CD122 with modified ICD-STAT signaling

Abstract

We provide a modified human CD122 that, upon binding to a cognitive IL2 ligand, can stimulate robust and sustained STAT3 and STAT5 signaling. [Solution] This disclosure relates to a modified human CD122 comprising one or more STAT3 binding motifs. In some embodiments, the modified human CD122 is a modified orthogonal human CD122 which can be selectively activated by cognitive orthogonal IL2. When the modified human CD122 binds to a cognitive IL2 ligand, it can stimulate robust and persistent STAT3 and STAT5 signaling.

Description

Technical Field

[0001] Related Applications This application claims the priority and the benefit of U.S. Provisional Application No. 62 / 961,157, filed on January 14, 2020. The entire content of the provisional application is incorporated herein by reference for all purposes.

Brief Description of the Drawings

[0002] [Figure 1] Schematic of an orthogonal CD122 (IL2Rb) polypeptide associated with the cell membrane (left), and one configuration of a representative embodiment of a genetically engineered receptor of the present disclosure in which the intracellular domain of an orthogonal CD122 peptide containing the STAT3 motif YRHQ is added to the carboxy terminus via a glycine-glycine linker (right). [Figure 2] Results of a FACS study confirming efficient transduction and expression of the indicated orthogonal CD122 (hoRb, middle panel) and orthogonal CD122 containing an additional STAT3 motif (right panel) being efficiently introduced and expressed in 3F8 cells. [Figure 3] Results of a FACS study assessing the relative proportion of 3F8 T cell clones transfected with the indicated orthogonal CD122 (hoRb) and orthogonal CD122 containing an additional STAT3 motif having increased expression of CD25 and CD122 in response to an orthogonal ligand. [Figure 4]PhosphoSTAT3 (pSTAT3) levels in CD4+ T cells (top left) and CD8+ T cells (top right) engineered to express an orthogonal hoRb receptor construct containing an IL2 wtIL2Rb ICD (black square) in response to wtIL2, a wt IL2R ICD (gray circle) in response to administration of orthogonal ligand STK-009, and an IL2 wtIL2Rb ICD containing an additional STAT3 motif in response to administration of orthogonal ligand STK-009; and phosphoSTAT3 (pSTAT3) levels in CD4+ T cells (bottom left) and CD8+ T cells (top right) engineered to express an orthogonal hoRb receptor construct containing an IL2 wtIL2Rb ICD (black square) in response to wtIL2, a wt IL2R ICD (gray circle) in response to administration of orthogonal ligand STK-009, and an IL2 wtIL2Rb ICD containing an additional STAT3 motif in response to administration of orthogonal ligand STK-009. This provides the results of evaluating phosphoSTAT5 (pSTAT5) levels in T cells (bottom right). [Figure 5] pERK signaling levels in CD4+ T cells (top left) and CD8+ T cells (top right) engineered to express an orthogonal hoRb receptor construct containing an IL2 wtIL2Rb ICD (black square) in response to wtIL2, a wt IL2R ICD (gray circle) in response to administration of orthogonal ligand STK-009, and an IL2 wtIL2Rb ICD containing an additional STAT3 motif in response to administration of orthogonal ligand STK-009; and pERK signaling levels in CD4+ T cells (bottom left) and CD8+ T cells (top right) engineered to express an orthogonal hoRb receptor construct containing an IL2 wtIL2Rb ICD (black square) in response to wtIL2, a wt IL2R ICD (gray circle) in response to administration of orthogonal ligand STK-009, and an IL2 wtIL2Rb ICD containing an additional STAT3 motif in response to administration of orthogonal ligand STK-009. This provides the results of evaluating pS6K (pSTAT5) levels in T cells (bottom right). [Figure 6]This paper provides graphs showing the results of evaluating the cytotoxicity of CD19 CAR T cell constructs containing an orthogonal CD122(hoRb) receptor with an additional STAT3 signaling motif compared to CD19 CAR T cell constructs containing orthogonal CD122(hoRb) with a wild-type CD122 intracellular domain, at various effector:target (E:T;CAR T:Raji tumor cells) ratios ranging from 10:1 to 0.1:1. It demonstrates that CD19 CAR T cell constructs expressing hoRb with an intracellular domain (ICD) expressing a STAT3 motif exhibit improved cytotoxicity against Raji tumor cells compared to CD19 CAR T cells containing orthogonal CD122(hoRb) with a wild-type CD122 intracellular domain. [Background technology]

[0003] Background of the Invention The controlled manipulation of the differentiation, development, and proliferation of cells, particularly genetically modified immune cells, is of significant clinical interest. T cells have been genetically modified for use in therapeutic applications such as recognizing and killing cancer cells, intracellular pathogens, and cells involved in autoimmunity. The use of genetically modified cell therapy in cancer treatment is facilitated by the selective activation and proliferation of modified T cells that are induced to provide specific functions and selectively attack cancer cells. In some examples of adoptive immunotherapy, T cells are isolated from the patient's blood, treated ex vivo, and reinjected into the patient. Therefore, compositions and methods that enable the selective activation of targeted modified cell populations are desired.

[0004] A challenge in manufacturing cell therapy products is that such "living drugs" require strict control of their environment to maintain their viability and functionality. In fact, isolated cells, whether patient-derived (autologous) or from a single donor source (allogeneic), begin to rapidly lose function once removed from the patient or from controlled culture conditions. If the health and function of the isolated cells can be successfully maintained while they are out of the patient or controlled culture conditions, then those isolated cells can be returned to the production flow of cell products or to function for reintroduction into patients.

[0005] Furthermore, a challenge associated with the clinical application of modified T-cell therapy is selectively stimulating these modified cells to maximize their therapeutic efficacy. A typical means of maintaining an activated modified T-cell product is systemic administration of cytokines such as IL-2. However, systemic administration of IL-2 is associated with nonspecific stimuli beyond the modified cell population, particularly at high doses, and is associated with serious toxicity in human patients. Moreover, IL-2 has a short in vivo lifespan, requiring frequent administration to maintain the modified T cells in an activated state. Modified cells from the initial administration of the initial population may be detectable for months or even years after administration of the modified cell product, but a significant proportion of these modified cells become quiescent and require reactivation to exhibit significant therapeutic effects. Consequently, a challenge in cell-based therapies is to confer desired controllable behavior to the transplanted cells, which is protected from endogenous signaling pathways, does not affect untargeted endogenous cells, and can be selectively controlled after the modified cell population is administered to the patient.

[0006] CD122 is a component of the medium-affinity and high-affinity IL2 receptor complex. CD122 contains a native STAT5 recognition motif. Upon IL2 binding, the receptor activates JAK (kinase), which phosphorylates specific tyrosine molecules in the intracellular domain of CD122. Phosphorylated CD122 recruits and phosphorylates STAT5 (STAT5A and / or STAT5B). Subsequently, phosphorylated STAT5 dimerizes and translocates to the nucleus, activating the transcription of target genes that play crucial roles in various pathways, from innate and adaptive immunity to cell proliferation, differentiation, and survival. Basham et al., Nucleic Acids Res. 2008 Jun; 36(11): 3802-3818 (Non-patent Literature 1).

[0007] In recent years, cell therapies using T cells transformed with recombinant CD122 have been developed. Sockolosky et al. (Science (2018) 359: 1037-1042 (Non-Patent Literature 2)) and Garcia et al. (US Patent Application Publication US2018 / 0228841A1 (Patent Literature 1), published August 16, 2018) describe an orthogonal IL2 / CD122 ligand / receptor system that facilitates the selective stimulation of cells engineered to express orthogonal CD122. Contact between modified T cells expressing orthogonal CD122 and the corresponding orthogonal ligand ("orthogonal IL2") of such orthogonal CD122 enables the specific activation of such modified T cells. In particular, this orthogonal IL2 receptor-ligand complex results in the selective expansion of cells engineered to express the orthogonal receptor in mixed populations of cells, especially mixed populations of T cells.

[0008] Orthogonal IL2s with reduced affinity for unmodified medium-affinity (CD122 / CD132) or high-affinity (CD25 / CD122 / CD132) IL2 receptor complexes are also useful for selectively targeting orthogonal IL2 activity to cells exhibiting high CD25 expression, for example, in the treatment of autoimmune diseases. Furthermore, orthogonal IL2s with significantly reduced affinity for the extracellular domain (ECD) of native wild-type CD122 but maintaining CD25 binding to the ECD can be used as competitive antagonists of wild-type IL2 by inhibiting the formation of high-affinity IL2 receptor complexes, potentially leading to their use in the treatment of autoimmune diseases or graft-versus-host (GVH) diseases. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Garcia et al., U.S. Patent Application Publication US2018 / 0228841A1, published August 16, 2018. [Non-patent literature]

[0010] [Non-Patent Document 1] Basham et al., Nucleic Acids Res. 2008 Jun; 36(11): 3802-3818 [Non-Patent Document 2] Sockolosky et al., Science (2018) 359: 1037-1042 [Overview of the project]

[0011] Brief summary of the invention This disclosure is directed toward modified human CD122 that retains the STAT5 motif similar to natural human CD122 but is engineered to include one or more STAT3-binding motifs. In some embodiments, the presence of the STAT3-binding motif makes the modified CD122 more stable, which can promote sustained and robust STAT3 and STAT5 signaling when bound to a cognitive IL2 ligand. In some embodiments, the modified human CD122 is a modified orthogonal human CD122 that can be selectively activated by cognitive orthogonal IL2. Thus, this disclosure also provides compositions and methods for the selective activation of immune cells engineered to express modified orthogonal human CD122 in order to promote stable and sustained IL2-mediated signaling. The methods described herein can be used to effectively treat patients requiring IL2 therapy without causing the serious adverse effects associated with standard IL2 therapy.

[0012] This application incorporates, by reference to, the entirety of the disclosures in WO 2019 / 104092 and US 2018-0228842 A1).

[0013] In some embodiments, the disclosure provides a polynucleotide encoding a modified human CD122, wherein the modified human CD122 comprises one or more STAT3-binding motifs. In some embodiments, the modified human CD122 comprises orthogonal human CD122 or native human CD122 fused to one or more STAT3-binding motifs. In some embodiments, the orthogonal human CD122 is modified in one or more residues selected from R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and Q214 compared to native human CD122. In some embodiments, the orthogonal human CD122 is modified in H133 and Y134 compared to native human CD122. In some embodiments, human CD122 is linked to two or three STAT3-binding motifs.

[0014] In some embodiments, the modified human CD122 comprises a sequence that is at least 90% identical to SEQ ID NO: 1, and the modified human CD122 binds to a natural IL2 polypeptide or an orthogonal IL2 polypeptide. In some embodiments, one or more STAT3 binding motifs comprise the sequence YX1X2Q, where X1 and X2 are any amino acids. In some embodiments, 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. In some embodiments, the STAT3 recognition motif is selected from the group consisting of YLRQ (SEQ ID NO: 11), YLKQ (SEQ ID NO: 12), YRHQ (SEQ ID NO: 13), YLRQ (SEQ ID NO: 14), YFKQ (SEQ ID NO: 15), YLPQ (SEQ ID NO: 16), YMPQ (SEQ ID NO: 17), and YDKPH (SEQ ID NO: 18).

[0015] In some embodiments, one or more STAT3-binding motifs are optionally fused to the C-terminus of the intracellular domain of native human CD122 or orthogonal human CD122 via a linker. In some embodiments, at least one of the STAT3-binding motifs is located between positions 355 and 364 corresponding to native human CD122, where the amino acid sequence of YFTY, YDPY, or YSEE of native human CD122 is replaced by at least one of the STAT3-recognizing motifs. In some embodiments, the linker comprises a dinucleotide (GG)n, where n is 1 to 10.

[0016] In some embodiments, the modified human CD122 is further modified compared to natural human CD122 in one or more residues selected from R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and Q214.

[0017] In some embodiments, the modified CD122 comprises an amino acid sequence comprising a linker and at least one STAT3 binding motif, wherein the amino acid sequence is selected from the group consisting of GGYLRQ (SEQ ID NO:3), GGYLKQ (SEQ ID NO: 4), GGYRHQ (SEQ ID NO: 5), GGYLRQ (SEQ ID NO: 6), GGYFKQ (SEQ ID NO: 7), GGYLPQ (SEQ ID NO: 8), GGYMPQ (SEQ ID NO: 9), and GGYDKPH (SEQ ID NO: 10).

[0018] Also provided herein is an expression vector comprising the polynucleotide described in any one of the above embodiments.

[0019] Also provided herein is an expression vector comprising the polynucleotide described in any one of the above embodiments. In some embodiments, the cell further expresses a chimeric antigen receptor (CAR), wherein the cell is a human immune cell. In some embodiments, the CAR is selected from the group consisting of CD19 CAR and BCMA CAR.

[0020] Also provided herein is a kit for the selective activation of a receptor in a cell, the kit comprising: (a) a cell expressing a modified human CD122 encoded by a polynucleotide disclosed herein; and (b) a human IL2 polypeptide. In some embodiments, the modified human CD122 comprises the native human CD122 encoded by SEQ ID NO: 1, and the human IL2 polypeptide is the native human IL2 polypeptide encoded by SEQ ID NO: 2. In some embodiments, the modified human CD122 comprises an orthogonal human CD122, and the human IL2 polypeptide is an orthogonal human IL2 polypeptide, and the orthogonal human IL2 polypeptide preferentially binds to the modified human CD122 as compared to the native human CD122.

[0021] In some embodiments, the orthogonal human IL2 polypeptide includes at least one amino acid substitution at residues T51 and R81 at the position corresponding to natural human CD122, with an amino acid other than the amino acid of the natural human IL2 polypeptide, or includes alanine at position M23 corresponding to natural human CD122, and includes amino acid substitutions at each of positions E15, H16, L19, and D20 corresponding to natural human CD122.

[0022] In some embodiments, the orthogonal human IL2 polypeptide comprises one or more amino acid substitutions corresponding to positions in native human IL2 selected from the following: [E15D, E15T, E15A, E15S], [H16N, H16Q], [L19V, L19I, L19A], [D20L, D20M], [Q22S, Q22T, Q22E, Q22K, Q22E], [M23A, M23W, M23H, M23Y, M23F, M23Q, M23Y], [G27K, G27S], [R81D, R81Y], [N88E, N88Q], [T51I].

[0023] In some embodiments, the modified human CD122 is expressed by mammalian cells. In some embodiments, the mammalian cells are immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are chimeric antigen receptor (CAR)-T cells.

[0024] Also provided is a method for stimulating immune cells expressing a modified human CD122 containing one or more STAT3-binding motifs, the method comprising contacting the immune cells with a human IL2 polypeptide. In some embodiments, the stimulation is performed ex vivo. In some embodiments, the stimulation is performed in vivo. In some embodiments, the modified human CD122 comprises orthogonal human CD122 or native human CD122 fused to one or more STAT3-binding motifs. In some embodiments, at least one of the STAT3-binding motifs is located between positions 381 and 390 corresponding to native human CD122, where the YFTY, YDPY, or YSEE amino acid sequence of native human CD122 is replaced by at least one of the STAT3-binding motifs. In some embodiments, the method comprises introducing immune cells expressing a modified human CD122 containing one or more STAT3-binding motifs into an organism, and administering a human IL2 polypeptide to the organism to activate an immune response in the organism. In some embodiments, the modified human CD122 comprises orthogonal human CD122, the human IL2 polypeptide is orthogonal human IL2 polypeptide, and the orthogonal human IL2 polypeptide preferentially binds to and activates the modified human CD122 rather than natural human CD122. In some embodiments, one or more STAT3-binding motifs comprise the sequence YX1X2Q, where X1 and X2 are arbitrary amino acids. In some embodiments, 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. In some embodiments, orthogonal human CD122 contains a sequence selected from the group consisting of GGYLRQ (SEQ ID NO: 2), GGYLKQ (SEQ ID NO: 3), GGYRHQ (SEQ ID NO: 4), GGYLRQ (SEQ ID NO: 5), GGYFKQ (SEQ ID NO: 6), GGYLPQ (SEQ ID NO: 7), GGYMPQ (SEQ ID NO: 8), and GGYDKPH (SEQ ID NO: 9). In some embodiments, the immune cells are T cells. In some embodiments, the T cells are CAR-T cells.In some embodiments, the immune cells are CD8+ T cells, and the individual has cancer. In some embodiments, the immune cells are Treg cells, and the individual has an autoimmune disease. In some embodiments, the individual has a viral, bacterial, or fungal infection. [Modes for carrying out the invention]

[0025] Detailed description of the invention definition To facilitate understanding of this disclosure, certain terms and phrases are defined below and throughout this Spec. The definitions set forth herein are not limiting and should be read with the knowledge of those skilled in the art in mind.

[0026] Before describing the methods and compositions of the present invention, it should be understood that the present invention is not limited to the specific methods or compositions described, and that such methods or compositions may naturally vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0027] Where a range of values ​​is provided, it is understood that each value interposing between the upper and lower limits of that range is also specifically disclosed, to the extent of one-tenth of the lower limit, unless otherwise explicitly indicated in the context. Each subrange between any stated or interposing value within the stated range and any other stated or interposing value within that stated range is included in the present invention. The upper and lower limits of these subranges may be independently included in or excluded from that range, and each range where either or both of the upper and lower limits are included in that subrange, or neither, is also included in the present invention, subject to any particularly excluded upper / lower limits within the stated range. Where the stated range includes either or both of the upper and lower limits, the range excluding either or both of the upper and lower limits that they include is also included in the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but only a number of possible and preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and explain methods and / or materials relating to the references of those publications.

[0029] It should be noted that the singular forms “a,” “an,” and “the” as used herein and in the appended claims include multiple references unless otherwise explicitly indicated by the context. For example, “a cell” includes multiple such cells, and “the peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0030] The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention does not have prior rights to such publications on the grounds of prior invention. Furthermore, the publication dates provided may differ from the actual publication dates and may need to be verified individually.

[0031] Unless otherwise specified, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius (°C), and pressure is atmospheric pressure or close to it. The following standard abbreviations are used: bp = base pair; kb = kilobase; pl = picoliters; s or sec = seconds; min = minutes; h or hr = hours; aa = amino acids; kb = kilobases; nt = nucleotides; pg = picograms; ng = nanograms; μg = micrograms; mg = milligrams; g = grams; kg = kilograms; dl or dL = deciliters; μl or μL = microliters; ml or mL = milliliters; l or L = liters; μM = micromoles; mM = millimoles; M = moles; kDa = k Rodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = once daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high-performance liquid chromatography; BW = body weight; U = unit; ns = not statistically significant; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; NHS = N-hydroxysuccinimide; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Dulbecco's modified Eagle medium; GC = genome copy; EDTA = ethylenediaminetetraacetic acid.

[0032] Throughout this disclosure, references to amino acids will be made according to one-letter or three-letter codes. For the reader's convenience, the one-letter and three-letter amino acid codes are shown in Table 1 below: (Table 1) Abbreviations of amino acids TIFF2026053539000002.tif96129

[0033] Standard methods in molecular biology are described in scientific literature (see, for example, 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 discusses cloning and DNA mutation introduction in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), complex carbohydrate and protein expression (Vol. 3), and bioinformatics (Vol. 4)). Scientific literature describes methods for purifying proteins, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as methods for chemical analysis, chemical modification, post-translational modification, fusion protein creation, and protein glycosylation (see, for example, Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).

[0034] Unless otherwise indicated, the following terms shall have the meanings set forth below. Other terms are defined elsewhere throughout this specification.

[0035] As used herein, the term “activate” is used to reflect the biological effect of an agonist ligand binding to a receptor or receptor complex. For example, the binding of an IL2 agonist to the IL2 receptor is said to “activate” the signaling of that receptor, producing one or more intracellular biological effects (e.g., phosphorylation of STAT5).

[0036] As used herein, the term “activity” is used to describe the properties of a molecule relating to a test system or biological function, such as the degree to which the molecule binds to another molecule. Examples of such biological functions include, but are not limited to, the catalytic activity of a biological substance; the ability to stimulate intracellular signaling, gene expression, and cell proliferation; and the ability to modulate immunological activity such as inflammatory responses. “Activity” is generally expressed as the biological activity per unit of administered substance, e.g., [catalytic activity] / [mg protein], [immune activity] / [mg protein], international units (IU) of activity, [STAT5 or STAT3 phosphorylation] / [mg protein], [T cell proliferation] / [mg protein], plaque-forming units (pfu), etc. The term “proliferative activity” encompasses the activity that promotes cell division, including unregulated cell division as seen in neoplastic diseases, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis, and angiogenesis.

[0037] The terms “administer” and “administer” are interchangeable herein and refer to the act of bringing cells, tissues, organs, or bodily fluids of a subject into contact with a substance (e.g., orthogonal IL-2, CAR-T cells, chemotherapeutic agents, antibodies, or modulators, or pharmaceutical formulations comprising one or more of the aforementioned) in vitro, in vivo, or ex vivo. Administration of a substance can be achieved by any of the various methods permitted in the Art, including, but not limited to, local, intravascular injection (including intravenous or intra-arterial injection), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, percutaneous, transmucosal, iontophoretic delivery, lymphatic infusion, intragastric infusion, intraprostatic infusion, intracapsular infusion (e.g., bladder), respiratory inhaler, intraocular injection, intraperitoneal injection, intrafocal injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), and others. The term "administration" includes not only the contact of a substance with cells, tissues, or organs, but also, if the liquid is in contact with cells, the contact of a substance with a liquid.

[0038] As used herein, the term "affinity" refers to the degree of specific binding of a first molecule (e.g., ligand) to a second molecule (e.g., receptor), and is measured by binding kinetics, expressed as Kd, which is the ratio of the dissociation constant (Koff) between the molecule and its target to the association constant (Kon) between the molecule and its target.

[0039] As used herein, the terms “biological sample” or “sample” refer to a sample obtained from or derived from a subject. For example, a biological sample includes substances selected from the group consisting of body fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluids (e.g., vitreous humor, aqueous humor), lymph, lymph node tissue, spleen tissue, bone marrow, and immunoglobulin concentrates derived from one or more of these tissues. In some embodiments, a sample is obtained from a subject exposed to a therapeutic treatment regimen including a pharmaceutical formulation of orthogonal IL-2, such as repeated exposure to the same drug. In other embodiments, a sample is obtained from a subject that has not recently been exposed to orthogonal IL-2, or from a subject prior to a planned administration of orthogonal IL-2.

[0040] As used herein, the terms “chimeric antigen receptor” and “CAR” refer to a chimeric polypeptide comprising multiple functional domains, interchangeably used and positioned from the amino terminus to the carboxyl terminus of the sequence: (a) an antigen-binding domain (ABD), (b) a transmembrane domain (TD), and (c) one or more cytoplasmic signaling domains (CSD); where the aforementioned domains may optionally be linked by one or more spacer domains. The CAR may also further comprise a signal peptide sequence, which is typically removed during post-translational processing and presentation of the CAR on the cell surface of cells transformed with an expression vector containing the nucleic acid sequence encoding the CAR. CARs useful for carrying out the methods of the present invention can be prepared according to principles well known in the art. For example, see Eshhaar et al. U.S. No. 7,741,465 B1, published June 22, 2010; Sadelain, et al (2013) Cancer Discovery 3(4):388-398; 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. Examples of commercially available CAR-T cell products that can be modified to incorporate the orthogonal receptor of the present invention include axicabtagene ciloleucel (marketed as Yescarta® and available from Gilead Pharmaceuticals) and tisagenlecleucel (marketed as Kymriah® and available from Novartis).

[0041] As used herein, the terms “chimeric antigen receptor T cells” and “CAR-T cells” refer to T cells that are interchangeably used and have been recombinantly modified to express a chimeric antigen receptor. As used herein, CAR-T cells may be engineered to express an orthogonal CD122 polypeptide.

[0042] As used herein, the terms “interleukin-2” or “IL2” refer to the naturally occurring IL2 polypeptide possessing IL2 activity. In some embodiments, IL2 refers to mature wild-type human IL2. Mature wild-type human IL2 (hIL2) exists as a 133-amino acid polypeptide (excluding a signal peptide consisting of an additional 20 N-terminal amino acids), 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: TIFF2026053539000003.tif11128

[0043] For use herein, the residue numbering is the same as that of SEQ ID NO:2, based on the IL2 sequence UniProt ID P60568, excluding the signal peptide.

[0044] As used herein, the term "natural human CD122" refers to naturally occurring human CD122, including its naturally occurring variants. The amino acid sequence of one naturally occurring human CD122 variant is as follows: TIFF2026053539000004.tif45128

[0045] As used herein, the term "human CD122" may refer to natural human CD122 or an ortholog of orthogonal human CD122. As used herein, the numbering of residues for human CD122 is based on SEQ ID NO: 1.

[0046] As used herein, the terms “human orthogonal CD122” or “orthogonal human CD122” refer to a variant of the natural CD122 polypeptide that is interchangeable and can specifically bind to at least one orthogonal IL2. In some embodiments, orthogonal human CD122 includes amino acid substitutions at the histidine 133 (H133) and tyrosine 134 (Y134) positions of the ECD of the hCD122 polypeptide. In some embodiments, orthogonal CD-122 includes an amino acid substitution from histidine at position 133 to aspartic acid (H133D), glutamic acid (H133E), or lysine (H133K), and / or an amino acid substitution from tyrosine at position 134 to phenylalanine (Y134F), glutamic acid (Y134E), or arginine (Y134R). In some embodiments, the orthogonal CD122 is an hCD122 molecule having amino acid substitutions H133D and Y134F.

[0047] As used herein, the term "modified human CD122" refers to a protein comprising human CD122 and one or more STAT3-binding motifs. Human CD122 may be human orthogonal CD122 or natural human CD122. In some embodiments, modified human CD122 retains the STAT5 motif YYLSL (SEQ ID NO: 20) as in natural human CD122.

[0048] As used herein, the term "modified human orthogonal CD122" refers to one type of modified human CD122 protein comprising human orthogonal CD122 and one or more STAT3-binding motifs. In some embodiments, the modified CD122 retains the STAT5 motif YLSL (SEQ ID NO: 20).

[0049] As used herein, the term “derived” means, in relation to an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence “derived” from an IL2 polypeptide), that a polypeptide or nucleic acid has a sequence based on the sequence of a reference polypeptide or nucleic acid (e.g., a naturally occurring IL2 polypeptide or nucleic acid encoding IL2), and does not mean to limit to the source or method from which the protein or nucleic acid is made. For example, the term “derived” includes homologs or variants of a reference amino acid sequence or DNA sequence.

[0050] 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) located outside the plasma membrane of a cell. An ECD can include the entire extracellular portion of a transmembrane protein, a cell surface or membrane-bound protein, a secreted protein, or a cell surface target protein.

[0051] The term "IL2 activity" refers to one or more biological effects on cells in response to contact with an effective amount of IL2 polypeptide. IL2 activity can be measured, for example, in a cell proliferation assay using CTLL 2 mouse 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 is approximately 2.1 × 10⁴ IU / μg and is calibrated against the recombinant human IL2 WHO international standard (NIBSC code: 86 / 500). In some embodiments, for example, if the IL2 orthogonal polypeptide of interest exhibits (or is engineered to have) reduced affinity for CD25, IL2 activity may be evaluated in human cells, such as YT cells, which do not require CD25 to provide IL2 receptor-mediated signaling and can rather signal via the medium-affinity CD122 / CD132 receptor. The orthogonal human IL2 of this disclosure, when evaluated at similar concentrations in equivalent assays, may have less than 20%, or about 10%, or about 8%, or about 6%, or about 4%, or about 2%, or about 1%, or about 0.5%, of the activity of wild-type mature human IL2, which is the WHO international standard (NIBSC code: 86 / 500).

[0052] As used herein, the term "requiring treatment" means a judgment made by a physician or other healthcare provider regarding a subject that the subject requires treatment or may benefit from treatment. This judgment is based on a variety of factors within the physician's or healthcare provider's area of ​​expertise.

[0053] As used herein, the term “intracellular domain of modified CD122” or “ICD” refers to a portion of a transmembrane orthogonal receptor located on the plasma membrane side of a cell expressing such a receptor. This ICD may contain one or more “proliferation signaling domains” or “PSDs,” which refer to protein domains that signal the cell to enter mitosis and initiate cell proliferation. Examples include, but are not limited to, Janus kinases, such as JAK1, JAK2, JAK3, Tyk2, Ptk-2, homologous members of the Janus kinase family from other mammalian or eukaryotic species, IL2 receptor β-chains and / or γ-chains, and other subunits, or portions, modifications, or combinations thereof, from proteins of the cytokine receptor superfamily that can interact with Janus kinase family proteins to transmit signals. Examples of signaling include phosphorylation of one or more STAT molecules, including but not limited to one or more of STAT1, STAT3, STAT5a, and / or STAT5b.

[0054] As used herein, the term "ligand" refers to a molecule that exhibits specific binding to a receptor and alters the biological activity of that receptor, thereby altering the activity of the receptor to which it binds. In one embodiment, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor. As used herein, the term "ligand" includes both natural and synthetic ligands. "Ligands" also include small molecules, such as peptide mimes of cytokines and peptide mimes of antibodies. The ligand-receptor complex is called a "ligand-receptor complex."

[0055] The "percentage of sequence identity" is determined by comparing two optimally aligned sequences in a comparison window, where the portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Substantial identity of amino acid sequences usually means at least 40% sequence identity. The identity percentage of a polypeptide can be any integer between 40% and 100%, for example, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, "substantially similar" polypeptides share sequences as described above, except that non-identical residue positions may differ due to conserved amino acid substitutions. Conserved amino acid substitutions mean the interchangeability of residues with similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Examples of groups of conserved amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

[0056] Suitable algorithms for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analysis is publicly available online through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). This algorithm first involves identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequence, match or satisfy a certain positive threshold score T. T is called the neighbor word score threshold (Altschul et al., see above). These initial adjacent word hits act as seeds to initiate a search for longer HSPs containing them. Word hits are extended in both directions along each sequence to the extent that the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction is stopped if: the cumulative alignment score falls by quantity X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses, by default, a word length of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, (1989)) alignment (B) of 50, an expected value (E) of 10, M=5, N=-4, and a comparison of both strands.

[0057] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, if the smallest sum probability in the comparison of a test nucleic acid and a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0058] In this specification, when used in relation to the structure of a polypeptide, “N-terminus” (or “amino-terminus”) and “C-terminus” (or “carboxyl-terminus”) refer to the amino-terminus and carboxyl-terminus at the outermost ends of the polypeptide, respectively, while the terms “N-terminal” and “C-terminal” refer to the relative positions in the amino acid sequence of the polypeptide toward the N-terminus and C-terminus, respectively, and may include residues at the N-terminus and C-terminus, respectively. “Immediately N-terminal” or “immediately C-terminal” refers to the position of the first amino acid residue relative to the second amino acid residue when the first and second amino acid residues are covalently bonded to provide a continuous amino acid sequence.

[0059] The terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-exclusive examples of polynucleotides include linear and cyclic nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, and primers.

[0060] In this specification, the term “functionally linked” is used to refer to a relationship between nucleic acid sequences that encode different functions when combined into a single nucleic acid sequence, such that, when introduced into a cell, they provide a nucleic acid that can influence the transcription and / or translation of a particular nucleic acid sequence within the cell. For example, the DNA of a signal sequence is functionally linked to the DNA of a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is functionally linked to a coding sequence if it influences the transcription of the coding sequence; or a ribosome binding site is functionally linked to a coding sequence if it is positioned to facilitate translation. Generally, “functionally linked” means that the linked DNA sequences are contiguous, and in the case of a secretion leader, they are contiguous and have matching reading phases. However, certain genetic elements, such as enhancers, do not need to be contiguous with respect to the sequence in which they exert their effect.

[0061] As used herein, the terms “polypeptide,” “peptide,” and “protein” are interchangeable herein and refer to polymeric forms of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified polypeptide backbone. These terms include, but are 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, and fusion proteins of immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments).

[0062] As used herein, the terms “prevent,” “prevention,” and “prevention” generally refer to a course of action initiated with respect to an individual who is predisposed to a particular disease, disorder, or condition due to genetic, empirical, or environmental factors, in order to temporarily or permanently prevent, suppress, block, or reduce (for example, determined by the absence of clinical symptoms) or delay the onset of such disease, disorder, condition, or its symptoms. In specific cases, the terms “prevent,” “prevention,” and “prevention” are also used to refer to delaying the progression of a disease, disorder, or condition from its current state to a more harmful state.

[0063] As used herein, the term “receptor” refers to a polypeptide having a domain that specifically binds to a ligand, and the binding of the ligand results in a change in at least one biological property of the polypeptide. In some embodiments, the receptor is a “soluble” receptor that is not bound to the cell surface. The soluble form of hCD25 is an example of a soluble receptor that specifically binds to hIL2. In some embodiments, the receptor is a cell surface receptor comprising an extracellular domain (ECD) and a membrane-bound domain that plays a role in fixing 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), linked by a membrane-bound domain commonly called a transmembrane domain (TM). When a ligand binds to a receptor, a conformational change occurs in the receptor, resulting in a measurable biological effect. In some examples, if the receptor is a transmembrane polypeptide comprising an ECD, TM, and ICD, when a ligand binds to the ECD, a measurable intracellular biological effect occurs, mediated by one or more domains of the ICD in response to the ligand's binding to the ECD. In some embodiments, receptors are components of multi-component complexes that facilitate intracellular signaling. For example, ligands may bind to cell surface molecules that are not involved in any intracellular signaling on their own, but upon ligand binding, they promote the formation of heteromultimers such as heterodimers (e.g., medium-affinity CD122 / CD132 IL2 receptors), heterotrimers (e.g., high-affinity CD25 / CD122 / CD132 hIL2 receptors), or homomultimers (homodimer, homotrimer, homotetramer) complexes, resulting in the activation of intracellular signaling cascades (e.g., the Jak / STAT pathway).

[0064] As used herein, the terms “recombinant” or “engineered” refer to polypeptides produced using recombinant DNA technology. Recombinant DNA techniques and protocols are well known in the art.

[0065] The term “response,” for example, the “response” of a cell, tissue, organ, or organism, encompasses changes in biochemical or physiological behavior, such as changes in concentration, density, adhesion, or migration within a biological compartment, gene expression rates, or differentiation state, which correlate with activation, stimulation, or treatment, or with internal mechanisms such as genetic programming. In certain contexts, terms such as “activation” and “stimulation” refer to the activation of a cell that is controlled not only by external or environmental factors but also by internal mechanisms; on the other hand, terms such as “inhibition” and “downregulation” refer to the opposite effect.

[0066] As used herein, the term “specifically binds” refers to the degree of selectivity or affinity with which one molecule binds to another. In the context of binding pairs (e.g., ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pairs), the first molecule of the binding pair can be said to bind specifically to the 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. The first molecule of the binding pair can be said to bind specifically to the second molecule of the binding pair if its affinity for the second molecule is at least twice, at least five times, at least ten times, at least twenty times, or at least one hundred times higher than its affinity for other components present in the sample. In a specific embodiment where the first molecule of the binding pair is an antibody, if the equilibrium dissociation constant between the antibody and the second molecule of the binding pair, as measured by, for example, Scatchard analysis (Munsen, et al. 1980 Analyt. Biochem. 107:220-239), is greater than approximately 10⁶ M, or greater than approximately 10⁸ M, or greater than approximately 10¹⁰ M, or greater than approximately 10¹¹ M, or greater than approximately 10¹⁰ M, or greater than approximately 10¹² M, then the antibody specifically binds to the second molecule of the binding pair (e.g., a protein, antigen, ligand, or receptor). In one embodiment, where the ligand is orthogonal IL2 and the receptor is orthogonal CD122 ECD, orthogonal IL2 specifically binds if the equilibrium dissociation constant of the IL2 ortholog / orthogonal CD122 ECD is greater than approximately 10⁵ M, or greater than approximately 10⁶ M, or greater than approximately 10⁷ M, or greater than approximately 10⁸ M, or greater than approximately 10⁹ M, or greater than approximately 10¹¹ M. Specific binding can be evaluated using techniques known in the art, including but not limited to competitive ELISA, BIACORE® assays, and / or KINEXA® assays.

[0067] The terms “recipient,” “individual,” “subject,” and “patient” are used interchangeably herein and refer to any mammalian subject, in particular human, to whom diagnosis, treatment, or therapy is desired. “Mammal” for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock and farm animals, as well as zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, and pigs. In some embodiments, mammal is human.

[0068] As used herein, the term “T-cell” or “T cell” is used in its conventional sense, referring to lymphocytes that differentiate in the thymus, possessing specific cell surface antigen receptors, and some regulating the initiation or suppression of cellular and humoral immunity, or lysing antigen-carrying 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., TH1, TH2, TH9, TH11, TH22, TFH; regulatory T cells, e.g., TR1, Treg, inducible Treg; memory T cells, e.g., central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TILs), and genetically modified variants of such T cells, e.g., CAR-T cells, recombinant modified TILs, and TCR-transformed cells.

[0069] As used herein, the term "therapeutic dose" refers to the amount of a drug administered to a subject, either alone or as part of a pharmaceutical composition or therapeutic regimen, in a single dose or as part of a series of doses, that produces a detectable positive effect on the symptoms, aspects, or characteristics of a disease, disorder, or condition. The therapeutic dose can be determined by measuring the relevant physiological effects and may be adjusted in relation to the administration regimen and in accordance with diagnostic analysis of the subject's condition, etc. Evaluation parameters for determining the therapeutic dose of a drug are determined by a physician using diagnostic criteria recognized in the art, including but not limited to the following indicators: age, weight, sex, general health status, ECOG score, observable physiological parameters, blood concentration, blood pressure, electrocardiogram, computed tomography, X-ray, etc. Alternatively, or additionally, to determine whether a therapeutically effective amount of the drug has been administered to the subject, other parameters commonly evaluated in clinical practice can be monitored, such as: body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or symptoms, appearance or characteristics of the disease, disorder or condition, biomarkers (inflammatory cytokines, IFN-□, granzymes, etc.), decrease in serum tumor markers, improvement in Response Evaluation Criteria In Solid Tumors (RECIST), improvement in Immune-Related Response Criteria (irRC), extension of survival, extension of progression-free survival, extension of time to progression, increase in time to treatment failure, extension of event-free survival, extension of time to the next treatment, improvement in objective response rate, improvement in duration of response, reduction in tumor burden, and complete response, partial response, disease stabilization, etc., which clinicians in this field trust to evaluate the improvement of the subject's condition in response to drug administration.As used herein, the terms “complete response (CR),” “partial response (PR),” “stable (SD),” and “progression (PD)” for targeted lesions, and “complete response (CR),” “incomplete response / stable (SD),” and “progression (PD)” for non-targeted lesions are understood to be as defined in the RECIST criteria. As used herein, the terms “irimmune-related complete response (irCR),” “irimmune-related partial response (irPR),” “irimmune-related progression (irPD),” and “irimmune-related stable (irSD)” are defined according to the immune-related response criteria (irRC). As used herein, the term “irRC” refers to a system for evaluating the response to immunotherapy, such as that described in Wolchok, et al. (2009) Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-Related Response Criteria, Clinical Cancer Research 15(23): 7412-7420. The therapeutically effective dose may be adjusted over the course of treatment in relation to the administration regimen and / or assessment of the subject's condition and variations in the aforementioned factors. In one embodiment, the therapeutically effective dose is the amount of the drug, when used alone or in combination with other drugs, that does not cause irreversible serious adverse events during administration to a mammalian subject.

[0070] The terms “to treat,” “to treat,” and “treatment” refer to a course of action initiated with respect to a subject after a disease, disorder, condition, or symptom has been diagnosed or observed in that subject, in order to temporarily or permanently eliminate, reduce, suppress, alleviate, or improve at least one of the underlying causes of the disease, disorder, or condition that is afflicting the subject, or at least one of the symptoms associated with the disease, disorder, or condition. This course of action includes a course of action taken with respect to a subject suffering from a disease that results in the suppression of the disease in that subject (e.g., prevention of the onset of the disease, disorder, or condition, or improvement of one or more of the symptoms associated with it).

[0071] As used herein, the terms “regulatory T cells” or “Treg cells” refer to a type of CD4+ T cell capable of suppressing the response of other T cells, including but not limited to effector T cells (Teff). Treg cells are characterized by the expression of CD4, the α-subunit of the IL2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)). “Conventional CD4+ T cells” refers to CD4+ T cells other than regulatory T cells.

[0072] Wild-type: In this specification, "wild-type," "WT," or "natural" means the amino acid or nucleotide sequence found in nature, including allele mutations. WT proteins, polypeptides, antibodies, immunoglobulins, IgG, etc., have an amino acid or nucleotide sequence that has not been modified by human intervention.

[0073] Modified Human CD122 This disclosure provides a modified human CD122 comprising one or more STAT3-binding motifs in addition to the native STAT5-recognition motif. The inclusion of the additional STAT3-binding motifs enhances signaling and stabilizes the IL2 response.

[0074] STAT protein and STAT3 binding motif STAT proteins, after phosphorylation of a conserved tyrosine residue at their C-terminus, translocate to the nucleus, where they act as transcription activators, binding to DNA and activating the transcription of target genes. Hennighausen L, Robinson GW. Genes Dev. 2008; 22:711-21. This family includes seven STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, which function in a variety of pathways, from innate and adaptive immunity to cell proliferation, differentiation, and survival. Basham et al., Nucleic Acids Res. 2008 Jun; 36(11): 3802-3818.

[0075] STAT-binding motifs are typically present on cytokine receptors. When cytokines bind to these motifs, Janus kinase (JAK) family tyrosine kinases are activated, and these tyrosine kinases phosphorylate specific tyrosine molecules in the receptor's intracellular domain. The phosphorylated receptor then recruits STAT to the STAT-recognition motif on the receptor, leading to its phosphorylation. The phosphorylated STAT dimerizes and translocates to the nucleus, activating the transcription of important genes; Hennighausen L, Robinson GW. Interpretation of cytokine signaling through the transcription factors STAT5A and STAT5B. Genes Dev. 2008;22:711-21.

[0076] Of these STAT proteins, STAT5 can be activated by cytokines such as IL2, IL-4, IL-7, IL-9, IL-15, and IL21, via their binding to their cognitive receptors. Lara E. Kallal & Christine A. Biron (2013) Changing partners at the dance, JAK-STAT, 2:1, e23504, DOI: 10.4161 / jkst.23504, Page 2, Col. 2. Activated STAT5 can recruit STAT5 by targeting genes such as recognition motifs, thereby activating the transcription of genes such as Cis, spi2.1, and Socs-1. Basham et al., Nucleic Acids Res. 2008 Jun; 36(11): 3802-3818. For example, the modified CD122 of this disclosure, also known as the β-receptor of IL2, contains a STAT5 recognition motif and can recruit and activate STAT5.

[0077] The STAT5 motif has the sequence YX1X2L (SEQ ID NO: 19). X1 and X2 can be any native amino acids. In some cases, X1 and X2 are the same amino acid residue. In other cases, X1 and X2 are different amino acid residues. In one embodiment, STAT5 has the sequence YLSL (SEQ ID NO: 20).

[0078] The STAT3 binding motif is not present in natural human CD122. It is typically found on receptors that bind to IL-6, IL-10, IL-21, IFNαβ, IFNγ, and IFNλ. When activated, STAT3 targets Bcl-XL, survivin, cyclin D1, and activated c-myc. Lara E. Kallal & Christine A. Biron (2013) Changing partners at the dance, JAK-STAT, 2:1, e23504, DOI: 10.4161 / jkst.23504, page 3. STAT3 is activated via tyrosine phosphorylation by various cytokines (their receptors share the gp130 chain), including IL-6 and IL-21, oncostatin M (OSM), and leukemia suppressor (LIF) [2]. STAT3 is involved in a variety of biological functions, including oncogenesis, angiogenesis, tumor metastasis, and anti-apoptosis. See Wei Sun et al., FEBS Lett. 2006 Oct 30;580(25):5880-4. Epub 2006 Oct 2. and Fukada et al. Immunity, 1 November 1996, 449-460 Vol. 5, issue 5. Thus, STAT3 signaling confers anti-apoptotic effects to cells, and as a result, prolongs the half-life of cells expressing modified human CD122.

[0079] In this disclosure, human CD122 (including an intact STAT5 motif) is modified to introduce one or more STAT3 binding motifs, and the modified human CD122 thus produced retains a STAT5 recognition motif and acquires one or more STAT3 binding motifs.

[0080] In some embodiments, the modified CD122 may contain one, two, three, or more STAT3 binding motifs. The STAT3 recognition motif has the sequence YX1X2Q (SEQ ID NO: 21). In some embodiments, 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. In some embodiments, the STAT3 sequence is selected from the group consisting of YLRQ (SEQ ID NO: 11); YLKQ (SEQ ID NO: 12); YRHQ (SEQ ID NO: 13); YLRQ (SEQ ID NO: 14); YFKQ (SEQ ID NO: 15); YLPQ (SEQ ID NO: 16); YMPQ (SEQ ID NO: 17) and YDKPH (SEQ ID NO: 18).

[0081] Human CD122 (including natural human CD122 and human CD122 orthologs) In addition to the STAT3 binding motif, the modified human CD122 includes human CD122, which may be natural human CD122 or human orthogonal CD122.

[0082] In some embodiments, the modified human CD122 includes orthogonal human CD122. This orthogonal human CD122 is created by mutating residues of natural CD122 so that it specifically binds to orthogonal IL2 but not to natural IL2. For example, the binding affinity to orthogonal IL2 is higher, for example, 2, 3, 4, 5, 10 or more times the affinity of natural IL2 to natural CD122. In some embodiments, the affinity of orthogonal IL2 to cognitive orthogonal CD122 is comparable to the affinity of natural IL2 to natural CD122, for example, having an affinity of at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100% of the binding affinity of natural CD122 to natural IL2. In some cases, orthogonal CD122 is modified in one or more residues selected from R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and Q214 compared to natural human CD122. In some embodiments, orthogonal human CD122 is modified at H133 and Y134. In some embodiments, orthogonal human CD122 includes substitutions at H133D and Y134F. In some embodiments, orthogonal human CD122 includes amino acid substitutions at Q70, T73, H133, and Y134 compared to the natural human CD122 protein. In some embodiments, orthogonal human CD122 includes amino acid substitutions at H133 and Y134. In some embodiments, amino acid substitutions are made for acidic amino acids, such as aspartic acid and / or glutamic acid. Specific amino acid substitutions, compared to natural human CD122, include, but are not limited to, Q70Y;T73D;T73Y;H133D;H133E;H133K;Y134F;Y134E;Y134R. The selection of orthologous cytokines may vary depending on the selection of orthologous receptors.

[0083] In some embodiments, in addition to having the above substitutions relative to natural human CD122, the orthogonal human CD122 has a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the full-length sequence of natural CD122 (SEQ ID NO: 2).

[0084] Orthogonal IL2: When using modified orthogonal human CD122, orthogonal IL2 can be used to facilitate the selective stimulation of cells engineered to express the modified orthogonal CD122. Contact between T cells engineered to express the modified orthogonal CD122 and the corresponding orthogonal ligand of the orthogonal CD122 enables the specific activation of such engineered T cells. In particular, this orthogonal IL2 receptor-ligand complex provides selective expansion (proliferation) of cells engineered to express the orthogonal receptor in mixed cell populations, especially mixed T cell populations. The IL2 orthogonal ligand provides selective binding and signaling via the receptor, which includes the extracellular domain of the CD122 orthogonal receptor, particularly the extracellular domain of human CD122 containing the amino acid substitutions H133D and Y134F. The IL2 activity of orthogonal IL2 against cells expressing wild-type CD122 is significantly reduced compared to the activity of orthogonal IL2 against cells expressing orthogonal CD122. Therefore, selective activation and / or expansion of cells engineered to express receptors containing the extracellular domain of orthogonal IL2 is provided using orthogonal IL2 against engineered cell populations.

[0085] Orthogonal IL2, by incorporating modifications to its primary structure, provides a polypeptide variant exhibiting the following properties: (a) significantly reduced affinity for its native CD122 (i.e., the native receptor for native IL2 from which orthogonal IL2 is derived); and (b) specific binding to modified orthogonal CD122, which is a variant of native CD122. When orthogonal IL2 binds to orthogonal CD122 (expressed on the surface of cells modified by recombinant DNA techniques to incorporate a nucleic acid sequence encoding the orthogonal receptor, functionally linked to regulatory elements that result in orthogonal receptor expression in recombinantly modified cells), activated orthogonal CD122 initiates signaling transmitted via native cellular components, providing biological activity that mimics the native response of the cognitive but is specific to the recombinantly modified cell population expressing the orthogonal receptor. In some embodiments of the present invention, the ortholog exhibits significant selectivity for orthogonal CD122 compared to the native CD122 receptor, and optionally, significantly reduced potency for native CD122. Selectivity is generally assessed by activity, which is measured in assays characteristic of activity induced in response to ligand / receptor binding. In some embodiments, the ortholog IL2 exhibits a difference of at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 100-fold, or at least 200-fold in EC50 measured in the same assay.

[0086] IL2 orthologs exhibit specific binding to the extracellular domain of the ortholog CD122, for example, human CD122 with modifications incorporated at positions 133 and 134.

[0087] IL2 Ortholog In various embodiments, the method of this disclosure includes the use of an orthogonal IL2 comprising the amino acid sequence of Formula 1 below: TIFF2026053539000005.tif73128In formula, • AA1 is either A (wild type) or deleted; • AA2 is either P (wild type) or deleted; AA3 is either T (wild type), C, A, G, Q, E, N, D, R, K, or P, or it is deleted; • AA4 is either S (wild type) or deleted; • AA5 is either S (wild type) or deleted; • AA6 is either S (wild type) or deleted; • AA7 is either T (wild type) or deleted; • AA8 is either K (wild type) or deleted; • AA9 is either K (wild type) or deleted; AA13 is either Q (wild type) or W, or deleted; • AA14 is either L (wild type), M, or W, or deleted; AA15 is either E (wild type), K, D, T, A, S, Q, or H, or it is deleted; AA16 is either H (wild type), N, or Q, or it is deleted; AA18 is L (wild type), R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D, or T; AA19 is either L (wild type), A, V, or I, or deleted; AA20 is either D (wild type), T, S, M, or L, or deleted; AA22 is either Q (wild type), F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, or F, or is deleted; AA23 is either M (wild type), A, W, H, Y, F, Q, S, V, L, or T, or it is deleted; AA27 is either G (wild type), K, or S, or deleted; AA38 is R (wild type), W, or G; AA39 is M (wild type), L, or V; AA42 is either F (wild type) or K; • AA51 is either T (wild type) or I, or deleted; AA55 is either H (wild type) or Y; AA74 is Q (wild type), N, H, or S; AA80 is L (wild type), F, or V; AA81 is either R (wild type), I, D, Y, or T, or deleted; AA85 is either L (wild type) or V; AA86 is either I (wild type) or V; • AA88 is either N (wild type), E, ​​or Q, or deleted; AA89 is either I (wild type) or V; AA91 is V (wild type), R, or K; AA92 is either I (wild type) or F; AA97 is either K (wild type) or Q; AA104 is either M (wild type) or A; AA109 is a non-natural amino acid with D (wild type), C, or activated side chains; AA113 is either T (wild type) or N; AA125 is C (wild type), A, or S; • AA126 is Q (wild type), H, M, K, C, D, E, G, I, R, S or T; and / or AA130 is S (wild type), T, or R.

[0088] In some embodiments, the present disclosure provides orthogonal IL2, which is an hIL2 polypeptide comprising the following set of amino acid modifications: TIFF2026053539000006.tif112128

[0089] Cys125: In some embodiments, the present disclosure provides orthogonal IL2 that facilitates recombinant expression in bacterial cells by removing the unpaired cysteine ​​residue at position 125 and / or removing the N-terminal Met and alanine at position 1 of the directly expressed IL2 polypeptide by post-translational processing with endogenous bacterial proteases. When one amino acid is missing, it is called "des". In some embodiments, the cysteine ​​at position 125 is replaced with alanine or serine (C125A or C125S). Such mutations are commonly used to avoid protein misfolding when recombinantly expressed in bacteria and isolated from inclusion bodies. For example, "des-Ala1" means that alanine at position 1 is absent in the IL2 polypeptide. In some embodiments, the orthogonal IL2 of the present invention comprises one of the following sets of amino acid modifications: TIFF2026053539000007.tif121128

[0090] Mutations that increase CD122 affinity In some embodiments, orthogonal IL2 comprises one or more mutations in the hIL2 sequence at a location in contact with hCD122, or at a location in the hIL2 sequence that alters the orientation of another location in contact with CD122, resulting in an orthogonal IL2 with increased affinity for CD122. IL2 residues known to be involved in the 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, orthogonal IL2 comprises one or more of the following amino acid substitutions: Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, I92F, or a combination thereof. In some embodiments, orthogonal IL2 includes one or more amino acid substitutions: L80F, R81D, L85V, I86V, and I92F. In some embodiments, orthogonal IL2 includes one or more amino acid substitutions: N74Q, L80F, R81D, L85V, I86V, I89V, and I92F. In some embodiments, orthogonal IL2 includes one or more amino acid substitutions: Q74N, L80V, R81T, L85V, I86V, and I92F. In some embodiments, orthogonal IL2 includes one or more amino acid substitutions: Q74H, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the orthogonal IL2 includes one or more amino acid substitutions: Q74S, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the orthogonal IL2 includes one or more amino acid substitutions: Q74N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the orthogonal IL2 includes one or more amino acid substitutions: Q74S, R81T, L85V, and I92F. In some embodiments, the orthogonal IL2 includes [L80F-R81D-L85V-I86V-I92F]. In some embodiments, the disclosure provides an orthogonal IL2 including one of the following sets of amino acid modifications: TIFF2026053539000008.tif59132

[0091] In some embodiments, the ortholog contains a substitution L85V that has been identified as increasing the affinity of IL2 to CD122. In some embodiments, the present disclosure provides an orthogonal IL2 which is an hIL2 polypeptide comprising one of the following sets of amino acid modifications: TIFF2026053539000009.tif52128

[0092] Adjustment of CD25 affinity In some embodiments, orthogonal IL2 includes one or more mutations that reduce affinity to CD25 at the location of the IL2 sequence in contact with CD25, or at the location of the IL2 sequence that alters the orientation of other locations in contact with CD25. These mutations may be located within 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 be in contact with CD25 are K35, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, V69, L72, and Y107. In some embodiments, the orthogonal IL2 of the present disclosure includes one or more point variants of R38A, F41A and F42A (Suave, et al (1991) PNAS(USA) 88:4636-4640); P65L (Chen et al. Cell Death and Disease (2018) 9:989); F42A / G / S / T / Q / E / N / R / K, Y45A / G / S / T / Q / E / N / D / R / K / and / or L72G / A / S / T / Q / E / N / D / R / K (Ast, et al. U.S. Patent Application Publication No. 2012 / 0244112A1, published September 27, 2012, and U.S. Patent No. 9266938B2, issued February 23, 2016). Certain substitution combinations have been shown to reduce binding to CD25. In some embodiments, the orthogonal IL2 of this disclosure comprises one or more of the following sets of substitutions: [R38A-F42A-Y45A-E62A]; [F42A-Y45A-L72G] (Roche RG7461 (RO6874281)); and / or [T41P-T51P] (Chang, et al (1995) Molecular Pharmacology 47:206-211), as described in Carmenate, et al (2013) J Immunol 190:6230-6238. In some embodiments, this disclosure provides an orthogonal IL2 which is an hIL2 polypeptide comprising one of the following sets of amino acid modifications: TIFF2026053539000010.tif52128

[0093] In some aspects of the present invention, orthogonal IL2 includes one or more mutations that alter binding to CD132 at the location of the IL2 sequence that contacts CD132, or at the location of the IL2 sequence that alters the orientation of other locations that contact CD132. An example of orthogonal IL2 includes one or more mutations that alter binding to CD132 at the location of the IL2 sequence that contacts CD132, or at the location of the IL2 sequence that alters the orientation of other locations that contact CD122. IL2 residues considered to contact CD132 include Q11, L18, Q22, E110, N119, T123, Q126, S127, I129, S130, and T133. In some embodiments, IL2 includes a modification to L18, and 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.

[0094] In some embodiments, the present disclosure provides an orthogonal IL2 which is an hIL2 polypeptide comprising one of the following sets of amino acid modifications: TIFF2026053539000011.tif148128

[0095] When produced by direct recombination in the absence of a leader sequence in a bacterial expression system, the endogenous protease deletes the N-terminal Met-Ala1 residue, resulting in a "desAla1" orthogonal IL2. In some embodiments, this disclosure provides an orthogonal IL2 which is an hIL2 polypeptide comprising one of the following sets of amino acid modifications: TIFF2026053539000012.tif182128

[0096] Conservative amino acid substitutions In addition to the modifications described above that contribute to the activity and selectivity of orthogonal IL2 for the CD122 orthogonal receptor, orthogonal IL2 may include one or more modifications to its primary structure that have minimal effect on IL2 activity. In some embodiments, the orthogonal IL2 of this disclosure may further include one or more conserved amino acid substitutions within the wild-type IL-2 amino acid sequence. Such conserved substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and those described by Argos in EMBO J., 8:779-785 (1989). Conservative substitutions are generally made according to the following chart shown in Table 3.

[0097] (Table 3) Examples of conservative amino acid substitutions TIFF2026053539000013.tif95134

[0098] Substantial changes in function or immunological specificity can be achieved by selecting non-conservative amino acid substitutions from those shown in Table 3. For example, substitutions that significantly affect the structure of the polypeptide backbone, or substitutions that disrupt secondary or tertiary elements, can be made, including substitutions of amino acids with small uncharged side chains (e.g., glycine) with amino acids with large, bulky charged side chains (asparagine). In particular, substitutions of IL2 residues involving amino acids that interact with one or more of CD25, CD122, and / or CD123 can be found in the crystal structure of IL2 bound to its receptor, as described.

[0099] In addition to the modifications described above that contribute to the activity and selectivity of orthogonal IL2 for the CD122 orthogonal receptor, orthogonal IL2 may include one or more modifications to its primary structure. Such modifications to the primary structure may optionally include further modifications that do not substantially reduce the IL2 activity of orthogonal IL2, and include, but are not limited to, the following substitutions: N30E, K32E, N33D, P34G, T37I, M39Q, F42Y, F44Y, P47G, T51I, E52K, L53N, Q57E, M104A (see U.S. Patent No. 5,206,344).

[0100] Removal of glycosylated sites The orthogonal IL2 of this disclosure may include modifications that eliminate the O-glycosylation site at position Thr3 in order to promote the production of aglycosylated orthogonal IL2 when expressed in mammalian cells such as CHO cells or HEK cells. Therefore, in certain embodiments, the orthogonal IL2 includes modifications that remove the O-glycosylation site of IL-2 at the position corresponding to residue 3 of human IL-2. In one embodiment, the modification that removes the O-glycosylation site of IL-2 at the 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 loss of biological activity (see U.S. Patent No. 5,116,943; Weiger et al., (1989) Eur. J. Biochem., 180:295-300). In a specific embodiment, the modification is the amino acid substitution T3A. In some embodiments, the present disclosure provides orthogonal IL2, which is an hIL2 polypeptide comprising one of the following sets of amino acid modifications: TIFF2026053539000014.tif162128

[0101] Orthogonal IL2s can include deletions of the first two amino acids (desAla1-desPro2) and substitutions of Thr3 glycosylation by a cysteine ​​residue to promote selective N-terminal modifications, particularly PEGylation of the sulfhydryl group of cysteine ​​(see, for example, Katre, et al., U.S. Patent No. 5,206,344, issued April 27, 1993). In some embodiments, the present disclosure provides orthogonal IL2s that are hIL2 polypeptides comprising one of the following sets of amino acid modifications: TIFF2026053539000015.tif80128

[0102] Oxidation-stable M104A The orthogonal IL2 disclosed herein may optionally further include modifications at position M104, in one embodiment, substitution of methionine 104 with an alanine residue (M104A) to provide a more oxidation-resistant orthologue (see Koths, et al., U.S. Patent No. 4,752,585, issued June 21, 1988).

[0103] Deletion of the N-terminus When produced by direct recombination in the absence of a leader sequence in a bacterial expression system, the endogenous protease deletes the N-terminal Met-Ala1 residue, resulting in the "desAla1" orthogonal IL2. The orthogonal IL2 disclosed herein may include deletion of the first two amino acids (desAla1-desPro2) and substitution of Thr3 glycosylation by a cysteine ​​residue (T3C) to promote N-terminal modification, particularly PEGylation of the sulfhydryl group of cysteine ​​(see, for example, Katre, et al., U.S. Patent No. 5,206,344, issued April 27, 1993).

[0104] Orthogonal IL2 may further involve the removal of an N-terminal amino acid at one or more positions, such as positions 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. In some embodiments, the disclosure provides an orthogonal IL2 which is an hIL2 polypeptide comprising one of the following sets of amino acid modifications: TIFF2026053539000016.tif142154

[0105] Minimizing vascular leak syndrome In some aspects of this disclosure, orthogonal IL2s include amino acid substitutions to avoid vascular leak syndrome, a significant dose-limiting side effect of IL2 therapy in humans, without substantial loss of efficacy. See Epstein, et al., U.S. Patent No. 7,514,073B2, issued April 7, 2009. Examples of such modifications included in the orthogonal IL2s of this disclosure include one or more of R38W, R38G, R39L, R39V, F42K, and H55Y.

[0106] Affinity maturation: In some embodiments, orthogonal IL2 can undergo affinity maturation to enhance its activity toward orthogonal CD122. A "affinity-matured" polypeptide is a polypeptide having one or more modifications on one or more residues, resulting in an improved affinity of the orthogonal polypeptide toward the cognitive orthogonal receptor, or vice versa, compared to a parent polypeptide without such modifications. Affinity maturation can be performed to increase the binding affinity of orthogonal IL2 by at least about 10%, at least about 50%, at least about 100%, at least about 150%, or 1 to 5 times, compared to the "parent" polypeptide. While the genetically engineered orthogonal IL2 of the present invention activates its cognitive orthogonal receptor as described above, it exhibits significantly reduced binding and activation of the wild-type IL2 receptor when evaluated by ELISA and / or FACS analysis using sufficient amounts of the molecule under appropriate assay conditions.

[0107] Modification to extend the duration of action in vivo As described above, the compositions of the present disclosure include orthogonal IL2 modified to provide extended lifetime and / or extended duration of action in vivo. Such modifications for extended lifetime and / or duration of action include modifications to the primary sequence of orthogonal IL2, conjugation to a carrier molecule (e.g., albumin, acylation, PEGylation), and Fc fusion.

[0108] Sequence modification to extend the duration of action in vivo As described above, the term orthogonal IL2 includes modifications of orthogonal IL2 to provide in vivo lifetime extension and / or extension of duration of action in the target.

[0109] In some embodiments, orthogonal IL2 may include specific amino acid substitutions that result in extended in vivo lifetime. For example, Dakshinamurthi, et al. (International Journal of Bioinformatics Research (2009) 1(2):4-13) states that one or more substitutions of V91R, K97E, and T113N in the IL2 polypeptide result in IL2 variants with enhanced stability and activity. In some embodiments, the orthogonal IL2 of this disclosure includes one, two, or all three of the V91R, K97E, and T113N modifications.

[0110] Conjugates and carrier molecules In some embodiments, orthogonal IL2 is modified to provide the orthogonal IL2 with specific properties (e.g., extension of the duration of action in a target), which can be achieved by conjugation to a carrier molecule that provides desired pharmacological properties, such as extension of half-life. In some embodiments, orthogonal IL2 can be covalently bonded to the Fc domain of IgG, albumin, or other molecules for extending half-life, for example, by PEGylation, glycosylation, fatty acid acylation, etc., as known in the art.

[0111] Albumin Fusion In some embodiments, orthogonal IL2 is expressed as a fusion protein with an albumin molecule (e.g., human serum albumin) known in the art to promote long-term in vivo exposure.

[0112] In one aspect of the present invention, the hIL2 analog is conjugated to albumin, which is referred to herein as the “orthogonal IL2 albumin fusion.” The term “albumin,” as used in relation to the hIL2 analog albumin fusion, includes albumins such as human serum albumin (HSA), cynomolgus monkey (cyno) serum albumin, and bovine serum albumin (BSA). In some aspects, the HSA contains a C34S or K573P amino acid substitution compared to the wild-type HSA sequence. According to this disclosure, albumin may be conjugated to orthogonal IL2 at the carboxyl terminus, the amino terminus, both carboxyl and amino terminus, or internally (see, for example, U.S. Patents 5,876,969 and 7,056,701). The HSA-orthogonal IL2 polypeptide conjugates envisioned by this disclosure may utilize various forms of albumin, e.g., albumin secretory presequences and their variants, fragments and variants thereof, and HSA variants. Such forms generally retain one or more desired albumin activities. In additional embodiments, this disclosure includes a fusion protein comprising an hIL2 analog polypeptide directly or indirectly fused to albumin, albumin fragments, or albumin variants, where the fusion protein has higher plasma stability than the unfused drug molecule and / or retains the therapeutic activity of the unfused drug molecule. In some embodiments, the indirect fusion is brought about by a linker, such as a peptide linker or a modified version thereof, as will be discussed in more detail below.

[0113] Alternatively, the hIL2 analog albumin fusion contains orthogonal IL2, which is a fusion protein comprising an albumin-binding domain (ABD) polypeptide sequence and an orthogonal IL2 polypeptide. As suggested above, a fusion protein comprising an albumin-binding domain (ABD) polypeptide sequence and an hIL2 analog polypeptide can be achieved by genetic engineering, for example, such that a nucleic acid or fragment encoding an HSA is bound to one or more nucleic acids encoding an orthogonal IL2 sequence. In some embodiments, the albumin-binding peptide comprises the amino acid sequence DICLPRWGCLW (SEQ ID #6).

[0114] Orthogonal IL2 polypeptides can also be conjugated into larger, gradually metabolizing macromolecules such as: proteins; polysaccharides, e.g., Sepharose, agarose, cellulose, or cellulose beads; amino acid polymers, e.g., polyglutamic acid, polylysine; amino acid copolymers; inactivated viral particles; inactivated bacterial toxins, e.g., diphtheria, tetanus, cholera toxoids, or leucotoxins; inactivated bacteria, dendritic cells, thyroglobulin; tetanus toxoid; diphtheria toxoid; polyamino acids, e.g., poly(D-lysine:D-glutamic acid); rotavirus VP6 polypeptide; influenza virus hemagglutinin, influenza virus nucleoprotein; keyhole limpet hemocyanin (KLH); and hepatitis B virus core proteins and surface antigens. Such conjugated forms can, if desired, be used to create antibodies against the polypeptides of this disclosure.

[0115] In some embodiments, orthogonal IL2 may be conjugated (chemically or as a fusion protein) with XTEN to provide a duration extension similar to PEGylation, and may be produced as a recombinant fusion protein in Escherichia coli (E. coli). XTEN polymers suitable for use with the orthogonal IL2 of this disclosure are provided in Podust, et al. (2016) “Extension of in vivo half-life of biologically active molecules by XTEN protein polymers”, J Controlled Release 240:52-66 and Haeckel et al. (2016) “XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic” PLOS ONE | DOI:10.1371 / journal.pone.0157193 June 13, 2016. The XTEN polymer fusion protein may incorporate a protease-sensitive cleavage site, such as an MMP-2 cleavage site, between the XTEN polypeptide and orthogonal IL2.

[0116] Additional candidate components and molecules for conjugation include those suitable for isolation or purification. Certain non-limiting examples include molecules containing binding molecules, e.g., biotin (biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or solid supports (e.g., plastic or polystyrene beads, plates or beads, magnetic beads, test strips, membranes, etc.).

[0117] In some embodiments, IL-2 mutein may also be linked to additional therapeutic agents, such as therapeutic compounds, e.g., anti-inflammatory compounds or antitumor 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. Antimicrobial agents include aminoglycosides, e.g., gentamicin; antiviral compounds, e.g., rifampicin, 3'-azido-3'-deoxythymidine (AZT), asirovir, etc.; antifungal agents, e.g., azoles such as fluconazole, polyene macrolides such as amphotericin B, candicidine, etc.; and antiparasitic compounds, e.g., antimony agents, etc. Orthogonal IL2 may be conjugated to additional cytokines, such as CSF, GSF, GMCSF, TNF, erythropoietin; immunomodulatory factors or cytokines, such as interferons, interleukins; neuropeptides; reproductive hormones, such as HGH, FSH, LH; thyroid hormones; neurotransmitters, such as acetylcholine; hormone receptors, such as estrogen receptors. Also included are nonsteroidal anti-inflammatory drugs, such as indomethacin, acetylsalicylic acid, ibuprofen, sulindac, piroxicam, naproxen, and anesthetics or analgesics. Furthermore, radioisotopes, useful not only for treatment but also for diagnostic imaging, are also included.

[0118] The orthogonal IL2 of this disclosure can be chemically conjugated to such carrier molecules using well-known chemical conjugation methods. For this purpose, bifunctional crosslinking reagents, such as homofunctional and heterofunctional crosslinking reagents well-known in the art, can be used. The type of crosslinking reagent used depends on the properties of the molecule to be coupled to the IL-2 mutein and can be easily determined by those skilled in the art. Alternatively, or additionally, the orthogonal IL2 and / or the molecule to be conjugated can be chemically derivatized so that they can be conjugated in separate reactions, as is also well known in the art.

[0119] PEGylation: In some embodiments, orthogonal IL2 is conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful for carrying out the present invention include: polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymer of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyolefin alcohol, polysaccharides, poly-α-hydroxy acids, polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0120] In some embodiments, orthogonal IL2 is conjugated, or "PEGylated," to one or more polyethylene glycol molecules. The method or site of PEG binding to orthogonal IL2 may vary, but in certain embodiments, the PEGylation does not alter or only minimally alters the activity of orthogonal IL2.

[0121] In some embodiments, cysteine ​​(3TC) can be used instead of threonine at position 3 to promote N-terminal PEGylation using specific chemistry.

[0122] In some embodiments, selective PEGylation of orthogonal IL2 (e.g., by incorporating non-natural amino acids with side chains to facilitate selective PEG conjugation chemistry, as described in Ptacin, et al. (PCT International Application No. PCT / US2018 / 045257, filed August 3, 2018, and published February 7, 2019, as International Publication No. WO 2019 / 028419Al)) can be used to generate orthogonal IL2 with reduced affinity to one or more subunits of the IL2 receptor complex (e.g., CD25, CD132). For example, orthogonal IL2 in which non-natural amino acids having specific PEGylated moieties are incorporated into the sequence or residues of IL2 containing amino acids 34-45, 61-72, and 105-109, which have been identified to interact with CD25, generally provides orthogonal IL2 with reduced binding to CD25. Similarly, orthogonal IL2 obtained by incorporating non-natural amino acids having specific PEGylated moieties into the sequence or residues of IL2 containing amino acids 18, 22, 109, 126, or 119-133, which have been identified to interact with hCD132, provides orthogonal IL2 with reduced binding to hCD132.

[0123] In certain embodiments, an increase in half-life outweighs a decrease in biological activity. PEGs suitable for conjugation with polypeptide sequences are generally soluble in water at room temperature and have the general formula R(O-CH2-CH2)nO-R; where R is a hydrogen atom or a protecting group, such as an alkyl group or an alkanol group, and n is an integer from 1 to 1000. If R is a protecting group, it generally has 1 to 8 carbon atoms. The PEG conjugated to the polypeptide sequence may be linear or branched. Branched PEG derivatives, such as "star-PEG" and multi-armed PEG, are intended by this disclosure.

[0124] The molecular weight of PEG used in this disclosure is not limited to a specific range. The PEG component of PEG-orthogonal IL2 may have a molecular weight 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 weight 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. Linear or branched PEG molecules have molecular weights of approximately 2,000 to 80,000 daltons, or approximately 2,000 to 70,000 daltons, or approximately 5,000 to 50,000 daltons, or approximately 10,000 to 50,000 daltons, or approximately 20,000 to 50,000 daltons, or approximately 30,000 to 50,000 daltons, or approximately 20,000 to 40,000 daltons, or approximately 30,000 to 40,000 daltons. In one aspect of the present invention, the PEG is a 40kD branched PEG containing two 20kD arms.

[0125] This disclosure also envisions a composition of conjugates in which PEG has different n values, and therefore 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 proportion of conjugates with n=1 is 18-25%, the proportion of conjugates with n=2 is 50-66%, the proportion of conjugates with n=3 is 12-16%, and the proportion of conjugates with n=4 is up to 5%. Such compositions can be prepared by reaction conditions and purification methods known in the art. The conjugate fraction can be separated using chromatography, and then the fraction containing, for example, conjugates with a desired number of PEGs can be purified and identified from the unmodified protein sequence and conjugates with other numbers of PEGs.

[0126] PEGs suitable for conjugation with polypeptide sequences are generally soluble in water at room temperature and have the general formula R(O-CH2-CH2)nO-R; where R is a hydrogen atom or a protecting group, such as an alkyl group or alkanol group, and n is an integer from 1 to 1000. If R is a protecting group, it generally has 1 to 8 carbon atoms.

[0127] Two widely used first-generation activated monomethoxyPEGs (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., Dolence, et al. U.S. Patent No. 5,650,234), which preferentially react with lysine residues to form carbamate bonds, but are also known to react with histidine and tyrosine residues. The use of PEG aldehyde linkers targets a single site at the N-terminus of a polypeptide via reductive amination.

[0128] PEGylation most frequently occurs at the N-terminal □-amino group of a polypeptide, the ε-amino group of the side chain of a lysine residue, and the imidazole group of the side chain of a histidine residue. Since most recombinant polypeptides have a single α-amino group and numerous ε-amino and imidazole groups, a large number of positional isomers can be generated depending on the linker chemistry. Common PEGylation strategies known in the art can be applied herein.

[0129] PEG can be attached to the orthogonal IL2 of this disclosure via terminal reactive groups ("spacers") that mediate the binding between one or more free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. An example of a PEG having spacers that can bind to free amino groups is N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide.

[0130] In some embodiments, PEGylation of orthogonal IL2 is facilitated by incorporating a non-natural amino acid with a unique side chain to promote site-directed PEGylation. It is known in the art to incorporate non-natural amino acids into polypeptides to provide a functional moiety and achieve site-directed PEGylation of such polypeptides. See, for example, Ptacin, et al. (PCT International Application No. PCT / US2018 / 045257, filed 3 August 2018 and published 7 February 2019 as International Publication No. WO 2019 / 028419Al). In one embodiment of the present invention, a non-natural amino acid is incorporated at the D109 position of the orthogonal IL2. In one embodiment of the present invention, the orthogonal IL2 is PEGylated at position 109 of the orthogonal IL2 to a PEG molecule having a molecular weight of approximately 20 kD, or approximately 30 kD, or approximately 40 kD.

[0131] The PEG conjugate to the polypeptide sequence may be linear or branched. Branched PEG derivatives, such as "star PEG" and multi-arm PEG, are intended by this disclosure.Specific examples of PEG useful for carrying out the present invention include: 10kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF Corporation), 20kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF Corporation), 20kDa linear PEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF Corporation), 20kDa 2-arm branched PEG-aldehyde (this 20kDa PEG-aldehyde contains two 10kDa linear PEG molecules; e.g., Sunbright® GL2-200AL3, NOF Corporation), 20kDa 2-arm branched PEG-NHS ester (this 20kDa PEG-NHS ester contains two 10kDa linear PEG molecules; e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF Corporation), 40kDa 2-arm branched PEG-aldehyde (this 40kDa PEG-aldehyde contains two 20kDa linear PEG molecules; e.g., Sunbright® GL2-400AL3), 40kDa 2-arm branched PEG-NHS ester (this 40kDa PEG-NHS esters contain two 20kDa linear PEG molecules; e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2 (NOF Corporation), a linear 30kDa PEG-aldehyde (e.g., Sunbright® ME-300AL), and a linear 30kDa PEG-NHS ester.

[0132] As previously mentioned, PEG can be linked directly to orthogonal IL2 or via a linker molecule. Suitable linkers include "flexible linkers," which are generally long enough to allow some movement between the modified polypeptide sequence and the linked components and molecules. Linker molecules are generally about 6 to 50 atoms long. Linker molecules can also be, for example, arylacetylenes, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacitates, amino acids, or combinations thereof. A suitable linker can be easily selected and may be of any length if appropriate, for example, 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 30, 30 to 50, or more than 50 amino acids. Examples of flexible linkers include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Because glycine polymers and glycine-serine polymers are relatively unstructured, they can function as neutral tethers between components. Further examples of flexible linkers include glycine polymer (G)n, glycine-alanine polymer, alanine-serine polymer, and glycine-serine polymer. Because glycine polymers and glycine-serine polymers are relatively unstructured, they can function as neutral tethers between components. Multimers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) can be linked together to provide flexible linkers that can be used to conjugate heterogeneous amino acid sequences into polypeptides disclosed herein.

[0133] Furthermore, such linkers may be used to link orthogonal IL2 to additional heterologous polypeptide components, as described herein; the heterologous amino acid sequence may be a signal sequence and / or fusion partner, such as albumin, an Fc sequence, etc.

[0134] In one aspect of this disclosure, the orthogonal IL2 is the human orthogonal IL2 of formula 2: 40kD branch PEG-linker-desAla1-hIL2[E15S-H16Q-L19V-D20L-Q22K-M23A]-COOH.

[0135] In another aspect of the present invention, the orthogonal IL2 is the human orthogonal IL2 of formula 3: In the formula TIFF2026053539000017.tif18138, n = 0 or 1.

[0136] Acylation In some embodiments, the orthogonal IL2 of this 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. The myristoyl group is usually linked to the N-terminal glycine, but lysine can also be myristoylated. Palmitoylation is usually achieved by enzymatic modification of the free cysteine-SH group, for example by the DHHC protein that catalyzes S-palmitoylation. Palmitorylation of serine and threonine residues is usually achieved enzymatically using the PORCN enzyme.

[0137] Acetylation In some embodiments, IL-2 mutein is acetylated at the N-terminus by enzymatic reaction with an N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, IL-2 mutein is acetylated at one or more lysine residues by enzymatic reaction with, for example, a lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325 (5942):834L2 ortho840.

[0138] Fc fusion In some embodiments, IL-2 fusion proteins can incorporate an Fc region derived from an IgG subclass of an antibody lacking an IgG heavy chain variable region. The “Fc region” may be a native or synthetic polypeptide homologous to the IgG C-terminal domain produced by digesting IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. Mutant IL-2 polypeptides may contain the entire Fc region or a smaller portion (which retains the ability to extend the cyclic half-life of the chimeric polypeptide from which it is part). Furthermore, the full-length or fragmented Fc region may be a variant of the wild-type molecule; that is, they may contain mutations that may or may not affect the polypeptide's function; native activity is not always necessary or desirable, as will be further discussed below. In certain embodiments, IL-2 mutein fusion proteins (e.g., IL-2 partial agonists or antagonists described herein) may contain an IgG1, IgG2, IgG3, or IgG4 Fc region. Exemplary Fc regions may contain mutations that inhibit complement fixation and Fc receptor binding, or they may be soluble, i.e., they may be able to bind to complement or lyse cells via another mechanism such as antibody-dependent complement lysis (ADCC).

[0139] In some embodiments, orthogonal IL2 contains a functional domain of an Fc-fusion chimeric polypeptide molecule. Fc-fusion conjugates have been shown to extend the systemic half-life of biopharmaceuticals, potentially allowing for less frequent administration. Fc binds to neonatal Fc receptors (FcRn) on endothelial cells lining the inside of blood vessels. Once bound, the Fc-fusion molecule is protected from degradation and re-released into circulation, allowing it to circulate for a longer period. This Fc binding is thought to be the mechanism by which endogenous IgG maintains its long plasma half-life. Recent Fc-fusion technologies involve entangling a single copy of a biopharmaceutical to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to conventional Fc-fusion conjugates. A useful "Fc region" for the preparation of Fc fusions can be a native or synthetic polypeptide homologous to the IgG C-terminal domain produced by digesting IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. Orthogonal IL2 may provide the entire Fc region or a smaller portion (which retains the ability to extend the cyclic half-life of the chimeric polypeptide it is part of). Furthermore, the full-length or fragmented Fc region may be a variant of the wild-type molecule. In a typical presentation, each monomer of the dimeric Fc carries a heterologous polypeptide, which may be identical or different.

[0140] In some embodiments, when orthogonal IL2 is administered in the form of an Fc fusion, particularly when the polypeptide chains conjugated to each subunit of the Fc dimer are different, the Fc fusion can be manipulated to have a "knob-into-hole modification." Knob-into-hole modification is described in detail in Ridgway, et al. (1996) Protein Engineering 9(7):617-621 and U.S. Patent No. 5,731,168 issued March 24, 1998. Knob-into-hole modification refers to modification of the CH3 domain at the interface between two immunoglobulin heavy chains, where i) in the CH3 domain of the first heavy chain, an amino acid residue is replaced with an amino acid residue with a larger side chain (e.g., tyrosine or tryptophan) to create a protrusion ("knob") from the surface; and ii) in the CH3 domain of the second heavy chain, an amino acid residue is replaced with an amino acid residue with a smaller side chain (e.g., alanine or threonine) to create a cavity ("hole") at the interface of the second CH3 domain, within which the protruding side chain ("knob") of the first CH3 domain is received by the cavity of the second CH3 domain. In one embodiment, the "knob-into-hole modification" includes amino acid substitution T366W and optionally amino acid substitution S354C in one antibody heavy chain, and amino acid substitution T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. Furthermore, the Fc domain can be modified by introducing cysteine ​​residues at positions S354 and Y349, which results in a stabilizing disulfide crosslink between the two antibody heavy chains in the Fe region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole configuration is used to facilitate the expression of heterodimer polypeptide conjugates by facilitating the expression of a first polypeptide (e.g., orthogonal IL2) on a first Fc monomer with a "knob" modification and a second polypeptide on a second Fc monomer with a "hole" modification.

[0141] The Fc region can be either lytic or non-lytic, but is usually non-lytic. Non-lytic Fc regions generally lack a high-affinity Fc receptor binding site and a Clq binding site. The high-affinity Fc receptor binding site of mouse IgG Fc contains a Leu residue at position 235 of IgG Fc. Therefore, this Fc receptor binding site can be inhibited by mutation or deletion of 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 mouse Clq binding site can be functionally disrupted by mutation or deletion of the 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 inducing antibody-dependent complement lysis. In contrast, the soluble IgG Fc region possesses both a high-affinity Fc receptor binding site and a Clq binding site. The high-affinity Fc receptor binding site contains the Leu residue at position 235 of IgG Fc, while the Clq binding site contains the Glu 318, Lys 320, and Lys 322 residues of IgG 1. Soluble IgG Fc has wild-type residues or conserved amino acid substitutions at these sites. Soluble IgG Fc can target cells for antibody-dependent cytotoxicity or complement-dependent cytolysis (CDC). Appropriate mutations in 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).

[0142] In certain embodiments, the amino-terminus or carboxyl-terminus of the orthogonal IL2 of this disclosure can be fused with an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to extend the systemic half-life of biopharmaceuticals, thus allowing these biopharmaceuticals to be administered at lower doses. Fc binds to neonatal Fc receptors (FcRn) on endothelial cells lining the inside of blood vessels. Upon binding, the Fc fusion molecule is protected from degradation and re-released into circulation, allowing it to circulate for a longer period. This Fc binding is thought to be the mechanism by which endogenous IgG maintains its long plasma half-life. Recent Fc fusion technologies involve fused a single copy of a biopharmaceutical to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to conventional Fc fusion conjugates.

[0143] In some embodiments, the Fc domain monomer contains at least one mutation compared to the wild-type human IgG1, IgG2, or IgG4 Fc region, as described in U.S. Patent No. US10259859B2 (the entire teaching of which is incorporated herein by reference). As disclosed therein, the Fc domain monomer contains the following mutations: (a) One of the following amino acid substitutions compared to wild-type human IgG1: TIFF2026053539000018.tif38128 or (b)(i) N297A mutation compared to the human IgG1 Fc region; (ii) L234A, L235A, and G237A mutations compared 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 compared to the human IgG4 Fc region; or (Viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations compared to the human IgG4 Fc region.

[0144] In some embodiments, the Fc domain monomer includes the following mutations: (a) One of the following amino acid substitutions compared to wild-type human IgG1: TIFF2026053539000019.tif38128 and (b) The Fc domain monomer further comprises the following mutations: (i) N297A mutation compared to the human IgG1 Fc region; (ii) L234A, L235A, and G237A mutations compared 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 compared to the human IgG4 Fc region; or (Viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations compared to the human IgG4 Fc region.

[0145] In some embodiments, the polypeptide exhibits reduced phagocytosis in phagocytosis assays compared to polypeptides having a wild-type human IgG Fc domain. In some embodiments, an Fc domain monomer is ligated to a second polypeptide containing a second Fc domain monomer to form an Fc domain dimer.

[0146] Chimeric polypeptide / fusion protein In some embodiments, the orthogonal IL2 may include a functional domain of a chimeric polypeptide. The orthogonal IL2 fusion proteins of this disclosure can be readily prepared by recombinant DNA methodologies known in the art, and are prepared by constructing a recombinant vector containing a nucleic acid sequence that includes, in frame, a nucleic acid sequence encoding a fusion partner at either the N-terminus or C-terminus of the orthogonal IL2, and optionally, a nucleic acid sequence encoding a linker or spacer polypeptide, also in frame.

[0147] Flag tag In other embodiments, orthogonal IL2 may be modified to include an additional polypeptide sequence that functions as an antigenic tag, such as a FLAG sequence. The FLAG sequence 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 orthogonal IL2 polypeptide further includes a C-terminal c-myc epitope tag.

[0148] His tag In some embodiments, the orthogonal IL2 of the present invention (including fusion proteins of such orthogonal IL2) is expressed as a fusion protein with one or more transition metal chelate polypeptide sequences. Incorporation of such transition metal chelate domains facilitates purification by immobilized metal affinity chromatography (IMAC), as described in Smith, et al., U.S. Patent No. 4,569,794, issued February 11, 1986. Examples of transition metal chelate polypeptides useful for carrying out the present invention are described in Smith, et al. (cited above) and Dobeli, et al., U.S. Patent No. 5,320,663, issued May 10, 1995, the entire teachings of which are incorporated herein by reference. Specific transition metal chelate polypeptides useful for carrying out the present invention are peptides containing 3 to 6 consecutive histidine residues, such as 6-histidine peptide (His)6, often referred to in the art as "His tags."

[0149] Targeted orthogonal IL2 fusion proteins: In some embodiments, the orthogonal IL2 is provided as a fusion protein with a polypeptide sequence ("target-directing domain") to facilitate selective binding to a specific cell type or tissue (expressing cell surface molecules that specifically bind to the target-directing domain), and optionally, the fusion protein may incorporate a linker molecule of 1 to 40 (or 2 to 20, or 5 to 20, or 10 to 20) amino acids between the orthogonal IL2 sequence and the target-directing domain sequence.

[0150] In other embodiments, chimeric polypeptides can be constructed that contain mutant IL-2 and an antibody or its antigen-binding moiety. The antibody or antigen-binding component of the chimeric protein can function as a target-directed motif. For example, it can be used to localize the chimeric protein to a specific cell subset or target molecule. Methods for constructing cytokine-antibody chimeric polypeptides are described, for example, in U.S. Patent No. 6,617,135.

[0151] In some embodiments, the target-directing domain of an orthogonal IL2 fusion protein specifically binds to cell surface molecules of tumor cells. In one embodiment, where the ECD of the CAR in CAR-T cells specifically binds to CD-19, the orthogonal IL2 may be provided as a fusion protein with a CD-19 target-directing moiety. For example, in one embodiment, where the ECD of the CAR in CAR-T cells is an scFv molecule that provides specific binding to CD-19, the orthogonal IL2 may be provided as a fusion protein with a CD-19 target-directing moiety such as a single-chain antibody (e.g., scFv or VHH) that specifically binds to CD-19.

[0152] In some embodiments, the fusion protein comprises IL-2 mutein and anti-CD19 sdFv FMC63 (Nicholson, et al. (1997) Mol Immunol 34: 1157-1165). Similarly, in some embodiments in which the ECD of the CAR-T cell specifically binds to BCMA, the orthogonal IL2 is provided as a fusion protein with a BCMA-targeting moiety, such as an antibody containing a CDR of an anti-BMCA antibody described in Kalled, et al. (U.S. Patent No. 9,034,324 issued May 9, 2015) or an antibody containing a CDR described in Brogdon, et al. (U.S. Patent No. 10,174,095 issued January 8, 2019). In some embodiments, orthogonal IL2 is provided as a fusion protein with a GD2 target-directing moiety, such as an antibody containing a CDR as described in Cheung, et al. (U.S. Patent No. 9,315,585 issued April 19, 2016), or an antibody containing a CDR derived 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.

[0153] In another embodiment, the targeted orthogonal IL2 of this disclosure may be administered in combination with CAR-T cell therapy to provide targeted delivery of orthogonal IL2 to CAR-T cells based on the extracellular receptor of CAR-T cells, such as an anti-FMC63 antibody, thereby targeting IL2 activity to CAR-T cells and restoring depleted CAR-T cells in vivo. Consequently, embodiments of this disclosure include targeted delivery of orthogonal IL2 by conjugation of orthogonal IL2 to an antibody or ligand designed to interact with specific cell surface molecules of CAR-T cells. An example of such a molecule is anti-FMC63-orthogonal IL2.

[0154] In other embodiments, the chimeric polypeptide comprises a mutant IL-2 polypeptide and a heterologous polypeptide, the heterologous polypeptide functioning to enhance the expression of the mutant IL-2 polypeptide or induce its cellular localization, such as the Aga2p aglutinin subunit (see, for example, Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0155] Protein-transfer domain fusion protein In some embodiments, orthogonal IL2 further includes a "protein transduction domain" or "PTD." A PTD is a polypeptide, polynucleotide, carbohydrate, organic, or inorganic molecule that facilitates passage through lipid bilayers, micelles, cell membranes, organelle membranes, or vesicle membranes. When a PTD is incorporated into orthogonal IL2, its membrane translocation is enhanced. In some embodiments, the PTD is covalently linked to the amino or carboxyl terminus of orthogonal IL2. In some embodiments, the PTD is incorporated as part of a PTD-orthogonal IL2 fusion protein, either at the N-terminus or C-terminus of the molecule.

[0156] Exemplary transduction domains include, but are not limited to, the following: the smallest decapeptide transduction domains (corresponding to residues 47-57 of HIV-1 TAT); polyarginine sequences containing a sufficient number of arginine residues to induce cell entry (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); VP22 domains (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); Drosophila Antennapedia transduction domains (Noguchi et al. (2003) Diabetes 52(7):1732-1737); cleaved human calcitonin peptides (Trehin et al. (2004) Pharm. Research 21:1248-1256); and 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); Antennapediapeptide (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).

[0157] In some embodiments, IL-2 conjugates have plasma half-lives in human subjects exceeding 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.

[0158] Orthogonal IL2-specific configuration of modified CD122 The STAT3 binding motif may be located at the C-terminus of the intracellular domain of human CD122 (e.g., human orthogonal CD122 or natural human CD122) or as an internal sequence.

[0159] Embodiments of modified CD122 containing one or more STAT3-binding motifs fused to the C-terminus of human CD122 In some cases, the modified human CD122 contains one or more STAT3-binding motifs fused to the C-terminus of the intracellular domain of human CD122. Human CD122 may be natural human CD122 (SEQ ID NO: 1) or orthogonal human CD122 as disclosed above.

[0160] (Table 1) Exemplary fusion proteins, below: The term "ortho-CD122" in TIFF2026053539000020.tif64128 refers to any of the orthogonal human CD122 proteins disclosed herein.

[0161] In some cases, modified human CD122 contains a STAT3-binding motif linked to human CD122 via a linker. The linker may be derived from a naturally occurring protein or a synthetic sequence. Methods for designing linkers are well known in the art, for example, disclosed in Chen et al. Adv. Drug. Deliv. Rev. 2013 Oct 15; 65(10): 1357-1369, the relevant portion of which is incorporated herein by reference. In some cases, the linker consists of 1 to 20 amino acid residues, 1 to 10 amino acid residues, or 1 to 5 amino acid residues. In some embodiments, the linker comprises a dinucleotide Gn, where n may be 1 to 10 or 1 to 5, for example, 2 to 4.

[0162] Modified human CD122 may contain one or more STAT3 binding motifs and one or more linker sequences. The linker sequences may link human CD122 to one of the STAT3 binding motifs, or to individual STAT3 binding motifs. The one or more linker sequences may be the same or different sequences.

[0163] Modified CD122 embodiment containing a STAT3 recognition motif as the internal sequence of the intracellular domain of CD122 In some embodiments, one or more STAT3-binding motifs exist as internal (i.e., neither C-terminal nor N-terminal) sequences in CD122. Modified human CD122 of this configuration can be prepared by identifying a suitable region within the coding sequence of native human CD122 or human orthogonal CD122 that can be mutated to encode a STAT3-binding motif. In one embodiment, the region has a sequence similar to the STAT3-binding motif and includes, for example, a 4-nucleotide sequence beginning with a tyrosine residue. One such region in native human CD122 encodes the YFTYDPYSEE sequence located between positions 355 and 364 of the native human CD122 protein. In some embodiments, one or two of the YFTY, YDPY, or YSEE sequences contained in this region are replaced with a STAT3-recognizing motif to prepare the modified human CD122 disclosed herein.

[0164] Modified human CD122 can induce STAT3 and STAT5 signaling when bound to cognitive IL2 ligand, and this ability can be confirmed, for example, by detecting the phosphorylation of STAT3 and STAT5. For example, modified human CD122 can be introduced and expressed in T cells, and the phosphorylation of STAT3 and STAT5 can be detected using antibodies specific to phospho-STAT5 and phosphor-STAT3. One exemplary method for detecting the ability of recombinant proteins to induce STAT3 and STAT5 signaling is described in Kagoya et al. Nat Med. 2018 March; 24(3): 352-359. doi:10.1038 / nm.4478, p7.

[0165] Polynucleotides and expression vectors Polynucleotides encoding modified human CD122 can be prepared using techniques well known in the art. In some cases, modified human CD122 can be prepared by any conventional method, including recombinant or solid-phase synthesis. In some embodiments, modified human CD122 is prepared by recombinant methods. The coding nucleic acid sequence is inserted into an expression vector and introduced into the genetically engineered cell. DNA encoding modified human orthogonal CD122 is available from various sources designed during the genetic engineering process. Amino acid sequence variants of the natural human CD122 polypeptide for obtaining the modified human CD122 of this disclosure are prepared by introducing appropriate nucleotide changes into the coding sequence, as described herein. Such variants represent insertions, substitutions, and / or specific deletions of the described residues. Any combination of insertions, substitutions, and / or specific deletions is performed to arrive at a final construct; provided that the final construct retains the desired biological activity as defined herein.

[0166] To express a modified CD122, the nucleic acid encoding the modified CD122 is inserted into a replicable expression vector. Many such vectors are available. Vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Examples of vectors include viral vectors, plasmid vectors, and embedded vectors. Plasmids are an example of non-viral vectors. To facilitate transfection of target cells, target cells may be directly exposed to the non-viral vector under conditions that facilitate its uptake. Examples of conditions that facilitate the uptake of foreign nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g., Lipofectamine®, Thermo-Fisher Scientific), high salt levels, and magnetic fields (electroporation).

[0167] Modified CD122 can be synthesized not only by direct recombinant production, but also as a fusion polypeptide with a heterologous polypeptide (e.g., a signal sequence, or another polypeptide having a specific cleavage site at the N-terminus of a mature protein or polypeptide). Generally, the signal sequence may be a component of the vector or part of a coding sequence inserted into the vector. The selected heterologous signal sequence is preferably one that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For expression in mammalian cells, native signal sequences can be used, but signal sequences from other mammals may be suitable, such as signal sequences from secretory polypeptides of the same or related species, and viral secretion leaders, such as the herpes simplex gD signal.

[0168] Expression vectors are also provided that can be used to introduce polynucleotides encoding modified CD122 into cells. Various vectors are known in the art and can be used for this purpose; for example, viral vectors, plasmid vectors, and minicircle vectors. Expression vectors typically contain a selection gene, also called a selection marker. This gene encodes a protein necessary for the survival or growth of transformed host cells cultured in a selection medium. Host cells not transformed with a vector containing the selection gene cannot survive in the medium. Typical selection genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) proteins that complement nutritional deficiencies; or (c) proteins that supply essential nutrients not available from the complex medium.

[0169] An expression vector contains a promoter that is recognized by the host organism and functionally ligated to the coding sequence of an orthogonal protein. The promoter is an untranslated sequence (generally within approximately 100–1000 bp) located upstream (5') of the start codon of a structural gene, and it controls the transcription and translation of the specific nucleic acid sequence to which it is functionally ligated. Such promoters are typically classified into two classes: inducible and constitutive. Inducible promoters are those that initiate an increased level of transcription from the DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or changes in temperature. A large number of promoters recognized by various potential host cells are well known.

[0170] Transcription from vectors in mammalian host cells can be controlled by promoters obtained from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (e.g., human adenovirus serotype 5), bovine papillomavirus, arowanasarcoma virus, cytomegalovirus, retroviruses (e.g., mouse stem cell virus), hepatitis B virus, and most preferably Simian virus 40 (SV40); heterologous mammalian promoters, such as actin promoters, PGK (phosphoglycerate kinase), or immunoglobulin promoters; provided that such promoters are compatible with the host cell system. Early and late promoters of the SV40 virus can be conveniently obtained as SV40 restriction fragments, which also include the origin of replication of the SV40 virus.

[0171] Transcription in higher eukaryotes is often amplified by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA that act on promoters to increase their transcription, and are typically about 10–300 bp in length. Enhancers are relatively direction and position independent and can be found at the 5' and 3' positions of transcription units, within introns, and within the coding sequence itself. Many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers derived from eukaryotic viruses are commonly used. Examples include the SV40 enhancer at the late origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer at the late origin of replication, and the adenovirus enhancer. Enhancers can be spliced ​​into expression vectors at the 5' or 3' position of the coding sequence, but are preferably located at the 5' site from the promoter. Expression vectors used in eukaryotic host cells will also include sequences necessary for transcription termination and mRNA stabilization. Such sequences are commonly available from the 5', and sometimes 3', untranslated regions of eukaryotic or viral DNA or cDNA. Standard techniques are used to construct appropriate vectors containing one or more of the above components.

[0172] Suitable host cells for cloning or expressing the coding sequence of modified CD122 are the cells of the prokaryotes, yeasts, or higher eukaryotes mentioned above. In some embodiments, the host cell is a human immune cell, such as a T cell. In some embodiments, the host cell is a CAR-T cell, as further described below.

[0173] In some embodiments, the host cells are, for example, the following mammalian host cell lines: mouse L cells (LM[TK-],ATCC#CRL-2648), monkey kidney CV1 cell line transformed with SV40 (COS-7,ATCC CRL1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK,ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,ATCC CRL-1 587); human cervical cancer cells (HELA,ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat hepatocytes (BRL 3A,ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary gland tumors (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatocarcinoma cell line (Hep G2).

[0174] Host cells containing the coding sequence of modified CD122 can be cultured in conventional nutrient media, modified as appropriate for promoter induction, transformant selection, or amplification of the gene encoding the desired sequence. Mammalian host cells can be cultured in a variety of media. Commercial media such as Ham F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. Any of these media can be supplemented as needed with: hormones and / or other growth factors (e.g., insulin, transferrin, epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, phosphates), buffers (e.g., HEPES), nucleosides (e.g., adenosine, thymidine), antibiotics, trace elements, and glucose or equivalent energy sources. Other necessary supplements can also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used with the host cells selected for expression and will be apparent to those skilled in the art.

[0175] In some embodiments, host cells, such as T cells, are transformed with an expression vector containing a nucleic acid sequence encoding a modified CD122. An IL2 ligand (e.g., orthogonal IL2) may be used in a method for selectively expanding the culture of modified T cells (e.g., human T cells) that have been engineered to express the corresponding modified human CD122 (e.g., modified orthogonal CD122). T cells useful for engineering with the constructs described herein include naive T cells, central memory T cells, effector memory T cells, or combinations thereof. T cells for performing such genetic engineering may be collected from a subject or donor and isolated from a cell mixture by techniques for enriching the cells of interest, or they may be genetically engineered and cultured without isolation. Alternatively, T cells for genetic engineering may be isolated from other cells. Techniques that provide precise isolation include fluorescence-activated cell sorters. Cells can be selected from dead cells by using dyes associated with dead cells (e.g., propidium iodide). The isolated cells are collected in a suitable medium to maintain their viability, and typically a serum cushion is placed at the bottom of the collection tube. Various media are commercially available and can be used depending on the properties of the cells, such as dMEM, HBSS, dPBS, RPMI, and Iskoff medium, and are often supplemented with fetal bovine serum (FCS). The collected and optionally concentrated cell population may be used immediately for genetic modification or may be cryopreserved at liquid nitrogen temperature for reuse after thawing. Cells are usually stored in 10% DMSO, 50% FCS, and 40% RPMI 1640 medium.

[0176] Immune cells engineered to express modified CD122 This specification also provides immune cells expressing modified human CD122. Immune cells may be, for example, T cells (e.g., CD4+ cells, CD8+ cells) and NK cells. In some embodiments, T cells include, but are not limited to, the following cells: naive CD8+ T cells, cytotoxic CD8+ T cells, naive CD4+ T cells, helper T cells, e.g., TH1, TH2, TH9, TH11, TH22, TFH; regulatory T cells, e.g., TR1, Treg, inducible Treg; memory T cells, e.g., central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TILs), and genetically modified variants of the above T cells (including, but not limited to, CAR-T cells, recombinant modified TILs, TCR-transformed cells, etc.). In some embodiments, genetically modified cells include a complex mixture of immune cells, e.g., tumor-infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. For example, Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Seminars in Oncol. 42(4):626-39 “Adoptive Cell Therapy-Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial See NCT01174121, “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641-645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”.

[0177] CAR-T cells In some embodiments, genetically engineered immune cells also express chimeric antigen receptors (CARs). CARs enable immune cells to bind to antigens on diseased cells, such as tumor cells, and thus specifically kill those diseased cells. The antigen-binding domain (ABD) of a CAR may be monovalent or polyvalent and may contain one or more (e.g., 1, 2, or 3) polypeptide sequences (e.g., scFv, VHH, and / or ligands) that specifically bind to cell surface tumor antigens. In some embodiments, CARs containing tumor antigens and ABDs that selectively bind to such cell surface tumor antigens are known in the art (see, for example, Dotti, et al., Immunol Rev. 2014 January; 257(1)). The methods and compositions of this disclosure are useful in conjunction with CAR therapy in which the ABD of the CAR specifically binds to tumor antigens, including but not limited to CD123, CD19, CD20, BCMA, CD22, CD30, CD70, Lewis Y, GD3, GD3, mesothelin, ROR CD44, CD171, EGP2, EphA2, ErbB2, ErbB3 / 4, FAP, FAR, IL11Ra, PSCA, PSMA, NCAM, etc. Antibodies that react with these targets are well known in the literature, and those skilled in the art can isolate CDRs from such antibodies for the construction of polypeptide sequences of single-chain antibodies (e.g., scFv, CDR-grafted VHH, etc.) that can be incorporated into the ABD of the CAR.

[0178] In one aspect of the present invention, the genetically modified immune cells are T cells that express a modified CD122 and also express a chimeric antigen receptor. These cells are known as "CAR-T" cells.

[0179] CARs typically contain an antigen-binding domain (ABD) that can specifically bind to antigens expressed on the surface of target cells. The ABD can be any polypeptide that specifically binds to one or more antigens expressed on the surface of target cells. CARs may further contain a transmembrane domain that connects the ABD (or a linker, if used) to the intracellular cytoplasmic domain of the CAR. The transmembrane domain consists of any polypeptide sequence that is thermodynamically stable in the eukaryotic cell membrane. The transmembrane domain may be derived from the transmembrane domains of naturally occurring transmembrane proteins or it may be synthetic. When designing synthetic transmembrane domains, amino acids that favor α-helix structures are preferred. Useful transmembrane domains for constructing CARs consist of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, or 24 amino acids that are favorable for conformations having an α-helix secondary structure. Amino acids favorable for α-helix conformations are well known in the art. See, for example, Pace, et al. (1998) Biophysical Journal 75: 422-427. Amino acids particularly favorable for α-helix conformation include methionine, alanine, leucine, glutamic acid, and lysine. In some embodiments, the CAR transmembrane domain may be derived from a transmembrane domain of a type I transmembrane protein such as CD3ζ, CD4, CD8, or CD28.

[0180] The cytoplasmic domain of a CAR polypeptide comprises one or more intracellular signaling domains. In one embodiment, the intracellular signaling domain includes the cytoplasmic sequences of a T cell receptor (TCR) and a co-receptor that initiates signaling after antigen receptor engagement, its functional derivatives, and subfragments. A cytoplasmic signaling domain, such as one derived from the T cell receptor ζ chain, is used as part of the CAR to generate a stimulating signal for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with a target antigen. Examples of cytoplasmic signaling domains include, but are not limited to, the cytoplasmic domains of CD27, CD28 (cytoplasmic domain S), CD137 (also known as 4-1BB and TNFRSF9), CD278 (also known as ICOS), the p110α, β, and δ catalytic subunits of PI3 kinase, human CD3 ζ chain, CD134 (also known as OX40 and TNFRSF4) (including FcεR1γ and β chains, MB1(Igα) chain, B29(Igβ) chain, etc.), CD3 polypeptides (δ, Δ, and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell signaling such as CD2, CD5, and CD28.

[0181] In some embodiments, CARs can also provide a co-stimulatory domain. The term “co-stimulatory domain” refers to the stimulatory domain of a CAR, usually the endodomain, that provides a secondary nonspecific activation mechanism (through which primary specific stimuli are propagated). The co-stimulatory domain refers to the portion of a CAR that promotes the proliferation, survival, or development of memory cells. Examples of co-stimulation include antigen-nonspecific T cell co-stimulation after antigen-specific signaling via the T cell receptor, and antigen-nonspecific B cell co-stimulation after signaling via the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved are described in Chen & Flies. (2013) Nat Rev Immunol 13(4):227-42. In some aspects of this disclosure, CSD includes one or more members of the TNFR superfamily, CD28, CD137(4-1BB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1(CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or a combination thereof.

[0182] CARs are often referred to as first-generation, second-generation, third-generation, or fourth-generation. The term first-generation CAR refers to a CAR in which the cytoplasmic domain transmits signals from antigen binding via only a single signaling domain (e.g., a signaling domain derived from the high-affinity IgE receptor FcεR1□ or CD3ζ chain). This domain contains one or three immunoreceptor tyrosine-based activating recognition motifs (ITAMs) for antigen-dependent T cell activation. The ITAM-based activation signal gives T cells the ability to lyse target tumor cells and secrete cytokines in response to antigen binding. Second-generation CARs include a costimulatory signal in addition to the CD3ζ signal. Co-delivery of the delivered costimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transformed T cells. The costimulatory domain is typically proximal to the membrane relative to the CD3ζ domain. Third-generation CARs include a tripartite signaling domain, such as CD28, CD3ζ, OX40, or 4-1BB signaling regions. In the fourth generation, or "armored CARs," CAR T cells are further modified to express or block immunoenhancing molecules and / or receptors, such as IL-12, IL-18, IL-7, and / or IL-10; 4-1BB ligand, CD-40 ligand, etc.

[0183] Examples of intracellular signaling domains that can be incorporated into the CAR of the present invention include (amino to carboxy): CD3ζ;CD28-41BB-CD3ζ;CD28-OX40-CD3ζ;CD28-41BB-CD3ζ;41BB-CD-28-CD3ζ and 41BB-CD3ζ.

[0184] As used herein, the term CAR includes, but is not limited to, CAR variants such as split CARs, on-switch CARs, bispecific or tandem CARs, inhibitory CARs (iCARs), and induced pluripotent stem (iPS) CAR-T cells.

[0185] The term "split CAR" refers to a CAR in which the extracellular portion, ABD, and cytoplasmic signaling domain are located on two separate molecules. CAR variants also include on-switch CARs, which are conditionally activatable CARs, such as split CARs in which the conditional heterodimerization of the two parts of the split CAR is pharmacologically controlled. CAR molecules and their derivatives (i.e., CAR variants) are, for example, PCT application numbers US2014 / 016527, US1996 / 017060, US2013 / 063083; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304, the disclosures of which are incorporated herein by reference in their entirety.

[0186] The term "bispecific or tandem CAR" refers to a CAR that contains a secondary CAR-binding domain capable of amplifying or inhibiting the activity of a primary CAR.

[0187] The term “inhibitory chimeric antigen receptor” or “iCAR” is used interchangeably herein and refers to a CAR that, using dual antigen targeting, shuts down the activation of an active CAR via engagement with a second inhibitory receptor equipped with an inhibitory signaling domain in the secondary CAR-binding domain, thereby inhibiting primary CAR activation. Inhibitory CARs (iCARs) are designed to regulate CAR-T cell activity through activation of the signaling module of an inhibitory receptor. This approach combines the activities of two CARs, one of which generates a dominant-negative signal that limits the response of CAR-T cells activated by the activating receptor. When an iCAR binds to a specific antigen expressed only in normal tissue, it can switch off the response of a counter-activating activator CAR. In this way, iCAR-T cells can distinguish between cancer cells and healthy cells and reversibly block the function of transduction T cells in an antigen-selective manner. The CTLA-4 or PD-1 intracellular domain of an iCAR induces inhibitory signals to T lymphocytes, resulting in decreased cytokine production, reduced efficiency of target cell lysis, and altered lymphocyte motility.

[0188] The term "tandem CAR" or "TanCAR" refers to a CAR that mediates bispecific activation of T cells via the engagement of two chimeric receptors, designed to deliver stimulatory or co-stimulatory signals in response to the independent engagement of two different tumor-associated antigens.

[0189] Generally speaking, chimeric antigen receptor T cells (CAR-T cells) are T cells that have been recombinantly modified by introducing an expression vector encoding a CAR, substantially in accordance with the above instructions.

[0190] kit Furthermore, this disclosure provides kits that can be used to activate immune cells. In some embodiments, a vector is provided comprising a coding sequence encoding a modified human CD122, where the coding sequence is functionally linked to a promoter that is active in the desired cell. Various vectors are known in the art and can be used for this purpose; for example, viral vectors, plasmid vectors, and minicircle vectors, which are integrated into the genome of target cells or maintained as episomes. The vector encoding the modified CD122 can be provided as a kit in combination with a vector encoding a cytokine that binds to the receptor and activates it. In some embodiments, the coding sequence of the cytokine may be functionally linked to a high-expression promoter to optimize it for production. In other embodiments, a kit is provided in which a vector encoding an orthogonal receptor is provided together with a purified composition of the cognitive cytokine, packaged for administration to a patient, for example, in unit doses.

[0191] Activation methods using ExVivo In some embodiments, the Disclosure provides a method for stimulating immune cells engineered to express a modified human CD122 containing one or more STAT3-binding motifs, the method comprising contacting the immune cells with a human IL2 polypeptide bound to the modified human CD122, thereby stimulating the immune cells.

[0192] Also provided is a method for selectively activating immune cells engineered to express a modified human orthogonal CD122 from a mixture of these engineered immune cells and a cell population containing innate immune cells, the method comprising contacting the mixture of immune cells with orthogonal human IL2 that specifically binds to the modified orthogonal human CD122, thereby selectively activating the engineered immune cells.

[0193] Treatment method In some cases, a therapeutic method is provided, which involves introducing a population of cells engineered to express a modified human CD122 (e.g., modified orthogonal human CD122) as provided in this disclosure into a subject requiring it. The engineered cell population can be engineered ex vivo and is typically autologous or allogeneic with respect to the subject. After introducing the engineered cell population, the introduced cell population is contacted in vivo with a cognitive IL2 ligand (e.g., an IL2 ortholog).

[0194] In some cases, the subject has cancer, and the manipulated immune cells are CD8+ T cells expressing modified human CD122 (e.g., modified orthogonal human CD122). In other cases, the subject has an autoimmune disease, and the immune cells are Treg cells expressing modified human CD122. In other cases, the manipulated immune cells are CAR-T cells (e.g., CD8+ T cells or Treg cells expressing one or more CARs).

[0195] Introduction of immune cells expressing modified human CD122 In some embodiments, genetically modified T cells are allogeneic with respect to the individual being treated. Graham et al. (2018) Cell 7(10) E155. In some embodiments, allogeneic genetically modified T cells are fully HLA-matched. However, since not all patients have fully matched donors, cell products suitable for all patients regardless of HLA type offer an alternative.

[0196] Since cell products can consist of the subject's own T cells, the population of cells administered to the subject will inevitably fluctuate, and the response to such drugs may also vary. Therefore, continuous monitoring and management of therapy-related toxicity is necessary to determine the optimal concentration of the cells. Typically, at least 1□10⁶ cells / kg, at least 1□10⁷ cells / kg, at least 1□10⁸ cells / kg, at least 1□10⁹ cells / kg, at least 1□10¹⁰ cells / kg, or more will be administered, but this is usually limited by the number of T cells available at the time of collection. In some cases, the patient will undergo several rounds of pharmacological immunosuppression or B cell depletion before CAR-T cell therapy is administered. Genetically modified cells can be injected into the subject in any physiologically acceptable medium and via any appropriate route of administration, usually intravascularly, but they can also be introduced via other routes in which the cells can find a suitable site for proliferation.

[0197] If the T cells are allogeneic T cells, such cells can be modified to mitigate graft-versus-host disease. For example, the modified cells of this invention may be TCRαβ receptor knockouts achieved by gene editing technology. TCRαβ is a heterodimer, and both the α and β chains must be present for it to be expressed. Since there are two genes encoding the β chain, while a single gene encodes the α chain (TRAC), the TRAC locus KO was deleted for this purpose. Many different approaches have been used to achieve this deletion; for example, CRISPR / Cas9; meganucleases; and genetically modified I-CreI homing endonucleases. For example, see Eyquem et al. (2017) Nature 543:113-117 (TRAC coding sequence replaced by CAR coding sequence); and Georgiadis et al. (2018) Mol. Ther. 26:1215-1227 (CAR expression associated with TRAC disruption by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 without directly incorporating CAR into the TRAC locus). An alternative strategy to prevent GVHD is to modify T cells to express inhibitors of TCRαβ signaling, for example, by using cleaved forms of CD3ζ as TCR inhibitors.

[0198] Orthogonal IL-2 administration Cognate IL2 ligand can be delivered to a target by introducing a population of immune cells engineered to express modified human CD122, followed by administration of a nucleic acid construct encoding the cognitive IL2 ligand to the target to achieve continuous exposure of the target to the cognitive IL2 ligand. In some cases, the engineered immune cells express modified orthogonal human CD122, and a recombinant vector encoding the cognitive orthogonal IL2 is introduced to provide extended delivery of the orthogonal IL2 to the target and long-term activation of corresponding cells engineered to express the cognitive orthogonal receptor associated with such orthogonal IL2.

[0199] Non-viral: In one embodiment, orthogonal IL2 can be administered to a target in the form of a nucleic acid expression construct of orthogonal IL2 in a nonviral vector and may be provided in a nonviral delivery system. A nonviral delivery system is typically a complex for promoting the transduction of target cells by a nucleic acid cargo, in which the nucleic acid is complexed with substances such as cationic lipids (DOTAP, DOTMA), surfactants, biomaterials (gelatin, chitosan), metals (gold, magnetic iron), or synthetic polymers (PLG, PEI, PAMAM). Numerous embodiments of nonviral delivery systems are well known in the art, for example, lipid vector systems (Lee et al. (1997) Critical Reviews of Therapeutic Drug Carrier Systems 14:173-206); polymer-coated liposomes (Marin et al. U.S. Patent No. 5,213,804, issued May 25, 1993; Woodle, et al. U.S. Patent No. 5,013,556, issued May 7, 1991); cationic liposomes (Epand et al. U.S. Patent No. 5,283,185, issued February 1, 1994; Jessee, JA U.S. Patent No. 5,578,475, issued November 26, 1996; Rose et al. U.S. Patent No. 5,279,833, issued January 18, 1994; Gebeyehu et al. Examples include U.S. Patent No. 5,334,761, issued August 2, 1994. In one embodiment, the nucleic acid sequence in a nonviral vector system encoding the IL2 receptor is under the control of a regulatory promoter, an inducible promoter, a tissue-specific or tumor-specific promoter, or a temporally regulated promoter.

[0200] Virus vectors: In another embodiment, orthogonal IL2 can be administered to a subject in the form of a nucleic acid expression construct in a viral vector encoding orthogonal IL2. The terms “viral vector” and “virus” are used interchangeably herein and refer to any obligate intracellular parasite that does not have a protein synthesis mechanism or an energy production mechanism. The viral genome may be RNA or DNA contained within a protein-coated structure of a lipid membrane. The terms “virus” and “viral vector” are used interchangeably herein. Viruses useful for carrying out the present invention include recombinantly modified enveloped or unenveloped DNA and RNA viruses, preferably selected from the Baculoviridae, Parvoviridae, Picornaviridae, Herpesviridae, Poxviridae, or Adenoviridae families. Viruses may be modified by recombinant DNA technology to include the expression of an exogenous transgene (e.g., a nucleic acid sequence encoding orthogonal IL2) and may be manipulated to be replication-deficient, conditionally replicating, or replicative. Minimal vector systems may also be used in which the viral backbone contains only the sequences necessary for packaging the viral vector and optionally includes a transgene expression cassette. The term “replication-deficient” refers to a vector whose replication in wild-type mammalian cells is severely weakened. To produce such vectors in large quantities, producer cell lines are generally created by co-transfection with a helper virus or by genomically modifying them to compensate for the missing function. The term “replicative viral vector” refers to a viral vector capable of infection, DNA replication, packaging, and lysis of infected cells. The term “conditional replication viral vector” is used herein to refer to a replicative vector designed to achieve selective expression in a particular cell type. Such conditional replication can be achieved by functionally linking tissue-specific, tumor-specific, cell-type-specific, or other selectively induced regulatory sequences to an initial gene (e.g., the E1 gene in an adenovirus vector).Infection of a target with a recombinant virus or nonviral vector can provide long-term expression of orthogonal IL2 in the target, and can also provide continuous and selective maintenance of T cells engineered to express the CD122 orthogonal receptor. In one embodiment, the nucleic acid sequence in the viral vector system encoding the IL2 receptor is under the control of a regulatory promoter, an inducible promoter, a tissue-specific or tumor-specific promoter, or a temporally regulated promoter.

[0201] Pharmaceutical preparations Genetically modified T cells may be provided as pharmaceutical compositions suitable for therapeutic use, for example, in human treatment. Therapeutic formulations containing such cells may be frozen or prepared for administration in aqueous solution form using physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). The cells are formulated, administered, and managed in a manner consistent with appropriate medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammalian being treated, the individual patient's clinical condition, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals.

[0202] The cells may be administered by any suitable means, usually parenterally. Parenteral administration includes intramuscular, intravenous (bolus or slow infusion), intra-arterial, intraperitoneal, intrathecal, or subcutaneous administration. In a typical implementation, genetically modified T cells are injected into the subject, usually intravascularly, in a physiologically acceptable culture medium, but they may also be introduced into other convenient sites where the cells can find a suitable site for proliferation. Typically, at least 1 × 10⁵ cells / kg, at least 1 × 10⁶ cells / kg, at least 1 × 10⁷ cells / kg, at least 1 × 10⁸ cells / kg, at least 1 × 10⁹ cells / kg, or more will be administered, but this is usually limited by the number of T cells available at the time of collection.

[0203] For example, a typical dose range for genetically modified immune cells used in carrying out the method of the present invention is approximately 1 × 10⁵ to 5 × 10⁸ live cells / kg of target body weight per course of treatment. As a result, when adjusted for body weight, a typical dose range of live cells in a human subject is approximately 1 × 10⁶ to 1 × 10¹³ live cells, or approximately 5 × 10⁶ to 5 × 10¹² live cells, or approximately 1 × 10⁷ to 1 × 10¹² live cells, or approximately 5 × 10⁷ to 1 × 10¹² live cells, or approximately 1 × 10⁸ to 1 × 10¹² live cells, or approximately 5 × 10⁸ to 1 × 10¹² live cells, or approximately 1 × 10⁹ to 1 × 10¹² live cells. In one embodiment, the cell dose is in the range of 2.5 to 5 × 10⁹ live cells per course of treatment.

[0204] A course of treatment may consist of a single dose or multiple doses over a period of time. In some embodiments, cells are administered in a single dose. In some embodiments, cells are administered in two or more divided doses over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. The amount of genetically modified cells administered in such divided dosing protocols may be the same in each dose or provided at different levels. Multi-day dosing protocols over a period of time may be provided by a person skilled in the art (e.g., a physician) by monitoring the administration of cells, taking into account the adverse effects of the treatment and the subject's response to the treatment, including their modulation, as described above.

[0205] The compositions and methods of this disclosure also provide a method for treating a subject with T-cell therapy (particularly CAR T-cell therapy) without prior lymphocyte depletion. Lymphocyte depletion is typically performed on a subject in combination with CAR T-cell therapy. This is because the subsequent administration of a mixed cell population, and the administration of nonspecific agents (e.g., IL-2) to expand the genetically modified cell population in the subject in combination with the administration of the cell therapy product, can result in significant systemic toxicity (including cytokine release syndrome or "cytokine storm") resulting from the widespread proliferation and activation of immune cells due to the administration of agents that cause widespread activation, as well as from the presence of a significant proportion of non-modified cells in the cell therapy product itself. The methods and compositions of this disclosure avoid this significant hurdle by: providing a substantially purified population of modified cells with virtually no contamination from non-modified cells when the aforementioned ex vivo method is employed; and / or selective activation and expansion of modified T cells by the orthogonal IL2 of the present invention, resulting in a substantial reduction in the off-target effects of nonspecific growth agents such as IL2 (or either one of these).

[0206] For example, in current clinical practice of CAR-T cell therapy, CAR-T cells are generally administered in combination with lymphocyte depletion (e.g., by administration of alemtuzumab (monoclonal anti-CD52), purine analogs, etc.) to promote CAR-T cell expansion prior to host immune recovery. In some embodiments, CAR-T cells can be modified to develop resistance to alemtuzumab. In one aspect of the present invention, lymphocyte depletion currently employed in connection with CAR-T therapy can be avoided or mitigated by the CAR-T cells of the present invention that express orthogonal ligands. As described above, lymphocyte depletion is commonly employed to enable CAR-T cell expansion. However, this lymphocyte depletion is also associated with a major side effect of CAR-T cell therapy. Since orthogonal ligands provide a means of selectively expanding a specific T cell population, the need for lymphocyte depletion before administration of CAR-T cells expressing orthogonal ligands can be reduced. The method of the present invention makes it possible to perform CAR-T cell therapy without or with reduced lymphocyte depletion before administration of CAR-T cells expressing orthogonal ligands.

[0207] In one embodiment, the present disclosure provides a method for treating subjects suffering from a disease, disorder, or condition (e.g., cancer) suitable for treatment with CAR-T cell therapy, by administering CAR-T cells expressing an orthogonal ligand in the absence of lymphocyte depletion prior to administration of the orthogonal ligand CAR-T. In one embodiment, the disclosure provides a method for treating a mammal suffering from a disease or disorder associated with the presence of an abnormal cell population (e.g., a tumor) characterized by the expression of one or more surface antigens (e.g., tumor antigens), the method comprising: (a) obtaining a biological sample containing T cells from an individual; (b) enriching the biological sample for the presence of T cells; (c) transfecting the T cells with one or more expression vectors comprising a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding an orthogonal CD122 receptor, wherein the antigen-targeting domain of the CAR is capable of binding to at least one antigen present on the abnormal cell population; (d) expanding the population of CAR-T cells expressing the orthogonal receptor ex vivo with orthogonal IL2; (e) administering a pharmaceutically effective amount of orthogonal receptor-expressing CAR-T cells to a mammal; and (f) regulating the proliferation of orthogonal CD122 receptor-expressing CAR-T cells by administering a therapeutically effective amount of orthogonal IL2 that selectively binds to the orthogonal CD122 receptor expressed on the CAR-T cells. In one embodiment, the method is associated with mammalian lymphocyte depletion or immunosuppression before initiating a course of CAR-T cell therapy. In another embodiment, the method is carried out in the absence of mammalian lymphocyte depletion and / or immunosuppression.

[0208] Combination therapy: The compositions and methods of this disclosure can be used in combination with additional therapeutic agents. For example, if the disease, disorder, or condition to be treated is a neoplastic disease (e.g., cancer), the methods of this disclosure may be used in combination with conventional chemotherapeutic agents or other biological anticancer agents, such as checkpoint inhibitors (e.g., PD1 or PDL1 inhibitors) or therapeutic monoclonal antibodies (e.g., Avastin, Herceptin).

[0209] Examples of chemical agents that have been proven useful in the art for the treatment of neoplastic diseases include, but are not limited to, abitrexate, adriamycin, adorucil, amsacrin, asparaginase, anthracycline, azacitidine, azathioprine, vicunu, blenoxane, busulfan, bleomycin, camptosal, camptothecin, carboplatin, carmustine, serubidin, chlorambucil, cisplatin, cladribine, cosmegen, cytarabine, cytosar, cyclophosphamide, cytoxan, dactinomycin, docetaxel, doxorubicin, daunorubicin, ellence, elspar, epirubicin, etoposide, fludarabine, fluorouracil, fludarabine, gemcitabine, gemzar, hicamtin, hydroxy Urea, hydra, idamycin, idarubicin, ifosfamide, ifex, irinotecan, lanvis, leukeran, leustatin, matulane, mechloretamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, mitramycin, mutamycin, mylosar, navelbine, nipent, novantrone, oncovin, oxaliplatin, paclitaxel, paraplatin, pentostatin, platinol, plicamycin, procarbazine, prinetol, laritrexed, taxotere, taxol, teniposide, thioguanine, tomdex, topotecan, barbicin, vervan, bepecid, vinblastine, vindesine, vincristine, vinorelbine, VP-16, and vumon.

[0210] The compositions of this disclosure may be administered in combination with one or more additional therapeutic agents selected from the group consisting of: tyrosine kinase inhibitors, e.g., imatinib mesylate (trade name: Gleevec®, also known as STI-571), gefitinib (Iressa®, also known as ZD1839), erlotinib (trade name: Tarceva®), sorafenib (Nexavar®), sunitinib (Sutent®), dasatinib (Sprycel®), lapatinib (Tykerb®), nilotinib (Tasig na(registered trademark), bortezomib (Velcade(registered trademark)), Jakafi(registered trademark) (ruxolitinib); Janus kinase inhibitors, e.g., tofacitinib; ALK inhibitors, e.g., crizotinib; Bcl-2 inhibitors, e.g., ovatocrax, venclexta, gossypol; FLT3 inhibitors, e.g., midostaurin (Rydapt(registered trademark)); IDH inhibitors, e.g., AG-221; PARP inhibitors, e.g., iniparib, olaparib; PI3K inhibitors, e.g., perifosine; VEGF receptor 2 inhibitors, e.g., apatinib; AN-152 (AEZS-108), doxorubicin linked to [D-Lys(6)]-LHRH; Braf inhibitors, e.g., vemurafenib, dabrafenib, LGX818; MEK inhibitors, e.g., trametinib; CDK inhibitors, e.g., PD-0332991, LEE011; Hsp90 inhibitors, e.g., sarinomycin; and / or small molecule drug conjugates, e.g., vintafolide; serine / threonine kinase inhibitors, e.g., temsirolimus (Torisel®), everolimus (Afinitor®), vemurafenib (Zelboraf®), trametinib (Mekinist), dabrafenib (Tafinlar®).

[0211] In some embodiments, particularly when the tumor antigen binding portion of the CAR is directed towards BCMA, genetically modified CAR-T cells are administered in combination with a γ-secretase inhibitor (GSI), as described in Pont, et al. (2019) “γ-secretase inhibition increases efficacy of BCMA-specific chimeric antigen receptor T cells in multiple myeloma” Blood doi.org / 10.1182 / blood.2019000050.

[0212] Examples of biopharmaceuticals that have been proven useful in the art for the treatment of neoplastic diseases include, but are not limited to, the following: cytokines or cytokine antagonists, e.g., IL-12, INFα, or anti-epidermal growth factor receptors; radiotherapy; irinotecan; tetrahydrofolate antimetabolites, e.g., pemetrexed; antibodies against tumor antigens; monoclonal antibody-toxin complexes; T-cell adjuvants; bone marrow transplantation; antigen-presenting cells (e.g., dendritic cell therapy); antitumor vaccines; replicating viruses; signaling inhibitors (e.g., Gleeve c(registered trademark) or Herceptin(registered trademark), or immunomodulators, cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade(registered trademark), Enbrel(registered trademark)), interferon-β1a (Avonex(registered trademark)), interferon-β1b (Betaseron(registered trademark)), and one or more of the aforementioned combinations implemented in known chemotherapy regimens readily understood by clinicians in the art.

[0213] Tumor-specific monoclonal antibodies that can be administered in combination with genetically modified cells include, but are not limited to, rituximab (trade names: MabThera or Rituxan), alemtuzumab, panitumumab, and ipilimumab (Yervoy).

[0214] In some embodiments, the compositions and methods of this disclosure can be used in combination with immune checkpoint therapies. Examples of immune checkpoint therapies include those that inhibit the binding of PD1 to PDL1 and / or PDL2. PD1 inhibitors against PDL1 and / or PDL2 are well known in the art. Examples of commercially available monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2 include nivolumab (Opdivo®, BMS-936558, MDX1106, commercialized by Bristol Myers Squibb, Inc. (Princeton, NJ)), pembrolizumab (Keytruda®, MK-3475, lambrolizumab, commercialized by Merck and Company, Inc. (Kenilworth, NJ)), and atezolizumab (Tecentriq®, Genentech / Roche, Inc., South San Francisco, CA). Examples of additional PD1 inhibitory antibodies include, but are not limited to, durvalumab (MEDI4736, Medimmune / AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, Bristol Myers Squibb), avelumab (MSB0010718C, Merck Serono / Pfizer), and SHR-1210 (Incyte). Additional antibody PD1 pathway inhibitors are described in U.S. Patent No. 8,217,149 (Genentech), issued July 10, 2012; U.S. Patent No. 8,168,757 (Merck Sharp and Dohme), issued May 1, 2012; U.S. Patent No. 8,008,449 (Medarex), issued August 30, 2011; and U.S. Patent No. 7,943,743 (Medarex), issued May 17, 2011. Furthermore, small molecule PD1 inhibitors against PDL1 and / or PDL2 are known in the art.For example, see Sasikumar, et al. WO2016142833A1 and Sasikumar, et al. WO2016142886A2, BMS-1166 and BMS-1001 (Skalniak, et al (2017) Oncotarget 8(42): 72167-72181). [Examples]

[0215] Example 1: Generation of CD19 CAR_T2A_hoRb and CD19 CAR_T2A_hoRB_GGYRHQ T cells The CD19 chimeric antigen receptor protein (hereinafter referred to as "CD19_28z") used in this study has the following structure: GMCSF receptor signal peptide, FMC63 anti-CD19 scFv, AAA linker, CD28 hinge / transmembrane / costimulatory domain, and CD3ζ. The amino acid sequence of CD19_28z is as follows: TIFF2026053539000021.tif59150

[0216] The orthogonal T cells used in these implementations were produced by techniques known in the art by transfecting isolated T cells with lentiviral vectors; the aforementioned CD19_28z CAR, T2A linker polypeptide, and human orthogonal CD122 orthogonal receptor (hoCD122) were based on the amino acid sequence: It has the code TIFF2026053539000022.tif128150 and is also called CD19 CAR_T2A_hoRb. For hoRB with wild-type ICD, hoRB has the following amino acid sequence: It has TIFF2026053539000023.tif128150.

[0217] Using a T2A linker makes it possible to create orthogonal CAR-T cells using a single lentiviral plasmid.

[0218] Primary CD4 and CD8 enriched T cells were stimulated with anti-CD3 and anti-CD28 antibodies and cultured in WT IL-2 for up to 2 days after transduction. Lentiviruses encoding CD19 CAR_T2A_hoRb or CD19 CAR_T2A hoRb_GGYRHQ were introduced into the cells. After 2 days, the cells were washed and switched to T cell proliferation medium containing 40Kd PEGylated orthogonal ligand STK-009 (100 nM). The cells were maintained in T cell medium + STK-009 at a cell density of 1E6 cells / ml until day 10, when the cells were harvested.

[0219] Example 2: Orthogonal ligand STK-009 The orthogonal cognitive ligand (hereinafter STK-009) of the hoCD122 receptor used in these studies is a compound of hIL2 mutein modified by adding a 40kD branched (2×20kD) PEG molecule containing an aldehyde linker at the N-terminal proline residue, amino acid substitutions L18R, Q22E, and Q126K (numbered according to mature wild-type hIL2), and a 40kDa PEG-aldehyde (Sunbright® GL2-400AL3, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA) containing a 40kDa branched (2×20kD) PEG molecule, a 40kDa two-arm branched PEG-aldehyde, and two 20kDa linear PEG molecules.

[0220] Example 3: Phosphotransduction assay: T cells stably transduced with CD19 CAR_T2A_hoRb or CD19 CAR_T2A hoRb_GGYRHQ were rested for 1 hour in RPMI medium + 10% FBS. The cells were then stimulated for 20 minutes by dose titration with either WT IL-2 or STK-009 and stained for various phosphorylated antigens (phospho-antigens). The cells were then subjected to flow cytometry analysis. The results of these studies are shown in Figures 4 and 5 of the attached diagrams. As shown, adding the STAT3 motif to the hoCD122 receptor in CAR T cells results in increased activation of pERK, pS6K, and a higher baseline of pSTAT3.

[0221] Example 4: Cytotoxic co-culture assay T cells stably transduced with CD19 CAR_T2A_hoRb or CD19 CAR_T2A hoRb_GGYRHQ were co-cultured overnight with the CD19 target cell line Raji-luciferase in the indicated effector:target cell (E:T) ratio. Cell viability was assessed by the conversion of D-luciferin to light and read using a bioluminescent plate reader. Data are presented as specific lysis, calculated as 100% × (natural death RLU - test RLU) / (natural death RLU - maximum killing RLU). The results of this study are shown in Figure 6 of the attached diagrams. This data demonstrates that adding the STAT3 motif to the orthoreceptor of CAR T cells yields superior cytotoxic activity.

[0222] The beneficial functional effects of adding a STAT3 signaling domain to the intracellular domain of the human ortho-Rb (hoRb, hoCD122) receptor were demonstrated in a series of studies evaluating the effects of cells expressing the wt hoCD122 receptor and cells expressing a modified hoCD122 receptor with a STAT3 signaling domain (YRHQ) incorporated into the carboxyl terminus of the hoCD122 ICD. A diagram illustrating the receptor structure is shown in Figure 1 of the attached drawings. Cell lines stably expressing hoRB receptors, including wild-type ICD and STAT3-modified ICD, were created and tested with wild-type IL2 and the orthogonal ligand STK-009 (Example 2). As shown in Figures 2 and 3, the hoRB receptor, including wild-type ICD and STAT3-modified ICD, was functional in the modified cells. Cells expressing the receptor were evaluated for the expression of various phosphorylation ligands substantially according to Example 3. The results are shown in Figures 4 and 5. As shown in Figures 4 and 5, adding a STAT3 motif to the hoCD122 receptor on CAR T cells increased pERK and pS6K activation and elevated baseline levels of pSTAT3.

[0223] To evaluate the enhanced effects of CAR-T cells expressing STAT3-modified orthogonal CD122, CAR-T cells were evaluated in cytotoxicity experiments using the Raji tumor cell line. The effect of STAT3 on the cytotoxicity of CD19 CAR T cell constructs containing an orthogonal CD122(hoRb) receptor with an additional STAT3 signaling motif was compared with CD19 CAR T cell constructs containing orthogonal CD122(hoRb) with a wild-type CD122 intracellular domain at various effector:target (E:T;CAR T:Raji tumor cells) ratios, substantially following the teachings of Example 4. The results of this study are shown in Figure 6 of the attached drawings. The data shown in Figure 6 demonstrate that CD19 CAR T cell constructs expressing hoRb with a modified intracellular domain (ICD) containing a STAT3 motif exhibited improved cytotoxicity against Raji tumor cells compared with CD19 CAR T cells containing orthogonal CD122(hoRb) with a wild-type CD122 intracellular domain. The aforementioned data demonstrate the beneficial properties of the STAT3 motif-modified receptor described herein.

[0224] Having described the present invention in detail above, it will be clear to those skilled in the art that various modifications and alterations can be made without departing from the spirit or scope of the invention.

[0225] Array description: TIFF2026053539000024.tif218147TIFF2026053539000025.tif20432SEQ ID NO: 22 (Natural human CD122, full-length protein) The first underlined region is a signal peptide. TIFF2026053539000026.tif91149

Claims

1. A polynucleotide encoding a modified human CD122, wherein the modified human CD122 comprises one or more STAT3-binding motifs.

2. The polynucleotide according to claim 1, comprising orthogonal human CD122 or natural human CD122 fused to one or more STAT3-binding motifs, wherein the modified human CD122 is fused to one or more STAT3-binding motifs.

3. The polynucleotide according to claim 2, wherein orthogonal human CD122 is modified with one or more residues selected from R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and Q214 compared to natural human CD122.

4. The polynucleotide according to claim 2, wherein the orthogonal human CD122 is modified at H133 and Y134 compared to natural human CD122.

5. The polynucleotide according to claim 1, wherein human CD122 is linked to two or three STAT3-binding motifs.

6. The polynucleotide according to claim 1, wherein the modified human CD122 comprises a sequence that is at least 90% identical to SEQ ID NO: 1, and the modified human CD122 is bound to a natural IL2 polypeptide or an orthogonal IL2 polypeptide.

7. The polynucleotide according to claim 1, wherein one or more STAT3-binding motifs comprise the sequence YX1X2Q, where X1 and X2 are any amino acids.

8. The polynucleotide according to claim 7, wherein 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.

9. The STAT3 recognition motif is (a) YLRQ (SEQ ID NO: 11), (b) YLKQ (SEQ ID NO: 12), (c) YRHQ (SEQ ID NO: 13), (d) YLRQ (SEQ ID NO: 14), (e) YFKQ (SEQ ID NO: 15), (f) YLPQ (SEQ ID NO: 16), (g) YMPQ (SEQ ID NO: 17), and (h) YDKPH (SEQ ID NO: 18) A polynucleotide according to claim 7, selected from the group consisting of the following.

10. The polynucleotide according to claim 1, wherein one or more STAT3-binding motifs are optionally fused via a linker to the C-terminus of the intracellular domain of native human CD122 or orthogonal human CD122.

11. The polynucleotide according to claim 1, wherein at least one STAT3-binding motif is located between positions 355 and 364 corresponding to natural human CD122, and the YFTY, YDPY, or YSEE amino acid sequence of natural human CD122 is replaced by at least one STAT3-recognizing motif.

12. The polynucleotide according to claim 11, wherein the modified human CD122 is further modified with respect to one or more residues selected from R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and Q214, compared to natural human CD122.

13. The polynucleotide according to claim 10, wherein the linker comprises a dinucleotide GG.

14. Modified CD122 comprises an amino acid sequence containing a linker and at least one STAT3 binding motif, and said amino acid sequence (i) GGYLRQ (SEQ ID NO: 3), (j) GGYLKQ (SEQ ID NO: 4), (k) GGYRHQ (SEQ ID NO: 5), (l) GGYLRQ (SEQ ID NO: 6), (m) GGYFKQ (SEQ ID NO: 7), (n) GGYLPQ (SEQ ID NO: 8), (o) GGYMPQ (SEQ ID NO: 9), and (p)GGYDKPH (SEQ ID NO: 10) A polynucleotide according to claim 13, selected from the group consisting of the following.

15. An expression vector comprising a polynucleotide according to any one of claims 1 to 14.

16. A cell comprising the polynucleotide described in any one of claims 1 to 14.

17. The cell according to claim 16, further expressing a chimeric antigen receptor (CAR) and being a human immune cell.

18. The cell according to claim 17, wherein the CAR is selected from the group consisting of CD19 CAR and BCMA CAR.

19. A kit for selectively activating receptors in cells, (a) Cells expressing a modified human CD122 encoded by the polynucleotide described in claim 1, and (b) Human IL2 polypeptide The kit includes the above.

20. The kit according to claim 19, wherein the modified human CD122 comprises natural human CD122 encoded by SEQ ID NO: 1, and the human IL2 polypeptide is natural human IL2 polypeptide encoded by SEQ ID NO:

2.

21. The kit according to claim 19, wherein the modified human CD122 comprises orthogonal human CD122, and the human IL2 polypeptide is an orthogonal human IL2 polypeptide, and the orthogonal human IL2 polypeptide preferentially binds to the modified human CD122 compared to natural human CD122.

22. The kit according to claim 21, wherein the orthogonal human IL2 polypeptide contains at least one amino acid substitution with an amino acid other than the amino acid of the natural human IL2 polypeptide at residues T51 and R81 at the position corresponding to natural human CD122, or contains alanine at position M23 corresponding to natural human CD122, and contains amino acid substitutions at each of positions E15, H16, L19 and D20 corresponding to natural human CD122.

23. The kit according to claim 19, wherein the orthogonal human IL2 polypeptide comprises one or more amino acid substitutions corresponding to positions in natural human IL2, selected from [E15D, E15T, E15A, E15S], [H16N, H16Q], [L19V, L19I, L19A], [D20L, D20M], [Q22S, Q22T, Q22E, Q22K, Q22E], [M23A, M23W, M23H, M23Y, M23F, M23Q, M23Y], [G27K, G27S], [R81D, R81Y], [N88E, N88Q], and [T51I].

24. The kit according to claim 19, wherein a modified human CD122 is expressed by mammalian cells.

25. The kit according to claim 24, wherein the mammalian cells are immune cells.

26. The kit according to claim 25, wherein the immune cells are T cells.

27. The kit according to claim 26, wherein the T cells are chimeric antigen receptor (CAR)-T cells.

28. A method for stimulating immune cells expressing modified human CD122 containing one or more STAT3-binding motifs, comprising the step of contacting the immune cells with a human IL2 polypeptide.

29. The method according to claim 28, wherein the stimulation is performed ex vivo.

30. The method according to claim 28, wherein the stimulation is performed in vivo.

31. The method according to claim 28, wherein the modified human CD122 comprises orthogonal human CD122 or natural human CD122 fused to one or more STAT3-binding motifs.

32. The method according to claim 28, wherein at least one STAT3-binding motif is located between positions 381 and 390 corresponding to natural human CD122, and the YFTY, YDPY, or YSEE amino acid sequence of natural human CD122 is replaced by at least one STAT3-binding motif.

33. A step of introducing immune cells expressing modified human CD122 containing one or more STAT3 binding motifs into an organism, and A step of administering human IL2 polypeptide to the individual, thereby activating the immune response in the individual. The method according to claim 30, including the method described in claim 30.

34. The method according to claim 33, wherein the modified human CD122 comprises orthogonal human CD122, the human IL2 polypeptide is an orthogonal human IL2 polypeptide, and the orthogonal human IL2 polypeptide preferentially binds to the modified human CD122 rather than to natural human CD122, thereby activating the modified human CD122.

35. The method according to claim 28, wherein one or more STAT3-binding motifs comprise the sequence YX1X2Q, where X1 and X2 are any amino acids.

36. The method according to claim 35, wherein 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.

37. Orthogonal human CD122, GGYLRQ (SEQ ID NO: 2), GGYLKQ (SEQ ID NO: 3), GGYRHQ (SEQ ID NO: 4), GGYLRQ (SEQ ID NO: 5), GGYFKQ (SEQ ID NO: 6), GGYLPQ (SEQ ID NO: 7), GGYMPQ (SEQ ID NO: 8), and GGYDKPH (SEQ ID NO: 9) The method according to claim 34, comprising an array selected from the group consisting of the following.

38. The method according to claim 33, wherein the immune cell is a T cell.

39. The method according to claim 38, wherein the T cell is a CAR-T cell.

40. The method according to any one of claims 33 to 39, wherein the immune cells are CD8+ T cells and the individual has cancer.

41. The method according to any one of claims 33 to 39, wherein the immune cells are Treg cells and the individual has an autoimmune disease.

42. The method according to any one of claims 33 to 39, wherein the individual has a viral, bacterial or fungal infection.

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