in Wadhwa, et al. (2013) "The 2nd International standard for Interleukin-2 (IL2) Report of a collaborative study" Journal of Immunological Methods 397:1-7. In the context of the present disclosure, it should be noted that the murine IL2 receptor functions differently from the human IL2 receptor, particularly with respect to the requirement of all components of the trimeric receptor complex to effect intracellular signaling (e.g., STAT5 phosphorylation). See, for example, 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 required for formation of the functional mouse IL2 receptor" European J Immunology 25(11)3001-5.Thus, when assessing the activity of hIL2 variants, particularly with respect to affinity for CD25 or activation of cells with respect to CD25 status, the use of human cells or systems that recapitulate the biology of the human low-, medium-, and high-affinity IL2 receptors and receptor complexes is preferred; molecules that exhibit selective binding or activation in murine test systems (e.g., in vitro test systems using murine cells or in vivo in mice) may not recapitulate such selective activity in human systems (e.g., in vitro test systems using human cells or in vivo in human subjects).

[0174] Selective As used herein, the term "selective" refers to the property of an agent to preferentially bind to and / or activate a particular cell type. In some embodiments, the present disclosure provides IL2 variants (IL2 orthologs) that selectively bind to engineered CD122 ECD polypeptides such that, in response to binding to a receptor comprising such a cognate CD122 ECD polypeptide, cells expressing a receptor comprising such a CD122 ECD polypeptide are activated. In some embodiments, the present disclosure provides hIL2 orthologs that are selective in that they exhibit preferential activation of immune cells expressing the hoCD122 receptor. Selectivity is typically assessed by activity measured in an assay characteristic of the activity induced in response to ligand / receptor binding. In some embodiments, the selectivity of an IL2 ortholog is measured by comparing the activation of cells expressing CD25 (e.g., YTCD25 or YTCD25 cells) with the activation of cells exhibiting significantly lower (preferably undetectable) levels of CD25 (e.g., YTCD25 or YTCD25 cells). In some embodiments, selectivity is measured by the activation of T cells expressing relatively high levels of CD25 (e.g., Tregs) with T cells expressing relatively low levels of CD25 (e.g., unstimulated CD8 T cells).

[0175] Significantly reduced binding:As used herein, the term "exhibiting significantly reduced binding" is used in reference to the affinity of a variant ligand (e.g., an ortholog) binding to a modified form of a receptor (e.g., orthogonal CD122) compared to the binding of the variant ligand to the naturally occurring form of the receptor. In some embodiments, a ligand (e.g., an ortholog) exhibits significantly reduced binding to the naturally occurring form of the ligand if the orthogonal ligand binds to the naturally occurring form of the receptor with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the affinity of the naturally occurring ligand. Similarly, an orthogonal receptor exhibits significantly reduced binding with respect to the native form of the ligand if the native form of the ligand binds to the orthogonal form of the receptor with an affinity that is less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of that of the naturally occurring receptor.

[0176] Specific binding to: As used herein, the term "specifically bind" refers to the degree of affinity with which one molecule binds to another molecule. In the context of binding pairs (e.g., ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pairs), a first molecule of the binding pair is said to specifically bind to a second molecule of the binding pair if the first molecule of the binding pair does not bind in significant amounts to other components present in the sample other than the second molecule. A first molecule of the binding pair is said to specifically bind to a second molecule of the binding pair if the affinity of the first molecule for the second molecule is at least 2-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 20-fold greater, or alternatively at least 100-fold greater than the affinity of the first molecule for other components present in the sample. In certain embodiments where the first molecule of the binding pair is an antibody, the antibody binds to the second molecule of the binding pair (e.g., a protein, antigen, ligand, or receptor) such that the equilibrium dissociation constant between the antibody and the second molecule of the binding pair is about 10 6Over M or about 10 8 Over M or about 10 10 Over M or about 10 11 Over M or about 10 10 Over M, about 10 12 M or greater indicates specific binding, as determined, for example, by Scatchard analysis (Munsen, et al. (1980) Analyt. Biochem. 107:220-239). In one embodiment, where the ligand is an IL2 ortholog and the receptor comprises an orthogonal CD122 ECD, the IL2 ortholog has an equilibrium dissociation constant of about 10 for IL2 ortholog / orthogonal CD122 ECD. 5 Over M or about 10 6 Over M or about 10 7 Over M or about 10 8 Over M or about 10 9 Over M or about 10 10 Over M or about 10 11 Specifically binds when the nucleotide sequence is greater than M. Specific binding can be assessed using techniques known in the art, including, but not limited to, competitive ELISA assays, radioactive ligand binding assays (e.g., saturation binding, Scatchard plots, non-linear 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 commercially available instruments such as the Biacore 8+, Biacore S200, and Biacore T200 from GE Healthcare Bio-Sciences (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA 01752)); solution-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 filter assays)).

[0177] subject The terms "recipient," "individual," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. A "mammal," for purposes of treatment, refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0178] suffering As used herein, the term "suffering" refers to the determination made by a physician about a subject that the subject needs treatment or will benefit from treatment, based on available information accepted in the art for identifying a disease, disorder or condition, including but not limited to X-rays, CT scans, conventional laboratory diagnostic tests (such as blood counts), genomic data, protein expression data, and immunohistochemistry. The term "suffering" is typically used in conjunction with a specific disease state, for example, "suffering from a neoplastic disease" refers to a subject who has been diagnosed with a neoplasm.

[0179] Virtually pure As used herein, the term "substantially pure" indicates that a component (e.g., a polypeptide) constitutes more than about 50% of the total content of the composition, typically more than about 60% of the total polypeptide content. More typically, "substantially pure" refers to a composition in which at least 75%, at least 85%, at least 90% or more of the total composition is the component of interest. In some cases, the polypeptide will constitute more than about 90%, or more than about 95% of the total content of the composition.

[0180] T-cells:As used herein, the term "T-cell" or "T cell" is used in its conventional sense to refer to lymphocytes that differentiate in the thymus and bear specific cell surface antigen receptors, including those that control the initiation or suppression of cellular and humoral immunity and those that lyse antigen-containing cells. In some embodiments, T cells include, but are not limited to, naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, e.g., T H 1. T H 2. T H 9. T H 11. T H 22, T FH ;regulatory T cells, e.g. R 1, Tregs, inducible Tregs; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor infiltrating lymphocytes (TILs), and engineered variants of such T cells, including but not limited to CAR-T cells, recombinantly modified TILs, and TCR-engineered cells.

[0181] Therapeutically effective doseThe phrase "therapeutically effective amount" is used herein in reference to administering to a subject an active ingredient, 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 capable of having any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition when administered to a subject. A therapeutically effective amount can be determined by measuring the relevant physiological effect, and the amount can be adjusted in conjunction with a dosing regimen and depending on the diagnostic analysis of the subject's condition, etc. The evaluation parameters for determining a therapeutically effective amount of an active ingredient are determined by a physician using diagnostic criteria accepted in the art, including, but not limited to, age, weight, sex, overall health, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, X-ray, and other evidence. Alternatively, or additionally, other parameters commonly assessed in a clinical setting, such as body temperature, heart rate, normalization of blood chemistries, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or characteristic of a disease, disorder, or condition, biomarker (e.g., inflammatory cytokines, IFN-γ, granzymes, etc.), reduction in serum tumor markers, improvement in Response Evaluation Criteria in Solid Tumors (RECIST), improvement in immune-related response criteria (irRC), increased survival, prolonged progression-free survival, prolonged time to progression, increased time to treatment success, prolonged event-free survival, prolonged time to initiation of next treatment, improved objective response rate, improved duration of response, reduction in tumor burden, complete response, partial response, stable disease, etc. (relying on a clinician skilled in the art to assess the improvement of a subject's condition in response to administration of the agent) can be monitored to determine whether a therapeutically effective amount of an agent has been administered to a subject. As used herein, the terms "complete response (CR)," "partial response (PR)," "stable disease (SD)," and "progressive disease (PD)" for target lesions, and the terms "complete response (CR)," "incomplete response / stable disease (SD)," and "progressive disease (PD)" for non-target lesions, are understood to be defined by RECIST criteria.As used herein, the terms "immune-related complete response (irCR)," "immune-related partial response (irPR)," "immune-related progressive disease (irPD)," and "immune-related stable disease (irSD)" are as defined according to the immune-related response criteria (irRC). As used herein, the term "immune-related response criteria (irRC)" refers to the evaluation system for 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. A therapeutically effective amount can be adjusted throughout the course of treatment of a subject in conjunction with the dosing regimen and / or evaluation of the subject's condition and variations in the aforementioned factors. In one aspect, a therapeutically effective amount is an amount of an agent that, when used alone or in combination with another agent, does not result in irreversible serious adverse events during administration to a mammalian subject.

[0182] Transmembrane domainThe term "transmembrane domain" or "TM" refers to a domain of a membrane-spanning polypeptide (e.g., a membrane-spanning polypeptide such as CD122 or CD132 or CAR) that is embedded in the cell membrane when the membrane-spanning polypeptide is associated with the cell membrane and that is peptidyl-linked to the extracellular domain (ECD) and intracellular domain (ICD) of the membrane-spanning polypeptide. The transmembrane domain can be homologous (naturally associated) or heterologous (not naturally associated) to either or both of the extracellular domain and / or intracellular domain. In some embodiments, the transmembrane domain is the transmembrane domain naturally associated with the ECD domain of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, the transmembrane domain is the transmembrane domain naturally associated with the ICD domain of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, the transmembrane domain is the transmembrane domain naturally associated with a growth signaling domain. In some embodiments, the transmembrane domain is the transmembrane domain naturally associated with a different protein. Alternatively, the transmembrane domain of the orthogonal receptor can be an artificial amino acid sequence that crosses the plasma membrane. In some embodiments, the transmembrane domain of the orthogonal receptor is the transmembrane domain normally 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 from a first parent receptor and a second extracellular domain from a second, different parent receptor, the transmembrane domain of the chimeric receptor is the transmembrane domain normally associated with either the ICD or ECD of the parent receptor from which the chimeric receptor is derived.

[0183] TreatThe terms "treat," "treating," "treatment," and the like refer to a course of action (such as administering IL2, CAR-T cells, or a pharmaceutical composition comprising same) initiated with respect to a subject after a disease, disorder, or condition, or a symptom thereof, has been diagnosed, observed, etc. in a subject, in order to eliminate, reduce, inhibit, alleviate, or ameliorate, either temporarily or permanently, at least one underlying cause of the disease, disorder, or condition afflicting the subject, or at least one symptom associated with such disease, disorder, or condition. Treatment includes a course of action taken with respect to a subject suffering from a disease that results in the inhibition of the disease in the subject (e.g., preventing the onset of the disease, disorder, or condition or ameliorating one or more symptoms associated therewith).

[0184] Treg cells or regulatory T cells The term "regulatory T cells" or "Treg cells," as used herein, refers to CD4 T cells that are capable of suppressing the responses of other T cells, including, but not limited to, effector T cells (Teff). + This refers to a type of T cell. Treg cells are characterized by the expression of CD4, the α-subunit of the IL2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)). + "T cells" refers to CD4 T cells other than regulatory T cells. + It stands for T cells.

[0185] variantThe terms "protein variant" or "variant protein" or "variant polypeptide" are used interchangeably herein and refer to a polypeptide that differs from a parent polypeptide by virtue of at least one amino acid modification. The parent polypeptide can be a naturally occurring or wild-type (WT) polypeptide, or can be a modified version of a WT polypeptide. The term variant polypeptide can refer to the polypeptide itself, a composition comprising the polypeptide, or a nucleic acid sequence encoding it. In some embodiments, a variant polypeptide contains about 1 to about 10 amino acid modifications relative to the parent polypeptide, alternatively about 1 to about 5 amino acid modifications relative to the parent, alternatively about 1 to about 3 amino acid modifications relative to the parent, alternatively 1 to 2 amino acid modifications relative to the parent, or alternatively a single amino acid modification relative to the parent. A variant can be at least about 99% identical, alternatively at least about 98% identical, alternatively at least about 97% identical, alternatively at least about 95% identical, or alternatively at least about 90% identical to the parent polypeptide from which it is derived.

[0186] Wild type: As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc., has an amino acid sequence or nucleotide sequence that has not been artificially modified.

[0187] Description of Specific Aspects Adoptive Cell Therapy: "Adoptive cell therapy" or simply "cell therapy" refers to the administration of externally engineered cells, particularly immune cells. One form of adoptive cell therapy uses externally engineered lymphocytes isolated from tumor tissue (referred to as "tumor-infiltrating lymphocytes" or "TILs"). Although such TILs are exposed to tumor antigens and can attack tumor cells, they are thought to be either scarce or "exhausted," and therefore unable to independently eliminate tumors. In TIL therapy, isolated TILs are cultured ex vivo to expand their numbers, exposed to activating agents, and reinfused into the patient from whom the cells were isolated (referred to as "autologous cell therapy"). TIL cell therapy has been reported as an effective treatment for neoplastic diseases in human subjects. See, e.g., U.S. Patent No. 5,126,132A to Rosenberg, issued June 30, 1992, and Spiess, et al (1987) J Natl Cancer Inst 79:1067-1075.

[0188] Currently, human TIL cell therapy consists of ex vivo expansion of TILs obtained from resected tumor material, adoptive transfer into the subject after the subject has undergone a lymphodepletion regimen, and subsequent support of the adoptively transferred cells with interleukin-2 (IL-2). The lymphodepletion regimen depletes Tregs, eliminating a cellular "sink" and is often characterized as a "manufacturing chamber" for the adoptively transferred cells. Systemic administration of IL-2 supports the in vivo persistence of reinfused TILs. In typical clinical practice, immediately after TIL infusion, patients receive high-dose IL-2 (720,000 IU / kg) intravenously every 8 hours until maximal tolerance. This subsequent support with IL-2 is thought to further enhance TIL survival and clinical efficacy.

[0189] Although clinical efficacy has been demonstrated, TIL cell therapy is associated with significant toxicity, primarily resulting from lymphodepletion preparative regimens resulting in pancytopenia and febrile neutropenia, as well as supportive care with high-dose IL-2 after re-administration of enriched TIL cell populations. The effects of high-dose IL-2 typically used in supportive regimens for adoptive cell therapy have been widely reported to result in significant toxicity. The most common side effects resulting from the use of IL-2 supportive care after 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.

[0190] TIL cell therapy also faces challenges arising from the ex vivo expansion of isolated T cells. Ex vivo expansion of TILs is carried out for a significant period of time in the presence of high-dose IL-2. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances B cell growth, and activates monocytes and natural killer cells. However, during the TIL expansion process, there is interclonal competition with different T-cell clones, although the frequency may increase or decrease. While it is desirable for the final TIL product to be administered to be as enriched as possible for tumor-specific clones, the nonspecific nature of hIL-2 may not provide selective support to T-cell clones that have experienced tumor-specific antigens. Therefore, the most effective tumor-reactive T-cell clones may be outcompeted and diluted during the ex vivo expansion phase due to the nonspecific T-cell proliferation effect of hIL-2. As a result, the TIL cell product to be reinfused into a subject may contain a suboptimal fraction (e.g., less than 60%, or less than 50%, or less than 40%, or less than 30%) of anti-tumor TILs (cells that have experienced tumor antigens) relative to the total number of cells in the cell product to be reinfused into the subject. In addition, the degree of T-cell differentiation of T cells after an ex vivo stimulation procedure may affect the in vivo survival, proliferation capacity, and efficacy of TILs after reinfusion. Li, et al. (2010) J Immunol. 2010;184:452-465. The high potency of IL2 and the effect of exposing cultured TILs to high doses of IL2 have been associated with the terminal differentiation of T cells cultured ex vivo in its presence, as well as autoimmune mediation and transplant rejection, in addition to other in vivo side effects.

[0191] Li et al. state: An important question that emerged from our study is whether other methods of performing REP can be used to generate post-REP T cells with a "younger" phenotype associated with maintained CD28 expression and other effector-memory markers that allow for better in vivo persistence during ACT. In other words, can we get the best of both worlds by generating large numbers of tumor-reactive cytotoxic T cells while maintaining a memory phenotype that favors continued cell division and long-term survival in vivo? Li et al., p. 465. Furthermore, Li et al. suggest that the terminal differentiation state of TILs obtained from current ex vivo protocols involving IL2 is problematic for current TIL therapy, and suggest that other cytokines, such as IL-15 or IL-21, may be used to circumvent the effects of IL-2 in ex vivo preparations of TILs. Nevertheless, support of TILs using high-dose IL2 therapy after ACT is associated with improved therapeutic outcomes. However, high-dose IL2 therapy is associated with significant toxicity in human subjects, which, as mentioned above, is one of the major challenges facing TIL therapy. Therefore, it would be desirable to provide a method for supporting the ex vivo viability and / or proliferation of tumor antigen-experienced T cells without driving the desired tumor antigen-experienced T cell population toward differentiation and / or exhaustion.

[0192] Engineered Cell Therapy In addition to TIL cell therapy, and inspired in part by the demonstrated ability of the human immune system to eliminate tumors, a wide variety of approaches are being investigated to engineer immune cells with particularly desirable properties. One such category of engineered immune cells that has been clinically proven and approved for human use is the use of immune cells engineered to express chimeric antigen receptors (CARs). CAR T cell therapy in early clinical trials for patients with pre-B-cell acute lymphoblastic leukemia (ALL) or B-cell lymphomas has been revolutionary and suggested a potential treatment option for patients refractory to standard treatments. These early trials led to the rapid FDA approval of anti-CD19 CAR T cell products for both acute lymphoblastic leukemia (ALL) and certain types of B-cell lymphoma. Initial clinical responses in the treatment of hematologic malignancies with CAR T therapy have been very promising, with reported initial response rates of over 90% in human subjects, which has stimulated significant research into the development of CAR-T, with a wide variety of CAR-T approaches in various stages of preclinical and clinical development for the treatment of a wide variety of neoplastic diseases.

[0193] The main targeting and activation component of CAR-T cells is the CAR, a multifunctional polyprotein typically containing a tumor antigen-specific targeting domain, additional structural (e.g., hinge, transmembrane) domains, and an intracellular signaling domain. A wide variety of CAR designs have been proposed in the literature and are often classified as first-, second-, third-, or fourth-generation CARs, primarily based on the architecture of the signaling domain. The term first-generation CAR refers to CARs whose intracellular domain transmits signals from antigen binding through only one signaling domain, such as a signaling domain derived from a high-affinity receptor for IgE FcεR1γ or CD3ζ chain. The intracellular signaling domain contains one or three immunoreceptor tyrosine-based activation motifs (ITAMs) for antigen-dependent T-cell activation. ITAM-based activation signals confer T-cells the ability to lyse target tumor cells and secrete cytokines in response to antigen binding. Second-generation CARs contain costimulatory signals in addition to the CD3ζ signal. The simultaneous delivery of a costimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells. The costimulatory domain is usually membrane-proximal compared to the CD3ζ domain. Third-generation CARs contain a tripartite signaling domain, including, for example, CD28, CD3ζ, OX40, or 4-1BB signaling regions. In fourth-generation or "armored CARs," CAR T-cells are further modified to express or block molecules and / or receptors that enhance immune activity, such as the expression of IL-12, IL-18, IL-7, and / or IL-10; 4-1BB ligand, CD-40 ligand.

[0194] While the intracellular signaling domain is important for the activation and proliferation of engineered CAR-T cells, the extracellular targeting domain (or ABD), which defines the targeting of CAR-T and its counterparts, has clinical applications. The extracellular targeting antigen-binding domain (ABD) typically comprises an antibody or antibody fragment (e.g., scFv or VHH) that specifically binds to a cell surface antigen (either a peptide or a carbohydrate) characteristic of neoplastic cells, providing selective targeting of CAR-T cells. To minimize potential side effects and toxicity of CAR-T cells, including autoimmune reactions, when selecting tumor antigens for targeting, it is preferable that such antigens be found in much greater abundance on tumor cell types than on normal cells in the subject to be treated. Examples of such tumor antigens and their clinical therapeutic targets for which antibody binding molecules have been identified include CD19 (e.g., hematological malignancies, e.g., ALL, CLL, B-cell lymphoma), CD20 (e.g., refractory or relapsed CD20), and CD21 (e.g., CD21). +B-cell lymphoma), BCMA (e.g., multiple myeloma; Carpenter, et al. (2013) Clin Cancer Res; 19(8); 2048-60), CD22 (B-cell malignancies, including pediatric B-cell precursor ALL, as described in Pan, et al. (2019) Leukemia 33, 2854-2866), CD30 (e.g., CD30+ lymphomas, including Hodgkin lymphoma; Grover, (2019) BMC Cancer 19, 203), CD70 (e.g., acute myeloid leukemia (AML; Sauer, et al. (2019) Blood 134 (Supplement_1): 1932), Lewis Y (e.g., AML; Ritchie, et al. (2013) Molecular Therapy 21(11): 2122-9), GD2 (e.g., glioma; Mount, et al (2018) Nat Med 24, 572-579), GD3 (e.g., metastatic melanoma and neuroectodermal tumors; Agnes, et al. (2010) DOI: 10.1158 / 1078-0432.CCR-10-0043), mesothelin (e.g., mesothelioma, lung, pancreatic, breast, ovarian and other solid tumors; Beatty, et. Al., (2014) Cancer Immunol Research 2(2)), ROR-1 (e.g., chronic lymphocytic leukemia; Aghebati-Maleki, et al (2017) Biomedicine and Pharmacology 88: 814-822), CD44 (e.g., AML and multiple myeloma; Casuccia, et al (2013) Blood 122 (20): 3461-3472), CD171 (e.g., neuroblastoma; Kunkele, et al (2017) Clin Cancer Research 23(2): 466-477); EGP2, EphA2 (e.g., glioblastoma; Yi, et al (2018) Molecular Therapy: Methods & Clinical Development 9:70-80), ErbB2, ErbB3 / 4, FAP, FAR These include, but are not limited to, IL11Ra, PSCA (prostate cancer), PSMA (prostate cancer), NCAM, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A) PSA, CEA, VEGF, VEGF-R2, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa), and Wnt1 antigen CD123. Additionally, ABDs can be multivalent in that they have the ability to bind more than one antigen, and in particular, can have specificity for more than one tumor antigen (e.g., CD19 and CD20 as described in Zah, et al (2016) Cancer Immunol Res;4(6);498-508; CD19 and CD22 as described in Tu, et al (2019) Frontiers in Oncology 9:1350).

[0195] One specific example of such an antigen is the 95 kDa glycoprotein CD19. CD19 is expressed by most B-cell lymphomas, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and some acute myeloid leukemia (AML), but CD19 is not present on most normal tissues other than normal B cells. While multiple CAR-T product candidates are in various stages of clinical development, the anti-CD19 CAR cell products axicabtagene ciloreucel (sold by Gilead Pharmaceuticals under the trade name Yescarta®) and tisagenlecleucel (sold by Novartis under the trade name Kymriah®) are currently the only CAR-T cell therapies approved for human use by major regulatory agencies, and a wide variety of CAR-T therapies are in various stages of preclinical and clinical development for the treatment of a wide variety of neoplastic diseases.

[0196] While initial clinical responses to the treatment of neoplastic disease with CAR T therapy have been very promising, with reported initial response rates of over 90% in human subjects, a growing body of literature on clinical experience with approved anti-CD19 CAR agents has revealed that the majority of subjects treated with such agents experience relapse of their disease state. The lack of durable responses in such patients is primarily due to poor CAR T cell persistence after administration of CAR T cell products and / or cancer cell resistance resulting from antigen loss or modulation. Administration of IL2 at doses tolerated by patients fails to provide long-term selective maintenance of activated populations of adoptively transferred cells, leading to recurrence and relapse of neoplastic disease.

[0197] The present disclosure provides methods and compositions that overcome these problems and opens up new possibilities for the use of engineered adoptive cell therapies, including CAR T therapy, particularly in the treatment of solid tumors where persistence of engineered cells is particularly important.

[0198] It is widely accepted that adoptively transferred human immune cells lose their activity relatively rapidly after administration. Therefore, typical approaches to address this rapid loss of function include (a) administering excessively high doses of cell therapy to maximize exposure of the cell therapy to the tumor before the cells lose efficacy, and / or (b) systemic administration of HD-hIL2 therapy to attempt to support the efficacy of the adoptively transferred cells. Both of these approaches exhibit significant toxicity. The toxicity associated with HD-hIL2 therapy has already been discussed above. High doses of engineered cell therapy agents are associated with life-threatening cytokine release syndrome (CRS). Currently available products have demonstrated CRS of all grades in many treated subjects, and CRS of grade 3 or higher in the majority of patients. Significant neurotoxicity is also observed in many patients. However, lower doses of cell therapy agents have been associated with significantly reduced clinical outcomes. Additionally, many patients who initially appeared to respond well to cell therapy relapse, primarily due to the lack of durability of cell therapy products. Currently, it has been reported that approximately 60% of patients treated with existing CD-19 cell therapy agents relapse. Byrne M, et al (2019) Biology of Blood and Marrow Transplantation 25(11):344-251.

[0199] As demonstrated by experimental results described in more detail below, administration of orthogonal cells in combination with an orthogonal ligand addresses many of the current challenges of cell therapy and provides an improved method of treating diseases amenable to cell therapy, including but not limited to: Compositions and methods that allow for the selective expansion in vivo of populations of adoptively transferred human immune cells without significant systemic off-target activation of other immune cells; Compositions and methods that support the persistence of activated orthogonal cells in adoptively transferred human immune cells without significant supportive agent-associated toxicity; · Compositions and methods for achieving in vivo therapeutic efficacy of a cell therapy product in the treatment of neoplastic disease in mammalian subjects using initial doses of the cell therapy agent that are significantly (10-1000 fold) lower than current doses of similar cell therapy products that have been reported to be ineffective; Compositions and methods that allow for the maintenance of therapeutically effective levels of orthogonal immune cells over extended periods of time through the regular administration of orthogonal ligands; Compositions and methods that allow for the treatment of recurrence of neoplastic conditions by administration of an orthogonal ligand to restore the effectiveness of previously administered orthogonal cells, without the need for administration of additional engineered orthogonal cells; Compositions and methods that provide selective modulation of orthogonal cell activity and proliferation, thereby allowing temporary or permanent cessation of a subject's exposure to an activated form of an orthogonal cell therapy by removal of the activating orthogonal ligand and without the need to further engineer the cells to contain a "kill switch" or use immunodepleting or immunosuppressive treatment regimens; and cellular responses without enhancing proliferation of adoptively transferred cells in a mammalian subject following administration of the cellular product, without significant toxicity. Compositions and methods that avoid the need for prior immunodepletion of a subject prior to adoptive cell therapy; · pharmaceutical formulations of orthogonal ligands, especially orthogonal ligands with an extended duration of action that allows for less frequent dosing of the supporting orthogonal ligand; Compositions and methods that provide demonstrable therapeutic anti-tumor efficacy superior to existing cell therapy agents for similar indications; and Compositions and methods that provide enhanced anti-tumor efficacy when combined with adjunctive therapeutic agents.

[0200] A series of experiments were performed to demonstrate the utility and functionality of the compositions and methods of the present disclosure in treating mammalian subjects suffering from neoplastic disease states, particularly in mammalian subjects undergoing orthogonal systems in the context of adoptive cell therapy. The utility of the compositions and methods of the present disclosure was evaluated in a series of experiments as further detailed in the accompanying Examples and data provided in the accompanying Figures.

[0201] Briefly, a series of in vitro and in vivo experiments were performed on a representative human IL2 orthologue of Formula 1, i.e., containing a set of amino acid substitutions numbered according to wt hIL2: [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125A], and the following amino acid sequence: This was performed using the hIL2 variant (sometimes referred to herein as "orthoIL2" and "hoIL2"), having TIFF2026041861000008.tif26128.

[0202] To confirm the selective binding properties of STK-007, Biacore surface plasmon resonance studies were performed to confirm that STK-007 retained significant binding to the CD25 (IL2Ra) and CD132 (IL2Rg) components of the IL2 receptor, demonstrating specific binding to hoRb, while wild-type hIL2 did not significantly bind to hoRb. Briefly, C-terminal HIS-tagged versions of wtCD25, wtCD122, hoCD122, and wtCD132 were prepared and immobilized on an anti-HIS capture chip, and binding was assessed using Biacore by flowing a solution of STK-007 and wt hIL2 molecules (Shenandoah Biotechnology, Inc.) over the immobilized receptor subunits. The results of this experiment are shown in Figure 1 of the accompanying drawings. Panels A, C, E, and G represent the binding of STK-007 to wtCD25, wtCD122, wtCD132, and hoCD122, respectively. Panels B, D, F, and H represent the binding of wtIL2 to wtCD25, wtCD122, wtCD132, and hoCD122, respectively. The data presented show that STK-007 retains similar binding to wt hIL2 to wtCD25 and wtCD132, but retains significantly lower affinity for wt CD122. However, STK-007 binds to hoCD122 with an affinity similar to that of wtIL2 to wtCD122 (panel G). Similarly, wt hIL2 demonstrates similar binding to wtCD25, wt CD122, and wtCD132, with significantly lower affinity for hoCD122.

[0203] For in vivo studies, the STK-007 molecule was synthesized using the following structure: TIFF2026041861000009.tif20128 was modified at its N-terminus by the addition of a 40 kDa branched PEG molecule, thereby yielding the following structure, hereinafter referred to as STK-009: 40kD-PEG-linker-desAla1-hIL2[E15S-H16Q-L19V-D20L-Q22K-M23A]-COOH[2] The representative orthogonal cell line used in these studies has the following amino acid sequence: The CD19 orthogonal CAR-T cells were engineered to express the orthogonal CD122 (IL2Rb) receptor (sometimes referred to herein as "hoCD122" or "hoRb" (human ortho receptor beta)) bearing TIFF2026041861000010.tif66128. The extracellular domain of the hoRb receptor contains two amino acid substitutions, H133D and Y134F (numbered according to wt hCD122).

[0204] The exemplary CAR used in these studies contains the FMC63 anti-CD19 scFv as the antigen-binding domain, the CD28 transmembrane and costimulatory domains, and the CD3z domain (Figure 2, panel A), and has the amino acid sequence: TIFF2026041861000011.tif41129 is an anti-CD19 chimeric antigen receptor.

[0205] As illustrated in Figure 1A, CD19_28z CAR, T2A peptide and hoRb sequence: Nucleic acid sequences encoding TIFF2026041861000012.tif87129 were synthesized. These constructs were synthesized and cloned downstream of the EF1α promoter in the third-generation pl4Syn lentiviral backbone (Alstem Bio). Following isolation and stimulation, "CD19_28z orthoCAR" T cells, also referred to as SYNCAR-001, were generated.

[0206] In the disseminated model, as demonstrated by the data provided in Figure 3, administration of PBS failed to control tumor burden (Figure 3, panel A, group 1, and Figure 3, panel B, top left), resulting in the need to sacrifice animals due to toxicity approximately on day 21 of the study. In contrast, administration of CD19_28z orthoCAR T cells led to antitumor responses in 4 / 8 mice (Figure 3, panel A, group 2, and Figure 3, panel B, top right). Combination treatment of CD19_28z orthoCAR T cells with STK-009 at both dose levels (Figure 3, panel A, 1 μg (group 3), and Figure 3, panel B, bottom right) and 2 μg (group 4), and Figure 3, panel B, bottom left)) provided additional antitumor function compared to CD19_28z orthoCAR T cell treatment alone.

[0207] In addition, the data provided in Figure 4 from the rechallenge model described in Example 8 demonstrate that STK009 rechallenge can restore the anti-tumor activity of CAR T cells, even in the absence of prolonged antigen or tumor ligand exposure.

[0208] The relapse model described in Example 9, together with the data provided in Figure 5, demonstrates that administration of STK-009 alone can achieve anti-tumor activity of CAR-T cells in animals that have relapsed from a prior course of treatment.

[0209] Additionally, as discussed in Example 11 and provided in Figure 6, orthogonal CAR-T cells contacted with an orthogonal ligand (STK-009) retained the SCM phenotype in vivo, demonstrating enhanced persistence of CAR T cells, providing a more durable anti-tumor effect.

[0210] Of particular note are the data obtained from the solid tumor model discussed in Example 12 and provided in Figures 7-10. This data demonstrates that orthogonal CAR-T cells in combination with their orthogonal cognate ligands can induce responses in solid tumors and increase CAR-T cell infiltration into solid tumors, demonstrating that this system is useful in treating solid tumors that were not previously treatable using adoptive cell transfer protocols such as CAR T.

[0211] These data demonstrate improved persistence of therapeutic engineered cells expressing orthogonal receptors (e.g., hoCART, hoTIL), the ability to selectively and potently activate engineered orthogonal immune cells in a dose-dependent manner in response to contact with the cognate orthogonal ligand, the specificity of the ligand for cells expressing the orthogonal ligand, and significantly reduced toxicity, without specific off-target toxicity as typically observed with administration of nonspecific T cell expansion agents such as hIL2, a well-documented source of toxicity in adoptive cell therapy protocols.

[0212] The selectivity of the orthogonal ligand for the orthogonal receptor results in a molecule with low in vivo toxicity. This was demonstrated in a non-human primate model toxicity study in which animals were exposed to high doses of a long-acting PEGylated orthogonal hIL2 molecule as described herein. Despite the persistence of the orthogonal IL2 compound at high doses, as observed in this study, no significant toxicity was observed at doses significantly greater than the therapeutically effective dose (likely at least 10- to 100-fold greater). The selectivity and efficacy of the orthogonal ligand to selectively activate therapeutic cells allows adoptive cell therapy to be used in a prophylactic manner, in conjunction with BCMA CAR T cells, to prevent disease progression, such as in the treatment of indolent precancerous conditions such as smoldering multiple myeloma.

[0213] The orthogonal ligands were successful in re-enervating the CAR T cells, conferring the ability to reconstitute the therapeutic efficacy of the engineered cells without re-administration of the cells, demonstrating the utility of this technology in preventing recurrence, relapse, and metastasis of neoplastic disease.

[0214] In some aspects, the present disclosure provides methods of treating a disease, disorder, or condition in a human subject, comprising: (a) administering to the subject a therapeutically effective amount of an orthogonal ligand; and (b) administering to the subject a mammalian immune cell comprising a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more expression control elements such that the mammalian immune cell expresses the orthogonal hCD122 receptor.

[0215] The present disclosure provides methods and compositions for treating a subject suffering from a neoplastic disease by preventing recurrence of the neoplastic disease by administering to the subject a maintenance therapy comprising co-administration of an orthogonal CD122 receptor and a chimeric antigen receptor (whose extracellular domains specifically bind to a tumor antigen) with an orthogonal IL2 ligand, and periodic administration of an orthogonal IL2 ligand of Formula 1, wherein the orthogonal ligand used in the treatment phase is the same as or different from the orthogonal ligand used in the maintenance phase. In some embodiments, the orthogonal ligand is modified to extend its half-life. In one embodiment, the orthogonal ligand is a pegylated fusion protein, or the like. In one embodiment, the orthogonal ligand comprises a 40 kD N-terminal PEG moiety. In some embodiments of the method, the orthogonal ligand is administered in a first treatment phase at a concentration above sufficient to result in expansion (e.g., >EC 10 PRO ) below concentrations sufficient to induce significant differentiation (e.g., <EC 90 ACT) ligand for a period of at least 24 hours, optionally for 30, 60, 90 or more days. In some embodiments, the maintenance phase optionally includes administration of an orthogonal ligand of Formula 1 sufficient to induce orthoCAR T activation, resulting in serum levels above the concentration for activation (e.g., >EC 50 ACT ) for a period of at least 24 hours. In some embodiments of the method, the orthogonal ligand is provided to the subject by administration of a recombinant viral or non-viral vector comprising a nucleic acid encoding an orthogonal IL2 ligand of Formula 1. In some embodiments of the method, the neoplastic disease is selected from a solid tumor and a hematological malignancy. In some embodiments of the method, the method further comprises one or more adjunctive anti-neoplastic agents during the treatment and / or maintenance phases. In some embodiments of the method, the adjunctive anti-neoplastic agents during the treatment and / or maintenance phases can be the same or different. In some embodiments of the method, the adjunctive neoplastic agents are selected from the group consisting of adjunctive biologics, including but not limited to chemotherapeutic agents, small molecules, checkpoint inhibitors (anti-PD1, Keytruda, Opdivo), anti-tumor antigen antibodies (Herceptin), and / or physical methods (surgery, radiation, etc.). In some embodiments, the orthogonal receptor is orthogonal CD122 comprising one or more STAT3 binding motifs.

[0216] Prevention of metastasis The present disclosure provides methods and compositions for treating a subject suffering from a neoplastic disease by preventing metastasis of the neoplastic disease by administering to the subject a maintenance therapy comprising the simultaneous administration of a plurality of engineered T cells expressing an orthogonal CD122 receptor and a chimeric antigen receptor (whose extracellular domains specifically bind to a tumor antigen) with an orthogonal IL2 ligand of Formula 1, and periodic administration of the orthogonal IL2 ligand of Formula 1, wherein the orthogonal ligand used in the treatment phase is the same as or different from the orthogonal ligand used in the maintenance phase. In some embodiments, the orthogonal ligand is modified to extend its half-life. In one embodiment, the orthogonal ligand is a pegylated fusion protein, or the like. In one embodiment, the orthogonal ligand comprises a 40 kD N-terminal PEG moiety. In some embodiments of the method, the orthogonal ligand is administered in a first treatment phase at a concentration above sufficient to result in expansion (e.g., >EC 10 PRO ) below concentrations sufficient to induce significant differentiation (e.g., <EC 90 ACT ) ligand for a period of at least 24 hours, optionally for 30, 60, 90 or more days. In some embodiments, the maintenance phase optionally includes administration of an orthogonal ligand of Formula 1 sufficient to induce orthoCAR T activation, resulting in serum levels above the concentration for activation (e.g., >EC 50 ACT) for a period of at least 24 hours. In some embodiments of the method, the orthogonal ligand is provided to the subject by administration of a recombinant viral or non-viral vector comprising a nucleic acid encoding an orthogonal IL2 ligand of Formula 1. In some embodiments of the method, the neoplastic disease is selected from a solid tumor and a hematological malignancy. In some embodiments of the method, the method further comprises one or more adjunctive anti-neoplastic agents during the treatment and / or maintenance phases. In some embodiments of the method, the adjunctive anti-neoplastic agents during the treatment and / or maintenance phases can be the same or different. In some embodiments of the method, the adjunctive neoplastic agents are selected from the group consisting of adjunctive biologics, including but not limited to chemotherapeutic agents, small molecules, checkpoint inhibitors (anti-PD1, Keytruda, Opdivo), anti-tumor antigen antibodies (Herceptin), and / or physical methods (surgery, radiation, etc.). In some embodiments, the orthogonal receptor is orthogonal CD122 comprising one or more STAT3 binding motifs.

[0217] CD19 CAR for hematologic malignancies: In one aspect, the present disclosure provides a method of treating or preventing a hematological malignancy in a subject in need thereof by administering a therapeutically effective amount of an orthogonal CD19 CAR in combination with administration of a therapeutically effective amount of an orthogonal IL2 ligand of Formula 1.

[0218] In one aspect, the present disclosure provides an orthogonal CD19 CAR for use in a method of preventing recurrence and / or metastasis in a subject suffering from a hematological neoplastic disease, comprising the step of administering a plurality of engineered T cells expressing an orthogonal CD122 polypeptide and a chimeric antigen receptor (the extracellular domain of which specifically binds CD19) in combination with administration of an orthogonal IL2 ligand of Formula 1. In some aspects, the CAR is SYNCAR-001 as described herein.

[0219] In some embodiments, the present invention provides methods for preventing relapse after a partial or complete response to an initial orthogonal CD19 CAR-T / orthogonal IL2 ligand treatment phase in a subject previously treated with orthogonal CD19 CAR T cells in combination with an orthogonal IL2 ligand, comprising administration of a maintenance therapy course comprising periodic administration of an orthogonal IL2 ligand of Formula 1 at a concentration lower than that administered during the treatment phase. The orthogonal ligand administered during the treatment phase can be the same or different from the orthogonal ligand administered during the maintenance phase.

[0220] In some embodiments, the hematological malignancy is a relapsed or refractory hematological malignancy, including but not limited to relapsed or refractory non-Hodgkin's lymphoma, relapsed or refractory myeloma, relapsed or refractory large B-cell lymphoma, and relapsed or refractory mantle cell lymphoma. A relapsed hematological malignancy (e.g., relapsed myeloma) involves a situation in which a patient has successfully completed an initial course of treatment but the disease has reappeared. A refractory hematological malignancy refers to a disease that progresses despite aggressive treatment. A patient suffering from refractory myeloma is said to have had primary refractory myeloma if the disease does not respond to chemotherapy and continues to progress, and is said to have secondary refractory patients if they had an initial response at the start of treatment but the treatment is no longer effective.

[0221] In some embodiments, the orthogonal ligand is modified to enhance half-life. In one embodiment, the orthogonal ligand is a pegylated Fc fusion protein, albumin fusion, or the like. In one embodiment, the orthogonal ligand comprises a 40 kD N-terminal PEG moiety.

[0222] In some embodiments of the method, the orthogonal ligand is administered at a concentration above that sufficient to cause expansion (e.g., >EC 10 PRO ) below concentrations sufficient to induce significant differentiation (e.g., <EC 90 ACT) ligand for a period of at least 24 hours, or for a period of 1 week, or for a period of 2 weeks, or for a period of at least 30 days, or for a period of at least 60 days, or for a period of at least 90 or more days. In some embodiments, the maintenance phase optionally includes administration of an orthogonal ligand of Formula 1 sufficient to induce orthoCAR T activation, resulting in serum levels above the concentration for activation (e.g., >EC 50 ACT ) for a period of at least 24 hours.

[0223] In some embodiments of the method, the orthogonal ligand is provided to the subject by administration of a recombinant viral or non-viral vector comprising a nucleic acid encoding an orthogonal IL2 ligand of Formula 1. In some embodiments of the method, the neoplastic disease is selected from a solid tumor and a hematological malignancy.

[0224] In some embodiments of the method, the method further comprises one or more adjunctive anti-neoplastic agents during the treatment and / or maintenance phases. In some embodiments of the method, the adjunctive anti-neoplastic agents during the treatment and / or maintenance phases can be the same or different. In some embodiments of the method, the adjunctive anti-neoplastic agents are selected from the group consisting of chemotherapeutic agents, small molecules, checkpoint inhibitors (anti-PD1, Keytruda, Opdivo), adjunctive biologics including but not limited to anti-tumor antigen antibodies (Herceptin), and / or physical methods (surgery, radiation, etc.). In some embodiments, the orthogonal receptor is orthogonal CD122 comprising one or more STAT3 binding motifs. In some embodiments, the extracellular domain of the CAR comprises one or more antibodies selected from the group consisting of an antibody that specifically binds to CD19 comprising the CDRs or FMC63.

[0225] In some embodiments, the hematological neoplastic disease is selected from acute lymphoblastic leukemia (ALL), including Philadelphia chromosome-positive ALL, chronic lymphocytic leukemia (CLL), and B-cell lymphoma. In some embodiments, the method further comprises co-administration of one or more chemotherapeutic agents.

[0226] When the hematologic malignancy is ALL, the adjunctive agent can be vincristine or vincristine liposome (Marqibo), daunorubicin (daunomycin) or doxorubicin (Adriamycin), cytarabine (cytosine arabinoside, ara-C), L-asparaginase or PEG-L-asparaginase (pegaspargase or Oncaspar), 6-mercaptopurine (6-MP), methotrexate, cyclophosphamide, prednisone, dexamethasone, delarabine (Arranon). When the hematologic malignancy is Philadelphia chromosome-positive ALL, the adjunctive agents can be imatinib (Gleevec®), dasatinib (Sprycel®), nilotinib (Tasigna®), ponatinib (Iclusig®), and bosutinib (Bosulif®).

[0227] When the hematological malignancy is CLL, the adjunctive agent may be a fludarabine-containing regimen, including but not limited to "FCR" (fludarabine, cyclophosphamide and rituximab) and FR (fludarabine and rituximab), a pentostatin-based treatment regimen, including but not limited to PCR (pentostatin, cyclophosphamide and rituximab), alemtuzumab (Campath®), chlorambucil, chlorambucil in combination with obinutuzumab (Gazyva® anti-CD20 Mab), tyrosine kinase inhibitors, including but not limited to ibrutinib.

[0228] When the hematologic malignancy is refractory or relapsed CLL, the adjunctive agents are selected from one or more of: lenalidomide, ofatumumab, phosphoinositide 3-kinase (PI3K) inhibitors such as duvelisib and idelalisib; venetoclax alone or in combination with obinutuzumab or rituximab. When the hematologic malignancy is B-cell lymphoma, the adjunctive agent is selected from one or more of rituximab (Rituxan®), optionally in combination with cyclophosphamide; bendamustine in combination with obinutuzumab or rituximab; CHOP (cyclophosphamide, doxorubicin or hydroxydaunorubicin, vincristine (Oncovin®), and prednisone), optionally in combination with obinutuzumab or rituximab; CVP (cyclophosphamide, vincristine, prednisone), optionally in combination with obinutuzumab or rituximab; lenalidomide and rituximab; cyclophosphamide; chlorambucil; and ibritumomab tiuxetang (Zevalin®).

[0229] If the hematologic malignancy is Burkitt's lymphoma, adjunctive agents include CODOX-M (cyclophosphamide, doxorubicin, vincristine in combination with intrathecal methotrexate and cytarabine, followed by high-dose systemic methotrexate), optionally in combination with rituximab; dose-adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin), optionally in combination with rituximab; hyperCVAD (cyclophosphamide, vincristine, doxorubicin, and doxorubicin), optionally in combination with rituximab; and cytarabine, optionally in combination with rituximab; RICE (rituximab, ifosfamide, carboplatin, etoposide), optionally in combination with intrathecal methotrexate; RIVAC (rituximab, ifosfamide, cytarabine, etoposide), optionally in combination with intrathecal methotrexate and RGDP (rituximab, gemcitabine, dexamethasone, cisplatin); and cytarabine, optionally in combination with rituximab.

[0230] In some embodiments, the orthogonal receptor is orthogonal CD122, which contains one or more STAT3 binding motifs.

[0231] Examples of anti-CD19 CARs useful in practicing the methods of the present disclosure include the following constructs:

[0232] CD19_28z: Construct containing GMCSF receptor signal peptide, FMC63 scFv, AAA spacer, CD28 hinge and costimulatory domain and CD3 zeta: TIFF2026041861000013.tif47128

[0233] CD19_4-1bbz: Construct containing CD8a receptor signal peptide, FMC63 scFv, CD8 hinge and transmembrane domain, 4-1BB hinge and costimulatory domain and CD3 zeta: TIFF2026041861000014.tif47128

[0234] In an alternative embodiment, the CD19 CAR comprises the following amino acid sequence: TIFF2026041861000015.tif47128

[0235] In an alternative embodiment, the CD19 CAR comprises the following amino acid sequence: TIFF2026041861000016.tif41128

[0236] BCMA CAR: The present disclosure provides methods and compositions for treating a subject suffering from multiple myeloma by administering a plurality of engineered T cells expressing an orthogonal CD122 polypeptide and a chimeric antigen receptor (the extracellular domain of which specifically binds BCMA) in combination with an orthogonal IL2 ligand of Formula 1 for a first period (a treatment phase) until the subject achieves a partial or complete response, and administering to the subject a maintenance therapy comprising regular administration of the orthogonal IL2 ligand of Formula 1 for at least 30 days, optionally at least 90 days, optionally at least 180 days, or for a longer period as deemed necessary by a physician, to prevent metastasis of the neoplastic disease.

[0237] The present disclosure provides methods and compositions for achieving stringent complete responses in the treatment of subjects suffering from multiple myeloma by administering to the subject a maintenance therapy comprising the simultaneous administration of a plurality of engineered T cells expressing an orthogonal CD122 polypeptide and a chimeric antigen receptor (the extracellular domain of which specifically binds BCMA) with an orthogonal IL2 ligand of Formula 1, followed by periodic administration of the orthogonal IL2 ligand of Formula 1.

[0238] In the above-described methods, the orthogonal ligand used in the treatment phase may be the same as or different from the orthogonal ligand used in the maintenance phase. In some embodiments, the orthogonal ligand is modified to extend its half-life. In one embodiment, the orthogonal ligand is a pegylated fusion protein or the like. In one embodiment, the orthogonal ligand comprises a 40 kD N-terminal PEG moiety.

[0239] In some embodiments of the method, the orthogonal ligand is administered at a concentration above that sufficient to cause expansion (e.g., >EC 10 PRO ) below concentrations sufficient to induce significant differentiation (e.g., <EC 90 ACT) ligand for a period of at least 24 hours, optionally for 30, 60, 90 or more days. In some embodiments, the maintenance phase optionally includes administration of an orthogonal ligand of Formula 1 sufficient to induce orthoCAR T activation, resulting in serum levels above the concentration for activation (e.g., >EC 50 ACT ) for a period of at least 24 hours.

[0240] In some embodiments of the method, the orthogonal ligand is provided to the subject by administration of a recombinant viral or non-viral vector comprising a nucleic acid encoding an orthogonal IL2 ligand of Formula 1. In some embodiments of the method, the neoplastic disease is selected from a solid tumor and a hematological malignancy. In some embodiments of the method, the method further comprises administration of one or more adjunctive anti-neoplastic agents during the treatment and / or maintenance phases. In some embodiments of the method, the adjunctive anti-neoplastic agents during the treatment and / or maintenance phases can be the same or different. In some embodiments of the method, the adjunctive anti-neoplastic agents are selected from the group consisting of adjunctive biologics, including but not limited to chemotherapeutic agents, small molecules, checkpoint inhibitors (anti-PD1, Keytruda, Opdivo), anti-tumor antigen antibodies (Herceptin), and / or physical methods (surgery, radiation, etc.). In some embodiments, the orthogonal receptor is orthogonal CD122 comprising one or more STAT3 binding motifs.

[0241] In some embodiments, adjunctive agents useful in the treatment of multiple myeloma comprise one or more agents selected from the group consisting of thalidomide, lenalidomide, dexamethasone, bortezomib, vincristine, doxorubicin, dexamethasone, melphalan, carfilzomib, cyclophosphamide, cisplatin, etoposide, bortezomib, prednisone, daratumumab, carfilzomib, and ixazomib. In some embodiments, adjunctive agents useful in the treatment of multiple myeloma include theortezomib / lenalidomide / dexamethasone; bortezomib / cyclophosphamide / dexamethasone; bortezomib / doxorubicin / dexamethasone; carfilzomib / lenalidomide / dexamethasone; ixazomib / lenalidomide / dexamethasone; bortezomib / dexamethasone; bortezomib / thalidomide / dexamethasone; lenalidomide / dexamethasone; dexamethasone and ixazomib / lenalidomide / dexamethasone.

[0242] The present disclosure provides methods and compositions for treating a subject suffering from smoldering multiple myeloma by administering to the subject a maintenance therapy comprising administering to the subject a plurality of engineered T cells expressing an orthogonal CD122 polypeptide and a chimeric antigen receptor (CAR), wherein the extracellular domain of the CAR specifically binds BCMA, in combination with an orthogonal IL2 ligand of Formula 1, followed by periodic administration of the orthogonal IL2 ligand of Formula 1. The present disclosure provides methods and compositions for inhibiting the progression of smoldering multiple myeloma to multiple myeloma in a subject afflicted with smoldering multiple myeloma by administering to the subject a maintenance therapy comprising concurrent administration of a plurality of engineered T cells expressing an orthogonal CD122 polypeptide and a chimeric antigen receptor (CAR), wherein the extracellular domain of the CAR specifically binds BCMA, and an orthogonal IL2 ligand of Formula 1, and periodic administration of the orthogonal IL2 ligand of Formula 1. In some embodiments, the ABD of the CAR specifically binds BCMA.The method further includes one or more adjunctive anti-neoplastic agents administered to the subject during the treatment and / or maintenance phase, wherein adjunctive agents useful in the treatment of multiple myeloma are one or more selected from the group consisting of thalidomide, lenalidomide, dexamethasone, bortezomib, vincristine, doxorubicin, dexamethasone, melphalan, carfilzomib, cyclophosphamide, cisplatin, etoposide, bortezomib, prednisone, daratumumab, carfilzomib, and ixazomib, or bortezomib / lenalidomide / dexamethasone; bortezomib / cyclophosphamide / dexamethasone; bortezomib / doxorubicin / dexamethasone; carfilzomib / lenalidomide Combination treatment regimens of chemotherapeutic agents for use in the treatment of multiple myeloma include: nalidimide / dexamethasone; ixazomib / lenalidomide / dexamethasone; bortezomib / dexamethasone; bortezomib / thalidomide / dexamethasone; lenalidomide / dexamethasone; dexamethasone / thalidomide / cisplatin / doxorubicin / cyclophosphamide / etoposide / bortezomib (VTD-PACE); lenalidomide / low-dose dexamethasone; daratumumab / bortezomib / melphalan / prednisone; carfilzomib / lenalidomide / dexamethasone; carfilzomib / cyclophosphamide / dexamethasone; and ixazomib / lenalidomide / dexamethasone.

[0243] Examples of anti-BCMA CARs useful in the practice of the present invention include the following constructs:

[0244] BCMA4_41bbz CAR: Construct containing CD8a receptor signal peptide, BCMA4 scFv, CD8 hinge and transmembrane domain, 4-1BB hinge and costimulatory domain and CD3 zeta: TIFF2026041861000017.tif46128 This can be co-expressed with the ortho-CD122 receptor using a T2a linker, with the following amino acid sequence: TIFF2026041861000018.tif92128

[0245] GSI5021_41bbz CAR: TIFF2026041861000019.tif41128 This can be co-expressed with the ortho-CD122 receptor using a T2a linker, with the following amino acid sequence: TIFF2026041861000020.tif92128

[0246] B2121 BCMA_41bbz)CAR TIFF2026041861000021.tif41128 This can be co-expressed with the ortho-CD122 receptor using a T2a linker, with the following amino acid sequence: TIFF2026041861000022.tif92128

[0247] BMCA10_41bbz CAR TIFF2026041861000023.tif41128 This can be co-expressed with the ortho-CD122 receptor using a T2a linker, with the following amino acid sequence: TIFF2026041861000024.tif87128

[0248] GD2 CAR The present disclosure provides methods and compositions for treating a subject suffering from a neoplastic disease of neuroectodermal origin (including human neuroblastoma and melanoma) or high-risk osteosarcoma by administering to the subject a maintenance therapy comprising the simultaneous administration of a plurality of engineered T cells expressing an orthogonal CD122 polypeptide and a chimeric antigen receptor (CAR), wherein the extracellular domain of the CAR specifically binds GD2, and an orthogonal IL2 ligand of Formula 1, followed by periodic administration of the orthogonal IL2 ligand of Formula 1. The present disclosure provides methods and compositions for preventing metastasis or recurrence of neoplastic diseases of neuroectodermal origin (including human neuroblastoma and melanoma) by administering to the subject a maintenance therapy comprising concurrent administration of a plurality of engineered T cells expressing an orthogonal CD122 polypeptide and a chimeric antigen receptor (CAR), wherein the extracellular domain of the CAR specifically binds BCMA, and an orthogonal IL2 ligand of Formula 1, and periodic administration of the orthogonal IL2 ligand of Formula 1. In some embodiments, the extracellular domain of the CAR specifically binds GD2. In some embodiments, the ABD of the CAR comprises an antibody that specifically binds to GD2 comprising the CDRs, or one or more of the antibodies 3F8, hu14.18, 14G2a, optionally co-administered with chemotherapeutic agents including cisplatin, doxorubicin, cyclophosphamide, and epipodophyllotoxins such as teniposide and etoposide.

[0249] PSMA CAR for prostate cancer Prostate cancer is the second most common malignancy in men worldwide, with an estimated 1.1 million new cases per year. It is responsible for 307,000 deaths, making it the fifth leading cause of cancer death. Localized primary tumors can be successfully treated by surgery or local radiation therapy, but these methodologies have not provided satisfactory results in advanced stages of the disease.

[0250] Prostate-specific membrane antigen (PSMA) is expressed on the surface of prostate cancer cells at all tumor stages, and is thought to be an ideal target for antigen-redirected immunotherapy, as it shows increased expression in the more severe androgen-independent and metastatic stages of the disease. A variety of antibodies targeting PSM have been described in the literature, which can be modified for use in conjunction with CARs, including, but not limited to, J591, 3D8, D2B, and 3 / F11.

[0251] Numerous CAR T cells targeting PSMA are in clinical development (see, for example, NCT01140373, NCT01929239, and NCT03089203). However, the oncolytic efficacy of these PSMA CAR T cells remains uncertain. In particular, engineered T cells expressing first-generation CARs using 3D8 or J591 scFv ABDs have shown low efficacy due to the lack of persistence of PSMA CAR T cells. While second- and third-generation CARs have been tested in the treatment of prostate cancer, their success has remained low, requiring high doses of CAR T or multiple CAR T infusions. Alzubi, et al. (2020) Molecular Therapy Oncolytics 18:226-235.

[0252] As discussed above, the compositions and methods of the present disclosure provide the ability to overcome the lack of persistence observed in conventional CAR therapy and attributed to the lack of efficacy of PSMA CARs in clinical practice. The methods and compositions of the present disclosure allow clinicians to maintain CARs in an active state for extended periods of time, thereby enabling much greater exposure ("area under the curve" or AUC) than conventional CAR T cell therapy, and therefore provide a means to treat cancers previously resistant to CAR T therapy, particularly solid tumors that require prolonged exposure to CARs for effective treatment, achieved through extravasation and penetration into the solid tumor environment. Conventional non-orthogonal CAR T cells cannot provide the required sustained exposure without excessive toxicity or multiple administrations of CAR T cells.

[0253] The present disclosure provides a method of treating prostate cancer, optionally preventing recurrence or relapse, comprising administration of a therapeutically effective amount of orthogonal PSMA CAR T cells in combination with an orthogonal ligand of Formula 1.

[0254] In some embodiments, the present disclosure provides orthogonal PSMA CAR T cells comprising a PSMA CAR: PSMA_28z: an anti-PSMA CAR comprising CD8a signal peptide, deimmunized J591 scFv, AAA spacer, CD28 hinge / transmembrane / co-stimulatory domain and CD3 zeta: TIFF2026041861000025.tif41128 This can be co-expressed with the ortho-CD122 receptor using a T2a linker, with the following amino acid sequence: TIFF2026041861000026.tif87128

[0255] In some embodiments, the PSMA CAR is PSMA_4-1BBz:deimmunized J591 scfv; CD8a signal peptide, CD8 hinge and transmembrane domain and 4-1bb costimulatory domain and CD3z: TIFF2026041861000027.tif42128 This can be co-expressed with the ortho-CD122 receptor using a T2a linker, with the following amino acid sequence: TIFF2026041861000028.tif87128

[0256] GPC3 CAR In some embodiments, the present disclosure provides orthogonal GPC3 CAR T cells. In some embodiments, the methods and compositions of the present disclosure are useful in treating cancers that express GPC3, including, but not limited to, liver cancer. Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Glypican-3, a cell surface glycoprotein, is overexpressed in HCC tissue but not in healthy adult liver, thus providing a useful targeting domain for the ABD of CARs. Various GPC targeting domains may be used in CAR construction and incorporated into orthogonal cells for use in the treatment of liver cancer in combination with an IL2 ortholog of Formula 1. Examples of GPC3 CARs that can be used in the preparation of orthogonal GPC3 CAR T cells include those with the amino acid sequence: CAR GPC3_28z with TIFF2026041861000029.tif41128, and amino acid sequence Examples include, but are not limited to, GPC3_4-1BB having TIFF2026041861000030.tif41128.

[0257] In some embodiments, the present disclosure provides orthogonal HPV-16 E6 TCR cells for use in treating HPV-associated tumors. An example of an HPV-16 E6 CAR that can be incorporated into the orthogonal cells of the present disclosure includes an HPV-16 E6 CAR having the sequence: TIFF2026041861000031.tif64128 or Examples include, but are not limited to, CAR having the sequence TIFF2026041861000032.tif50128.

[0258] GD2 CAR for neuroblastoma GD2 is highly expressed by almost all neuroblastomas, most melanomas and retinoblastomas, and many Ewing's sarcomas, and is also expressed to a lesser extent by small cell lung cancer, glioma, osteosarcoma, and soft tissue sarcoma. Therefore, GD2 has been identified as a target for CAR T cell therapy, and various GD2 CARs are known in the art. However, persistence remains a challenge in the treatment of tumors expressing GD2. Therefore, if the duration of action of orthogonal immune cells can be extended by administering ortholigands, this will be of great help in the development of GD2 cell therapy. In some embodiments, the present invention provides ortho-GD2-targeted immune cells. In one embodiment, the targeting domain of orthogonal GD2 immune cells, such as orthogonal GD2 CAR-T cells, has the amino acid sequence: Incorporating 14g2a scFv with TIFF2026041861000033.tif25128.

[0259] Making cells into a more homogenous cell product: The present disclosure provides methods for preparing a cell product comprising an engineered immune cell type, wherein the engineered cell type is an immune cell recombinantly modified to express a receptor comprising the extracellular domain of an orthogonal CD122 polypeptide, and the cell product comprises at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90% of the engineered immune cells, the method comprising culturing to obtain a population of immune cells, and transselectively expanding the engineered immune cells expressing the orthogonal CD122 receptor in the mixed cell population of the engineered cell product. Currently, the greatest obstacle to the success of adoptive cell therapy and CAR-T therapy is the high rate of disease relapse, particularly due to the low persistence of engineered CAR T cells.

[0260] The selectivity of the molecules allows for the ex vivo use of orthogonal ligands to selectively expand engineered cells in a mixed cell population. Therefore, the techniques described herein are useful in preparing cell therapy products that are significantly enriched for therapeutic cells. Conventional nonspecific ex vivo stimulation with IL2 without sorting leads to cell populations for patient administration that provide a relatively small proportion (10%-20%) of the desired TIL or CAR cells. Orthogonal IL2 can be used to selectively activate CARs during preparation, enabling the creation of cell products with a significantly higher percentage of ex vivo therapeutic hoCARs or hoTILs. These more homogeneous cell products can be used in the treatment of neoplastic diseases, resulting in greater efficacy and less toxicity.

[0261] In some aspects, the present disclosure provides a method of treating a disease, disorder, or condition in a subject suffering from the disease, disorder, or condition, comprising: a. administering to a subject engineered mammalian cells comprising a nucleic acid sequence encoding a transmembrane receptor molecule comprising the extracellular domain (ECD) of orthogonal hCD122 operably linked to one or more expression control elements capable of effecting expression and surface display of the ECD of the transmembrane receptor molecule; and b. administering to said subject a therapeutically effective dose of a hIL2 ortholog of Formula #1. A method is provided by

[0262] In some aspects, the present disclosure provides a method for preparing an engineered T cell product comprising at least 20% hoCD122 T cells, the method comprising: a. isolating a population of T cells from a mammalian subject; b. contacting the isolated population of T cells ex vivo with a recombinant vector comprising a nucleic acid sequence encoding hoCD122 operably linked to one or more expression control sequences to facilitate expression in mammalian T cells under conditions that allow for uptake of the recombinant vector by the T cells; c. contacting the isolated population of T cells with an effective amount of the hIL2 ortholog of claim 1. The present invention provides a method comprising:

[0263] In some aspects, the present disclosure provides a cell population comprising at least 20% engineered hoCD122 T cells.

[0264] The present disclosure further provides methods for producing the hIL2 orthologs of the present invention. In particular, the present disclosure provides recombinant expression vectors comprising a nucleic acid sequence encoding a hIL2 ortholog operably linked to control elements to provide for expression of the nucleic acid sequence encoding the hIL2 ortholog in a host cell.

[0265] The present disclosure further provides a composition comprising a mixed cell population comprising at least 10%, alternatively at least 20%, alternatively at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70% T cells (e.g., T cells, CD8+ T cells, Tregs, TILs, NK cells, TCR-modified cells, CAR-T cells, etc.), wherein the T cells are recombinantly modified to express an orthogonal hCD122 receptor polypeptide. The present disclosure further provides a method of making a pharmaceutically acceptable dosage form of an engineered cell therapy product, wherein the dosage form comprises a population of T cells, wherein the population of T cells is substantially enriched for one or more species of engineered T cells, and the engineered T cells express a receptor comprising an extracellular domain of an hCD122 orthogonal polypeptide, the method comprising ex vivo culturing the population of T cells, comprising engineered T cells that express a receptor comprising an extracellular domain of an hCD122 orthogonal polypeptide, in the presence of an hIL2 ortholog of the invention, for a period of time sufficient to enrich the cell population for one or more such engineered T cells.

[0266] In some embodiments, the present disclosure provides a recombinant vector comprising a nucleic acid sequence encoding a hIL2 ortholog described herein operably linked to regulatory elements to facilitate expression and secretion of the hIL2 ortholog from mammalian cells, and administered to a subject to provide in situ expression of the hIL2 ortholog. In some embodiments, the recombinant vector is administered intratumorally to a subject suffering from cancer. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) or recombinant adenovirus (rAd), e.g., in some embodiments, a replication-deficient adenovirus derived from human adenovirus serotypes 3 and / or 5. In some embodiments, the replication-deficient adenovirus has one or more modifications to the E1 region that disrupt the virus's ability to initiate the cell cycle and / or apoptotic pathway. Replication-deficient adenoviral vectors can optionally contain deletions in the E3 domain. In some embodiments, the adenovirus is a replication-competent adenovirus. In some embodiments, the adenovirus is a replication-competent recombinant virus that has been engineered to selectively replicate in neoplastic cells.

[0267] IL2 orthologs Nomenclature: The present disclosure provides various polypeptide ligands of IL2 receptor polypeptide variants. The following nomenclature is used herein to refer to substitutions, deletions, or insertions: Residues may be designated herein by the single-letter or three-letter amino acid code of the naturally occurring amino acid found in the wild-type molecule, followed by the IL2 amino acid position of the mature IL2 molecule; for example, "Cys125" or "C125" refers to a cysteine ​​residue at position 125 of the wild-type hIL2 molecule. With respect to IL2 orthologs, substitutions are designated herein by the single-letter amino acid code, followed by the IL2 amino acid position, followed by the single-letter amino acid code of the substituted amino acid. For example, an IL2 ortholog having the modification "K35A" refers to a substitution of the lysine (K) residue at position 35 of the wild-type IL2 sequence with an alanine (A) residue at this position. Deletions of amino acid residues are referred to as "des," followed by the amino acid residue and its position in SEQ ID NO:4. For example, the term "des-Ala1" or "desA1" refers to a deletion of an alanine at position 1 of the polypeptide of the wild-type IL2 sequence. Similarly, with respect to amino acid substitutions in orthogonal CD122, the amino acid substitution is designated herein by the single-letter amino acid code of the naturally occurring amino acid, followed by the number of its position in the wild-type IL2 sequence, followed by the single-letter amino acid code of the amino acid substituted at that position. For example, in an hCD122 ortholog in which the tyrosine residue at position 134 is replaced with a phenylalanine residue, the substitution is abbreviated as "Y134F."

[0268] Orthologs (cognate ligands for receptors containing the orthogonal CD122 ECD): In some embodiments, the present disclosure provides methods of use of IL2 orthologs that are cognate ligands for receptors comprising an orthogonal CD122 ECD. In some embodiments, the IL2 ortholog is a ligand for orthogonal CD122, the ICD of which comprises one or more STAT3 binding motifs. In some embodiments, the term IL2 ortholog refers to a hIL2 variant that is a ligand for a receptor comprising an extracellular domain of human CD122 comprising an amino acid substitution at positions H133 and / or Y134, the ICD of which optionally comprises one or more STAT3 binding motifs. In some embodiments, the IL2 ortholog is a cognate ligand for a receptor comprising an extracellular domain of human CD122 comprising an amino acid substitution at positions H133 and / or Y134. In some embodiments, the IL2 ortholog is a ligand for a receptor comprising an extracellular domain of human CD122 comprising an amino acid substitution at positions H133 and / or Y134, the ICD of which optionally comprises one or more STAT3 binding motifs. In some embodiments, the IL2 ortholog is a ligand for an orthogonal human CD122 that comprises amino acid substitutions at positions H133 and Y134. In some embodiments, the IL2 ortholog is a ligand for an orthogonal CD122 that comprises amino acid substitutions H133D and Y134F. In some embodiments, an ortholog of the present disclosure is a hIL2 ortholog that is the cognate ligand for a receptor that comprises the extracellular domain of the hCD122 molecule that comprises amino acid substitutions at positions H133 and Y134, the ICD of which comprises one or more STAT3 binding motifs.

[0269] In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising an extracellular domain of an hCD122 molecule comprising an amino acid substitution at position H133. In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising an extracellular domain of an hCD122 molecule comprising an amino acid substitution at position H133, the ICD of which comprises one or more STAT3 binding motifs. In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising an extracellular domain of an hCD122 molecule comprising an amino acid substitution at position H133D. In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising an extracellular domain of an hCD122 molecule comprising an amino acid substitution at position H133D, the ICD of which comprises one or more STAT3 binding motifs. In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising an extracellular domain of an hCD122 molecule comprising an amino acid substitution at position Y134. In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising an extracellular domain of an hCD122 molecule comprising an amino acid substitution at position Y134, the ICD of which comprises one or more STAT3 binding motifs. In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising an extracellular domain of an hCD122 molecule comprising an amino acid substitution at position Y134F. In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising an extracellular domain of an hCD122 molecule comprising an amino acid substitution at position Y134F, the ICD of which comprises one or more STAT3 binding motifs.

[0270] In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising the extracellular domain of an hCD122 molecule comprising an amino acid substitution at position Y134, the ICD of which comprises one or more STAT3 binding motifs. In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of a receptor comprising the extracellular domain of orthogonal human CD122 comprising the amino acid substitutions H133D and Y134F. In some embodiments, an ortholog of the present disclosure is an hIL2 ortholog that is the cognate ligand of orthogonal human CD122 comprising the amino acid substitutions H133D and Y134F.

[0271] IL2 Orthologs (Formula #1): In one aspect, the disclosure provides hIL2 orthologs, the amino acid sequence of which has at least 80%, 90%, 95%, 98%, 99% or 100% identity to the polypeptide set forth in Formula #1 (SEQ ID NO: 50): TIFF2026041861000034.tif64150 formula: AA1 is A (wild type) or deleted; AA2 is P (wild type) or deleted; AA3 is T (wild type), C, A, G, Q, E, N, D, R, K, P, or a deletion; AA4 is S (wild type) or deleted; AA5 is S (wild type) or deleted; AA6 is S (wild type) or deleted; AA7 is T (wild type) or deleted; AA8 is K (wild type) or deleted; AA9 is K (wild type) or deleted; AA13 is Q (wild type), W, or deletion; AA14 is L (wild type), M, W, or deleted; AA15 is E (wild type), K, D, T, A, S, Q, H, or deletion; AA16 is H (wild type), N or Q or deletion; 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 deletion; AA20 is D (wild type), T, S, M, L, or deletion; 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 a deletion; AA23 is M (wild type), A, W, H, Y, F, Q, S, V, L, T, or deletion; AA27 is G (wild type), K, S, or deletion; 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 deleted 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 deletion AA85 is L (wild type) or V; AA86 is I (wild type) or V; 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.

[0272] In some aspects, the present disclosure provides a method for treating a cancer cell comprising: TIFF2026041861000035.tif107128 provides a hIL2 ortholog, which is a hIL2 polypeptide comprising a set of amino acid modifications.

[0273] Cys125: In some embodiments, the present disclosure provides hIL2 orthologs that facilitate recombinant expression in bacterial cells by eliminating the unpaired cysteine ​​residue at position 125 and / or by eliminating the N-terminal Met of directly expressed IL2 polypeptides and 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). Such mutations are typically used to avoid protein misfolding when recombinantly expressed in bacteria and isolated from inclusion bodies.

[0274] In some embodiments, an IL2 ortholog of the present invention is selected from the group consisting of: Contains one of the sets of amino acid modifications in TIFF2026041861000036.tif121128.

[0275] Mutations to increase CD122 affinity In some embodiments, hIL2 orthologs contain one or more mutations at positions in the hIL2 sequence that contact hCD122 or that alter the orientation of other positions that contact CD122, resulting in an IL2 ortholog with increased affinity for CD122. IL2 residues that have been identified as being involved in binding of IL2 to CD122 include L12, Q13, H16, L19, D20, M23, Q74, L80, R81, D84, L85, I86, S87, N88, I89, V91, I92, and E95. In some embodiments, IL2 orthologs contain 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, an IL2 ortholog comprises one or more of the amino acid substitutions: L80F, R81D, L85V, I86V, and I92F. In some embodiments, an IL2 ortholog comprises one or more of the amino acid substitutions: N74Q, L80F, R81D, L85V, I86V, I89V, and I92F. In some embodiments, an IL2 ortholog comprises one or more of the amino acid substitutions: Q74N, L80V, R81T, L85V, I86V, and I92F. In some embodiments, an IL2 ortholog comprises one or more of the amino acid substitutions: Q74H, L80F, R81D, L85V, I86V, and I92F. In some embodiments, an IL2 ortholog comprises one or more of the following amino acid substitutions: Q74S, L80F, R81D, L85V, I86V, and I92F. In some embodiments, an IL2 ortholog comprises one or more of the following amino acid substitutions: Q74N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, an IL2 ortholog comprises one or more of the following amino acid substitutions: Q74S, R81T, L85V, and I92F. In some embodiments, an IL2 ortholog comprises [L80F-R81D-L85V-I86V-I92F]. In some embodiments, the present disclosure provides an IL2 ortholog comprising: TIFF2026041861000037.tif59131 provides hIL2 orthologs containing one of the sets of amino acid modifications.

[0276] In some embodiments, the ortholog contains the substitution L85V, which has been identified as increasing the affinity of IL2 for CD122. TIFF2026041861000038.tif52128 provides a hIL2 ortholog, which is a hIL2 polypeptide comprising one of the set of amino acid modifications of TIFF2026041861000038.tif52128.

[0277] Modifications to modulate CD25 affinity In some embodiments, IL2 orthologs contain one or more mutations at positions in the IL2 sequence that contact CD25 or that alter the orientation of other positions that contact CD25, resulting in reduced affinity for CD25. The mutations can be in or near regions known to be in close proximity to CD25 based on published crystal structures (Wang, et al. Science 310:1159 2005). IL2 residues thought to contact CD25 include K35, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, V69, L72, and Y107. In some embodiments, an IL2 ortholog of the present disclosure comprises one or more of the following point mutations: R38A, F41A, and F42A (Suave, et al (1991) PNAS (USA) 88:4636-4640); P65L (Chen et al. Cell Death and Disease (2018) 9:989); F42A / G / S / T / Q / E / N / R / K, Y45A / G / S / T / Q / E / N / D / R / K / and / or L72G / A / S / T / Q / E / N / D / R / K (U.S. Patent Application Publication No. 2012 / 0244112A1 to Ast et al., published September 27, 2012; U.S. Patent No. 9,266,938B2, published February 23, 2016). Certain combinations of substitutions have been identified as reducing binding to CD25. In some embodiments, the IL2 orthologs of the present disclosure comprise one or more of the following sets of substitutions as described in Carmenate, et al (2013) J Immunol 190:6230-6238: [R38A-F42A-Y45A-E62A]; [F42A-Y45A-L72G] (Roche RG7461 (RO6874281); and / or [T41P-T51P] (Chang, et al (1995) Molecular Pharmacology 47:206-211). In some embodiments, the present disclosure provides IL2 orthologs comprising one or more of the following sets of substitutions: TIFF2026041861000039.tif52128 provides a hIL2 ortholog, which is a hIL2 polypeptide comprising one of the set of amino acid modifications of TIFF2026041861000039.tif52128.

[0278] Modifications to modulate CD132 affinity In some embodiments of the present invention, IL2 orthologs contain one or more mutations at positions in the IL2 sequence that contact CD132 or that alter the orientation of other positions that contact CD132, resulting in altered binding to CD132. Exemplary IL2 orthologs contain one or more mutations at positions in the IL2 sequence that alter the orientation of other positions that contact CD132 or that contact CD122, resulting in altered binding to CD132. IL2 residues that are thought to contact CD132 include Q11, L18, Q22, E110, N119, T123, Q126, S127, I129, S130, and T133. In some embodiments, IL2 comprises a modification at L18, wherein 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.

[0279] In some aspects, the present disclosure provides a method for treating a cancer cell comprising: TIFF2026041861000040.tif148128 provides a hIL2 ortholog, which is a hIL2 polypeptide comprising one of the set of amino acid modifications.

[0280] When recombinantly produced in a bacterial expression system directly in the absence of a leader sequence, an endogenous protease results in the deletion of the N-terminal Met-Ala1 residues to provide a "desAla1" IL2 ortholog. TIFF2026041861000041.tif183128 is provided, which is a hIL2 polypeptide comprising one of the set of amino acid modifications of TIFF2026041861000041.tif183128.

[0281] Conservative amino acid substitutions In addition to the aforementioned modifications that contribute to the activity and selectivity of IL2 orthologs for the CD122 orthogonal receptor, IL2 orthologs may contain one or more modifications in their primary structure that have minimal effect on the activity of IL2. In some embodiments, IL2 orthologs of the present disclosure may further contain one or more conservative amino acid substitutions within the wild-type IL-2 amino acid sequence. Such conservative substitutions include those described in *The Atlas of Protein Sequence and Structure* 5 (1978) by Dayhoff and *EMBO J., 8:779-785 (1989) by Argos. Conservative substitutions are generally made according to the following chart, depicted as Table XXX.

[0282] Table X: Exemplary Conservative Amino Acid Substitutions TIFF2026041861000042.tif135134

[0283] Substantial changes in function or immunological identity can be made by selecting amino acid substitutions that are less conservative than those shown in Table 3. For example, substitutions can be made that more significantly affect the structure of the polypeptide backbone or disrupt secondary or tertiary elements, including substitutions of amino acids with small, uncharged side chains (e.g., glycine) for large, bulky, charged side chains (asparagine). In particular, substitutions of such IL2 residues involving amino acids that interact with one or more of CD25, CD122, and / or CD123 can be determined from the crystal structure of IL2 associated with its receptor, as described.

[0284] In addition to the aforementioned modifications that contribute to the activity and selectivity of IL2 orthologs for the CD122 orthogonal receptor, IL2 orthologs can contain one or more modifications to their primary structure. The modifications to the primary structure as provided above can optionally further include modifications that do not substantially reduce the IL2 activity of the IL2 ortholog, including, but not limited to, the following substitutions: N30E; K32E; N33D; P34G; T37I, M39Q, F42Y, F44Y, P47G, T51I, E52K, L53N, Q57E, M104A (see U.S. Pat. No. 5,206,344).

[0285] Removal of glycosylation sites The IL2 orthologs of the present disclosure can contain a modification at Thr3 that eliminates an O-glycosylation site to facilitate production of a deglycosylated IL2 ortholog when the IL2 ortholog is expressed in mammalian cells, such as CHO or HEK cells. Thus, in certain embodiments, the IL2 ortholog contains a modification at a position corresponding to residue 3 of human IL-2 that eliminates the O-glycosylation site of IL-2. In one embodiment, the modification that eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2 is an amino acid substitution. 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. Pat. No. 5,116,943; Weiger et al., (1989) Eur. J. Biochem., 180:295-300). In specific embodiments, the modification is the amino acid substitution T3A. TIFF2026041861000043.tif162128 provides a hIL2 ortholog, which is a hIL2 polypeptide comprising one of the set of amino acid modifications.

[0286] IL2 orthologs can include deletion of the first two amino acids (desAla1-desPro2) as well as selective N-terminal modifications, particularly substitution of the Thr3 glycosylation with a cysteine ​​residue, which facilitates PEGylation of the sulfhydryl group of cysteine ​​(see, e.g., U.S. Patent No. 5,206,344, issued April 27, 1993, to Katre et al.). In some embodiments, the present disclosure provides: 10 provides a hIL2 ortholog, which is a hIL2 polypeptide comprising one of the set of amino acid modifications of TIFF2026041861000044.tif80128.

[0287] Oxidation-stabilized M104A: IL2 orthologs can optionally further include a modification at position M104, in one embodiment a substitution of methionine 104 with an alanine residue (M104A), to provide a more oxidation-resistant ortholog (see U.S. Patent 4,752,585 to Koths et al., issued June 21, 1988).

[0288] N-terminal deletion: When recombinantly produced directly in a bacterial expression system in the absence of a leader sequence, an endogenous protease results in the deletion of the N-terminal Met-Ala residues, providing a "desAla" IL2 ortholog. IL2 orthologs can include deletion of the first two amino acids (desAla-desPro), as well as N-terminal modifications, notably the replacement of the Thr3 glycosylation with a cysteine ​​residue (T3C), which facilitates PEGylation of the sulfhydryl group of the cysteine ​​(see, e.g., U.S. Patent No. 5,206,344, issued April 27, 1993, to Katre et al.).

[0289] The IL2 ortholog may further comprise the elimination of N-terminal amino acids at one or more of positions 1-9, alternatively 1-8, alternatively 1-7, alternatively 1-6, alternatively 1-5, alternatively 1-4, alternatively 1-3, alternatively 1-2. Provided is an hIL2 ortholog that is an hIL2 polypeptide comprising one of the sets of amino acid modifications of 141153 of TIFF2026041861000045.

[0290] Modifications to minimize vascular leak syndrome In some aspects of the present disclosure, the IL2 orthologs include amino acid substitutions to avoid vascular leak syndrome, a substantially negative dose-limiting side effect of the use of IL2 therapy in humans, without substantial loss of efficacy. See U.S. Patent No. 7,514,073B2 to Epstein et al., published April 7, 2009. Examples of such modifications included in the IL2 orthologs of the present disclosure include one or more of R38W, R38G, R39L, R39V, F42K, and H55Y.

[0291] Affinity maturation: In some aspects, the IL2 orthologs may be affinity matured to enhance their activity with respect to orthogonal CD122. An "affinity matured" polypeptide has one or more mutations in one or more residues that result in an improvement in the affinity (or vice versa) of the orthogonal polypeptide for its cognate orthogonal receptor as compared to the parent polypeptide that does not carry the mutation. Affinity maturation can be performed to increase the binding affinity of the IL2 ortholog by at least about 10%, or at least about 50%, or at least about 100%, or at least about 150%, or 1 to 5-fold as compared to the "parent" polypeptide. The engineered IL2 orthologs of the invention activate their cognate orthogonal receptors as described above, but have significantly reduced binding and activation of the wild-type IL2 receptor as evaluated by ELISA and / or FACS analysis using a sufficient amount of the molecule under appropriate assay conditions.

[0292] Modifications to extend duration of action in vivo As discussed above, the compositions of the present disclosure include IL2 orthologs that have been modified to provide an extended in vivo lifespan and / or extended duration of action in a subject. Such modifications to provide an extended lifespan and / or duration of action include primary sequence modifications of the IL2 ortholog, conjugation to a carrier molecule (e.g., albumin, acylation, PEGylation), and Fc fusion.

[0293] Sequence modifications to extend duration of action in vivo As discussed above, the term IL2 ortholog includes modifications of IL2 orthologs to provide an extended in vivo lifespan and / or extended duration of action in a subject.

[0294] In some embodiments, IL2 orthologs may contain certain amino acid substitutions that confer extended in vivo life span. For example, Dakshinamurthi et al. (International Journal of Bioinformatics Research (2009) 1(2):4-13) state that one or more of the substitutions V91R, K97E, and T113N in an IL2 polypeptide will result in an IL2 variant possessing enhanced stability and activity. In some embodiments, an IL2 ortholog of the present disclosure contains one, two, or all three of the V91R, K97E, and T113N modifications.

[0295] Conjugates and Carrier Molecules In some embodiments, an IL2 ortholog is modified to provide the IL2 ortholog with a particular property (e.g., an extended duration of action in a subject), which may be achieved through conjugation to a carrier molecule to provide a desired pharmacological property, such as an extended half-life. In some embodiments, an IL2 ortholog 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.

[0296] Albumin fusions In some embodiments, the IL2 ortholog is expressed as a fusion protein with an albumin molecule (eg, human serum albumin), which is known in the art to facilitate extended in vivo exposure.

[0297] In one embodiment of the present invention, a hIL2 ortholog is conjugated to albumin, herein referred to as an "IL2 ortholog albumin fusion." The term "albumin" when used in the context of a hIL2 ortholog albumin fusion includes albumins such as human serum albumin (HSA), cynomolgus monkey serum albumin, and bovine serum albumin (BSA). In some embodiments, the HSA contains the amino acid substitution C34S or K573P compared to the wild-type HSA sequence. According to the present disclosure, albumin can be conjugated to a hIL2 ortholog at the carboxy terminus, amino terminus, both the carboxy terminus and amino terminus, and internally (see, e.g., USP 5,876,969 and USP 7,056,701). The HSA-hIL2 ortholog polypeptide conjugates contemplated by the present disclosure can use various forms of albumin, such as the albumin secretory presequence and its variants, fragments and variants, and HSA variants. Such forms generally retain one or more desired albumin activities. In additional embodiments, the present disclosure includes fusion proteins comprising hIL2 ortholog polypeptides fused directly or indirectly to albumin, albumin fragments, albumin variants, and the like, wherein the fusion proteins have greater plasma stability than the unfused drug molecule and / or the fusion proteins retain the therapeutic activity of the unfused drug molecule. In some embodiments, the indirect fusion is achieved by a linker, such as a peptide linker or modified version thereof, as discussed in more detail below.

[0298] Alternatively, a hIL2 ortholog albumin fusion comprises an IL2 ortholog that is a fusion protein comprising an albumin binding domain (ABD) polypeptide sequence and an IL2 ortholog polypeptide. As alluded to above, a fusion protein comprising an albumin binding domain (ABD) polypeptide sequence and an hIL2 ortholog polypeptide can be achieved, for example, by genetic engineering, such that a nucleic acid encoding a HAS, or a fragment thereof, is joined to a nucleic acid encoding one or more IL2 ortholog sequences. In some embodiments, the albumin-binding peptide comprises the amino acid sequence DICLPRWGCLW (SEQ ID NO:22).

[0299] IL2 ortholog polypeptides can also be conjugated to large, slowly metabolized macromolecules, such as proteins; polysaccharides such as sepharose, agarose, cellulose, or cellulose beads; polymeric amino acids such as polyglutamic acid or polylysine; amino acid copolymers; inactivated virus particles; inactivated bacterial toxins such as toxoids derived from diphtheria, tetanus, cholera, or leukotoxin molecules; inactivated bacteria, dendritic cells, thyroglobulin; tetanus toxoid; diphtheria toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); rotavirus VP6 polypeptide; influenza virus hemagglutinin, influenza virus nucleoprotein; keyhole limpet hemocyanin (KLH); and hepatitis B virus core protein and surface antigen. Such conjugated forms can be used, if desired, to produce antibodies against the polypeptides of the present disclosure.

[0300] In some embodiments, IL2 orthologs can be conjugated (either chemically or as a fusion protein) with XTEN, which provides an extended duration of action due to PEGylation, and produced as a recombinant fusion protein in E. coli. XTEN polymers suitable for use with the IL2 orthologs of the present disclosure are provided in Podust, et al. (2016) "Extension of in vivo half-life of biologically active molecules by XTEN protein polymers," J Controlled Release 240:52-66 and Haeckel et al. (2016) "XTEN as a 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 can be a fusion protein that can include a protease-sensitive cleavage site, such as an MMP-2 cleavage site, between the XTEN polypeptide and the IL2 ortholog.

[0301] Additional candidate components and molecules for conjugation include those suitable for isolation or purification. Specific, non-limiting examples include molecules that contain binding molecules, such as biotin (biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or solid supports (including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes).

[0302] In some embodiments, the IL-2 muteins may also be linked to additional therapeutic agents, including therapeutic compounds such as anti-inflammatory compounds or anti-neoplastic agents, therapeutic antibodies (e.g., Herceptin), immune checkpoint modulators, immune checkpoint inhibitors (e.g., anti-PD1 antibodies), and cancer vaccines, as described elsewhere in this disclosure. Antibacterial agents include aminoglycosides, including gentamicin; antiviral compounds, such as rifampicin, 3'-azido-3'-deoxythymidine (AZT) and acyclovir; antifungal agents, such as azoles, including fluconazole; plyre macrolides, such as amphotericin B and candicidin; antiparasitic compounds, such as antimonial agents; and the like. IL2 orthologs may be conjugated to additional cytokines, such as CSF, GSF, GMCSF, TNF, erythropoietin, immunomodulators, or cytokines such as interferons or interleukins, neuropeptides, reproductive hormones such as HGH, FSH, or LH, thyroid hormones, neurotransmitters such as acetylcholinesterase inhibitors, and hormone receptors such as estrogen receptors. Also included are nonsteroidal anti-inflammatory drugs, such as indomethacin, acetylsalicylic acid, ibuprofen, sulindac, piroxicam, and naproxen, as well as anesthetics or analgesics. Also included are radioisotopes, such as those useful for imaging as well as therapy.

[0303] The IL2 orthologs of the present disclosure can be chemically conjugated to such carrier molecules using well-known chemical conjugation methods. Bifunctional cross-linking reagents, such as homofunctional and heterofunctional cross-linking reagents, well known in the art, can be used for this purpose. The type of cross-linking reagent to be used depends on the nature of the molecule to be coupled to the IL-2 mutein and can be easily identified by those skilled in the art. Alternatively or additionally, the IL2 ortholog and / or the molecule intended to be conjugated thereto can be chemically derivatized so that the two can be conjugated in separate reactions, which is also well known in the art.

[0304] PEGylation: In some embodiments, the IL2 ortholog is conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of the present invention include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), polyolefin alcohols, polysaccharides, poly-alpha-hydroxy acids), polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0305] In some embodiments, the IL2 ortholog is conjugated to one or more polyethylene glycol molecules or "PEGylated." The method or site of attachment of the PEG to the IL2 ortholog can vary, but in certain embodiments, the PEGylation does not alter, or only minimally alters, the activity of the IL2 ortholog.

[0306] In some embodiments, certain chemistries may be used to substitute cysteine ​​for threonine at position 3 (3TC) to facilitate N-terminal PEGylation.

[0307] In some embodiments, selective PEGylation of IL2 orthologs (e.g., by incorporation of unnatural amino acids with side chains that facilitate selective PEG conjugation chemistry), as described in PCT International Application No. PCT / US2018 / 045257 to Ptacin et al. (filed August 3, 2018, published February 7, 2019 as International Publication No. WO 2019 / 028419A1), can be used to generate IL2 orthologs with reduced affinity for one or more subunits of the IL2 receptor complex (e.g., CD25, CD132). For example, hIL2 orthologs incorporating unnatural amino acids with specific PEGylatable moieties at sequences or residues of IL2 identified as interacting with CD25, including amino acids 34-45, 61-72, and 105-109, typically provide IL2 orthologs with reduced binding to CD25. Similarly, hIL2 orthologs incorporating unnatural amino acids with specific PEGylatable moieties at sequences or residues of IL2 identified as interacting with hCD132, including amino acids 18, 22, 109, 126, or 119-133, provide IL2 orthologs with reduced binding to hCD132.

[0308] In certain embodiments, the increase in half-life is greater than any decrease in biological activity. PEG suitable for conjugation to polypeptide sequences is generally soluble in water at room temperature and has the general formula R(O-CH-CH). n OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, the protecting group generally has 1 to 8 carbons. The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives "star PEG" and multi-arm PEG are contemplated by the present disclosure.

[0309] The molecular weight of PEG used in the present disclosure is not limited to any particular range. The PEG component of a PEG-IL2 ortholog can have a molecular mass of greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular mass is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. The linear or branched PEG molecule has a molecular weight of about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, alternatively about 30,000 to about 40,000 daltons. In one embodiment of the invention, the PEG is a 40 kD branched PEG containing two 20 kD arms.

[0310] The present disclosure also contemplates compositions of conjugates in which PEGs have different n values, and thus various different PEGs are present in specific ratios. For example, some compositions contain a mixture of conjugates where n=1, 2, 3, and 4. In some compositions, the percentage of conjugates where n=1 is 18-25%, the percentage of conjugates where n=2 is 50-66%, the percentage of conjugates where n=3 is 12-16%, and the percentage of conjugates where n=4 is up to 5%. Such compositions can be produced using reaction conditions and purification methods known in the art. Chromatography can be used to separate conjugate fractions, and then fractions containing, for example, conjugates with a desired number of PEGs attached can be identified and purified to remove unmodified protein sequences and conjugates with other numbers of PEGs.

[0311] PEG suitable for conjugation to polypeptide sequences is generally soluble in water at room temperature and has the general formula R(O-CH-CH) n OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, the protecting group generally has 1 to 8 carbons.

[0312] 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 carbonate PEG (BTC-PEG; see, e.g., U.S. Pat. No. 5,650,234 to Dolence et al.), which react preferentially with lysine residues to form carbamate linkages but are also known to react with histidine and tyrosine residues. The use of PEG-aldehyde linkers targets a single site on the N-terminus of a polypeptide through reductive amination.

[0313] Pegylation most frequently occurs at the α-amino group at the N-terminus of polypeptide, the epsilon amino group on the side chain of lysine residue, and the imidazole group on the side chain of histidine residue.Most recombinant polypeptides have a single α-amino group and many epsilon amino groups and imidazole groups, so depending on linker chemistry, multiple positional isomers can be produced.General pegylation strategies known in the art can be applied herein.

[0314] PEG can be attached to an IL2 ortholog of the present disclosure via a terminal reactive group ("spacer") that mediates a bond between one or more free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. PEGs with spacers that can be attached to free amino groups include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide.

[0315] In some embodiments, PEGylation of IL2 orthologs is facilitated by the incorporation of unnatural amino acids with unique side chains that facilitate site-specific PEGylation. The incorporation of unnatural amino acids into polypeptides to provide functional moieties for achieving site-specific PEGylation of such polypeptides is known in the art. See, e.g., PCT International Application No. PCT / US2018 / 045257 to Ptacin et al. (filed August 3, 2018, published February 7, 2019 as International Publication No. WO 2019 / 028419A1). In one embodiment, an IL2 ortholog of the invention incorporates an unnatural amino acid at position D109 of the IL2 ortholog. In one embodiment of the invention, the IL2 ortholog is PEGylated at position 109 of the IL2 ortholog with a PEG molecule having a molecular weight of about 20 kD, alternatively about 30 kD, or alternatively about 40 kD.

[0316] The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives "star PEG" and multi-arm PEG are contemplated by the present disclosure.In a specific embodiment, PEG useful in the practice of the present invention includes 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601). Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 10 kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF), 20 kDa linear PEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), 20 kDa two-arm branched PEG-aldehyde, i.e., a 20 kDa PEG containing two 10 kDa linear PEG molecules. PEG-aldehyde (e.g., Sunbright® GL2-200AL3, NOF), 20 kDa two-arm branched PEG-NHS ester, i.e., a 20 kDa PEG-NHS ester comprising two 10 kDa linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), 40 kDa two-arm branched PEG-aldehyde, i.e., a 40 kDa PEG-aldehyde comprising two 20 kDa linear PEG molecules (e.g., Sunbright® GL2-400AL3), 40 kDa two-arm branched PEG-NHS ester, i.e., a 40 kDa PEG-NHS ester comprising two 20 kDa linear PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30 kDa PEG-aldehyde (eg, Sunbright® ME-300AL), and linear 30 kDa PEG-NHS ester.

[0317] As mentioned above, PEG can be attached to the IL2 ortholog directly or via a linker molecule. Suitable linkers generally include "flexible linkers" of sufficient length to allow some movement between the modified polypeptide sequence and the linked components and molecules. Linker molecules are generally about 6-50 atoms in length. Linker molecules can also be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable linkers can be readily selected and can be of any suitable length, such as 1 amino acid (e.g., 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), glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, or other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and therefore can serve as neutral tethers between components. Additional examples of flexible linkers include glycine polymers (G) n Examples of linker sequences include glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers. Glycine and glycine-serine polymers are relatively unstructured and therefore can serve as neutral tethers between components. Multimers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of these linker sequences can be linked together to provide flexible linkers that can be used to conjugate heterologous amino acid sequences to the polypeptides disclosed herein.

[0318] Additionally, such linkers may be used to link an IL2 ortholog to additional heterologous polypeptide components as described herein, where the heterologous amino acid sequence may be a signal sequence and / or a fusion partner such as albumin, Fc sequence, etc.

[0319] In one embodiment of the present disclosure, the IL2 ortholog is a human IL2 ortholog of the following structure: [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

[0320] In another embodiment of the invention, the IL2 orthologue is a human IL2 orthologue of the following structure: TIFF2026041861000046.tif18136 where n=0 or 1.

[0321] Acylation In some embodiments, the IL2 orthologs of the present disclosure can be acylated by conjugation to a fatty acid molecule, as described in Resh (2016) Progress in Lipid Research 63: 120-131. Examples of fatty acids that can be conjugated include myristic acid, palmitic acid, and palmitoleic acid. Myristoylate is typically linked to the N-terminal glycine, although lysine can also be myristoylated. Palmitoylation is typically achieved by enzymatic modification of free cysteine ​​-SH groups; for example, DHHC proteins catalyze S-palmitoylation. Palmitoylation of serine and threonine residues is typically achieved enzymatically using the PORCN enzyme.

[0322] Acetylation In some embodiments, the IL-2 mutein is acetylated at the N-terminus by an enzymatic reaction with an N-terminal acetyltransferase, e.g., acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, the IL-2 mutein is acetylated at one or more lysine residues, e.g., by an enzymatic reaction with a lysine acetyltransferase. See, e.g., Choudhary et al. (2009) Science 325 (5942):834L2 ortho840.

[0323] Fc fusion In some embodiments, an IL-2 fusion protein may incorporate an Fc region derived from an IgG subclass of antibody lacking the IgG heavy chain variable region. The "Fc region" can be a naturally occurring or synthetic polypeptide homologous to the IgG C-terminal domain generated by papain digestion of IgG. IgG Fc has a molecular weight of approximately 50 kDa. A mutant IL-2 polypeptide can contain the entire Fc region or a smaller portion thereof that retains the ability to extend the circulating half-life of the chimeric polypeptide to which it belongs. In addition, full-length or fragmented Fc regions can be variants of the wild-type molecule. That is, they can contain mutations that may or may not affect the function of the polypeptide; as described further below, native activity is not required or desired in either case. In certain embodiments, an IL-2 mutein fusion protein (e.g., an IL-2 partial agonist or antagonist as described herein) comprises an IgG1, IgG2, IgG3, or IgG4 Fc region. Exemplary Fc regions can contain mutations that inhibit complement fixation and Fc receptor binding, or can be lytic, i.e., capable of binding complement or lysing cells via another mechanism such as antibody-dependent complement lysis (ADCC).

[0324] In some embodiments, the IL2 ortholog comprises a functional domain of an Fc-fused chimeric polypeptide molecule. Fc-fusion conjugates have been shown to increase the systemic half-life of biologics, thereby allowing biopharmaceuticals to be administered less frequently. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels. Upon binding, the Fc-fusion molecule is protected from degradation and re-released into the circulation, allowing the molecule to remain in circulation longer. This Fc binding is thought to be the mechanism by which endogenous IgG maintains its long plasma half-life. More recent Fc-fusion technology optimizes the pharmacokinetic and pharmacodynamic properties of biologics compared to traditional Fc-fusion conjugates by linking only a small portion of the biologic to the Fc region of an antibody. The "Fc region" useful in preparing Fc fusions can be a naturally occurring or synthetic polypeptide homologous to the IgG C-terminal domain, which is generated by papain digestion of IgG. IgG Fc has a molecular weight of approximately 50 kDa. An IL2 ortholog may provide the entire Fc region, or a smaller portion thereof that retains the ability to extend the circulating half-life of the chimeric polypeptide to which it is a part. In addition, the full-length or fragmented Fc region can be a variant of the wild-type molecule. In a typical implementation, each monomer of a dimeric Fc carries a heterologous polypeptide, which may be the same or different.

[0325] In some embodiments, when an IL2 ortholog is to be administered in the format of an Fc fusion, particularly in situations where the polypeptide chains conjugated to each subunit of the Fc dimer are different, the Fc fusion may be engineered to possess a "knob-into-hole modification." Knob-into-hole modifications are described in more detail in Ridgway, et al. (1996) Protein Engineering 9(7):617-621 and U.S. Patent No. 5,731,168, issued March 24, 1998. A knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domains, in which i) in the CH3 domain of a first heavy chain, an amino acid residue is replaced with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan) to create a protrusion ("knob") from the surface, and ii) in the CH3 domain of a second heavy chain, an amino acid residue is replaced with an amino acid residue having a smaller side chain (e.g., alanine or threonine), thereby creating a cavity ("hole") within the interface in the second CH3 domain, within which the protruding side chain ("knob") of the first CH3 domain is accommodated by the cavity in the second CH3 domain. In one embodiment, a "knob-into-hole modification" comprises the amino acid substitution T366W and, optionally, the amino acid substitution S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V, and, optionally, Y349C in the other antibody heavy chain. Additionally, the Fc domain may be modified by the introduction of cysteine ​​residues at positions S354 and Y349, which result in stable disulfide bridges between the two antibody heavy chains in the Fe region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole format is used to facilitate the expression of a first polypeptide (e.g., an IL2 ortholog) on ​​a first Fc monomer bearing a "knob" modification and a second polypeptide on a second Fc monomer bearing a "hole" modification, facilitating the expression of heterodimeric polypeptide conjugates.

[0326] Fc regions can be "lytic" or "nonlytic," but are typically nonlytic. Nonlytic Fc regions typically lack high-affinity Fc receptor binding and Clq binding sites. The high-affinity Fc receptor binding site of murine IgG Fc contains a Leu residue at position 235 of IgG Fc. Therefore, the Fc receptor binding site can be disrupted by mutating or deleting Leu 235. For example, substituting Leu 235 with Glu inhibits the ability of the Fc region to bind to the high-affinity Fc receptor. The murine Clq binding site can be functionally disrupted by mutating or deleting Glu 318, Lys 320, and Lys 322 residues of IgG. For example, substituting Glu 318, Lys 320, and Lys 322 with Ala residues prevents IgG1 Fc from directing antibody-dependent complement lysis. In contrast, the soluble IgG Fc region has a high-affinity Fc receptor binding site and a Clq binding site. The high-affinity Fc receptor binding site contains a Leu residue at position 235 of IgG Fc, and the Clq binding site contains Glu318, Lys320, and Lys322 residues of IgG1. Soluble IgG Fc has wild-type residues or conservative amino acid substitutions at these sites. Soluble IgG Fc can target cells for antibody-dependent cellular cytotoxicity or complement-directed cytolysis (CDC). Suitable mutations for human IgG are also known (see, e.g., Morrison et al., The Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2:125, 1994).

[0327] In certain embodiments, the amino or carboxyl terminus of an IL2 ortholog of the present disclosure can be fused to an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half-life of biologics, thus allowing biopharmaceuticals to be administered less frequently. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels, and upon binding, the Fc fusion molecule is protected from degradation and re-released into the circulation, allowing the molecule to remain in circulation longer. This Fc binding is thought to be the mechanism by which endogenous IgG maintains its long plasma half-life. Furthermore, recent Fc fusion technology optimizes the pharmacokinetic and pharmacodynamic properties of biologics compared to traditional Fc fusion conjugates by linking only a small portion of the biologic to the Fc region of an antibody.

[0328] In some embodiments, the Fc domain monomer comprises at least one mutation compared to a wild-type human IgG1, IgG2, or IgG4 Fc region, as described in U.S. Patent No. US10259859B2, the teachings of which are incorporated herein by reference in their entirety. As disclosed herein, the Fc domain monomer comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N , L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368 Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D39 9K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; or (b)(i) N297A mutation compared to the human IgG1 Fc region; (ii) L234A, L235A, and G237A mutations relative to the human IgG1 Fc region; (iii) L234A, L235A, G237A, and N297A mutations compared to the human IgG1 Fc region; (iv) N297A mutation compared to the human IgG2 Fc region; (v) A330S and P331S mutations compared to the human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations compared to the human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations relative to the human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations compared to the human IgG4 Fc region.

[0329] In some embodiments, the Fc domain monomer comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L35 IT, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q. K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; and (b) The Fc domain monomer further comprises: (i) N297A mutation compared to the human IgG1 Fc region; (ii) L234A, L235A, and G237A mutations relative to the human IgG1 Fc region; (iii) L234A, L235A, G237A, and N297A mutations compared to the human IgG1 Fc region; (iv) N297A mutation compared to the human IgG2 Fc region; (v) A330S and P331S mutations compared to the human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations compared to the human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations relative to the human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations compared to the human IgG4 Fc region.

[0330] In some embodiments, the polypeptide exhibits reduced phagocytosis in a phagocytosis assay compared to a polypeptide having a wild-type human IgG Fc region. In some embodiments, the Fc domain monomer is linked to a second polypeptide comprising a second Fc domain monomer to form an Fc domain dimer.

[0331] Chimeric Polypeptides / Fusion Proteins In some embodiments, an IL2 ortholog can comprise a functional domain of a chimeric polypeptide. IL2 ortholog fusion proteins of the present disclosure may be readily produced by recombinant DNA methodology by techniques known in the art by constructing a recombinant vector comprising a nucleic acid sequence that comprises an IL2 ortholog encoding a nucleic acid sequence in-frame with a nucleic acid sequence encoding a fusion partner at either the N- or C-terminus of the IL2 ortholog, optionally further comprising a nucleic acid sequence encoding a linker or spacer polypeptide in-frame.

[0332] Flag tag In other embodiments, IL2 orthologs can be modified to contain an additional polypeptide sequence that functions as an antigen tag, such as a FLAG sequence, which is recognized by a biotinylated, highly specific anti-FLAG antibody, as described herein (see, e.g., Blanar et al. (1992) Science 256:1014 and LeClair, et al. (1992) PNAS-USA 89:8145). In some embodiments, the IL2 ortholog polypeptide further comprises a C-terminal c-myc epitope tag.

[0333] His tag In some embodiments, the IL2 orthologs of the present invention (including fusion proteins of such IL2 orthologs) are expressed as fusion proteins with one or more transition metal chelating polypeptide sequences. The incorporation of such transition metal chelating domains facilitates purification by immobilized metal affinity chromatography (IMAC), as described in U.S. Pat. No. 4,569,794 to Smith et al., issued February 11, 1986. Examples of transition metal chelating polypeptides useful in the practice of the present invention are described in Smith et al., supra, and U.S. Pat. No. 5,320,663 to Dobeli et al., issued May 10, 1995, the entire teachings of which are incorporated by reference. A particular transition metal chelating polypeptide useful in the practice of the present invention is a peptide containing three to six consecutive histidine residues (SEQ ID NO: 51), such as the six-histidine peptide (His) (SEQ ID NO: 52), often referred to in the art as a "His tag."

[0334] Targeted IL2 ortholog fusion proteins: In some embodiments, the IL2 ortholog is provided as a fusion protein having a polypeptide sequence ("targeting domain") to facilitate selective binding to a particular cell type or tissue expressing a cell surface molecule that specifically binds to the targeting domain, optionally incorporating a linker molecule of 1 to 40 (alternatively, 2 to 20, alternatively, 5 to 20, alternatively, 10 to 20) amino acids between the IL2 ortholog sequence and the sequence of the targeting domain of the fusion protein.

[0335] In another embodiment, chimeric polypeptides can be produced that contain orthogonal IL-2 and an antibody or its antigen-binding portion. The antibody or antigen-binding component of the chimeric protein can serve as a targeting moiety. For example, this 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, for example, in U.S. Patent No. 6,617,135.

[0336] In some embodiments, the targeting domain of the IL2 ortholog fusion protein specifically binds to a cell surface molecule of a tumor cell. In one embodiment in which the ECD of the CAR of a CAR-T cell specifically binds to CD-19, the IL2 ortholog can be provided as a fusion protein with a CD-19 targeting moiety. For example, in one embodiment in which the ECD of the CAR of a CAR-T cell is an scFv molecule that provides specific binding to CD-19, the IL2 ortholog is provided as a fusion protein with a CD-19 targeting moiety, such as a single-chain antibody (e.g., scFv or VHH) that specifically binds to CD-19.

[0337] In some embodiments, the fusion protein comprises an IL-2 mutein and anti-CD19 sdFv FMC63 (Nicholson, et al. (1997) Mol Immunol 34: 1157-1165). Similarly, in some embodiments where the ECD of the CAR of a CAR-T cell specifically binds BCMA, the IL2 ortholog is provided as a fusion protein with a BCMA-targeting moiety, such as an antibody comprising the CDRs of an anti-BMCA antibody as described in Kalled et al. (U.S. Patent 9,034,324 issued May 9, 2015) or an antibody comprising the CDRs as described in Brogdon et al. (U.S. Patent No. 10,174,095 issued January 8, 2019). In some embodiments, the IL2 ortholog is provided as a fusion protein with a GD2 targeting moiety, such as an antibody comprising CDRs as described in Cheung et al. (U.S. Patent No. 9,315,585 issued April 19, 2016), or CDRs from ME36.1 (Thurin et al., (1987) Cancer Research 47:1229-1233), 14G2a, 3F8 (Cheung, et al., 1985 Cancer Research 45:2642-2649), hu14.18, 8B6, 2E12, or ic9.

[0338] In an alternative embodiment, the targeted IL2 orthologs of the present disclosure can be administered in combination with CAR-T cell therapy to provide targeted delivery of the IL2 ortholog to CAR-T cells based on their extracellular receptors, e.g., by anti-FMC63 antibodies, to target IL2 activity to CAR-T cells and restore exhausted CAR-T cells in vivo. Thus, embodiments of the present disclosure include targeted delivery of IL2 orthologs by conjugating such IL2 orthologs to antibodies or ligands designed to interact with specific cell surface molecules on CAR-T cells. An example of such a molecule would be an anti-FMC63-hIL2 ortholog.

[0339] In other embodiments, the chimeric polypeptide comprises a mutant IL-2 polypeptide and a heterologous polypeptide, such as an Aga2p agglutinin subunit, that functions to enhance expression or direct cellular localization of the mutant IL-2 polypeptide (see, e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0340] Protein transduction domain fusion proteins: In some embodiments, the IL2 ortholog further comprises a "protein transduction domain" or "PTD." A PTD is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic molecule that facilitates crossing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. Incorporation of a PTD into an IL2 ortholog facilitates the molecule's crossing of the membrane. In some embodiments, the PTD is covalently linked to the amino or carboxy terminus of the IL2 ortholog. In some embodiments, the PTD is incorporated at either the N- or C-terminus of the molecule as part of a PTD-IL2 ortholog fusion protein.

[0341] Exemplary protein transduction domains include a minimal decapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT); a polyarginine sequence containing a sufficient number of arginine residues to direct cell entry (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008), transportan (described in Wierzbicki, et al., (2014) Folio Histomchemica et Cytobiologica 52(4): 270-280 and Pooga, et al. (1998) FASEB J 12(1)67-77 and commercially available from AnaSpec under catalog number AS-61256); KALA (described in Wyman et al., (1997) Biochemistry 36(10) 3008-3017 and commercially available from AnaSpec under catalog number AS-65459); antennapedia peptide (described in Pietersz et al., (2001) Vaccine 19:1397 and commercially available from AnaSpec under catalog number AS-61032); TAT 47-57 (commercially available from AnaSpec under catalog number AS-60023).

[0342] In some embodiments, the IL-2 conjugate comprises a human subject plasma half-life of greater than 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, or 30 days.

[0343] In some embodiments, when an IL2 ortholog is to be administered in the format of an Fc fusion, particularly in situations where the polypeptide chains conjugated to each subunit of the Fc dimer are different, the Fc fusion may be engineered to possess a "knob-into-hole modification." Knob-into-hole modifications are described in more detail in Ridgway, et al. (1996) Protein Engineering 9(7):617-621 and U.S. Patent No. 5,731,168, issued March 24, 1998. A knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domains, in which i) in the CH3 domain of a first heavy chain, an amino acid residue is replaced with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan) to create a protrusion ("knob") from the surface, and ii) in the CH3 domain of a second heavy chain, an amino acid residue is replaced with an amino acid residue having a smaller side chain (e.g., alanine or threonine), thereby creating a cavity ("hole") within the interface in the second CH3 domain, within which the protruding side chain ("knob") of the first CH3 domain is accommodated by the cavity in the second CH3 domain. In one embodiment, a "knob-into-hole modification" comprises the amino acid substitution T366W and, optionally, the amino acid substitution S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V, and, optionally, Y349C in the other antibody heavy chain. Additionally, the Fc domain may be modified by the introduction of cysteine ​​residues at positions S354 and Y349, which result in stable disulfide bridges between the two antibody heavy chains in the Fe region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole format is used to facilitate the expression of a first polypeptide (e.g., an IL2 ortholog) on ​​a first Fc monomer bearing a "knob" modification and a second polypeptide on a second Fc monomer bearing a "hole" modification, facilitating the expression of heterodimeric polypeptide conjugates.

[0344] Synthesis of IL2 orthologs The IL2 orthologs of the present disclosure can be produced by conventional methods for constructing polypeptides, including recombinantly or by solid phase synthesis.

[0345] Solid phase chemical synthesis: In addition to producing mutant polypeptides through expression of nucleic acid molecules altered by recombinant molecular biology techniques, the subject IL2 orthologs can be chemically synthesized. Chemically synthesized polypeptides are routinely produced by those skilled in the art. Chemical synthesis involves direct synthesis of peptides by chemical means from a protein sequence encoding an IL2 ortholog exhibiting the described properties.

[0346] In some embodiments, the IL2 orthologs of the present disclosure can be prepared by chemical synthesis. Chemical synthesis of IL2 orthologs can proceed via liquid phase or solid phase. Solid-phase peptide synthesis (SPPS) allows the incorporation of unnatural amino acids and / or peptide / protein backbone modifications. Various forms of SPPS are available for synthesizing the IL2 orthologs of the present disclosure and are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero JA et al., (2005) Protein Pept Lett. 12:723-8). During chemical synthesis, alpha functions and any reactive side chains can be protected with acid-labile or base-labile groups that are stable under conditions that link amide bonds but can be easily cleaved without harming the formed peptide chain.

[0347] In solid-phase synthesis, either the N- or C-terminal amino acid can be coupled to a suitable support material. Suitable support materials are inert to the reagents and reaction conditions of the stepwise condensation and cleavage reactions of the synthetic process and are insoluble in the reaction medium used. Examples of commercially available support materials include styrene / divinylbenzene copolymers modified with reactive groups and / or polyethylene glycol; chloromethylated styrene / divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene / divinylbenzene copolymers; and the like. The sequential coupling of protected amino acids can be carried out according to conventional methods in peptide synthesis, typically using an automated peptide synthesizer.

[0348] At the end of solid-phase synthesis, the peptide is cleaved from the support material with concomitant cleavage of the side-chain protecting groups. The resulting peptide can be purified by a variety of chromatographic methods, including but not limited to hydrophobic adsorption chromatography, ion exchange chromatography, partition chromatography, high performance liquid chromatography (HPLC), and reverse-phase HPLC.

[0349] Recombinant production: Alternatively, the IL2 orthologs of the present disclosure are produced by recombinant DNA technology. In a typical implementation of recombinant production of a polypeptide, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector appropriate for the host cell in which expression is to be achieved, and the nucleic acid sequence is operably linked to one or more expression control sequences encoded by the vector and functional in the target host cell. If the polypeptide incorporates a secretory leader sequence (signal peptide), the recombinant protein can be recovered through disruption of the host cell or from the cell culture medium. The recombinant protein can be purified and concentrated for further use, including incorporation. Processes for recombinant production of IL2 polypeptides are known in the art and are described in U.S. Patent No. 4,604,377 to Fernandes and Taforo, issued August 5, 1986, and IL2 orthologs are described in U.S. Patent No. 4,512,584 to Mark et al., issued May 21, 1985, and U.S. Patent No. 4,401,756 to Gillis, issued August 30, 1983, the entire teachings of which are incorporated herein by reference.

[0350] Construction of nucleic acid sequences encoding IL2 orthologues In some embodiments, the IL2 ortholog is produced by recombinant methods using a nucleic acid sequence encoding the IL2 ortholog (or a fusion protein comprising the IL2 ortholog). The nucleic acid sequence encoding the desired IL2 ortholog can be synthesized by chemical means using an oligonucleotide synthesizer.

[0351] Nucleic acid molecules are not limited to sequences that encode polypeptides; they can also include some or all of the non-coding sequences upstream or downstream from the coding sequence (e.g., the coding sequence for IL2). Those skilled in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. These can be produced, for example, by treating genomic DNA with restriction endonucleases or by performing polymerase chain reaction (PCR). When the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0352] Nucleic acid molecules encoding IL2 orthologs (and fusions thereof) can contain naturally occurring sequences or sequences that differ from those naturally occurring but encode the same polypeptide due to the degeneracy of the genetic code. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., produced by phosphoramidite base synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. In addition, nucleic acid molecules can be double-stranded or single-stranded (i.e., either the sense strand or the antisense strand).

[0353] Nucleic acid sequences encoding IL2 orthologs may be obtained from various sources that provide custom nucleic acid sequences. Amino acid sequence variants of IL2 polypeptides for producing IL2 orthologs of the present disclosure are prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code, as is well known in the art. Such variants represent residue insertions, substitutions, and / or specified deletions, as described. Any combination of insertions, substitutions, and / or specified deletions can be used to arrive at a final construct, provided that the final construct possesses the desired biological activity, as defined herein.

[0354] Methods for constructing DNA sequences encoding IL2 orthologs and expressing them in appropriately transformed hosts include, but are not limited to, PCR-assisted mutagenesis. Mutations consisting of deletions or additions of amino acid residues to the IL2 polypeptide can also be generated using standard recombinant techniques. In the case of deletions or additions, the nucleic acid molecule encoding IL2 is optionally digested with an appropriate restriction endonuclease. The resulting fragments can be expressed directly or further manipulated, for example, by ligation to a second fragment. Ligation can be facilitated when the two ends of the nucleic acid molecule contain overlapping complementary nucleotides, although blunt-ended fragments can also be ligated. PCR-generated nucleic acids can also be used to generate various mutant sequences.

[0355] The IL2 orthologs of the present disclosure can be produced recombinantly not only by direct recombinant production but also as fusion polypeptides with heterologous polypeptides, such as signal sequences or other polypeptides with specific cleavage sites at the N- or C-terminus of the mature IL2 ortholog. Generally, the signal sequence can be a component of the vector or can be part of the coding sequence inserted into the vector. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In some embodiments, the signal sequence is the signal sequence naturally associated with the IL2 ortholog (i.e., the human IL2 signal sequence). The inclusion of a signal sequence depends on whether it is desired to secrete the IL2 ortholog from the recombinant cell in which it is produced. If the selected cell is prokaryotic, it is generally preferred that the DNA sequence not encode a signal sequence. If the selected cell is eukaryotic, it is generally preferred that a signal sequence be encoded, with the wild-type IL2 signal sequence being most preferably used. Alternatively, heterologous mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, such as the herpes simplex gD signal, may be suitable. When the recombinant host cell is a yeast cell such as Saccharomyces cerevisiae, the alpha mating factor secretory signal sequence can be used to achieve extracellular secretion of an IL2 ortholog into the culture medium, as described in U.S. Patent No. 7,198,919 B1, issued April 3, 2007, to Singh.

[0356] When an IL2 ortholog to be expressed is expressed as a chimera (e.g., a fusion protein comprising an IL2 ortholog and a heterologous polypeptide sequence), the chimeric protein can be encoded by a hybrid nucleic acid molecule comprising a first sequence encoding all or a portion of the IL2 ortholog and a second sequence encoding all or a portion of the heterologous polypeptide. For example, the IL2 ortholog of the pr...

Claims

1. Orthogonal human CAR-T cells, wherein the cells are (A) A first nucleic acid sequence encoding a signal peptide for cell surface expression and an orthogonal hCD122 receptor polypeptide, wherein the orthogonal hCD122 receptor polypeptide comprises an extracellular domain, a transmembrane domain, and an intracellular domain. (i) The extracellular domain has the following amino acid sequence: AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLM APISLQVVHVETHRCNISWEISQASDFFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT (SEQ ID NO:6) Includes, and, (ii) The intracellular domain of the orthogonal hCD122 receptor polypeptide is selected from the group consisting of the intracellular domains of human CD122, hIL4Ra, hIL7Ra, hIL9R, and IL21R. The first nucleic acid sequence, and (B) A second nucleic acid sequence encoding a signal peptide and a chimeric antigen receptor (CAR) for cell surface expression, wherein the CAR comprises an antigen-binding domain (ABD), an optional hinge region, a transmembrane domain, and an intracellular signaling domain (ISD), (i) The antigen-binding domain of the CAR comprises a polypeptide that specifically binds to GPC3, and (ii) The intracellular signaling domain of the CAR comprises a human CD3 ζ chain signaling domain and (a) a CD28 costimulatory domain or (b) a 4-1BB costimulatory domain, Second nucleic acid sequence The orthogonal human CAR-T cells, including the orthogonal human CAR-T cells.

2. The orthogonal human CAR-T cell according to claim 1, wherein the intracellular domain of the orthogonal hCD122 receptor polypeptide is the human CD122 intracellular domain.

3. The orthogonal human CAR-T cell according to claim 1, wherein the intracellular domain of the orthogonal hCD122 receptor polypeptide is the hIL9R intracellular domain.

4. The orthogonal hCD122 receptor polypeptide has the amino acid sequence: AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQA SDFFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKF FSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHL (SEQ ID NO:7) Orthogonal human CAR-T cells according to claim 2, comprising polypeptides.

5. The intracellular domain of the orthogonal hCD122 receptor polypeptide further comprises at least one STAT3 signaling motif, wherein the STAT3 signaling motif comprises a polypeptide of formula YX 1 X 2 Q (SEQ ID NO: 21), where, Y is the amino acid tyrosine, X1 is selected from the group consisting of L, R, F, and M, and X2 is selected from the group consisting of R, K, H, and P. Orthogonal human CAR-T cells according to claim 4.

6. The orthogonal human CAR-T cell according to claim 5, wherein the polypeptide of formula YX 1 X 2 Q (SEQ ID NO: 21) is a polypeptide selected from the group consisting of YLRQ (SEQ ID NO: 24); YLKQ (SEQ ID NO: 25); YRHQ (SEQ ID NO: 26); YLRQ (SEQ ID NO: 24); YFKQ (SEQ ID NO: 28); YLPQ (SEQ ID NO: 16); YMPQ (SEQ ID NO: 17); and YDKPH (SEQ ID NO: 18).

7. The orthogonal human CAR-T cell according to any one of claims 1 to 6, wherein the polypeptide that specifically binds to GPC3 comprises a single-domain antibody.

8. The orthogonal human CAR-T cell according to claim 7, wherein the single-domain antibody is scFv.

9. The second nucleic acid sequence encoding a signal peptide and a chimeric antigen receptor (CAR) is an amino acid sequence: DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGG SGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWG QGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 45) Orthogonal human CAR-T cells according to claim 1, comprising polypeptides.

10. The second nucleic acid sequence encoding a signal peptide and a chimeric antigen receptor (CAR) is an amino acid sequence: DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGG GSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTY WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 46) Orthogonal human CAR-T cells according to claim 1, comprising polypeptides.

11. The orthogonal human CAR-T cell according to any one of claims 1 to 10, wherein the T cell is selected from the group consisting of tumor-infiltrating lymphocytes (TILs), CD8+ T cells, CD25+CD8+ T cells, NK cells, CD4+ T cells, and Treg cells.

12. The orthogonal human CAR-T cell according to claim 11, wherein the T cell is a tumor-infiltrating lymphocyte (TIL).

13. The orthogonal human CAR-T cell according to claim 11, wherein the T cell is a CD8+ T cell.

14. The orthogonal human CAR-T cell according to claim 11, wherein the T cell is a CD25+CD8+ T cell.

15. The orthogonal human CAR-T cell according to claim 11, wherein the T cell is an NK cell.

16. A pharmaceutical composition for treating a neoplasm in a human subject, comprising a therapeutically effective amount of orthogonal human CAR-T cells according to any one of claims 1 to 15, wherein the pharmaceutical composition is used in combination with a therapeutically effective amount of a pharmaceutical preparation comprising an hIL2 ortholog (hoIL2), and the hoIL2 polypeptide is It has at least 95% identity to a polypeptide containing the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 5), and The following set of amino acid modifications: [E15S-H16Q-L19V-D20L-Q22K-M23A-C125S]; [E15S-H16Q-L19V-D20L-Q22K-M23A-C125A]; [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A]; [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125A-Q126H]; [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125A-Q126H]; [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125A-Q126H]; [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125S]; [desAla1-desPro2-E15S-H16Q-L19V-D20L-Q22K-M23A]; [desAla1-desPro2-desThr3-E15S-H16Q-L19V-D20L-Q22K-M23A]; [desAla1-desPro2-desThr3-desSer4-E15S-H16Q-L19V-D20L-Q22K-M23A]; [desAla1-desPro2-desThr3-desSer4-desSer5-E15S-H16Q-L19V-D20L-Q22K-M23A]; and [desAla1-desPro2-desThr3-desSer4-desSer5-desSer6-E15S-H16Q-L19V-D20L-Q22K-M23A] Having one of the following, The aforementioned pharmaceutical composition.

17. The pharmaceutical composition according to claim 16, wherein the hoIL2 is modified to extend the duration of action of hoIL2 in vivo in human subjects.

18. The pharmaceutical composition according to claim 17, wherein the IL2 ortholog is a PEGylated hoIL2 having the following structure: [PEG]-[linker] n-[hoIL2] During the ceremony, PEG is a linear or branched polyethylene glycol molecule having an average molecular weight of 2,000–80,000 daltons, or 2,000–70,000 daltons, or 5,000–50,000 daltons, or 10,000–50,000 daltons, or 20,000–50,000 daltons, or 30,000–40,000 daltons, and n = 0 or 1.

19. The pharmaceutical composition according to claim 18, wherein the PEGylated hoIL2 ortholog is a compound of the following formula: [PEG]-[Linker] n -[desAla1-hIL2 E15S-H16Q-L19V-D20L-Q22K-M23A] In the formula, n = 0 or 1.

20. The pharmaceutical composition according to claim 19, wherein the IL2 ortholog comprises the following structure: 40kD-PEG-(linker)n-PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRM LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL KGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 23) In the formula, 40kD-PEG is a branched polyethylene glycol having an average molecular weight of 40,000 daltons, and n = 1.

21. The pharmaceutical composition according to any one of claims 16 to 20, wherein the subject is treated with a lymphocyte depletion regimen before administration of the orthogonal human CAR-T cells.

22. The pharmaceutical composition according to claim 21, wherein the lymphocyte depletion regimen comprises the administration of fludarabine and cyclophosphamide to the subject.

23. The pharmaceutical composition according to any one of claims 16 to 22, wherein the dose of orthogonal human CAR-T cells administered to the subject is used to be 100,000 to 1,000,000 orthogonal human CAR-T cells per kilogram of body weight of the subject.

24. The pharmaceutical composition according to any one of claims 16 to 22, wherein hoIL2 is used to be administered to the subject regularly over a period of at least one month in order to maintain a level of 10,000 to 1,000,000 activated orthogonal human CAR-T cells per kilogram of body weight of the subject.

25. A pharmaceutical composition for treating the recurrence of a neoplasm after treatment with the pharmaceutical composition according to any one of claims 16 to 22, The pharmaceutical formulation contains an effective therapeutic dose of hoIL2, and The administration of hoIL2 is given to the patient such that, without administering an additional amount of orthogonal human CAR-T cells, the administration of hoIL2 induces the activation and / or proliferation of orthogonal human CAR-T cells previously administered to the patient. The aforementioned pharmaceutical composition.

26. A pharmaceutical composition for use in a method for treating a neoplasm in a human subject, wherein the pharmaceutical composition comprises an orthogonal ligand and the method comprises the following steps: (i) A step of administering a therapeutically effective dose of the orthogonal ligand to the subject, (ii) The next step of administering to the subject an orthogonal human CAR-T cell according to any one of claims 1 to 15 within a period of 1 to 2 weeks, wherein the orthogonal ligand is an allogeneic hIL2 ligand of the orthogonal CD122 receptor of the orthogonal CAR-T cell. (iii) administering the orthogonal ligand of (i) and the orthogonal human CAR-T cells of (ii) to the subject until a lack of evidence of neoplastic disease is observed, and (iv) The step of administering to the subject a therapeutically effective amount of the orthogonal ligand in a dose sufficient to maintain a level of circulating orthogonal CAR-T cells sufficient to maintain immune surveillance of neoplasms.

27. ​​A pharmaceutical composition for use in a method for treating a neoplastic disease in a human subject, wherein the pharmaceutical composition comprises orthogonal human CAR-T cells as described in any one of claims 1 to 15, and the method comprises the following steps: (i) The step of administering an effective therapeutic dose of an orthogonal ligand to the subject, (ii) Next, administer the orthogonal human CAR-T cells to the subject within a period of 1 to 2 weeks, wherein the orthogonal ligand is an allogeneic hIL2 ligand of the orthogonal CD122 receptor of the orthogonal CAR-T cells. (iii) administering the orthogonal ligand of (i) and the orthogonal human CAR-T cells of (ii) to the subject until a lack of evidence of neoplastic disease is observed, and (iv) The step of administering to the subject a therapeutically effective amount of the orthogonal ligand in a dose sufficient to maintain a level of circulating orthogonal CAR-T cells sufficient to maintain immune surveillance of neoplasms.

28. A recombinant expression vector comprising a nucleic acid sequence comprising a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence from 5' to 3', (A) The first nucleic acid sequence encodes the orthogonal hCD122 receptor polypeptide described in claim 1, (B) The second nucleic acid sequence encodes a T2A polypeptide, (C) The third nucleic acid sequence encodes a signal peptide for cell surface expression and the CAR described in claim 1, The recombinant expression vector wherein the nucleic acid sequence is functionally linked to one or more functional expression regulatory sequences in human cells.

29. The intracellular domain of the orthogonal receptor polypeptide further comprises at least one STAT3 signaling motif, wherein the STAT3 signaling motif comprises a polypeptide of formula YX 1 X 2 Q, where, Y is the amino acid tyrosine, X1 is selected from the group consisting of L, R, F, and M, and X2 is selected from the group consisting of R, K, H, and P. The recombinant expression vector according to claim 28.

30. The recombinant expression vector according to claim 29, wherein the polypeptide of formula YX 1 X 2 Q is a polypeptide selected from the group consisting of YLRQ (SEQ ID NO: 24); YLKQ (SEQ ID NO: 25); YRHQ (SEQ ID NO: 26); YLRQ (SEQ ID NO: 24); YFKQ (SEQ ID NO: 28); YLPQ (SEQ ID NO: 16); YMPQ (SEQ ID NO: 17); and YDKPH (SEQ ID NO: 18).

31. The polypeptide comprising a signal peptide and a chimeric antigen receptor (CAR) is as follows: DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGG SGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWG QGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 45) or DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGG GSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTY WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 46) The recombinant expression vector according to claim 28, comprising:

32. A recombinant expression vector according to any one of claims 28 to 31, which is a viral vector.

33. The recombinant expression vector according to claim 32, wherein the viral vector is a lentiviral vector.

34. The recombinant expression vector according to claim 32, wherein the viral vector is a retroviral vector.

35. A method for preparing a cell product enriched with orthogonal human CAR-T cells, comprising the following steps: (a) Transduction of a certain amount of T cells isolated from a human subject with a recombinant expression vector according to any one of claims 28 to 34, and (b) A step of ex vivo contacting the amount of T cells isolated from step (a) with an amount of hoIL2 sufficient to induce proliferation of the cells transduced by the contact of step (a), the contact step being carried out over a period of time such that the transduced cells constitute at least 20% of the cells in the population.

36. (a) A first nucleic acid sequence encoding a signal peptide for cell surface expression and an orthogonal CD122 receptor polypeptide, wherein the orthogonal CD122 receptor polypeptide comprises: AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQAS WACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQV VHVETHRCNISWEISQASDFFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLET LTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGA FGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSS SFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFH LPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRD DLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDL VDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSL QELQGQDPTHLV (SEQ ID NO: 29) Furthermore (b) A second nucleic acid sequence encoding a polypeptide comprising a signal peptide for cell surface expression and a GPC3 CAR, wherein the polypeptide comprises: DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGG SGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWG QGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 45) or DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGG GSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTY WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 46) Human orthogonal GPC3 CAR-T cells, including The orthogonal CD122 receptor polypeptide and the GPC3 CAR are expressed by human orthogonal GPC3 CAR-T cells, and the first nucleic acid sequence and the second nucleic acid sequence are functionally linked to one or more functional expression regulatory elements in human immune cells so that the extracellular domains of the orthogonal CD122 receptor polypeptide and the GPC3 CAR are presented on the cell surface. The aforementioned human orthogonal GPC3 CAR-T cells.

37. A pharmaceutical composition for treating a neoplastic disease in human subjects, comprising the following formula: 40kD-PEG-(linker)n-PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRM LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL KGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 23) hIL2 orthologue, wherein 40kD-PEG is a branched polyethylene glycol having an average molecular weight of 40,000 daltons, and n=1, The pharmaceutical composition comprising a therapeutically effective amount of human orthogonal GPC3 CAR-T cells according to claim 36, which is used in combination with a therapeutically effective amount of a pharmaceutical preparation comprising the above.

38. The pharmaceutical composition according to claim 37, used in combination with a therapeutically effective amount of an adjunct agent.

39. The pharmaceutical composition according to claim 38, wherein the auxiliary substance is an anti-PD1 or anti-PDL1 checkpoint inhibitor antibody.

40. The pharmaceutical composition according to any one of claims 37 to 39, wherein the subject is treated with a lymphocyte depletion regimen prior to the administration of human orthogonal GPC3 CAR-T cells.

41. The pharmaceutical composition according to any one of claims 37 to 39, to be used in the absence of preceding lymphocyte depletion.

42. The pharmaceutical composition according to any one of claims 37 to 41, wherein the subject is periodically exposed to a therapeutically effective amount of a pharmaceutical formulation containing an hIL2 ortholog, such that 10,000 to 1,000,000 human orthogonal GPC3 CAR-T cells are maintained in the subject for a period of at least two months.

43. The pharmaceutical composition according to any one of claims 37 to 42, wherein the neoplastic disease is a hematological malignancy.

44. The pharmaceutical composition according to claim 43, wherein the hematological malignancy is relapsed or refractory non-Hodgkin lymphoma, relapsed or refractory myeloma, relapsed or refractory large B-cell lymphoma, or relapsed or refractory mantle cell lymphoma.

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