Compositions and methods useful in the treatment of autoimmune disease

Abstract

The present disclosure relates to methods and compositions for the treatment of a mammalian subject suffering from lupus the method comprising, administering to said subject a therapeutically effective amount of an orthogonal CAR-T cell directed against a B-cell antigen in combination with an engineered orthogonal IL2 ligand that selectively activates orthogonal CAR T cell expressing an engineered orthogonal CD122 receptor. In some embodiments, the lupus is systemic lupus erythramatosis (SLE). In some embodiments, the lupus is lupus nephritis. In some embodiments the subject suffering from lupus is also suffering from cytopenia. In some embodiments the methods are practiced in the absence of prior lymphodepletion of the subject.

Description

Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT COMPOSITIONS AND METHODS USEFUL IN THE TREATMENT OF AUTOIMMUNE DISEASE CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application claims benefit of priority from United States Provisional Patent Application Serial No.63 / 569,630, filed March 25, 2024 and United States Provisional Patent Application Serial No.63 / 632,461, filed April 10, 2024, each of which is incorporated by reference for all purposes. BACKGROUND OF THE INVENTION

[0002] Systemic lupus erythematosus (SLE) is an autoimmune chronic inflammatory disease predominantly affecting women in the reproductive age range. The prevalence of SLE varies considerably by race, ethnicity, and geographical location Almaani, et al. (2017) Clin J Am Soc Nephrol.12(5):825-35). Prevalence of SLE in the United States has been reported to be between 20 to 150 cases per 100,000 (but as high as 406 per 100,000 among African American women) (Chakravarty et al. (2007) Arthritis Rheum.56(6):2092-4, Izmirly, et al. (2021) Arthritis Rheumatol.73(6):991-996). Historically, the disease has been associated with significant premature mortality and associated morbidity; although mortality rates have decreased over time, permanent organ damage such as end stage renal disease remains frequently associated with disease activity, occurring in up to 30% of patients with lupus nephritis (Fanouriakis, et al. (2021) Annals of the Rheumatic Diseases 80(1):14-25; Tektonidou, et al. (2016) Arthritis & Rheumatology 68(6):1432-1441). Due to the heterogenous nature of the manifestations of SLE, patient-specific treatment needs tend to be similarly heterogenous, contributing to an ongoing need for new therapeutic options.

[0003] Antimalarials and glucocorticoids are the mainstay treatments for patients with SLE. Multiple immunosuppressive and cytotoxic agents have also been utilized to treat SLE, including methotrexate, azathioprine, mycophenolate mofetil, and cyclophosphamide. However, these medications were not developed specifically to treat SLE, and their effectiveness and safety profiles are variable. Belimumab, was approved by the Food and Drug Administration (FDA) in 2011 Anifrolumab-fnia was approved by the FDA to treat SLE in 2021. The overall efficacy of these more recent targeted therapy approvals remains limited and more effective treatment options for SLE are needed.

[0004] CAR-T cell therapy is established as an efficacious treatment for a variety of hematological cancers including non-Hodgkin lymphoma (NHL), follicular lymphoma, and B-Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT cell acute lymphoblastic leukemia (B-ALL). In the current clinical practice of B-cell targeted CAR-T cell therapy in the treatment of hematological malignancies, the CAR-T cells are commonly administered to the subject following lymphodepletion.

[0005] Recently, anti-CD19 CAR-T cell therapies against B-cell antigens have been reported to be efficacious in the treatment of SLE. Mackensen, et al. (2022) Anti-CD19 CAR-T cell therapy for refractory systemic lupus erythematosus, Nature Medicine 28:2124–2132. Similar to existing treatment regimens for the treatment of hematological cancers, the Mackensen, et al. study involved the infusion of autologous T cells transduced with a lentiviral anti-CD19 CAR vector administered at a dose of 1 × 106CAR-T cells / kg body following lymphodepletion with fludarabine and cyclophosphamide.

[0006] CD19-specific CAR T cells ("CD19 CAR-Ts") have demonstrated efficacy in a variety of B cell malignancies. Presently, four CD19 CAR-Ts have been approved by the FDA for the treatment of B cell malignancies.

[0007] Clinical studies with CD19 CAR T cell therapies show that a significantly improved response correlates with CAR-T cell expansion and overall exposure (Mueller, et al. (2017) Blood 130(21):2317-2325), and the lack of complete response in subjects treated with CD19 CAR-Ts appears to be largely due to inadequate activation, expansion, and persistence of the CD19 CAR-T cells following their initial administration. Antigen-specific T cells require three distinct signals for their activation: TCR engagement of peptide-major histocompatibility complex (signal 1), co-stimulation (signal 2), and cytokines (signal 3). Current generation CD19 CAR-T cell therapies provide signals 1 and 2 but are dependent on endogenous cytokine production for signal 3 and insufficient cytokine support is an important limitation of existing CD19 CAR-T cell therapies.

[0008] To provide cytokine support, current CD19 CAR-T cell therapies typically involve lymphodepletion before CAR-T cell infusion. Lymphodepletion before CAR-T cell infusion leads to an increase in endogenous IL-7 and IL-15 and the increase in these endogenous cytokines provides cytokine support (signal 3) to facilitate T cell proliferation and is essential to efficacy. However, the elevation of endogenous IL-7 and IL-15 associated with lymphodepletion is short lived (typically less than one week) and the withdrawal of endogenous cytokine support leads to a rapid decrease in the population of the CAR-T cells. Endogenous cytokine secretion during CD19 CAR T therapy is also unpredictable and can lead to acutely to life-threatening cytokine release syndrome (CRS) and neurotoxicity. In some instances, CAR-T cells have been proposed that incorporate signal 3 directly into CAR-T cell design to improve survival and persistence and this remains an active area of research.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0009] As currently practiced, CAR-T cell therapy presents several challenges. Because CAR-T cells expand within the subject, this results in a highly variable relationship between dose and exposure. Identifying an optimal CAR T cell dose sufficient to facilitate CAR-T cell expansion and efficacy without triggering severe toxicities is difficult to determine. Additionally, as current autologous CAR-T cells are administered as a single infusion, there is no opportunity to adjust exposure once the therapy has been administered.

[0010] B-cell depletion as a therapeutic intervention has been evaluated across a range of autoimmune diseases. B cell depletion strategies employing anti-CD20 monoclonal antibodies (e.g., rituximab and obinutuzumab) have shown evidence of efficacy across a variety of autoimmune indications. These results implicate B cells as the central drivers of autoimmune disease. B cell depletion with anti-CD20 antibodies has demonstrated efficacy, to varying degrees. Improved tissue depletion of B-cells by type II monoclonal antibodies such as obinutuzumab suggest the potential to deliver greater efficacy compared to rituximab in autoimmune diseases.

[0011] CD19-targeted CAR-T cells directly and specifically deplete B cells in the circulation and tissues more effectively than anti-CD20 monoclonal antibodies. In addition to providing disease control, a deeper and broader depletion of B cells, including the disease-driving pathogenic B cells, may provide the prospect of an immune reset, an outcome which has been reported concurrent with deep and broad depletion of leukocytes (including B cells) following hematopoietic stem cell transplant.

[0012] Nonclinical in vivo studies of anti-CD19 CAR-T cell therapy in the autoimmune disease setting have supported this approach. Treatment with a murine anti-CD19 CAR-T cell therapy (composed of CD8+ T cells) in two spontaneous mouse models of lupus (NZB / W F1 and MRL / lpr mice) led to effective depletion of CD19+ B cells in the spleen, inflamed kidneys, and the bone marrow, which resulted in significant amelioration of anti-double-stranded DNA (dsDNA) antibody production and proteinuria, and improved survival of the mice (Kansal, et al. (2019) Sustained B cell depletion by CD19-targeted CAR T cells is a highly effective treatment for murine lupus, Science Translational Medicine 11:eaav1648). Similarly, a study in a mouse lupus model (MRL / lpr) showed that anti-CD19 CAR-T cells (composed of mixed CD8+ and CD4+ cells) reduced B cell counts, prolonged the life span of animals, and improved the disease in target tissues (Jin, et al. (2021) Therapeutic efficacy of anti-CD19 CAR-T cells in a mouse model of systemic lupus erythematosus, Cell Mol Immunol 18:1896–1903). However, similar to the human clinical experience, in both these studies the mice were lymphodepleted by total body irradiation prior to treatment. In Kansal, et al. mice were subjected to 5 Gy of total bodyAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT irradiation five days prior to the initiation of CAR-T cell treatment. In Jin, et al., mice were subjected to 1.5 Gy of total body irradiation one prior to the imitation of CAR-T cell treatment.

[0013] Preliminary results from an ongoing compassionate use program of a CD19 CAR T cell therapy (MBCAR-T19.1) in patients with SLE demonstrate activity in SLE. MBCAR-T19.1 is an autologous CD19 CAR-T, derived from CD4+ and CD8+ T cells isolated and purified from the patient’s peripheral blood mononuclear cells (PBMCs) and transduced with a lentiviral vector encoding the CAR that contains a CD19-recognizing scFv (FMC63) (Mackensen, supra). Seven patients with severe, refractory SLE (all with active kidney disease) were treated with lymphodepletion (LD) (cyclophosphamide and fludarabine) followed by a single infusion of 1×106CD19 CAR T cells / kg (Taubmann, et al. (2023) Ann Rheum Dis.82:93-94). With a median follow-up of 13 months, all 7 patients achieved and maintained drug-free remission as assessed by DORIS remission criteria. Grade 1 CRS was observed in 4 / 7 patients and no cases of immune effector cell-associated neurotoxicity syndrome (ICANS) were reported. These data were updated to include 15 patients with treatment resistant autoimmune disease including SLE (n=8), idiopathic inflammatory myopathy (n=3), and systemic sclerosis (n=4) (Schett, et al. (2023) Lancet.25;402(10416):2034-2044.).

[0014] A clinical trial in SLE was recently initiated with YTB323, a autologous CD19- directed CAR T-cell therapy (Dickinson, et al. (2023) Cancer Discovery 13(9):1982-1997). Preliminary results from the first three patients treated with YTB323 at a dose of 12.5×106cells after lymphodepletion suggest a favorable safety profile, successful CAR T cell expansion, B cell depletion, and evidence of efficacy accompanied by improvements in biomarkers including dsDNA, complement, and proteinuria. All 3 patients had grade 3 and 4 cytopenias related to lymphodepletion and 2 / 3 patients had Grade 1 and 2 CRS (Cortés Hernández, et al (2023) Arthritis Rheumatol.2023; 75 (suppl 9))Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT SUMMARY OF THE DISCLOSURE

[0015] The present disclosure provides methods and compositions useful in the treatment of lupus in a mammalian subject.

[0016] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus. In some embodiments, the method comprises administering to said subject a therapeutically effective amount of an orthogonal chimeric antigen receptor (CAR)-T cell in combination with an orthogonal IL2 ligand, wherein the CAR- T cell comprises a CAR, and wherein the CAR comprises an antigen binding domain (ABD), a transmembrane domain (TMD) and an intercellular domain (ICD), wherein the antigen binding domain (ABD) of the CAR of the orthogonal CAR-T cell selectively binds to at least one human B cell antigen, and wherein CAR-T cell comprises an orthogonal receptor, wherein the orthogonal receptor comprises an extracellular domain (ECD), a transmembrane domain (TMD) and an intercellular domain (ICD), wherein the ECD comprises an orthogonal CD122 ECD polypeptide. In some embodiments, the lupus is systemic lupus erythramatosis (SLE). In some embodiments, the lupus is lupus nephritis. In some embodiments the subject suffering from lupus is also suffering from cytopenia. In some embodiments the subject suffering from lupus has not received an immunodepletion treatment regimen prior to the initiation of treatment (e.g., with CAR-T cell therapy). In some embodiments, the subject suffering from lupus has not received a lymphodepletion treatment regimen, optionally within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day, prior to administering of the orthogonal CAR-T cell.

[0017] In some embodiments the CAR-T cell expresses a chimeric antigen receptor, the antigen binding domain (ABD) of the chimeric antigen receptor specifically binds to a B-cell antigen. In some embodiments, the antigen binding domain (ABD) of the chimeric antigen receptor specifically binds to a B-cell antigen is selected from the group consisting of CD19, CD20, CD22, BAFF, TACI and BCMA. In some embodiments, the antigen binding domain (ABD) of the chimeric antigen receptor specifically binds to the B-cell antigen CD19 (the CAR comprising this ABD is referred to as a CD19 CAR). In some embodiments, the ABD of the CD19 CAR comprises an anti-CD19 scFv. In some embodiments, the ABD of the CD19 CAR comprises an anti-CD19 scFv wherein the anti-CD19 scFv comprises a light chain polypeptideAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 26, a linker that joins the heavy and light chain domains of the scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 and a heavy chain sequence having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 27. In some embodiments, the ABD of the CD19 CAR comprises an anti-CD19 scFv having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 35.

[0018] In some embodiments the CAR-T cell expresses a chimeric antigen receptor, the antigen binding domain (ABD) of which specifically binds to a B-cell antigen, a CD3z stimulatory domain and a CD28 costimulatory domain. In some embodiments the CAR-T cell expresses a chimeric antigen receptor wherein the ABD of the CAR specifically binds to CD19 and the CAR is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 61.

[0019] In some embodiments the orthogonal CAR-T cell expresses a chimeric antigen receptor and an orthogonal receptor. In some embodiments, the orthogonal receptor is selected from the group consisting of an orthogonal hIL2 receptor, a chimeric orthogonal IL4R receptor, a chimeric orthogonal IL7R receptor, a chimeric orthogonal IL9 receptor, a chimeric orthogonal IL21 receptor. In some embodiments, the extracellular domain of the orthogonal hIL2 receptor, a chimeric orthogonal IL4R receptor, a chimeric orthogonal IL7R receptor, a chimeric orthogonal IL9 receptor, or chimeric orthogonal IL21 receptor comprises an orthogonal hCD122 ECD. In some embodiments the orthogonal hCD122 ECD comprises is variant polypeptide derived from the ECD of a human CD122 wherein the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequenceAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising one or more amino acid substitutions or deletions of amino acid residues selected from the group consisting of R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and / or Q214 (numbered in accordance with SEQ ID NO:3). In some embodiments the orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 hCD122 ECD having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising one or more amino acid substitutions or deletions of amino acid residues selected from the group consisting of H133 and Y134 (numbered in accordance with SEQ ID NO:3). In some embodiments, the one or more amino acid substitutions or deletions of amino acid residues at H133 and Y134 are amino acid substitutions selected from the group consisting of H133D, H133E, H133K, Y134F, Y134E, and Y134R (numbered in accordance with SEQ ID NO:3). In some embodiments. In some embodiments, the ECD of the orthogonal hCD122 comprises amino acid substitutions at positions H133 and Y134 are amino acid substitutions include an amino acid substitution at position H133 selected from the group consisting of H133D, H133E, and H133K, and an amino acid substitution at position H134 selected from the group consisting of Y134F, Y134E, and Y134R (numbered in accordance with SEQ ID NO:3). In some embodiments, the ECD of the orthogonal hCD122 comprises amino acid substitutions at positions H133 and Y134 wherein the substitution at H133 is H133D and the amino acid substitution at position H134 is Y134F (numbered in accordance with SEQ ID NO:3). In some embodiments, the ECD of the orthogonal hCD122 comprises a polypeptide having the amino acid sequence: AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPV SQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLM APISLQVVHVETHRCNISWEISQASDFFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT (SEQ ID NO:40) In some embodiments, the orthogonal hCD122 receptor is a polypeptide comprising the amino acid sequence: AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPV SQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLM APISLQVVHVETHRCNISWEISQASDFFERHLEFEARTLSPGHTWEEAPLLTLKQKAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWL GHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGD VQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLT SCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLS GEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDW DPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEF RALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO:39).

[0020] In some embodiments, the method comprises the administration of an orthogonal IL2 ligand in combination with the orthogonal CAR-T cell. In some embodiments, the orthogonal IL2 ligand comprises a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO: 12, further comprising amino acid substitutions at one or more of E15, H16, L19, D20, Q22, and M23 numbered in accordance with the mature human IL2 polypeptide (SEQ ID NO: 12), optionally further comprising: (a) one or more amino acid substitutions selected from the group consisting of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P, T3C N30E; K32E; N33D; P34G; T37I, R38W, R38G, M39Q, R39L, R39V, F42K, F42Y, F44Y, P47G, T51I, E52K, L53N, H55Y, Q57E, V91R, K97E M104A, T113N C125S, and C125A; and / or (b) a deletion of one or more N-terminal amino acids, the deletion of positions 1-9, alternatively positions 1-8, alternatively positions 1-7 alternatively positions 1-6, alternatively positions 1-5, alternatively positions 1-4, alternatively positions 1-3, alternatively positions 1-2, or alternatively position 1 numbered in accordance with SEQ ID NO: 12. In some embodiments, the orthogonal IL2 ligand comprises amino acid substitutions at positions one or more of positions E15, H16, L19, D20 Q22, and M23, wherein such substitution is a set of amino acid substitutions selected from the group consisting of: [E15S-H16Q-L19V-D20L-Q22K- M23A], [E15S-H16Q-L19V-D20L-Q22K]; [H16N, L19V, D20N, Q22T, M23H, G27K]; [E15D, H16N, L19V, D20L, Q22T, M23H]; [E15D, H16N, L19V, D20L, Q22T, M23A]; [E15D, H16N, L19V, D20L, Q22K, M23A]; [E15S; H16Q; L19V, D20T; Q22K, M23L]; [E15S; H16Q; L19V, D20T; Q22K, M23S]; [E15S; H16Q; L19V, D20S; Q22K, M23S]; [E15S; H16Q; L19I, D20S; Q22K; M23L]; [E15S; L19V; D20M; Q22K; M23S]; [E15T; H16Q; L19V; D20S; M23S]; [E15Q; L19V; D20M; Q22K; M23S]; [E15Q; H16Q; L19V; D20T; Q22K; M23V]; [E15H; H16Q; L19I; D20S; Q22K; M23L]; [E15H; H16Q; L19I; D20L; Q22K; M23T]; and [L19V; D20M; Q22N; M23S], numbered in accordance with the mature human IL2 polypeptide (SEQ ID NO: 12). In some embodiments, the orthogonal IL2 ligand comprises the amino acid substitutions [E15S-H16Q-L19V-D20L-Q22K-M23A], [E15S-H16Q-L19V-D20L-Q22K];Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT numbered in accordance with the mature human IL2 polypeptide (SEQ ID NO: 12). In some embodiments, the orthogonal IL2 ligand comprises a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43. In some embodiments, the orthogonal IL2 ligand comprises a polypeptide a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO: 43. In some embodiments, the orthogonal IL2 ligand comprises a polypeptide a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO: 41.

[0021] In some embodiments, the orthogonal IL2 ligand is modified to extend the duration of action of the molecule in a mammalian subject. In some embodiments, the modification is covalent linkage to one or more molecules selected from the group consisting of a carrier molecule, albumin, an Xten polymer, an anti-albumin antibody, an Fc domain, a fatty acid molecule. In some embodiments, the orthogonal IL2 ligand is covalently linked to a carrier molecule wherein the carrier molecule is a water-soluble polymer. In some embodiments, the orthogonal IL2 ligand is covalently linked to a polyethylene glycol (PEG). In some embodiments, the orthogonal IL2 ligand is covalently linked to a PEG wherein the PEG is a linear PEG, branched PEG, star-PEG, or multi-armed PEG having an average molecular mass from about 2 kDa to about 80 kDa, alternatively from about 2 kDa to about 70 kDa, alternatively from about 5 kDa to about 50 kDa, alternatively from about 5 kDa to about 50 kDa, alternatively from about 20 kDa to about 50 kDa, alternatively from about 30 kDa to about 50 kDa, alternatively from about 20 kDa to about 40 kDa, alternatively from about 5 kDa to about 10 kDa, alternatively from about 5 kDa to about 15 kDa, alternatively from about 5 kDa to about 20 kDa, from about 10 kDa to about 15 kDa, alternatively from about 10 kDa to about 20 kDa, alternatively from about 10 kDa to about 25 kDa or alternatively from about 10 kDa to about 30 kDa alternatively from about 30 kDa to about 40 kDa, alternatively about 5 kDa, alternatively about 10 kDa, alternatively about 15 kDa, alternatively about 20 kDa, alternatively about 25 kDa, alternatively about 30 kDa, alternatively about 35 kDa, alternatively about 40 kDa, alternatively about 45 kDa, alternatively about 50 kDa, alternatively about 55 kDa, alternatively about 60 kDa, alternatively about 65 kDa, alternatively about 70 kDa, alternatively about 75 kDa, or alternatively about 80 kDa. In some embodiments, the orthogonal IL2 ligand is covalently linked to a 40 kDa linear PEG. In some embodiments, the orthogonal IL2 ligand isAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT covalently linked to a branched PEG having an average molecular weight of about 40 kDa comprising two 20 kDa arms. In some embodiments, the orthogonal IL2 ligand is covalently linked to the PEG via a linker. In some embodiments, the orthogonal IL2 ligand is covalently linked to a compound of the structure: (PEG)-(linker)n-(PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 41). wherein n = 0 (absent) or 1 (present) and PEG is 40kDa-PEG, in some embodiments the branched 40kDa PEG comprising two 20kDa PEG arms, in some embodiments the PEG is covalently attached to the orthogonal hIL2 ligand via a linker.

[0022] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus the method comprising, administering to said subject a therapeutically effective amount of an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand, wherein the antigen binding domain (ABD) of the CAR of the orthogonal CAR-T cell selectively binds to at least one human B cell antigen, and wherein the extracellular domain of the orthogonal receptor of the orthogonal CAR-T cell comprises orthogonal receptor, wherein the subject is a human being and the orthogonal CAR-T cell is administered to the subject at a dose from 50x106to 150x106cells, alternatively from 70 x106to 120x106cells, alternatively from 90x106to 120x106cells, alternatively 90 x106cells, alternatively 100 x106cells, alternatively 110x106cells, alternatively 120x106cells, alternatively 120x106cells, alternatively 120x106cells, alternatively 120x106cells, alternatively 130x106cells, alternatively 140x106cells, or alternatively 150x106cells. In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus the method comprising, administering to said subject a therapeutically effective amount of an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand, wherein the antigen binding domain (ABD) of the CAR of the orthogonal CAR-T cell selectively binds to at least one human B cell antigen, and wherein the extracellular domain of the orthogonal receptor of the orthogonal CAR-T cell comprises orthogonal receptor, wherein the subject is a human being and the orthogonal CAR-T cell is administered to the subject at a dose from 1.0x106to 2x106cells per kg of bodyweight, alternatively from 1.5x106to 2x106cells per kg of bodyweight, alternatively 1x106cells per kg of bodyweight, alternatively 1.1x106cells per kg of bodyweight, alternatively 1.2 x106cells per kg of bodyweight, alternatively 1.3x106cells per kg of bodyweight, alternatively 1.4 x106cells per kg of bodyweight, alternatively 1.5x106cells per alternatively 1.6 x106cells per kg of bodyweight, alternatively 1.7x106cells per kg of bodyweight, alternatively 1.8 x106cells per kgAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT of bodyweight, alternatively 1.9x106cells per kg of bodyweight, alternatively 2x106cells per kg of bodyweight, alternatively 2.1x106cells per kg of bodyweight, alternatively 2.2x106cells per kg of bodyweight, alternatively 2.3x106cells per kg of bodyweight, alternatively 2.4x106cells per kg of bodyweight, alternatively 2.5x106cells per kg of bodyweight, or alternatively 3.0x106cells per kg of bodyweight.

[0023] In some embodiments, the orthogonal CAR-T cell is administered in combination with an orthogonal IL2 ligand wherein the orthogonal IL2 ligand is administered to the subject in one or more doses of approximately 0.05mg / kg to 1 mg / kg; alternatively from approximately 0.05mg / kg to 0.7 mg / kg; alternatively of approximately 0.05mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1mg / kg to 0.3 mg / kg wherein the orthogonal wherein is administered daily, twice weekly, thrice weekly, weekly, about every 10 days, or about every 14 days over a period of at least one month, optionally at least 2 months, optionally at least 3 months. In some embodiments, the orthogonal CAR-T cell is an orthogonal CD19 CAR-T cell wherein the CAR of the orthogonal CD19 CAR-T is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 61 and the orthogonal receptor is an orthogonal hIL2 receptor has the amino acid sequence having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: SEQ ID NO:39. In some embodiments. In some embodiments, the orthogonal CAR-T cell is an orthogonal CD19 CAR-T cell wherein the CAR of the orthogonal CD19 CAR-T is a polypeptide administered in combination of an orthogonal IL2 ligand of the structure: (PEG)-(linker)n-(PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 41). wherein n = 0 (absent) or 1 (present) and PEG is 40kDa-PEG. In some embodiments of the foregoing, the orthogonal IL2 ligand is administered daily, twice weekly, thrice weekly, weekly, about every 10 days, or about every 14 days, optionally for period of at least 10 weeks. In some embodiments of the foregoing methods, the orthogonal IL2 ligand is administered at an initial dose of approximately 0.05mg / kg to 0.5 mg / kg for a period of approximately 1 week, alternatively approximately 2 weeks followed by a maintenance dose 0.02 mg / kg to 0.3 mg / kg for a period of at least 2 weeks, alternatively at least 3 weeks, alternatively at least one month,Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT alternatively at least at least two months, alternatively at least three months. In some embodiments, the orthogonal IL2 ligand is administered to the subject at a dose and frequency sufficient to maintain a therapeutically effective quantity of orthogonal CAR-T cells in the subject wherein the therapeutically effective quantity of orthogonal CAR-T cells is from about 1x105cells per kg of bodyweight to about 3x106cells per kg of bodyweight, alternatively from about 2x105to about 3x106cells per kg of bodyweight, alternatively from about 2x105to about 2x106cells per kg of bodyweight, alternatively from about 3x105to about 2x106cells per kg of bodyweight, alternatively from about 5x105to about 1.5x106cells per kg of bodyweight, alternatively from about 5x105to about 1x106cells per kg of bodyweight, alternatively from about 1x106to about 2x106cells per kg of bodyweight, alternatively from about 1x106to about 1.3x106cells per kg of bodyweight, alternatively from about 1x106to about 1.5x106cells per kg of bodyweight, alternatively from about 1.3x106to about 1.7x106cells per kg of bodyweight, or alternatively from about 1.5x106to about 2x106cells per kg of bodyweight, alternatively about 1.2x106cells per kg of bodyweight, alternatively about 1.3x106cells per kg of bodyweight, alternatively about 1.4x106cells per kg of bodyweight, alternatively about 1.5x106cells per kg of bodyweight, alternatively about 1.6x106cells per kg of bodyweight, alternatively about 1.7x106cells per kg of bodyweight, alternatively about 1.8x106cells per kg of bodyweight, alternatively about 1.9x106cells per kg of bodyweight, alternatively about 2.0x106cells per kg of bodyweight, nd wherein the orthogonal IL2 ligand is administered to the subject in one or more doses of approximately 0.05mg / kg to 1 mg / kg; alternatively from approximately 0.05 mg / kg to 0.7 mg / kg; alternatively of approximately 0.05mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1mg / kg to 0.3 mg / kg wherein the orthogonal wherein is administered daily, twice weekly, thrice weekly, weekly, about every 10 days, or about every 14 days over a period of at least one month, optionally at least 2 months, optionally at least 3 months. In some embodiments, the T cell used to prepare the orthogonal CAR-T cell is a T cell obtained from the subject to be treated, i.e. and autologous CAR-T cell.

[0024] The methods of the present disclosure are useful in the treatment of lupus, including lupus nephritis. In some embodiments, the practice of the methods of the present disclosure result in a subject exhibiting an improvement in one or more lupus efficacy criteria in the subject compared to the subject prior to the initiation of treatment, wherein the lupus efficacy criteria are selected from the group consisting of: (a) remission of lupus per definition of remission in SLE (DORIS) criteria (van Vollenhoven, et al. (2021) Lupus Sci Med.8(1):e000538); (b) lupus low disease activity state (LLDAS) attainment rate (Franklyn (2016) Ann Rheum Dis.75(9):1615- 21.; (c) improvement in SLEDAI-2K scores; (d) improvement in BILAG individual systemAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT scores; (e) improvement in urine protein creatinine ratio (UPCR); (f) a decrease of ≥ 50% of Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) activity score (Albrecht, et al. (2005) J Invest Dermatol 125(5): 889- 894) in subjects with baseline activity score ≥ 10 prior to initiation of treatment; (g) complete renal response (CRR) defined as a UPCR < 0.5 and eGFR ≥ 60 mL / min per 1.73 m2or no confirmed eGFR decrease > 20%) in subjects with having a UPCR >1.0 mg / mg prior to the initiation of treatment; (h) decrease in total tender and swollen joint counts in subjects with ≥ 4 tender and swollen joints; (i) time to occurrence of a moderate or severe BILAG flare; (j) time to occurrence of a mild / moderate or severe flare as defined by SELENA-SLEDAI Flare index; (k) reduction in levels of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies; (l) reduction in levels of anti-nuclear antibodies (ANAs); (m) an ANA titer less than 1:80; (n) reduction in levels of anti-Smith antibodies; (o) reduction in level of complement factor C3, (p) reduction in level of complement factor C3C4) ; (q) improvement in HAQ-DI, SF-36; (r) improvement in FACIT-Fatigue Scale version 4 (Tennant, K. (2015) Supportive Care in Cancer 23(5):1355-1364); (s) reduction in disease activity as assessed by the UCSF / JHU Lupus Activity Index (LAI); reduction in disease activity as assessed by the SLE Disease Activity Index (SLEDAI); reduction in disease activity as assessed by the Systemic Lupus Activity Measure (SLAM) (Liang, et al (1989) Arthritis Rheum. 32(9):1107-18) or SLAM-R (Bae, et al (2001) Lupus 2001; 10: 405-9) and the reduction in disease activity as assessed by the British Isles Lupus Assessment Group (BILAG) Activity Index, wherein the improvement in one more efficacy criteria occurs within 14 days, 30 days, 60 days, 90 days, 12 weeks, alternatively within 24 weeks following administration of the orthogonal CAR-T cells, alternatively with 48 weeks following administration of the orthogonal CAR-T cells, or alternatively within 96 weeks following administration of the orthogonal CAR- T cells. In some embodiments, the subject

[0025] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus the method comprising, administering to said subject a therapeutically effective amount of an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand, wherein the antigen binding domain (ABD) of the CAR of the orthogonal CAR-T cell selectively binds to at least one human B cell antigen, and wherein the orthogonal IL2 ligand selectively binds to the extracellular domain of the orthogonal CD122 receptor, further wherein the subject is not treated with a lymphodepletion treatment regimen within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternativelyAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day prior to administering of the orthogonal CAR-T cell.

[0026] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus and one or more cytopenias, the method comprising, administering to said subject a therapeutically effective amount of an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand, wherein the antigen binding domain (ABD) of the CAR of the orthogonal CAR-T cell selectively binds to at least one human B cell antigen, and wherein the orthogonal IL2 ligand selectively binds to the extracellular domain of the orthogonal receptor, further wherein the subject is not treated with a lymphodepletion treatment regimen within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day prior to administering of the orthogonal CAR-T cell.

[0027] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus and one or more cytopenias, the method comprising, administering to said subject a therapeutically effective amount of an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand, wherein the antigen binding domain (ABD) of the CAR of the orthogonal CAR-T cell selectively binds to at least one human B cell antigen, and wherein the orthogonal IL2 ligand selectively binds to the extracellular domain of the orthogonal receptor wherein: (a) the orthogonal CAR-T cell is an autologous orthogonal CD19 CAR-T cell wherein the CD19 CAR-T cell expresses a CD19 CAR having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 61 and an orthogonal hCD122 receptor having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to the amino acid sequence of SEQ ID NO: 39; (b) the orthogonal IL2 ligand is a compound of the structure: (PEG)-(linker)n-(PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 41). wherein n = 0 (absent) or 1 (present) and PEG is 40kDa-PEG;Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT (c) the CD19 CAR-T cell is administered at a dose of from 1x105cells per kg of bodyweight to about 3x106cells per kg of bodyweight, alternatively from about 2x105to about 3x106cells per kg of bodyweight, alternatively from about 2x105to about 2x106cells per kg of bodyweight, alternatively from about 3x105to about 2x106cells per kg of bodyweight, alternatively from about 5x105to about 1.5x106cells per kg of bodyweight, alternatively from about 5x105to about 1x106cells per kg of bodyweight, alternatively from about 1x106to about 2x106cells per kg of bodyweight, alternatively from about 1x106to about 1.3x106cells per kg of bodyweight, alternatively from about 1x106to about 1.5x106cells per kg of bodyweight, alternatively from about 1.3x106to about 1.7x106cells per kg of bodyweight, or alternatively from about 1.5x106to about 2x106cells per kg of bodyweight, alternatively about 1.2x106cells per kg of bodyweight, alternatively about 1.3x106cells per kg of bodyweight, alternatively about 1.4x106cells per kg of bodyweight, alternatively about 1.5x106cells per kg of bodyweight, alternatively about 1.6x106cells per kg of bodyweight, alternatively about 1.7x106cells per kg of bodyweight, alternatively about 1.8x106cells per kg of bodyweight, alternatively about 1.9x106cells per kg of bodyweight, alternatively about 2.0x106cells per kg of bodyweight; (d) the orthogonal IL2 ligand is administered to the subject in one or more doses of approximately 0.05 mg / kg to 1 mg / kg; alternatively from approximately 0.05 mg / kg to 0.7 mg / kg; alternatively of approximately 0.05 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.3 mg / kg wherein the orthogonal wherein is administered daily, twice weekly, thrice weekly, weekly, about every 10 days, or about every 14 days over a period of at least one month, optionally at least 2 months, optionally at least 3 months; (e) the subject has not been treated with a lymphodepletion treatment regimen within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day, prior to the administering of the orthogonal CAR-T cell; and (f) the subject exhibits an improvement in one or more lupus efficacy criteria in the subject compared to the subject prior to the initiation of treatment, wherein the lupus efficacy criteria are selected from the group consisting of: (a) remission of lupus per definition of remission in SLE (DORIS) criteria (van Vollenhoven, et al. (2021) Lupus Sci Med. 8(1):e000538); (b) lupus low disease activity state (LLDAS) attainment rate (Franklyn (2016) Ann Rheum Dis.75(9):1615-21.; (c) improvement in SLEDAI-2K scores; (d) improvement inAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT BILAG individual system scores; (e) improvement in urine protein creatinine ratio (UPCR); (f) a decrease of ≥ 50% of Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) activity score (Albrecht, et al. (2005) J Invest Dermatol 125(5): 889- 894) in subjects with baseline activity score ≥ 10 prior to initiation of treatment; (g) complete renal response (CRR) defined as a UPCR < 0.5 and eGFR ≥ 60 mL / min per 1.73 m2or no confirmed eGFR decrease > 20%) in subjects with having a UPCR >1.0 mg / mg prior to the initiation of treatment; (h) decrease in total tender and swollen joint counts in subjects with ≥ 4 tender and swollen joints; (i) time to occurrence of a moderate or severe BILAG flare; (j) time to occurrence of a mild / moderate or severe flare as defined by SELENA-SLEDAI Flare index; (k) reduction in levels of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies; (l) reduction in levels of anti-nuclear antibodies (ANAs); (m) an ANA titer less than 1:80; (n) reduction in levels of anti-Smith antibodies; (o) reduction in level of complement factor C3, (p) reduction in level of complement factor C3C4) ; (q) improvement in HAQ-DI, SF-36; (r) improvement in FACIT- Fatigue Scale version 4 (Tennant, K. (2015) Supportive Care in Cancer 23(5):1355-1364); (s) reduction in disease activity as assessed by the UCSF / JHU Lupus Activity Index (LAI); reduction in disease activity as assessed by the SLE Disease Activity Index (SLEDAI); reduction in disease activity as assessed by the Systemic Lupus Activity Measure (SLAM) (Liang, et al (1989) Arthritis Rheum.32(9):1107-18) or SLAM-R (Bae, et al (2001) Lupus 2001; 10: 405-9) and the reduction in disease activity as assessed by the British Isles Lupus Assessment Group (BILAG) Activity Index wherein the improvement in the one more efficacy criteria occurs within 14 days, 30 days, 60 days, 90 days, 12 weeks, alternatively within 24 weeks following administration of the orthogonal CAR-T cells, alternatively with 48 weeks following administration of the orthogonal CAR-T cells, or alternatively within 96 weeks following administration of the orthogonal CAR-T cells. BRIEF DESCRIPTION OF THE FIGURES

[0028] Figure 1 of the attached drawing provides data from an experiment as more fully described in Example 4 relating to the administration of mouse orthogonal CD19 CAR-T cells (moCD19CAR-T) to C57 / B6 mice that had not been subjected to a lymphodepleting treatment regimen alone (moCD19CAR-T+PBS) and in combination with a mouse orthogonal IL2 ligand (moCD19CAR-T+3A1). As illustrated Figure 1, upper panel, the administration of the mouse orthogonal CD19 CAR-T cell (moCD19CAR-T) in combination with an murine orthogonal IL2 ligand (moCD19CAR-T+3A1) provided durable suppression of B cells (CD19+ cells, vertical axis) over time (horizontal axis) in contrast to treatment with orthogonal murine CD19 CAR-TAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT cells in the absence of the orthogonal mIL2 ligand (i.e., moCD19CAR-T + PBS) or an anti CD20 antibody. Figure 1, lower panel, provides data demonstrating that the circulating levels of the orthogonal murine CD19 CAR T cell (moCD19CAR-T), as determined by the presence of the Thy 1.1+ marker (vertical axis), is substantially increased and maintains a significant concentration over time (horizontal axis) when in administered in combination with the orthogonal mIL2 ligand (moIL2, 3A1) (open squares) when compared to the administration of orthogonal murine CD19 CART in the absence the orthogonal mIL2 ligand (moCD19CAR-T + PBS) (closed circles) in as more fully described in the Example 4. This data demonstrates that the administration of an orthogonal CAR-T cell to a mammalian subject that has not undergone a lymphodepleting treatment regimen in combination with an orthogonal ligand (moIL2, 3A1) wherein the ABD of the CAR is selectively binds to a B-cell antigen (CD19) and the orthogonal ligand selectively binds to the extracellular domain of an orthogonal receptor (moCD122) provides effective depletion of B-cells over an extended period of time greater than the analogous CAR-T cell alone or an anti-CD20 antibody and is able to maintain an elevated circulating level of the orthogonal CAR-T cell over an extended period of time greater than CAR-T cell in the absence of treatment in combination with an orthogonal ligand.

[0029] Figure 2, upper panel, of the attached drawing provides data showing levels of B cells (CD19+ cells, vertical axis) over time (horizontal axis) in mice in response to the treatment of a mouse orthogonal CAR-T cells (moCD19CAR-T) at various concentrations alone and in combination with the orthogonal murine IL2 ligand (moIL2) in accordance with the Example 4. Figure 2, lower panel, demonstrates that the circulating levels of the orthogonal murine CD19 CAR T cell (moCD19CAR-T), as determined by the presence of the Thy 1.1+ marker (vertical axis) over time (horizontal axis) in mice in response to the treatment of a mouse orthogonal CAR-T cells (moCD19CAR-T) at various concentrations alone and in combination with the orthogonal murine IL2 ligand (moIL2) as more fully described in Example 4.

[0030] Figure 3 upper panel illustrates the level of B cells (CD19+ cells, vertical axis) over time (horizontal axis) present in the MRL / lpr mouse when treated with the mouse orthogonal CAR-T cell (moCD19CAR-T) in combination with an orthogonal ligand (moIL2) and the mouse orthogonal CD19 CAR-T without the orthogonal mIL2 ligand (i.e., with PBS) in accordance with the Example 5. Figure 3 lower panel, illustrates the level of orthogonal murine CD19 CART cells (moCD19CAR-T) as determined by the presence of the Thy 1.1+ marker (vertical axis) over time (horizontal axis) present in the MRL / lpr mouse in response to treatment with a mouse orthogonal CAR-T cell (moCD19CAR-T) in combination with an orthogonal ligand (moIL2,Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT 3A1) and the orthogonal murine CD19 CART without the orthogonal mIL2 ligand (i.e., with PBS) as more fully described in Example 5.

[0031] Figure 4, upper panel, of the attached drawings illustrates the level of antinuclear antibodies (ANAs) as determined by optical density at a wavelength of 450 nm (vertical axis) present in the MRL / lpr mouse over time (horizontal axis) in response to treatment with a mouse orthogonal CAR-T cell (moCD19CAR-T) in combination with an mouse orthogonal ligand (moIL2, 3A1) and the orthogonal murine CD19 CAR-T without the mouse orthogonal mIL2 ligand (i.e., with PBS) in accordance with the Example 6. Figure 4 lower panel B of the attached drawings illustrates the level of anti-double stranded DNA antibodies (anti-dsDNA) as determined by optical density at a wavelength of 450 nm (vertical axis) present in the MRL / lpr mouse over time when treated with the mouse orthogonal CAR-T cell (moCD19CAR-T) in combination with an orthogonal ligand and the orthogonal murine CD19 CART (moCD19CAR- T) without the orthogonal mIL2 ligand (i.e., with PBS) in accordance with the Example 6. This data demonstrates that the administration of an orthogonal CAR-T cell (moCD19-CAR) to a mammalian subject suffering from lupus that has not undergone a lymphodepleting treatment regimen in combination with an orthogonal ligand (moIL2, 3A1) wherein the ABD of the CAR is selectively binds to a B-cell antigen (CD19) and the orthogonal ligand selectively binds to the extracellular domain of the orthogonal receptor (moCD122) provides enhanced reduction of anti- nuclear antibodies (upper panel) and anti-dsDNA antibodies (lower panel) relative to administration of the CAR-T cell alone.

[0032] Figure 5, upper panel, of the attached drawings illustrates the level of CD19+ B cells (vertical axis) over time (horizontal axis) present in the NZB / W mouse model when treated with the mouse orthogonal CAR-T cell (moCD19CAR-T) in combination with an orthogonal ligand (moIL2) and the mouse orthogonal CAR-T cell (moCD19CAR-T) without the orthogonal mIL2 ligand (i.e., with PBS) in accordance with the Example 7. Figure 5, lower panel, of the attached drawings illustrates the level of orthogonal murine CD19 CART cells (moCD19CAR-T) present in the NZB / W mouse model as determined by the Thy1.1+ marker (vertical axis) over time (horizontal axis) when treated with the mouse orthogonal CAR-T cell (moCD19CAR-T) in combination with an orthogonal ligand and the orthogonal murine CD19 CART without the orthogonal mIL2 ligand (i.e., with PBS) in accordance with the Example 7.

[0033] Figure 6 upper panel of the attached drawings illustrates the levels of anti-dsDNA antibodies (vertical axis) present in the NZB / W mouse model when treated with the mouse orthogonal CAR-T cell (moCD19CAR-T) in combination with a mouse orthogonal IL2 ligand (moIL2) and the orthogonal murine CD19 CART (moCD19CAR-T) without the orthogonalAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT mIL2 ligand (i.e., with PBS) in accordance with the Example 7. Figure 6, lower panel, of the attached drawings illustrates the levels of anti-nuclear antibodies (ANAs, vertical axis) present in the NZB / W mouse model when treated with the mouse orthogonal CAR-T cell (moCD19CAR- T) in combination with a mouse orthogonal IL2 ligand (moIL2) and the orthogonal murine CD19 CART (moCD19CAR-T) without the orthogonal mIL2 ligand (i.e., with PBS) in accordance with the Example 7. This data demonstrates that the administration of an orthogonal CAR-T cell (moCD19-CAR) to a mammalian subject suffering from lupus that has not undergone a lymphodepleting treatment regimen in combination with an orthogonal ligand (moIL2, 3A1) wherein the ABD of the CAR is selectively binds to a B-cell antigen (CD19) and the orthogonal ligand selectively binds to the extracellular domain of the orthogonal receptor (moCD122) provides a moIL2 dose-dependent reduction of anti-nuclear antibodies (lower panel) and anti- dsDNA antibodies (anal panel) relative to administration of the CAR-T cell alone.

[0034] Figure 7 of the attached drawings provides a graphical representation of the levels of urine protein (vertical axis) present in the various treatment groups (horizontal axis) of the NZB / W mouse lupus model when treated with PBS (p), 4x106mouse orthogonal CD19 CAR-T cells (moCD19CAR-T) alone (Ä), 4x106moCD19CAR-T or in combination with a mouse orthogonal IL2 ligand (moIL2) (¡), 1.25x106moCD19CAR-T or in combination moIL2 (s), and the parental NZB (£) and NZW(u) mice as more fully described in Example 8. Protein in the urine was determined in 10 month NZB / W old mice, 9 weeks after the start of treatment. This data demonstrates that the administration of an orthogonal CAR-T cell (moCD19-CAR) to a mammalian subject suffering from lupus that has not undergone a lymphodepleting treatment regimen in combination with an orthogonal ligand (moIL2, 3A1) wherein the ABD of the CAR is selectively binds to a B-cell antigen (CD19) and the orthogonal ligand selectively binds to the extracellular domain of the orthogonal receptor (moCD122) results in a reduction of urine protein levels comparable to that of a normal mammal (NZB and NZW parental) and superior to the administration of the CAR-T cell alone.

[0035] Figure 8 of the attached drawings provides a graphical representation of the percentage of CD25+CD127- Tregs in in a peripheral blood sample (vertical axis) in response to treatment with the multiple indicated treatment parameters (horizontal axis) in the NZB / W mouse lupus model as more fully described in Example 9 herein.

[0036] Figure 9 of the attached drawings provides a graphical representation of the percentage of CD80+ memory B cells as a percentage of total B cells in a peripheral blood sample (vertical axis) in response to treatment with the multiple indicated treatment parameters (horizontal axis) in the NZB / W mouse lupus model as more fully described in Example 9 herein.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0037] Figure 10 of the attached drawings provides a graphical representation of the percentage of CD138+ plasmablast cells as a percentage of total B cells in a peripheral blood sample (vertical axis) in response to treatment with the multiple indicated treatment parameters (horizontal axis) in the NZB / W mouse lupus model as more fully described in Example 9 herein.

[0038] Figure 11 of the attached drawings provides a graphical representation of the B cells per microliter (vertical axis) in the peripheral blood (Figure 11, upper left panel), B cells per 106lymph node cells (vertical axis) in the lymph nodes (Figure 11, upper right panel), B cells per 106spleen cells (vertical axis) in the spleen (Figure 11, lower left panel) and B cells per 106kidney cells (vertical axis) in the kidney (Figure 11, lower right panel) in response to treatment with the multiple indicated treatment parameters (horizontal axis) in the NZB / W mouse lupus model as more fully described in Example 10 herein.

[0039] Figure 12 of the attached drawings, left panel, provides a graphical representation of the level of moCD19 CAR-T cells per microliter of blood (vertical axis) in the peripheral blood in response to treatment with various treatment parameters (horizontal axis) in the NZB / W mouse lupus model as more fully described in Example 10 herein. Figure 12, of the attached drawings, right panel, provides a graphical representation of the level of moCD19 CAR-T cells per 106kidney cells (vertical axis) in the kidney in response to treatment with the multiple indicated treatment parameters (horizontal axis) in the NZB / W mouse lupus model as more fully described in Example 10 herein. DETAILED DESCRIPTION

[0040] To facilitate the understanding of present disclosure, certain terms and phrases are defined below as well as throughout the specification. The definitions provided herein are non- limiting and should be read in view of the knowledge of one of skill in the art.

[0041] Before the present methods and compositions are described, it is to be understood that this invention is not limited to a particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither orAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0043] The term “about” refers to a value that is plus or minus 10% of a numerical value described herein, such as plus or minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of numerical value described herein. The term “about” also applies to all numerical ranges described herein. All values described herein are understood to be modified by the term “about” whether or not the term “about” is explicitly recited in reference to a given value.

[0044] It should be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the peptide” includes reference to one or more peptides and equivalents thereof, e.g., polypeptides, known to those skilled in the art, and so forth.

[0045] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. All publications, patents, published patent applications, GenBank accession numbers and UniProt reference numbers mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0046] Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric. Standard abbreviations are used, including the following: bp = base pair(s); kb = kilobase(s); pl = picoliter(s); s or sec = second(s); min = minute(s); h or hr = hour(s); AA or aa = amino acid(s); kb = kilobase(s); nt = nucleotide(s); pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar; mM = millimolar; M = molar; kDa = kilodalton; i.m. = intramuscular(ly); i.p. = intraperitoneal(ly); SC or SQ = subcutaneous(ly); QD = daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = not statistically significant; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSAAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT = mouse serum albumin; DMEM = Dulbeco’s Modification of Eagle’s Medium; EDTA = ethylenediaminetetraacetic acid.

[0047] It will be appreciated that throughout this disclosure reference is made to amino acids according to the single letter or three letter codes. For the reader’s convenience, the single and three letter amino acid codes are provided in Table 1. Table 1. Naturally Occurring Amino Acids and Abbreviations n

[0048] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)). The scientificAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vols.1-2, John Wiley and Sons, Inc., NY).

[0049] Nomenclature of Amino Acid Substitutions and Deletions: The present disclosure provides variant polypeptides comprising amino acid substitutions relative to the wild-type or parent polypeptide. The following nomenclature is used herein to refer to substitutions, deletions, or insertions. Residues may be designated herein by the one-letter or three-letter amino acid code of the naturally occurring amino acid found in the wild-type molecule. Amino acid sequences of polypeptides are presented as from amino terminus to carboxy terminus. When a formula representing a polypeptide is provided (e.g., with multiple domains or subdomains) the formula is understood as representing the polypeptide from amino to carboxy terminus. Similarly, all nucleic acid sequences are presented from 5' to 3'.

[0050] hIL2 Residue Numbering: In the present disclosure, the numbering of amino acid residues of human IL2 is made in reference to the number of the residue of the mature wild type human IL2 (wt hIL2) as provided in (SEQ ID NO: 12). mIL2 Residue Numbering: In the present disclosure, the numbering of amino acid residues of mouse IL2 is made in reference to the number of the residue of the mature wild-type mouse IL2 (wt mIL2) as provided in (SEQ ID NO: 14). hCD122 ECD Residue Numbering: In the present disclosure, the numbering of amino acid residues of the extracellular domain of human CD122 is made in reference to the number of the residue of the ECD of the mature wild type human CD122 as provided in (SEQ ID NO: 3). hCD122 Residue Numbering: In the present disclosure, the numbering of amino acid residues of the human CD122 is made in reference to the number of the residue of the mature wild type human CD122 as provided in (SEQ ID NO: 2). mCD122 ECD Residue Numbering: In the present disclosure, the numbering of amino acid residues of the extracellular domain of mouse CD122 (mCD122) is made in reference to the number of the residue of the ECD of the mature wild type mouse CD122 as provided in (SEQ ID NO: 8). mCD122 Residue Numbering: In the present disclosure, the numbering of amino acid residues of the mouse CD122 (mCD122) is made in reference to the number of the residue of the mature wild-type mouse CD122 as provided in (SEQ ID NO: 7).

[0051] When referencing amino acid substitutions in a variant or mutein of a reference polypeptide, the amino acid substitution is designated by the one letter amino acid code followed by position of the residue in the reference polypeptide followed by the one letter amino acid code of the amino acid which is substituted at the position. For example, a hIL2 variant polypeptideAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT having an amino acid substitution of the naturally occurring leucine (L) residue at position 18 of the wt hIL2 reference sequence with an arginine (R) residue is denoted by the abbreviation “L18R”. A deletion of an amino acid reside is referred to as “des” or the symbol “^” followed by the amino acid residue and its position in reference sequence. For example, a hIL2 variant comprising a deletion of the N-terminal alanine (A) residue at position 1 of the mature wild-type human IL2 reference sequence may be referred to as “des-Ala1.” Additionally, in certain instances herein, a sequence is referred to as a “mature” molecule lacking a signal sequence so as to distinguish the polypeptide from the precursor molecule containing the signal peptide.

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated, the following terms are intended to have the meaning set forth below. Other terms are defined elsewhere throughout the specification.

[0053] Activate: As used herein the term “activate” is used in reference to a receptor or receptor complex to reflect a biological effect, directly and / or by participation in a multicomponent signaling cascade, arising from the binding of an agonist ligand to a receptor responsive to the binding of the ligand. The term activate is also used in reference to a cell that expresses a receptor wherein one more biological activities of the cell are modulated (e.g., T cell activation) in response to binding of a ligand for such receptor.

[0054] Activity: As used herein, the term “activity” is used with respect to a molecule to describe a property of the molecule with respect to a test system (e.g., an assay) or biological or chemical property (e.g., the degree of binding of the molecule to another molecule) or of a physical property of a material or cell (e.g., modification of cell membrane potential). Examples of such biological functions include but are not limited to catalytic activity of a biological agent, the ability to stimulate intracellular signaling, gene expression, cell proliferation, and the ability to modulate immunological activity such as inflammatory response. “Activity” is typically expressed as a level of a biological activity per unit of agent tested such as [catalytic activity] / [mg protein], [immunological activity] / [mg protein], international units (IU) of activity, [STAT3 phosphorylation] / [mg protein], [STAT5 phosphorylation] / [mg protein], [proliferation] / [mg protein], plaque forming units (pfu), etc. As used herein, the term proliferative activity refers to an activity that promotes cell proliferation and replication of a cell, including dysregulated cell division.

[0055] Administer / Administration / Administering: The terms “administration,” “administer,” and “administering” are used interchangeably herein to refer the act of contacting a subject, including contacting a cell, tissue, organ, or biological fluid of the subject in vitro, in vivo or exAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT vivo with an agent. Administration of an agent may be achieved through any of a variety of art recognized methods including but not limited to the topical administration, intravascular injection (including intravenous or intraarterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, transdermal, transmucosal, iontophoretic delivery, intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhalation (e.g., respiratory inhalers including dry-powder inhalers), intraocular injection, intraabdominal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intracerebroventricular injection (ICVI), and the like. The term “administration” includes contact of an agent to the cell, tissue or organ as well as the contact of an agent to a fluid, where the fluid is in contact with the cell, tissue or organ.

[0056] Agonist: As used herein, the term “agonist” refers a first agent that specifically binds a second agent (“target”) and interacts with the target to cause or promote an increase in the activation of the target. In some instances, agonists are activators of receptor proteins that modulate cell activation, enhance activation, sensitize cells to activation by a second agent, or up-regulate the expression of one or more genes, proteins, ligands, receptors, biological pathways, that may result in modulation of cell proliferation or pathways or the cell cycle. In some embodiments, an agonist is a modified form of a cognate ligand that binds to its cognate receptor and alters the state of the cognate receptor in a biological response that mimics the biological effect of the interaction of the naturally occurring cognate ligand with its cognate receptor. The term “agonist” includes partial agonists, full agonists and superagonists. An agonist may be described as a “full agonist” when such agonist which leads to a substantially full biological response (i.e., the response associated with the naturally occurring ligand / receptor binding interaction) induced by receptor under study, or a partial agonist. A “partial agonist” elicits only partial activation of the receptor relative to a full agonist. A “superagonist” can produce a maximal response greater than the endogenous agonist for the target receptor, and thus has an activity of more than 100% of the native ligand.

[0057] Corresponding To: As used herein, the terms “correspondence” or “corresponding to” in the context of an amino acid or nucleic acid sequence refers to the equivalent position of a reference sequence that is aligned with one or more other sequences to maximize the percentage of sequence identity. As used herein, the term “corresponding to” is used in the context of generating amino acid substitutions of a mutein from a first species to generate a mutein of another species such as muteins of human sequences for generating murine surrogate muteins.

[0058] Derived From: As used herein, the term “derived from” is used in the context of a variant polypeptide or nucleic acid to indicate that a variant polypeptide or nucleic acid has aAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT sequence that is based on but differs from that of a reference polypeptide or nucleic acid. The term derived and is not meant to be limiting as to the source or method by which the variant protein or nucleic acid is made.

[0059] GSA Linker: As used herein the term "GSA linker" refers collectively to polypeptides from 1-40 (alternatively 2-20, alternatively 5-20, alternatively 10-20) amino acids comprised of glycine (G) polymers, glycine-alanine (GA) polymers, alanine-serine (AS) polymers, glycine- serine (GS) polymers and glycine-serine-alanine (GSA) polymers, referred to collectively herein as "GSA linkers". In some embodiments, the GSA linker is a multimer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of individual linker sequences. In some embodiments, the GSA linker is a glycine polymer having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 ,18, 19, or 20 amino acids comprised of glycine residues. In some embodiments, the GSA linker is a glycine- serine polymer having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 ,18, 19, or 20 amino acids comprised of amino acids selected from the group consisting of glycine and serine. In some embodiments, the GSA linker is a polypeptide having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 ,18, 19, or 20 amino acids comprised of amino acids selected from the group consisting of glycine, serine, and alanine. In some embodiments, the GSA linker is a polymer of the structure (GGGGSm)n, (GGGSm)n, (GGGAm)n and (GGGGAm)n, and combinations thereof, where m, n, and o are each independently selected from 1, 2, 3 or 4. Exemplary glycine-serine linkers include but are not limited monomers, homopolymers or heteropolymers of: GGGGS (referred to as “G4S”), GGGGA (referred to as “G4A”), GGGS (referred to as “G3S”) and GGGA (referred to as “G3A”). In the construction of such GSA linkers, in some embodiments it is preferred to avoid inclusion of repeated “GSG” sequences which potentially may provide introduction of a non-naturally occurring glycosylation site. In some embodiments, the GSA linker a polypeptide of SEQ ID NO: 34.

[0060] Identity: The term “identity,” as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in each of the two molecules is occupied by the same amino acid or nucleotide, then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res.25: 3389-3402. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithmAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W of the query sequence, which either match or satisfy some positive-valued threshold score “T” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (the reward score for a pair of matching residues; always >0) and “N” (the penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: (a) the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or (b) the end of either sequence is reached. The BLAST algorithm parameters “W”, “T”, and “X” determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) functions similarly but uses as defaults a word size (“W”) of 28, an expectation (“E”) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, (1989) PNAS(USA) 89:10915-10919).

[0061] In Combination With: As used herein, the term “in combination with” as used in reference to the administration of multiple agents to a single subject refers to the administration of a first agent (e.g., an orthogonal CAR-T cell) and a second agent (e.g., an orthogonal ligand), optionally further administering an additional (i.e., third, fourth, fifth, etc.) supplementary agent to a subject (for example but not limited to, an adjuvant), simultaneously, contemporaneously or sequentially. The term “in combination with” shall also understood to apply to the situation where a first agent and a second agent are co-formulated in single pharmaceutically acceptable formulation and the co-formulation is administered to a subject. A first agent is considered to be administered in combination with a second agent if the first and second agents are administered simultaneously, contemporaneously, or sequentially. A first agent is administered “simultaneously” with a second agent if first and second agents are administered within about 30 minutes of each other. A first agent is administered “contemporaneously” with a second agent if first and second agents are administered within about 24 hours of each another, alternatively within about 12 hours of each other, alternatively within about 6 hours of each other, alternatively within about 2 hours of each other, or alternatively within about 60 minutes of each other.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0062] When administered sequentially, administration of the first agent may provide a therapeutic effect over an extended time and the administration of the second agent administered while the therapeutic effect of the first agent persists in the subject such that the second agent is considered to be administered in combination with the first agent, even though the first agent may have been administered at a point in time significantly distant (e.g. days or weeks) from the time of administration of the second agent. For example, in the case of molecules that have been designed to provide an extended duration of action in a subject (for example, by covalent linkage of the active agent to a carrier molecule) or agents that exhibit extended in vivo durations of action (e.g. a CAR-T cell), the administration of the first agent and the second agent may be separated by a significant period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or longer) may result in the maintenance of a therapeutically effective amount of the first agent over a period of weeks such that the administration of the second agent at time point days or weeks after the administration of the first agent such that the second agent would be considered to be administered in combination with the first agent. In some embodiments, the biological effect of the administration of a first therapeutic agent may persist for a period of time beyond the point where there is a detectable level of the first therapeutic agent in the subject. In certain instances, a first agent and a second agent(s) are administered sequentially, e.g., where one agent is administered to the subject at least 24 hours prior to the administration of the second agent. When a first agent and second agent are administered to a subject sequentially, the first agent is considered to be administered “in combination with” with the second agent if at least 10%, alternatively at least 20%, alternatively at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90%, alternatively at least 95% of the biological effect of the first agent persists in the subject at the time of administration of the second agent.

[0063] An orthogonal CAR-T cell expressing an orthogonal receptor comprising an orthogonal hCD122 ECD (e.g., SEQ ID NO:39) is considered to be administered in combination with an orthogonal hIL2 ligand (e.g. a human IL2 orthogonal ligand comprising the polypeptide of SEQ ID NO: 41) if the biological effect resulting from the administration of the orthogonal CAR-T cell persists in the subject at the time of administration of the orthogonal hIL2 ligand such that the therapeutic effects of the orthogonal CAR-T cell and orthogonal IL2 ligand overlap, whether or not there is a detectable level of the orthogonal CAR-T cell in the subject at the time of administration of the orthogonal IL2 ligand. Conversely, a orthogonal IL2 ligand is considered to be administered in combination with an orthogonal CAR-T cell if the biological effect resulting from the administration of the orthogonal hIL2 ligand persists in the subject at the time ofAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT administration of the orthogonal CAR-T cell such that the therapeutic effects of the orthogonal CAR-T cell and orthogonal hIL2 ligand overlap, whether or not there is a detectable level of the orthogonal hIL2 ligand remaining in the subject at the time of administration of the orthogonal CAR-T cell. The determination of whether that therapeutic effect of the first agent (e.g., orthogonal CAR-T cell or orthogonal hIL2 ligand) persists after the agent is no longer detectable can be established through the upregulation or down-regulation of biological markers that are characteristically modulated in response to the agent. One of skill in the art is capable of performing pharmacokinetic studies to determine the in vivo duration of action of the orthogonal CAR-T cells and orthogonal hIL2 ligand molecules described herein. Studies in primates, e.g., cynomolgus monkeys, chimpanzees, rhesus monkeys, can be used to provide information relating to the duration of action and toxicity indicative of the response in human subjects. In one embodiment, a therapeutically effective dose of the orthogonal hIL2 ligand in combination with an orthogonal CAR-T cell is established by evaluation of pharmacokinetic data, indicators of response and / or toxicity and other factors known to the clinician.

[0064] Isolated: As used herein the term “isolated” when used in reference to a molecule that, if naturally occurring, is in an environment different from that in which it naturally occurs. “Isolated” is meant to include molecule that are within samples that are substantially enriched for the molecule of interest and / or in which the molecule of interest is partially or substantially purified. Where the molecule is not naturally occurring, “isolated” indicates that the molecule has been separated from an environment in which it was synthesized. For example, a polypeptide may be isolated from a recombinant cell culture comprising cells engineered to express the polypeptide or by a solution resulting from solid phase or cell free synthesis.

[0065] Ligand: As used herein, the term “ligand” is used in its conventional sense to refer to a molecule that specifically binds a receptor (e.g., the extracellular domain of a receptor) and wherein such binding of the ligand to the receptor results in modulation of the receptor sufficient to effect a change in the activity of the receptor or results in a response (e.g., intracellular signaling, cellular activation and / or proliferation) in a cell that expresses the receptor. In some embodiments, a ligand is an agonist, partial agonist or superagonist of the receptor to which it exhibits specific binding.

[0066] Modulate: As used herein, the terms “modulate,” “modulation,” and the like refer to the ability of an agent, for example, a test agent, to cause a response, either positive or negative or directly or indirectly, in a system, including a biological system, or biochemical pathway.

[0067] Nucleic Acid: The terms “nucleic acid,” “nucleic acid molecule,” “polynucleotide,” and the like are used interchangeably herein to refer to a polymeric form of nucleotides of anyAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers and the like.

[0068] Operably Linked: The term “operably linked” is used herein to refer to the relationship between molecules, typically polypeptides or nucleic acids, which are arranged in a construct such that the functions of the component molecules are retained although the operable linkage may result in the modulation of the activity, either positively or negatively, of the individual components of the construct. For example, the operable linkage of a polyethylene glycol (PEG) molecule to a wild-type protein may result in a construct where the biological activity of the protein (e.g., Emax) is diminished relative to the to the wild-type molecule, however the two are nevertheless considered operably linked. When the term “operably linked” is applied to the relationship of multiple nucleic acid sequences, the multiple nucleic acid sequences when combined into a single nucleic acid molecule that, for example, when introduced into a cell using recombinant technology, provides a nucleic acid which is capable of effecting the transcription and / or translation of a particular nucleic acid sequence in a cell. For example, the nucleic acid sequence encoding a signal sequence may be considered operably linked to DNA encoding a polypeptide if it results in the expression of a preprotein whereby the signal sequence facilitates the secretion of the polypeptide; a promoter or enhancer is considered operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is considered operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, in the context of nucleic acid molecules, the term "operably linked" means that the nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader or associated subdomains of a molecule, contiguous and in reading phase. However, certain genetic elements such as enhancers may function at a distance and need not be contiguous with respect to the sequence to which they provide their effect but nevertheless may be considered operably linked.

[0069] Polypeptide: As used herein the terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones. The term polypeptide include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without N-terminal methionine residues; fusion proteins with amino acid sequences that facilitate purification such as chelating peptides; fusion proteins withAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT immunologically tagged proteins; fusion proteins comprising a peptide with immunologically active polypeptide fragment (e.g., diphtheria toxin or tetanus toxin fragments) and the like.

[0070] Prevent: As used herein the terms “prevent,” “preventing,” “prevention,” and the like refer to a course of action initiated with respect to a subject prior to the onset of a disease, disorder, condition or symptom thereof so as to prevent, suppress, inhibit or reduce, either temporarily or permanently, a subject’s risk of developing a disease, disorder, condition or the like (as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof. A course of action to prevent a disease, disorder or condition in a subject is typically applied in the context of a subject who is predisposed to developing a disease, disorder, or condition due to genetic, experiential, or environmental factors of developing a particular disease, disorder, or condition. In certain instances, the terms “prevent,” “preventing,” “prevention” are also used to refer to the slowing of the progression of a disease, disorder, or condition from an existing state to a more deleterious state. In some instances, “prevent” is used in the context of prevention of the recurrence of a disease or symptom thereof wherein a prior course of therapy may have partially or completely eliminated the evidence of the disease as measured by conventional clinical testing.

[0071] Receptor: As used herein, the term “receptor” refers to a polypeptide having a domain that specifically binds a ligand that binding of the ligand results in a change to at least one biological property of the polypeptide. In some embodiments, the receptor is a cell membrane associated protein that comprises an extracellular domain (ECD) and a membrane associated domain which serves to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a membrane spanning polypeptide comprising an intracellular domain (ICD) and extracellular domain (ECD) linked by a membrane spanning domain referred to as a transmembrane domain (TM). In some instances, the binding of a ligand to the ECD of the receptor results in a conformational change in the receptor resulting in a measurable biological effect such as a change in the activity of the receptor or the binding affinity of the receptor for another protein. In some instances, where the receptor is a membrane spanning polypeptide comprising an ECD, TMD and ICD, the binding of a ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to the binding of the ligand to the ECD. In some embodiments, a receptor is a component of a multi-component complex that facilitates intracellular signaling. For example, the ligand may bind a cell surface receptor that is not associated with any intracellular signaling alone but upon ligand binding facilitates the formation of a heteromultimeric (including heterodimeric, heterotrimeric, etc.) or homomultimeric (including homodimeric, homotrimeric, homotetrameric, etc.) complex that results in a measurable biological effect in the cell such as activation of an intracellular signaling cascade (e.g., theAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT Jak / STAT pathway). In some embodiments, a receptor is a membrane spanning single chain polypeptide comprising ECD, TM and ICD domains wherein the ECD, TM and ICD domains are derived from the same or differing naturally occurring receptor variants or synthetic functional equivalents thereof (referred to herein as a "chimeric receptor").

[0072] Recombinant: As used herein, the term “recombinant” is used as an adjective to refer to the method by which a polypeptide, nucleic acid, or cell was modified using recombinant DNA technology. A “recombinant protein” is a protein produced using recombinant DNA technology and is frequently abbreviated with a lower case “r” preceding the protein name to denote the method by which the protein was produced (e.g., recombinantly produced human interleukin-2 is commonly abbreviated “rhIL2” or “rhIL-2”). Similarly, a cell is referred to as a “recombinant cell” if the cell has been modified by the incorporation (e.g., transfection, transduction, infection) of exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids and the like) using recombinant DNA technology. The techniques and protocols for recombinant DNA technology are well known in the art such as those found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0073] Response: The term “response,” for example, of a cell, tissue, organ, or organism, encompasses a quantitative or qualitative change in a evaluable biochemical or physiological parameter, (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzymatic activity, level of gene expression, rate of gene expression, rate of energy consumption, level of or state of differentiation) where the change is correlated with the activation, stimulation, or treatment, with or contact with exogenous agents or internal mechanisms such as genetic programming. In certain contexts, the terms “activation,” “stimulation,” and the like refer to cell activation as regulated by internal mechanisms, as well as by external or environmental factors, whereas the terms “inhibition,” “down-regulation,” and the like refer to the opposite effects. A “response” may be evaluated in vitro such as through the use of assay systems, surface plasmon resonance, enzymatic activity, mass spectroscopy, amino acid or protein sequencing technologies. A “response” may be evaluated in vivo quantitatively by evaluation of objective physiological parameters such as body temperature, bodyweight, blood pressure, results of X-ray or other imaging technology or qualitatively through changes in reported subjective feelings of well-being, depression, agitation, or pain. In some embodiments, the level of activation of T cells in response to the administration of a test agent may be determined by flow cytometric methods. In some methods, a response can be measured by determining the level of T cell expansion, STAT (e.g., STAT3, STAT5) phosphorylation, in accordance with methods well known in the art. ForAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT example, the response of a T cell expressing the IL2 receptor subunits to being contacted with an IL2 ligand may be determined by the levels of intracellular phospho-STAT5 (pSTAT5) by techniques well known in the art.

[0074] Specifically Binds: As used herein the term “specifically binds” refers to the degree of affinity for which a first molecule exhibits with respect to a second molecule. In the context of binding pairs (e.g., ligand / receptor) a first molecule of a binding pair is said to specifically bind to a second molecule of a binding pair when the first molecule of the binding pair does not bind in a significant amount to other components present in the sample. A first molecule of a binding pair is said to specifically bind to a second molecule of a binding pair when the affinity of the first molecule for the second molecule is at least two-fold greater, alternatively at least five times greater, alternatively at least ten times greater, alternatively at least 20-times greater, or alternatively at least 100-times greater, or alternatively at least 500-times greater or alternatively at least 1000-times greater than the affinity of the first molecule for other components present in the sample. Specific binding may be assessed using techniques known in the art including but not limited to competition ELISA assays, radioactive ligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting programs and competition binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET); liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid phase ligand binding assays (e.g., multiwell plate assays, on- bead ligand binding assays, on-column ligand binding assays, and filter assays)) and surface plasmon resonance assays (see, e.g., Drescher et al., (2009) Methods Mol Biol 493:323-343 with commercially available instrumentation such as the Biacore 8K, Biacore 8K+, Biacore S200, Biacore T200 (Cytiva, 100 Results Way, Marlborough MA 01752).

[0075] Subject: The terms “recipient,” “individual,” “subject,” and “patient,” are used interchangeably herein and refer to any mammal for whom, the opinion of a skilled artisan (e.g., physician or veterinarian), treatment is desired. As used herein, the term mammal any animal classified as a mammal, including humans, mice, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments the term “subject” refers to a human being. In some embodiments, the human being is a female human being. In some embodiments, the human being is a male human being.

[0076] Suffering From: As used herein, the term “suffering from” refers to a determination made by a physician or other medical practitioner with respect to a subject based on the available objective or subjective information accepted in the field for the identification of a disease, disorder or condition including but not limited to physical manifestations observable by the physician asAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT well as diagnostic tests including, X-ray, PET scans, CT-scans, conventional laboratory diagnostic tests (e.g., blood count, etc.), genomic data, protein expression data, immunohistochemistry, that the subject requires or will benefit from treatment. The term suffering from is typically used in conjunction with a particular disease state such as “suffering from lupus” refers to a subject which has been diagnosed by a physician as having lupus.

[0077] T cell: As used herein the term “T cell” is used in its conventional sense to refer to a lymphocyte that differentiates in the thymus, possess specific cell-surface antigen receptors, and include some that control the initiation or suppression of cell-mediated and humoral immunity and others that lyse antigen-bearing cells. In some embodiments the a "T cell" is a cell selected from the group consisting of naïve CD8+ T cells, cytotoxic CD8+ T cells, naïve CD4+ T cells, helper T cells, e.g., TH1, TH2, TH9, TH11, TH22, TFH; regulatory T cells, e.g., TR1, Tregs, inducible Tregs; memory T cells, e.g., central memory T cells, effector memory T cells, NKT cells and engineered variants of such T cells including but not limited to CAR-T cells, recombinantly modified TILs and TCR-engineered cells. As used herein the name of molecule followed by a plus sign is used to designate that the expresses that molecule (e.g., a “CD25+ cell" is a cell that expresses CD25).

[0078] Terminus / Terminal: As used herein in the context of the structure of a polypeptide, “N- terminus” (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while the terms “N-terminal” and “C- terminal” refer to relative positions in the amino acid sequence of the polypeptide toward the N- terminus and the C-terminus, respectively, and can include the residues at the N-terminus and C- terminus, respectively. As used herein in the context of nucleic acids, the “5’-terminus” (or “five- prime terminus”) and “3’-terminus” (or “carboxyl terminus”) refer to the extreme ends of the nucleic acid sequence, respectively, while the terms “5’ to” and “3’ to” refer to relative positions in the nucleic acid sequence of the polypeptide toward the 5’-terminus and the 3’-terminus, respectively, and can include the residues at the 5’-terminus and 3’-terminus, respectively.

[0079] Therapeutically Effective Amount: As used herein to the phrase “therapeutically effective amount” refers to the quantity of an agent when administered to a subject, either alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a treatment regimen comprises multiple doses, provides a positive effect on any quantitative or qualitative symptom, aspect, or characteristic of a disease, disorder or condition. A therapeutically effective amount can be ascertained by measuring one or more relevant physiological effects, and it may be adjusted in connection with a dosing regimen and in response to diagnostic analysis of the subject’s condition. The parameters for evaluation to determine a therapeutically effectiveAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT amount of an agent are determined by the physician using art accepted diagnostic criteria including but not limited to indicia such as age, weight, sex, general health, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computerized tomography, X-ray, and the like. Alternatively, or in addition, other parameters commonly assessed in the clinical setting may be monitored to determine if a therapeutically effective amount of an agent has been administered to the subject such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or characteristic of lupus, biomarkers increase in duration of survival, extended duration of progression free survival, extension of the time to progression, increased time to treatment failure, extended duration of event free survival, extension of time to next treatment, improvement objective response rate, improvement in the duration of response, complete response, partial response, stable disease, and the like that that are relied upon by clinicians in the field for the assessment of an improvement in the condition of the subject in response to administration of an agent. In one embodiment, a therapeutically effective amount is an amount of an agent when used alone or in combination with another agent provides a positive effect on any quantitative or qualitative symptom, aspect, or characteristic of a disease, disorder or condition. In one embodiment, a therapeutically effective amount is an amount of an agent when used alone or in combination with another agent provides a positive effect on any quantitative or qualitative symptom, aspect, or characteristic of a disease, disorder or condition and does not result in non- reversible serious adverse events in the course of administration of the agent to the mammalian subject.

[0080] Treat: The terms “treat,” “treating,” treatment,” and the like refer to a course of action (such as administering to the subject a pharmaceutical composition comprising an orthogonal IL2 ligand) in combination with a pharmaceutical composition comprising a orthogonal CAR-T cell, optionally in combination with one or more supplementary agents, that is initiated with respect to a subject in response to a diagnosis that the subject is suffering from a disease, disorder or condition (e.g., lupus)the course of action being initiated so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of: (a) the underlying causes of such disease, disorder, or condition afflicting a subject; and / or (b) at least one of the symptoms associated with the disease, disorder or condition (e.g. lupus). In some embodiments, treating includes a course of action taken with respect to a subject suffering from lupus where the course of action results in the inhibition (e.g., arrests or slows the development of the lupus and / or one or more associated symptoms thereof,), prevents the recurrence of lupus or a lupus flare or ameliorates one or more symptoms associated with the presence of lupus in the subject.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0081] Variant: The terms “variant,” “protein variant,” or “variant protein” or “variant polypeptide” are used interchangeably herein to refer to a polypeptide that has an amino acid sequence that differs from a parent or reference polypeptide by virtue of at least one amino acid modification, substitution, or deletion relative to the parent or reference amino acid sequence. In some embodiments, the parent or reference polypeptide may be a naturally occurring or wild-type (WT) polypeptide. In some embodiments, the variant polypeptide comprises from about one to about ten, alternatively about one to about eight, alternatively about one to about seven, alternatively about one to about five, alternatively about one to about four, alternatively from about one to about three alternatively from one to two amino acid modifications, substitutions, or deletions, or alternatively a single amino acid amino acid modification, substitution, or deletion compared to the parent or reference polypeptide. In some embodiments, a variant polypeptide has at least about 99%, alternatively at least about 98%, alternatively at least about 97%, alternatively at least about 95%, or alternatively at least about 90% sequence identity to the parent or reference polypeptide from which the variant is derived.

[0082] Wild-Type: The terms “wild-type” or “WT” or “native” are used interchangeably herein in reference to a polypeptide or nucleic acid sequence herein to refer to a polypeptide having amino acid sequence or a nucleotide sequence, respectively, that is found in nature, including allelic variations. In some instances, there are multiple “wild-type” variants of particular protein with a single species being accepted by those of skill in the art as the “canonical” sequence. Unless a specific naturally occurring form of the protein is specified, the term “wild-type” is used to refer to the canonical sequence. A wild-type molecule (e.g. a protein, polypeptide, gene or nucleotide coding sequence) has a structure that has not been modified by the hand of man.

[0083] It will be understood that individual embodiments, which are separately described herein for clarity and brevity, can be combined without limitation. Thus, the present disclosure includes one or more, or all, combinations of the embodiments described herein as if each and every combination was individually and explicitly disclosed. This also applies to any and all sub- combinations of the embodiments disclosed herein, such that the present disclosure includes one or more, or all, sub-combinations of the embodiments described herein as if each and every sub- combination was individually and explicitly disclosed. The Orthogonal IL2 Ligand / Orthogonal CD122 Receptor System

[0084] A significant hurdle to the effectiveness of CAR-T cell therapies is sustaining the duration of viability (commonly referred as "persistence") of the CAR-T cells in a subject following administration. Typically, CAR-T cells are observed to be depleted or inactivatedAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT within a comparatively short period of time following administration. As a result, the period of time where the concentration of CAR-T cells associated with therapeutic efficacy is maintained in a subject is comparatively short.

[0085] A variety of strategies have been employed, alone or in combination, in an effort to extend CAR-T cell persistence and maximize the "area under the curve" to achieve greater therapeutic efficacy of the CAR-T cells. As previously discussed, lymphodepletion is associated with upregulation of IL7 and IL15 which provides an initial support of CAR-T cells. However, this effect is transient and frequently expires within a few days of completion of the lymphodepleting regimen. In contrast, the administration of the orthogonal CAR-T cell in combination with the administration of the orthogonal IL2 ligand, results in specific activation and proliferation of the orthogonal CAR-T cell in the subject which may be maintained in the subject over an extended period of time by periodic administration of the orthogonal IL2 ligand. The orthogonal IL2 ligand may be administered to the subject over an extended period of time providing prolonged specific support to the orthogonal CAR-T cell.

[0086] Another strategy to improve the persistence of CAR-T cells has been the administration of high dose IL2. T cells, including engineered T cells, are activated and proliferate in response to IL2 and therefore provides a potential intervention to extend CAR-T cell persistence. In clinical practice, the administration of CAR-T cells is frequently followed by the administration of Proleukin®, a hIL2 analog exhibiting activity comparable to wild type human IL2. However, the administration of high dose Proleukin results in non-specific activation of cells resulting in significant systemic toxicity (including cytokine release syndrome or “cytokine storm”) arising from its indiscriminate and widespread proliferation and activation of immune cells. As the orthogonal ligand exhibits minimal binding to the wild-type CD122, the administration of an orthogonal IL2 ligand does not result in significant off-target activation of other CD122+ cells (e.g., T cells) in the subject. The lack of indiscriminate off-target activation of T cells enables the administration of the orthogonal IL2 ligand without triggering toxicities associated with high dose IL2 therapies such as Proleukin. Additionally, as the orthogonal CD122 receptor exhibits minimal binding to wild-type IL2, orthogonal cells are not substantially activated by endogenous wild-type hIL2 produced by the subject.

[0087] The orthogonal IL2 ligand / orthogonal CD122 receptor system as described in more detail herein below provides a means to achieve extended persistence of a therapeutically effective amount of the orthogonal CAR-T cells in the subject by the administration to the subject of one or more doses of the orthogonal ligand in combination with the administration of the population of orthogonal CAR-T cells. The present disclosure further provides a method of extending of anAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT active form (“persistence”) of an orthogonal CAR-T cell in vivo in a mammalian subject by the administration to the subject an effective amount of an orthogonal IL2 ligand in combination with the orthogonal CAR-T cell. The present disclosure further provides a method of extending of an active form (“persistence”) of an orthogonal CAR-T cell in vivo in a mammalian subject by the administration to the subject an effective amount of an orthogonal IL2 ligand in combination with the orthogonal CAR-T cell wherein the subject has not been subjected to a lymphodepleting treatment regimen prior to the administration of the orthogonal CAR-T cells. Sockolosky, et al. (Science (2018) 359: 1037–1042), Garcia, et al. (United States Patent Application Publication US2018 / 0228841A1 published August 16, 2018), Garcia, et al., PCT International Application Number PCT / US2016 / 050511 published March 16, 2017 as WO / 2017 / 044464A1; Garcia, et al., PCT International Application No PCT / US2019 / 021451 published September 12, 2019 as WO2019173773A1 describe an orthogonal IL2 / CD122 ligand / receptor system to facilitate selective stimulation of cells engineered to express an orthogonal receptor, especially an orthogonal CD122. The present patent application incorporates by reference the disclosures of WO 2019 / 104092, WO2019173773A1, WO / 2017 / 044464A1 and US 2018-0228842 A1) in their entireties.

[0088] The administration of the orthogonal IL2 ligand in combination with (e.g. subsequent to) the administration of the CAR-T cells provides selective expansion of the initial population of administered orthogonal CAR-T cells. The ability to selectively expand the initial population of orthogonal CAR-T enables the administration of a lower initial dose of CAR-T cells relative to conventional non-orthogonal CAR-T cells and to treat subjects in the absence of prior lymphodepletion. Avoiding lymphodepletion improves the safety of the CAR-T cell therapy and expands the scope of patients to which the benefits of CAR-T cell therapy are available. Orthogonal CAR-T Cell

[0089] In one embodiment, the present disclosure provides for treating a mammalian subject suffering from lupus the method comprising, administering to said subject a therapeutically effective amount of an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand. As used herein the term "orthogonal CAR-T cell" refers to a CAR-T cell which expresses an orthogonal receptor. In some embodiments, the extracellular domain of the CAR of the orthogonal CAR-T cell selectively binds to at least one human B cell antigen. CAR-T cell

[0090] In some embodiments the T-cell expressing the orthogonal receptor is a T-cell (e.g., human T-cell) which has been modified to surface express a chimeric antigen receptor (a ‘CAR-Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT T’ cell). In one embodiment, the CAR-T cell is prepared using T-cells isolated from the subject to be treated also referred to as autologous CAR-T cells. In one embodiment, the modified T cell is an allogenic CAR-T cell. In some embodiments, the allogenic CAR-T is modified to remove the endogenous TCRa and TCRb functions.

[0091] In the context of the present disclosure, consistent with the nomenclature in the scientific literature, orthogonal CAR-T cells are denoted by the antigen(s) to which the antigen binding domain of the CAR of the orthogonal CAR-T cell specifically binds. For example, a "orthogonal CD19 CAR-T cell" is a orthogonal CAR-T cell wherein the ABD of the CAR selectively binds to the B-cell antigen CD19, an "orthogonal CD20 CAR-T cell" is a orthogonal CAR-T cell wherein the ABD of the CAR selectively binds to the B-cell antigen CD20, an "orthogonal CD22 CAR-T cell" is a orthogonal CAR-T cell wherein the ABD of the CAR selectively binds to the B-cell antigen CD22, an "orthogonal BCMA CAR-T cell" is a orthogonal CAR-T cell wherein the ABD of the CAR selectively binds to the B-cell antigen BCMA, and so forth. In those embodiments wherein the ABD of the CAR of the orthogonal CAR-T cell binds to more than one surface antigen, the antigens bound by the ABD are reflected in the naming convention. For example, an "orthogonal CD19 / CD20 CAR-T cell" is an orthogonal CAR-T cell wherein the ABD of the CAR selectively binds to both the B-cell antigen CD19 and the B-cell antigen CD20.

[0092] As used herein the term CAR-T cell refers to a T-cell that has been recombinantly modified to a chimeric antigen receptor ("CAR"). As used herein, the term “CAR” encompasses a chimeric polypeptide comprising multiple functional domains arranged from amino to carboxy terminus in the sequence: (a) an extracellular domain (ECD) comprising an antigen binding domain (ABD), and optionally comprising a “hinge” domain, (b) a transmembrane domain (TMD); and (c) an intracellular domain (ICD) comprising one or more cytoplasmic signaling domains (CSDs) wherein the foregoing domains may optionally be linked by one or more spacer domains.

[0093] CARs useful in the practice of the present invention are prepared in accordance with principles well known in the art. See e.g., Eshhaar et al. United States patent No 7,741,465 B1 issued June 22, 2010; Sadelain, et al (2013) Cancer Discovery 3(4):388-398 (The basic principles of chimeric antigen receptor (CAR) design); Campana and Imai (United States Patent No 8,399,645 issued March 19, 2013) ; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15 (Designing chimeric antigen receptors to effectively and safely target tumors); Gross, et al. (1989) PNAS(USA) 86(24):10024-10028 (Expression of immunoglobulin- T-cell receptor chimeric molecules as functional receptors with antibody-type specificity);Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT Curran, et al. (2012) J Gene Med 14(6):405-15; Curran, et al. (2012) J Gene Med 14(6):405-15; Brogdon, et al. (United States patent No 10.174,095 issued January 8, 2019); Guedan, et al. (2019) Engineering and Design of Chimeric Antigen Receptors Molecular Therapy: Methods & Clinical Development Vol.12: 145-156. Considerations regarding the construction of the CAR and of the functional domains thereof in the context of the present invention are discussed below. Antigen Binding Domain

[0094] As used herein, the term antigen binding domain (ABD) refers a polypeptide sequence of the ECD of a CAR wherein the polypeptide sequence exhibits specific binding to at least one cell surface antigen. The function of the ABD is to target the CAR-T cell to a specific target cell or tissue comprising cells which express the cell surface antigen. In some embodiments, the ABD comprises a polypeptide with two binding domains that selectively bind to the same antigen or two different antigens on the surface of the target cells. The ABD may be any polypeptide that specifically binds to one or more antigens expressed on the surface of a target cell. In some embodiments, the ABD of the CAR of the orthogonal CAR-T cell is "monovalent" or "monospecific." As used herein the terms "monovalent ABD" and "monospecific ABD" are used interchangeably to refer an ABD the exhibits selective binding to a single surface antigen of a cell. In some embodiments, the ABD of the CAR is multivalent (a "multivalent ABD"). As used herein the term multivalent ABD refers to an ABD the selectively binds to multiple (e.g., 2, 3, or 4) cell surface antigens. CAR-T cells comprising multivalent ABDs are well known in the art. See, e.g. Xie, et al (2022) Cancers 14:3020.

[0095] In some embodiments, the cell surface antigen to which the ABD of the orthogonal CAR-T cell selectively binds is a B-cell antigen. As used in the context of the present disclosure, the B-cell antigen is a surface protein preferentially, primarily, or exclusively expressed on B-cells ("B-cell antigen"). In some embodiments the B-cell antigen is selected from the group consisting of mouse CD19 (mCD19), human CD19 (hCD19), human CD20 (hCD20), human CD22 (hCD22), and human BCMA (hBCMA), B-cell-activating factor of the tumour-necrosis-factor family (BAFF) and TACI (transmembrane activator and CAML interactor (TACI), also known as tumor necrosis factor receptor superfamily member 13B (TNFRSF13B). In some embodiments ABD of the CAR of the orthogonal CAR-T cell is a monospecific ABD that selectively binds to a single B-cell antigen, wherein the B-cell antigen selected from the group consisting of the extracellular domains of mCD19, hCD19, hCD20, hCD22, BAFF, TACI and hBCMA. In some embodiments ABD of the CAR of the orthogonal CAR-T cell is a multivalent ABD that selectively binds to one cell surface antigen, alternatively two cell surface antigens, alternatively three cell surface antigens, or alternatively four cellAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT surface antigens. In some embodiments, the ABD of the CAR of the orthogonal CAR-T cell is a multivalent ABD that specifically binds to more than one cell surface antigen, wherein at least one of the cell surface antigens is a B-cell antigen. In some embodiments, the ABD of the CAR of the orthogonal CAR-T cell is a multivalent ABD that specifically binds to more than one cell surface antigen, wherein at least one of the cell surface antigens is a B-cell antigen selected from the group consisting of the extracellular domains of hCD19, hCD20, hCD22, BAFF, TACI and hBCMA. VHHs:

[0096] In some embodiments, the polypeptide sequence of the ECD of a CAR that exhibits specific binding to at one least cell surface antigen is a VHH. The term VHH refers to a fragment of a camelid heavy-chain antibody that exhibits specific binding to the antigen associated with the parent heavy chain antibody. The use of VHHs to facilitate targeting of CAR-T cells to a variety of targets has been described in the scientific literature (Bao, et al. (2021) Biomolecules 8(11):238; Nix and Wiita (2024) Cytotherapy 26(7):729-738; and Wang, et al (2021) Cancer cell international 21(1):450). In some embodiments, the VHH is a humanized VHH. In some embodiments, ABD of the CAR of the orthogonal CAR-T cell comprises a VHH that specifically binds to a B-cell antigen, wherein the B-cell antigen selected from the group consisting of the extracellular domains of hCD19, hCD20, hCD22, BAFF, TACI and hBCMA. VHHs may be obtained through immunization of a camel or llama with a preparation comprising a cell surface antigen or fragment thereof including but not limited to fragments the extracellular domain of the cell surface antigen, the preparation optionally comprising one or more adjuvants. In some embodiments, the preparation for immunization of the camel or llama comprises a fragment of the extracellular domain of hCD19, hCD20, hCD22, BAFF, TACI and hBCMA. See, e.g., Muyldermans, S. (2001) Reviews in Molecular Biotechnology 74: 277-302. VHHs that bind to CD19 are known in the art and orthogonal CD19 CARs useful in the practice of the present disclosure may be constructed using, for example, the VHHs as described in Banahashemi, et al. (2018) Development of specific nanobodies (VHH) for CD19 immuno-targeting of human B- lymphocytes, Iran J Basic Med Sci.21(5):455-464; Ganji, et al. (2023) Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display, Journal of Translational Medicine 21(1):891; and Li, et al (2020) Chinese Journal of Cellular and Molecular Immunology 36(11):1036-1043). scFvs

[0097] In some embodiments, the polypeptide sequence of the ECD of a CAR that exhibits specific binding to at one least cell surface antigen is a single chain Fv (ScFv). An ScFv is aAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT polypeptide comprised of the variable regions of the immunoglobulin heavy and light chain of an antibody covalently connected by a peptide linker (Bird, et al. (1988) Science 242:423-426; Huston, et al. (1988) PNAS(USA) 85:5879-5883; S-z Hu, et al. (1996) Cancer Research, 56, 3055-3061; Ladner, United States Patent No 4946778 issued August 7, 1990). The generation of ScFvs based on monoclonal antibody sequences is well known in the art. See, e.g. The Protein Protocols Handbook, John M. Walker, Ed. (2002) Humana Press Section 150 “Bacterial Expression, Purification and Characterization of Single-Chain Antibodies” Kipriyanov, S. In some embodiments, the scFv is a bivalent. See, e.g. Thirion, et al. (1996) European J. of Cancer Prevention 5(6):507-511; DeKruif and Logenberg (1996) J. Biol. Chem 271(13)7630-7634; and Kay, et al. United States Patent Application Publication Number 2015 / 0315566 published November 5, 2015. In some embodiments, the scFv is a humanized scFv. In some embodiments, ABD of the CAR of the orthogonal CAR-T cell comprises a scFv that specifically binds to a B-cell antigen, wherein the B-cell antigen selected from the group consisting of [the extracellular domains of] hCD19, hCD20, hCD22, BAFF, TACI and hBCMA.

[0098] In some embodiments the linker that joins the heavy and light chain domains of the scFv is a GSA linker of 4-20, alternatively 12-16, amino acids. In some embodiments the linker that joins the heavy and light chain domains of the scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. CD19 ScFv

[0099] In some embodiments the ABD of the CAR comprises an scFv that binds to CD19 (an "anti-CD19 scFv" or "CD19 scFv"). Examples of anti-CD19 scFvs that may be incorporated into the ABD of the CAR include but are not limited to FMC63, 1D3, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b (Kang, et al (2020) Int J Mol Sci 21(23):9163).

[0100] In some embodiments, anti-CD19 scFv comprises a light chain polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 26, a linker that joins the heavy and light chain domains of the scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequenceAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT identity, alternatively 100% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 and a heavy chain sequence having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 27. In some embodiments, the anti-CD19 scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 35.

[0101] In some embodiments, anti-CD19 scFv comprises a light chain polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 28, a linker that joins the heavy and light chain domains of the scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 and a heavy chain sequence having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 29. In some embodiments, the anti-CD19 scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 36.

[0102] In some embodiments, scFv or sdAb may be optimized for increased the binding affinity to the cell surface antigen. Techniques for optimization of the binding affinity well known in the art such as phage display and directed evolution. See, e.g. Barbas, et al. (1991) PNAS(USA) 88:7978-82; Ladner, et al. United States Patent No.5,223,409 issued June 29, 1993; Stemmer, W. (1994) Nature 370:389-91; Garrard United States Patent No 5,821,047 issued October 13,1998; Camps, et al. (2003) PNAS(USA) 100(17): 9727-32; Dulbecco United States Patent No 4,593,002 issued June 3, 1986; McCafferty United States Patent No 6,806,079 issued October 19, 2004; McCafferty, United States Patent No 7,635,666 issued December 22, 2009; McCafferty, United States Patent No.7,662,557 issued February 16, 2010; McCafferty,Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT United States Patent No.7,723,271 issued May 25, 2010; and / or McCafferty United States Patent No.7,732,377. Synthetic Peptides

[0103] In some embodiments, the polypeptide sequence of the ECD of a CAR that exhibits specific binding to at one least cell surface antigen is a synthetic peptide. Synthetic peptides that exhibit specific binding to one or more cell surface antigens are generated synthetically through the generation of peptide libraries and isolating compounds having the desired target cell antigen binding properties. The generation of and identification of synthetic peptides that exhibit specific binding to one or more cell surface antigens are described in the scientific literature. See, e.g. Wigler, et al. United States Patent No.6303313 B1 issued November 12, 1999; Knappik, et al., United States Patent No 6696248 B1 issued February 24, 2004, Binz, et al (2005) Nature Biotechnology 23:1257-1268; Bradbury, et al.(2011) Nature Biotechnology 29:245-254.

[0104] In some embodiments, the polypeptide sequence of the ECD of a CAR that exhibits specific binding to at one least cell surface antigen is CAR-T cell adapter. As used herein, by “CAR-T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR-T cell adapter will have to binding regions, one specific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR-T cell adapters include but are not limited to e.g., those described in Kim et al. (2015) J Am Chem Soc.137(8):2832-5; Ma et al. (2016) Proc Natl Acad Sci U S A.113(4):E450-8 and Cao et al. (2016) Angew Chem Int Ed Engl. 55(26):7520-4 Linkers / Hinge

[0105] CARs useful in the practice of the present invention may optionally include one or more polypeptide spacers linking the domains of the CAR, in particular the linkage between the ARD to the transmembrane spanning domain of the CAR. Although not an essential element of the CAR structure, the inclusion of a spacer domain is generally considered desirable to facilitate antigen recognition by the ARD. Moritz and Groner (1995) Gene Therapy 2(8) 539-546. As used in conjunction with the CAR-T T cell technology described herein, the terms “linker”, “linker domain” and “linker region” refer to an oligo- or polypeptide region from about 1 to 100 amino acids in length, which links together any of the domains / regions of the CAR of the disclosure. Linkers may be composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Certain embodimentsAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT comprise the use of linkers of longer length when it is desirable to ensure that two adjacent domains do not sterically interfere with each another.

[0106] There is no particular sequence of amino acids that is necessary to achieve the spacer function but the typical properties of the spacer are flexibility to enable freedom of movement of the ABD to facilitate targeting antigen recognition. Similarly, it has been found that there is there is substantial leniency in spacer length while retaining CAR function. Jensen and Riddell (2014) Immunol. Review 257(1) 127-144. Sequences useful as spacers in the construction of CARs useful in the practice of the present invention include but are not limited to the hinge region of IgG1, the immunoglobulin1CH2-CH3 region, IgG4 hinge-CH2-CH3, IgG4 hinge-CH3, and the IgG4 hinge. The hinge and transmembrane domains may be derived from the same molecule such as the hinge and transmembrane domains of CD8-alpha. Imai, et al. (2004) Leukemia 18(4):676-684. Transmembrane Domain

[0107] The transmembrane domain (TMD) of this comprised of any polypeptide sequence which is thermodynamically stable in a eukaryotic cell membrane. The transmembrane spanning domain may be derived from the transmembrane domain of a naturally occurring membrane spanning protein or may be synthetic. In designing synthetic transmembrane domains, amino acids favoring alpha-helical structures are preferred. Transmembrane domains useful in construction of CARs are comprised of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, or 24 amino acids favoring the formation having an alpha-helical secondary structure. Amino acids having a to favor alpha-helical conformations are well known in the art. See, e.g Pace, et al. (1998) Biophysical Journal 75: 422-427. Amino acids that are particularly favored in alpha helical conformations include methionine, alanine, leucine, glutamate, and lysine. In some embodiments, the CAR transmembrane domain may be derived from the transmembrane domain from type I membrane spanning proteins, such as CD3ζ, CD4, CD8, CD28, etc. In some embodiments, the TMD of the CAR of the orthogonal CAR-T cell comprises is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO:19). Intracellular Domain (ICD) of the CAR

[0108] The CAR of the orthogonal CAR-T cell comprises an intracellular domain (ICD) comprising one or more cytoplasmic signaling domains optionally linked by one or more spacer domains.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0109] In some embodiments, the ICD of the CAR of the orthogonal CAR-T cell comprises one or more intracellular signaling domains. In one embodiment, the intracellular signaling domains comprise the cytoplasmic sequences of the T-cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement and functional derivatives and sub-fragments thereof. A cytoplasmic signaling domain, such as those derived from the T cell receptor zeta-chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Examples of cytoplasmic signaling domains include but are not limited to the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also referred to as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also referred to as ICOS), p110α, β, or δ catalytic subunit of PI3 kinase, the human CD3 ζ- chain, cytoplasmic domain of CD134 (also referred to as OX40 and TNFRSF4), FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (δ, Δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28.

[0110] In some embodiments, the ICD of the CAR of the orthogonal CAR-T cell comprises a co-stimulatory domain. The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. The co-stimulatory domain refers to the portion of the CAR which enhances the proliferation, survival or development of memory cells. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co- stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co- stimulation, and the factors involved have been described in Chen & Flies. (2013) Nat Rev Immunol 13(4):227-42. In some embodiments of the present disclosure, the CSD comprises one or more of 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 combinations thereof.

[0111] In some embodiments, the ICD of the CAR of the orthogonal CAR-T cell comprises one or more intracellular signaling domains including but not limited to intracellular signaling domains comprising the CD3ζ signaling domain; and (amino to carboxy) the CD28 and CD3ζ signaling domains; the 41BB and CD3ζ signaling domains; the OX40 and CD3ζ signaling domains; the 41BB, CD28 and CD3ζ signaling domains; the OX40, CD28 and CD3ζ signaling domains; the ICOS, 41BB and CD3ζ signaling domains; and the CD28, 41BB and CD3ζAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT signaling domains. In some embodiments, the CD CD3ζ signaling domain contains mutations that inactivate the first and third ITAM of the CD3ζ signaling domain.

[0112] In some embodiments, the orthogonal CAR-T cell is an orthogonal CD19 CAR-T cell wherein the CAR of the orthogonal CD19 CAR-T is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 61. Orthogonal Receptor

[0113] The present disclosure provides a method to selectively activate immune cells, wherein the immune cell is recombinantly modified to express an orthogonal receptor the method comprising the step of contacting the cell with an orthogonal IL2 ligand. In some embodiments, the orthogonal receptor is selected from the group consisting of an orthogonal hIL2 receptor, a chimeric orthogonal IL4R receptor, a chimeric orthogonal IL7R receptor, a chimeric orthogonal IL9 receptor, a chimeric orthogonal IL21 receptor.

[0114] As used herein the term "orthogonal receptor" refers to polypeptide comprising an orthogonal CD122 ECD, a transmembrane domain (TMD) and an intracellular domain (ICD), wherein the binding of an orthogonal ligand to the orthogonal CD122 ECD results in intracellular signaling in a cell recombinantly modified to express the orthogonal receptor. Orthogonal CD122 ECD:

[0115] As used herein the term "orthogonal CD122 ECD" refers to the extracellular domain of an orthogonal receptor wherein the ECD of the orthogonal receptor wherein the orthogonal CD122 ECD comprises one or more amino acid modifications (e.g., deletions or substitutions) that modulate the binding of a wild type IL2 to the ECD of a wild-type CD122. Amino acids positions that modulate the binding wild type hIL2 to the ECD of wild-type hCD122 include but are not limited to amino acids at positions R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and / or Q214 (numbered in accordance with SEQ ID NO:3).

[0116] In some embodiments, the orthogonal CD122 ECD is an orthogonal human CD122 ECD ("hCD122 ECD").

[0117] In some embodiments orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3),Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT the polypeptide further comprising one or more amino acid substitutions or deletions of amino acid residues selected from the group consisting of R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and / or Q214 (numbered in accordance with SEQ ID NO:3).

[0118] In some embodiments orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising one or more amino acid substitutions or deletions of amino acid residues selected from the group consisting of Q70, T73, H133, and / or Y134 (numbered in accordance with SEQ ID NO:3).

[0119] In some embodiments orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 (hCD122) wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising one or more amino acid substitutions or deletions of amino acid residues selected from the group consisting of H133 and Y134 (numbered in accordance with SEQ ID NO:3).

[0120] In some embodiments the orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 (hCD122) wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising one or more amino acid substitutions selected from the group consisting of Q70Y, T73D, T73Y, H133D, H133E, H133K, Y134F, Y134E, and Y134R (numbered in accordance with SEQ ID NO:3).

[0121] In some embodiments the orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 (hCD122) wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising one or more amino acid substitutions selected fromAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT the group consisting of H133D, H133E, H133K, Y134F, Y134E, and Y134R (numbered in accordance with SEQ ID NO:3).

[0122] In some embodiments the orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 (hCD122) wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising an amino acid substitution at position H133 selected from the group consisting of H133D, H133E, and H133K, or an amino acid substitution at position H134 selected from the group consisting of Y134F, Y134E, and Y134R (numbered in accordance with SEQ ID NO:3).

[0123] In some embodiments the orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 (hCD122) wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising an H133D amino acid substitution or a Y134F amino acid substitution (numbered in accordance with SEQ ID NO:3).

[0124] In some embodiments orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 (hCD122) wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising an amino acid substitution at position H133 selected from the group consisting of H133D, H133E, and H133K, and an amino acid substitution at position H134 selected from the group consisting of Y134F, Y134E, and Y134R (numbered in accordance with SEQ ID NO:3).

[0125] In some embodiments the orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 (hCD122) wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising an H133D amino acid substitution and a Y134F amino acid substitution (numbered in accordance with SEQ ID NO:3).Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0126] In some embodiments the orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 (hCD122) the polypeptide comprising an H133D amino acid substitution and a Y134F amino acid substitution (numbered in accordance with SEQ ID NO:3) wherein the polypeptide has the amino acid sequence: AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTC ELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDF KPFENLRLMAPISLQVVHVETHRCNISWEISQASDFFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWS QPLAFRTKPAALGKDT (SEQ ID NO:40)

[0127] The ECD of the wild type hCD122 receptor comprises disulfide bonds between residues Cys10-Cys20, Cys33-Cys84 and Cys48-Cys60, numbered in accordance with the mature hCD122 (SEQ ID NO:3). When preparing an orthogonal hCD122 ECD, amino acid substitutions or deletions at positions C10, C20, C33, C48, C60 and C84 are not preferred.

[0128] The ECD of the wild type hCD122 receptor comprises N-linked glycosylation sites at N3, N17, N45 and N123. In some embodiments, the orthogonal hCD122 ECD may further comprise conservative amino acid substitutions (for example with alanine or isoleucine) at one or more positions selected from the group consisting of N3, N17, N45 and / or N123 numbered in accordance with the mature hCD122 (SEQ ID NO: 3). Orthogonal Murine CD122:

[0129] The experiments described in the Examples were conducted with a murine orthogonal CAR-T cell comprising an orthogonal mouse CD122 receptor and a mouse orthogonal IL2 ligand. In some embodiments, the orthogonal CD122 ECD is an orthogonal murine CD122 ECD ("orthogonal mCD122 ECD"). In some embodiments the orthogonal mCD122 ECD comprises a variant polypeptide derived from the ECD of a murine CD122 (mCD122) wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type mCD122 (SEQ ID NO:7), the polypeptide further comprising an H134D amino acid substitution and a Y135F amino acid substitution (numbered in accordance with the mature wild type murine CD122 ECD, SEQ ID NO:7). In some embodiments the orthogonal mCD122 ECD comprises a variant polypeptide derived from the ECD of a murine CD122 the polypeptide comprising an H134D amino acid substitution and a Y135F amino acid substitution (numbered in accordance with SEQ ID NO:7) wherein the polypeptide has the amino acid sequence:Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTC ELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCD FHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSDFIEPYLEFEARRRLL GHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSP WSQPLTFRTRPADPMKE (SEQ ID NO: 101) In one embodiment, the orthogonal mCD122 receptor is a polypeptide having the amino acid sequence: AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTC ELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCD FHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSDFIEPYLEFEARRRLL GHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSP WSQPLTFRTRPADPMKEILPMSWLRYLLLVLGCFSGFFSCVYILVKCRYL GPWLKTVLKCHIPDPSEFFSQLSSQHGGDLQKWLSSPVPLSFFSPSGPAPEI SPLEVLDGDSKAVQLLLLQKDSAPLPSPSGHSQASCFTNQGLKQHLPNAL EIESCQVYFTYDPCVEEEVEEDGSRLPEGSPHPPLLPLAGEQDDYCAFPPR DDLLLFSPSLSTPNTAYGGSRAPEERSPLSLHEGLPSLASRDLMGLQRPLE RMPEGDGEGLSANSSGEQASVPEGNLHGQDQDRGQGPILTLNTDAYLSL QELQAQDSVHLI (SEQ ID NO:67) In one embodiment, the orthogonal mIL2 ligand (moIL2) is a polypeptide having the amino acid sequence: APTSSSTSSSTAEAQQQQQQQQQQQQHLDNLLILLQVLLSRMENYRNLKL PRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFI SNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ (SEQ ID NO:69) Orthogonal hCD122 Receptor:

[0130] In some embodiments, the orthogonal receptor is an orthogonal hCD122 receptor. As used herein, the terms “orthogonal hCD122 receptor” and “orthogonal hIL2 receptor” are used interchangeably to refer to a orthogonal receptor comprising an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD), wherein: (a) the ECD is an orthogonal hCD122 ECD, (b) the TMD is a polypeptide derived from the TMD of wild type human CD122 (hCD122) the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequenceAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT identity to the TMD of wild type human CD122 (SEQ ID NO:4) and (c) the ICD is a polypeptide derived from the ICD of wild type human CD122 (hCD122) the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ICD of wild type human CD122 (SEQ ID NO:5).

[0131] In some embodiments, the orthogonal receptor is an orthogonal hIL2 receptor having the amino acid sequence: AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPV SQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLM APISLQVVHVETHRCNISWEISQASDFFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWL GHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGD VQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLT SCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLS GEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDW DPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEF RALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO:39).

[0132] In some embodiments, the ICD of the orthogonal hCD122 receptor comprises one or more STAT3 binding motifs. In some embodiments, the ICD of the orthogonal hCD122 receptor may comprise one, two, three, or more STAT3 binding motifs. In some embodiments, the STAT3 recognition motif of the formula YX1X2Q, wherein X1is an amino acid selected from the group consisting of L, R, F, M, and X2 is an amino acid selected from the group consisting of R, K, H, and P. In some embodiments, the STAT3 recognition motif has an amino acid sequence selected from the group consisting of: YLRQ (SEQ ID NO:89); YLKQ (SEQ ID NO: 90); YRHQ (SEQ ID NO: 91); YLRQ (SEQ ID NO: 92); YFKQ (SEQ ID NO: 93); YLPQ (SEQ ID NO: 94), YMPQ (SEQ ID NO: 95), and YDKPH (SEQ ID NO: 96). hCD122 receptors comprising one or more STAT3 binding motifs are described in PCT International Application Number PCT / US2021 / 013519 published on July 22, 2021 as WO 2021 / 146485A2.

[0133] In some embodiments, one or more STAT3 binding motifs are present as an internal (i.e., at neither C nor N terminus) sequence of the ICD of the orthogonal hCD122 receptor. A modified orthogonal CD122 of this configuration can be produced by identifying a suitable region within the ICD of the orthogonal hIL2 receptor amino acid sequence that can be mutatedAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT to create a STAT3 binding motif. In some embodiments, the present disclosure provides a human orthogonal CD122 wherein one of the endogenous STAT5 motifs of the hCD122 ICD is retained and at least one of the endogenous STAT5 motifs of the hCD122 ICD is modified to introduce one or more STAT3 binding motifs and the modified human CD122 so produced retains at least one STAT5 recognition motif and gains one or more STAT3 binding motifs. In one embodiment, the ICD of the orthogonal hIL2 receptor possesses sequences similar to the STAT3 binding motif that may readily be modified to create a STAT3 binding motif with minimal modification. For example, positions 355-364 of the wild type human CD122 protein (numbered in accordance with the full length hCD122 polypeptide) contains the amino acid sequence of YFTYDPYSEE (SEQ ID NO:97). In some embodiments, one or two of the YFTY (SEQ ID NO: 98) of SEQ ID NO:97; YDPY (SEQ ID NO: 99) of SEQ ID NO:97, or YSEE (SEQ ID NO: 100) of SEQ ID NO:97 are substituted with a STAT3 motif to produce a modified hCD122 ICD comprising a STAT3 binding motif.

[0134] In some embodiments, the ICD of the orthogonal hIL2 receptor comprises one or more STAT3 binding motifs fused to the C-terminus of the intracellular domain of a human CD122, wherein the STAT3 binding motif is linked to the C-terminus of the intracellular domain of the hCD122 orthogonal receptor via a polypeptide linker. Linkers can be derived from naturally occurring proteins or synthetic sequences. Methods for designing linkers are well- known in the art, for example, as disclosed in Chen, et al. (2013) Adv. Drug. Deliv. Rev. 65(10):1357–1369, the relevant portion thereof is herein incorporated by reference. In some embodiments the linker is a GSA linker.

[0135] Contacting a cell modified to express an orthogonal hIL2 receptor, the ICD of which comprises STAT3 and STAT5 motifs, with an orthogonal hIL2 ligand, results increased intracellular levels of phosphorylated STAT3 (pSTAT3) and phosphorylated STAT5 (pSTAT5) One exemplary method for detecting a recombinant protein’s ability to induce STAT3 and STAT5 signaling is described in Kagoya, et al. (2018) Nat Med.24(3):352–359.

[0136] In some embodiments, the present disclosure provides a method of stimulating an engineered cell expressing a modified orthogonal hCD122 orthogonal receptor wherein the ICD of the orthogonal receptor comprises one or more STAT3 binding motifs, the method comprising contacting the engineered T cell with a human orthogonal IL2 ligand by contacting the cell with an orthogonal hIL2 ligand, wherein the contacting results in increased intracellular levels of STAT3 and STAT5 in the cell. In some embodiments, this disclosure provides a method of stimulating a CAR-T cell expressing an orthogonal hCD122 receptor, the ICD comprising one or more STAT3 binding motifs, the method comprising contacting the CAR-T cell with a humanAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT orthogonal IL2 ligand wherein the contacting results in intracellular signaling resulting in activation and or proliferation of the engineered CAR-T cell. In some embodiments, this disclosure provides a method of increasing the intracellular levels of pSTAT3 and pSTAT5 in a CAR-T cell expressing a orthogonal hCD122 receptor, wherein the ICD of the orthogonal hCD122 receptor comprises one or more STAT3 binding motifs, the method comprising contacting the CAR-T cell with a human orthogonal IL2 ligand wherein the contacting results in increased the intracellular levels of pSTAT3 and pSTAT5 in the CAR-T cell. Chimeric Orthogonal Receptors

[0137] In some embodiments, the orthogonal receptor is a chimeric orthogonal receptor polypeptide comprising, from amino terminus to carboxy terminus, an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD) wherein: (a) the ECD of the chimeric orthogonal receptor comprises an orthogonal hCD122 polypeptide and (b) a polypeptide comprising the TMD and ICD polypeptides derived from a TMD and ICD, respectively, of a heterologous human common gamma chain receptor polypeptide such that contacting a cell expressing the chimeric orthogonal receptor with an orthogonal IL2 ligand results in intracellular signaling characteristic of the heterologous receptor. Chimeric orthogonal receptors useful in the practice of the present disclosure are described in PCT International Application Number PCT / US2020 / 050232 published March 18, 2021 as WO2021 / 050752A1.

[0138] In one embodiment, the chimeric orthogonal receptor is a "chimeric orthogonal IL4 receptor". The term "chimeric orthogonal IL4 receptor" refers to a orthogonal receptor comprising an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD), wherein: (a) the ECD is an orthogonal hCD122 ECD, (b) the TMD is a polypeptide derived from the TMD of the human IL4 receptor Type II receptor subunit a ("hIL4Ra") the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the TMD of wt hIL4Ra (SEQ ID NO:45) and (c) the ICD is a polypeptide derived from the ICD of hIL4Ra the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ICD of wt hIL4Ra (SEQ ID NO:46).Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0139] In one embodiment, the chimeric orthogonal hIL4 receptor comprises, from amino terminus to carboxy terminus: (a) the ECD of SEQ ID NO:40, (b) the TMD of SEQ ID NO: 45, and (c) the ICD of SEQ ID NO: 46.

[0140] In some embodiments, the chimeric orthogonal IL4 receptor is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 47.

[0141] In one embodiment, the chimeric orthogonal receptor is a "chimeric orthogonal IL7 receptor". The term "chimeric orthogonal IL7 receptor" refers to a orthogonal receptor comprising an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD), wherein: (a) the ECD is an orthogonal hCD122 ECD, (b) the TMD is a polypeptide derived from the TMD of the human IL7 receptor subunit a ("hIL7Ra") the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the TMD of wt hIL7Ra (SEQ ID NO:48) and (c) the ICD is a polypeptide derived from the ICD of hIL7Ra the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ICD of wt hIL7Ra (SEQ ID NO:49).

[0142] In some embodiments, the chimeric orthogonal IL7 receptor comprises, from amino terminus to carboxy terminus: (a) the ECD of SEQ ID NO: 40, (b) the TMD of SEQ ID NO: 48, and (c) the ICD of SEQ ID NO: 49.

[0143] In some embodiments, the chimeric orthogonal IL7 receptor is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 50.

[0144] In one embodiment, the chimeric orthogonal receptor is a "chimeric orthogonal IL9 receptor". The term "chimeric orthogonal IL9 receptor" refers to a orthogonal receptor comprising an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD), wherein: (a) the ECD is an orthogonal hCD122 ECD, (b) the TMD is a polypeptide derived from the TMD of the human IL9 receptor ("hIL9R") the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99%Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT sequence identity, alternatively 100% sequence identity to the TMD of wt hIL9R (SEQ ID NO:51) and (c) the ICD is a polypeptide derived from the ICD of hIL9Ra the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ICD of wild type hIL9R (SEQ ID NO:52).

[0145] In some embodiments, the chimeric orthogonal IL9 receptor comprises, from amino terminus to carboxy terminus: (a) the ECD of SEQ ID NO: 40, (b) the TMD of SEQ ID NO: 51, and (c) the ICD of SEQ ID NO: 52.

[0146] In some embodiments, the chimeric orthogonal IL9 receptor is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 53.

[0147] In one embodiment, the chimeric orthogonal receptor is a "chimeric orthogonal IL21 receptor". The term "chimeric orthogonal IL21 receptor" refers to a orthogonal receptor comprising an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD), wherein: (a) the ECD is an orthogonal hCD122 ECD, (b) the TMD is a polypeptide derived from the TMD of the human IL21 receptor ("hIL21R") the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the TMD of wt hIL21R (SEQ ID NO:54) and (c) the ICD is a polypeptide derived from the ICD of wt hIL21R the polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ICD of wt hIL21R (SEQ ID NO:55).

[0148] In some embodiments, the chimeric orthogonal IL21 receptor comprises, from amino terminus to carboxy terminus: (a) the ECD of SEQ ID NO: 40, (b) the TMD of SEQ ID NO: 54, and (c) the ICD of SEQ ID NO: 55.

[0149] In some embodiments, the chimeric orthogonal IL21 receptor is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 56.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT Orthogonal CAR-T Cells

[0150] In one embodiment of the invention the T-cell expressing the orthogonal receptor is a T-cell (e.g., human T-cell) which has been modified to surface express a chimeric antigen receptor (a ‘CAR-T’ cell). In one embodiment, the modified T cell is an allogenic CAR-T cell. In some embodiments, the allogenic CAR-T is modified to remove the endogenous TCRa and TCRb functions.

[0151] Orthogonal CAR-T cells of the present disclosure are prepared by: (a) obtaining a population of mammalian T-cells, (b) contacting the population of mammalian T cells with first nucleic acid sequence encoding an orthogonal receptor and a first nucleic acid sequence encoding CAR wherein the first and second nucleic acid sequences are operably linked to one or more expression control sequences operable in a mammalian T cell, under conditions wherein ^ 5%, alternatively ^ 10%, alternatively ^ 15%, alternatively ^ 20%, alternatively ^ 25%, alternatively ^ 30%, alternatively ^ 35%, alternatively ^ 40%, alternatively ^ 45%, alternatively ^ 50%, alternatively ^ 60%, alternatively ^ 70%, alternatively ^ 80%, or alternatively ^ 90% of the isolated population of mammalian T cells are transduced with the first and second nucleic acid sequence and express an orthogonal receptor and a CAR.

[0152] T-cells useful in the preparation of orthogonal CAR-T cells may be obtained from the mammalian subject to be treated (autologous) or may an allogeneic T cell. T cells can be obtained from a number of sources of the mammalian subject, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, and spleen tissue. T cells include naïve T-cells, central memory T-cells, effector memory T-cells or combination thereof. In one embodiment, T-cells are obtained by apheresis of peripheral blood. In some embodiments, the population of T cells comprises one or more of species human immune cells selected from the group consisting myeloid cells, lymphocytes, peripheral blood mononuclear cells (PBMCs), CD8+T cells, naïve CD8+T cells, cytotoxic CD8+T cells, CD25+CD8+ T cells, naïve CD4+T cells, helper T cells, e.g. TH1, TH2, TH9, TH11, TH22, TFH; regulatory T cells, e.g. TR1, natural TReg, inducible TReg; memory T cells, e.g. central memory T cells, effector memory T cells, NK T cells, gamma / delta T cells.

[0153] In some embodiments, the orthogonal CAR-T cells is a CAR-T cell which has been modified to express an orthogonal receptor. In some embodiments, the CAR-T cell is a commercially available CAR T including but not limited to commercially available CD19 CAR T cells such as axicabtagene ciloleucel (marketed as Yescarta® commercially available from Gilead Pharmaceuticals) and tisagenlecleucel (marketed as Kymriah® commercially available from Novartis). In some embodiments, the orthogonal CAR T cell is a CD-19 CAR T cellAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT selected from the group consisting of axicabtagene ciloleucel and tisagenlecleucel which further expresses an orthogonal receptor comprising a human orthogonal CD122 ECD having at least 90%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to SEQ ID NO:40, alternatively which further expresses a human orthogonal receptor having at least 90%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to SEQ ID NO: 39.

[0154] In some embodiments, the present disclosure provides an orthogonal Treg CAR-T cell. The present invention further provides a method of induce immune suppression in a subject by the administration a therapeutically effective amount of an orthogonal Treg CAR-T cell in combination with the administration of an orthogonal IL2 ligand sufficient to cause the proliferation and / or activation of the orthogonal Treg in the subject.

[0155] T cells collected from a subject or a donor may be separated from a mixture of cells by techniques that enrich for desired cells or may be engineered and cultured without separation. Alternatively, the T cells for engineering may be separated from other cells. In some embodiments, the T cell population is enriched for particular T cell subtypes. In some embodiments, the T cell population is enriched for one or more T cell subtypes selected from naïve CD8+T cells, cytotoxic CD8+T cells, naïve CD4+T cells, helper T cells, e.g. TH1, TH2, TH9, TH11, TH22, TFH; regulatory T cells, e.g. TR1, Tregs, inducible Tregs; memory T cells, e.g. central memory T cells, effector memory T cells, NK cells, and engineered variants of such T- cells including but not limited to TCR engineered cells. Techniques providing accurate separation include fluorescence activated cell sorters. The cells may be selected against dead cells by employing dyes associated with dead cells (e.g., propidium iodide). The separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., frequently supplemented with fetal calf serum (FCS). The collected and optionally enriched cell population may be used immediately for genetic modification or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0156] In some embodiments, the isolated T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680;Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7, 144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No.2006 / 0121005. Generally, the T cells are expanded by culturing the cells in contact with a surface providing an agent that stimulates a CD3 TCR complex associated signal (e.g., an anti- CD3 antibody) and an agent that stimulates a co-stimulatory molecule on the surface of the T cells (e.g., an anti-CD28 antibody). Conditions appropriate for T cell culture are well known in the art Lin, et al. (2009) Cytotherapy 11(7):912-922 (Optimization and validation of a robust human T-cell culture method for monitoring phenotypic and polyfunctional antigen-specific CD4 and CD8 T-cell responses); Smith, et al. (2015) Clinical & Translational Immunology 4:e31 published online 16 January 2015 (“Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement”). The orthogonal CAR-T cells are prepared by introducing the nucleic acid sequences encoding the orthogonal receptor and CAR as described below. The T cells and CAR-T cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2).

[0157] In some embodiments of the present disclosure, the orthogonal CAR-T cells or the present disclosure comprises mammalian T cell recombinantly modified to incorporate a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding an orthogonal receptor wherein the nucleic acid sequences encoding the CAR and orthogonal receptor are operably linked to one or more control elements functional in a mammalian T cell such that the orthogonal ligand and CAR are co-expressed on the mammalian T cell. In some embodiments, the nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding an orthogonal receptor are genomically integrated into the genome of the CAR-T cell. In some embodiments, the nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding an orthogonal receptor are maintained extrachromasomally.

[0158] Nucleic acids encoding the CAR and orthogonal receptor may be prepared by techniques known in the art. Due to the known degeneracy of the genetic code, multiple nucleic acid sequences encode the same the CAR or orthogonal receptor. The nucleic acid molecules encoding the orthogonal receptor and CAR may contain naturally occurring sequences or sequences that differ from those that occur naturally. A nucleic acid sequenced encoding the orthogonal receptor and CAR and / or orthogonal receptor may be synthesized by conventional means using an oligonucleotide synthesizer. In some embodiments, the nucleic acid sequence encoding the orthogonal receptor and / or CAR is codon optimized for expression in the host cell of employed for recombinant production. Techniques for codon optimization in a wide varietyAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT of expression systems, including mammalian, yeast and bacterial host cells, are well known in the and there are online tools to provide for a codon optimized sequences for expression in a variety of host cell types. See e.g. Hawash, et al., (2017) 9:46-53 and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols, edited by David Hacker (Human Press New York). Additionally, there are a variety of software tools that may be used in the preparation of codon optimized nucleic acid sequences. Services providing synthesis of nucleic acid sequences encoding the orthogonal receptormay be obtained from various commercial sources that provide custom made nucleic acid sequences.

[0159] In some embodiments, the nucleic acid sequences encoding the CAR and a nucleic acid sequence encoding an orthogonal receptor are provided on one or more vectors. In some embodiments, the vector is an expression vector comprising one or more control elements that are operably linked to the heterologous CAR and orthogonal receptor nucleic acid sequences.

[0160] The term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation, a nucleic acid encoding signal peptide is operably linked to a nucleic acid sequence encoding such polypeptide if it is expressed as a fusion protein and participates in directing the fusion protein to the cell membrane or in secretion of the polypeptide. Typically, nucleotide sequences that are operably linked are contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked yet physically distant and may even function in trans from a different allele or chromosome.

[0161] The term "control element" is herein to refer collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation which affect the replication, transcription and translation of the polypeptide coding sequence in a recipient cell. Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell.

[0162] The expression vector comprises the heterologous nucleic acid sequence encoding the orthogonal receptor and / or CAR is operably linked to a promoter sequence. The term "promoter" is used in its conventional sense to refer to a nucleotide sequence at which theAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT initiation and rate of transcription of a coding sequence is controlled. The promoter contains the site at which RNA polymerase binds and also contains sites for the binding of regulatory factors (such as repressors or transcription factors). Promoters can be naturally occurring or synthetic. The promoter can be constitutively active, activated in response to external stimuli (inducible), active in particular cell type or cell state (tissue specific) promoters, and / or regulatable promoters. The term "inducible promoter" refers to promoters that facilitate transcription of the Bioactive polypeptide preferably (or solely) under certain conditions and / or in response to external chemical or other stimuli. Examples of inducible promoters are known in the scientific literature (see, e.g., Yoshida et al., Biochem. Biophys. Res. Comm., 230:426-430 (1997); Iida et al., J. Virol., 70(9): 6054-6059 (1996); Hwang et al., J. Virol., 71(9): 7128-7131 (1997); Lee et al., Mol. Cell. Biol., 17(9): 5097-5105 (1997); and Dreher et al., J. Biol. Chem., 272(46): 29364- 29371 (1997). Examples of radiation inducible promoters include the EGR-1 promoter. Boothman. et al., volume 138, supplement pages 293 embodiment the promoter is the myoproliferative sarcoma virus (enhancer negative control region deleted dl587rev primer binding site substituted) (“MND”) promoter.

[0163] In some embodiments, the preparation of the orthogonal CAR-T cell transfecting a cell with an vector comprising a first nucleic acid sequence encoding a CAR and a second nucleic acid sequence encoding an orthogonal receptor, wherein the first nucleic acid sequence encoding the CAR further comprises a nucleic acid sequence encoding a first signal peptide and the second nucleic acid sequence encoding the orthogonal receptor further comprises a nucleic acid sequence encoding a second signal peptide, wherein the first and second signal peptides are the same or different.

[0164] The term "vectors" includes viral vectors and non-viral vectors. The term "non-viral vector" refers to an autonomously replicating, extrachromosomal circular DNA molecule, distinct from the normal genome and nonessential for cell survival under nonselective conditions capable of effecting the expression of a coding sequence in the target cell. Plasmids are examples of non-viral vectors. In order to facilitate transfection of the target cells, the target cell may be exposed directly with the non-viral vector may under conditions that facilitate uptake of the non- viral vector. Examples of conditions which facilitate uptake of foreign nucleic acid by mammalian cells are well known in the art and include but are not limited to chemical means (such as Lipofectamine®, Thermo-Fisher Scientific), high salt, magnetic fields (electroporation)

[0165] In some embodiments of the present disclosure, the vector is a viral vector. Example of viruses useful include recombinantly modified enveloped or non-enveloped DNA and RNA viruses. In some embodiments, the viral vector is derived from the genome of human or bovineAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT adenoviruses, vaccinia virus, lentivirus, herpes virus, adeno-associated virus, human immunodeficiency virus, sindbis virus, and retroviruses (including but not limited to Rous sarcoma virus), and hepatitis B virus. Retroviral or lentiviral expression vectors are preferred to transfect T-cells due to an enhanced efficacy of gene transfer to T-cells using these systems resulting in a decreased time for culture of significant quantities of T-cells for clinical applications. In particular, gamma retroviruses a particularly preferred for the genetic modification of clinical grade T-cells and have been shown to have therapeutic effect. Pule, et al. (2008) Nature Medicine 14(11):1264-1270. Similarly, self-inactivating lentiviral vectors are also useful as they have been demonstrated to integrate into quiescent T-cells. June, et al. (2009) Nat Rev Immunol 9(10):704-716. The nucleic acids encoding the CAR and orthogonal ligand of interest are inserted into such vectors to allow packaging of the gene construct, typically with accompanying viral genomic sequences, followed by infection of the T cells.

[0166] In some embodiments, the preparation of the orthogonal CAR-T cell transfecting a cell with an expression vector comprising a first nucleic acid sequence encoding a CAR and a second nucleic acid sequence encoding an orthogonal CD122 receptor, wherein the first nucleic acid sequence encoding the CAR further comprises a nucleic acid sequence encoding a first signal peptide and the second nucleic acid sequence encoding an orthogonal CD122 receptor further comprises a nucleic acid sequence encoding a first signal peptide, and wherein the first and second nucleic acid sequences are linked by a nucleic acid sequence encoding a viral 2A peptide wherein the nucleic acid sequence is operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0167] The expression vector encoding the CAR and / or orthogonal receptor may optionally provide one or more expression cassettes comprising a nucleic acid sequence encoding a “rescue” gene. A “rescue gene” is a nucleic acid sequence, the expression of which in the transduced cell renders the cell susceptible to killing by external factors or causes a toxic condition in the cell such that the cell is killed. Providing a rescue gene enables selective cell killing of transduced cells. The rescue gene provides a safety precaution when said constructs are incorporated into the cells of a mammalian subject to prevent undesirable spreading of transduced cells or the effects of replication competent vector systems. In one embodiment, the rescue gene is the thymidine kinase (TK) gene (see e.g. Woo, et al. U.S. Pat. No.5,631,236 issued May 20, 1997, and Freeman, et al. U.S. Pat. No.5,601,818 issued Feb.11, 1997) in which the cells expressing the TK gene product are susceptible to selective killing by the administration of ganciclovir. Alternatively, an inducible promoter may be operably linked to a proapoptoticAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT gene to facilitate targeted cell killing by the administration of an exogenous agent that induces expression from the inducible promoter.

[0168] In some embodiments, the orthogonal CAR-T cell further comprises a cell surface marker. The presence of a cell surface marker facilitates ready identification and quantification of the orthogonal CAR-T cells in vivo and ex vivo. In some embodiments, the cell surface marker is a protein expressed on the cell surface of the orthogonal CAR-T cell having an extracellular domain, wherein the extracellular domain comprises one or more antigens is detectable by an antibody.

[0169] The expression vector may optionally provide additional genes, such as those encoding drug resistance, can be included to allow selection or screening for the presence of the recombinant vector. Such additional genes can include, for example, genes encoding neomycin resistance, multi-drug resistance, thymidine kinase, beta-galactosidase, dihydrofolate reductase (DHFR), and chloramphenicol acetyl transferase. In some embodiments, the expression vector may optionally provide additional genes that provide "armoring" of the CAR-T cell including IL18 and cJUN.

[0170] In some embodiments, the orthogonal CAR-T cell is an orthogonal CD19 CAR-T cell comprising a first expression vector and a second expression vector the first expression vector comprising a nucleic acid sequence encoding a CD19 CAR operably linked to one or more regulatory control sequences operable in a mammalian T cell and second expression vector comprising a nucleic acid sequence encoding an orthogonal hCD122 receptor operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0171] In some embodiments, the preparation of the orthogonal CAR-T cell transfecting a cell with a first expression vector comprising a nucleic acid sequence encoding a CAR and second expression vector comprising a nucleic acid sequence encoding an orthogonal receptor.

[0172] In some embodiments, orthogonal CD19 CAR-T cell comprises a first expression vector comprising a nucleic acid sequence encoding a CD19 CAR having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 61 (hCD19 CAR without signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell and a second expression vector comprising a nucleic acid encoding an orthogonal receptor having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to an amino acid sequence selected from the groupAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT consisting of SEQ ID NO: 39 (hoCD122 without signal peptide), SEQ ID NO: 47 (chimeric orthogonal IL4 receptor), SEQ ID NO: 50 (chimeric orthogonal IL7 receptor), SEQ ID NO: 53 (chimeric orthogonal IL9 receptor) and SEQ ID NO: 56 (chimeric orthogonal IL21 receptor) operably linked to one or more regulatory control sequences operable in a mammalian T cell. In some embodiments, the first expression vector comprises a nucleic acid sequence encoding a CD19 CAR the nucleic acid sequence having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 62 (DNA encoding hCD19 CAR without signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell and the second expression vector comprises a nucleic acid sequence encoding an orthogonal CD122 receptor, the nucleic acid sequence having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 103 (DNA encoding hoCD122 without signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0173] In some embodiments, the first expression vector comprises a nucleic acid sequence encoding CD19 CAR receptor with a signal peptide having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 104 (hCD19 CAR with GMCSFR signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell and the second expression vector comprises a nucleic acid encoding an orthogonal CD122 receptor with a signal peptide having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 37 (hoCD122 with wt hCD122 signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell. In some embodiments, the first expression vector comprises a nucleic acid sequence coding CD19 CAR with a signal peptide, the nucleic acid sequence having ^ 90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 105 (DNA encoding hCD19 CAR with GMCSFR signal peptide) operably linked to one or more regulatoryAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT control sequences operable in a mammalian T cell and the second expression vector comprises a nucleic acid sequence encoding an orthogonal CD122 receptor with a signal peptide, the nucleic acid sequence having ^ 90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 38 (DNA encoding hoCD122 with wt hCD122 signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0174] In some embodiments, the preparation of the orthogonal CAR-T cell transfecting a cell with an expression vector, the expression vector comprising a first nucleic acid sequence encoding a signal peptide and the CAR and a second nucleic acid sequence encoding a signal peptide and the orthogonal receptor, wherein the first and second nucleic acid sequences are each operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0175] In some embodiments, the orthogonal CAR-T cell is prepared by transfecting a T cell with an expression vector, the expression vector comprising a nucleic acid sequence encoding (from 5'-3'): (a) a signal peptide, (b) a CAR; (c) a nucleic acid sequence encoding a 2A peptide; (d) a signal peptide; and (e) an orthogonal receptor, wherein the nucleic acid sequence is operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0176] In some embodiments, the first expression vector comprises a nucleic acid sequence encoding CD19 CAR receptor with a signal peptide having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 104 (hCD19 CAR with hGMCSFR signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell and the second expression vector comprises a nucleic acid encoding an orthogonal CD122 receptor with a signal peptide having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 37 (hoCD122 with wt hCD122 signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell. In some embodiments, the first expression vector comprises a nucleic acid sequence coding CD19 CAR with a signal peptide, the nucleic acid sequence having ^ 90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 105 (DNAAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT encoding hCD19 CAR with hGMCSFR signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell and the second expression vector comprises a nucleic acid sequence encoding an orthogonal CD122 receptor with a signal peptide, the nucleic acid sequence having ^ 90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 38 (DNA encoding hoCD122 with wt hCD122 signal peptide) operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0177] In some embodiments, the orthogonal CAR-T cell is prepared by transfecting a T cell with an expression vector, the expression vector a nucleic acid sequence encoding (from 5'-3'): (a) a signal peptide, (b) a CAR; (c) a nucleic acid sequence encoding a 2A peptide, the 2A peptide having an amino acid sequence selected from SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, (d) a signal peptide, (e) an orthogonal receptor having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 39 (hoCD122 without signal peptide), SEQ ID NO: 47 (chimeric orthogonal IL4 receptor), SEQ ID NO: 50 (chimeric orthogonal IL7 receptor), SEQ ID NO: 53 (chimeric orthogonal IL9 receptor) and SEQ ID NO: 56 (chimeric orthogonal IL21 receptor), wherein the nucleic acid sequence is operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0178] In some embodiments, the orthogonal CAR-T cell is prepared by transfecting a T cell with an expression vector, the expression vector comprising a nucleic acid sequence encoding (from 5'-3'): (a) a signal peptide, (b) a CD19 CAR having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to the amino acid sequence of SEQ ID NO:61; (c) a nucleic acid sequence encoding a 2A peptide, the 2A peptide having an amino acid sequence selected from SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, (d) a signal peptide, (e) an orthogonal receptor having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 39 (hoCD122 without signal peptide), SEQAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT ID NO: 47 (chimeric orthogonal IL4 receptor), SEQ ID NO: 50 (chimeric orthogonal IL7 receptor), SEQ ID NO: 53 (chimeric orthogonal IL9 receptor) and SEQ ID NO: 56 (chimeric orthogonal IL21 receptor), wherein the nucleic acid sequence is operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0179] In some embodiments, the orthogonal CAR-T cell is prepared by transfecting a T cell with an expression vector, the expression vector comprising a nucleic acid sequence encoding (from 5'-3'): (a) a signal peptide, (b) a CD19 CAR; (c) a nucleic acid sequence encoding a 2A peptide; (d) a signal peptide; and (e) an orthogonal receptor, the nucleic acid sequence having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to the nucleic acid sequence of SEQ ID:64, wherein the nucleic acid sequence is operably linked to one or more regulatory control sequences operable in a mammalian T cell.

[0180] In some embodiments, the orthogonal CAR-T cell orthogonal is a CD19 CAR-T cell wherein a T cell is recombinantly modified to express a CD19 CAR and an orthogonal CD122 receptor. In some embodiments, the orthogonal CAR-T cell is an orthogonal CD19 CAR-T cell wherein a T cell is a T cell recombinantly modified to express a CD19 CAR and an orthogonal human CD122 (hCD122) receptor. In one embodiment, the orthogonal CD19 CAR-T cell is recombinantly modified to express a CD19 CAR having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 61 (CD19 CAR without signal peptide) and an orthogonal hCD122 receptor having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 39 (hoCD122 without signal peptide), SEQ ID NO: 47 (chimeric orthogonal IL4 receptor), SEQ ID NO: 50 (chimeric orthogonal IL7 receptor), SEQ ID NO: 53 (chimeric orthogonal IL9 receptor) and SEQ ID NO: 56 (chimeric orthogonal IL21 receptor).

[0181] In some embodiments, orthogonal CD19 CAR-T cell comprises a first nucleic acid sequence encoding a CD19 CAR and second nucleic acid sequence encoding an orthogonal CD122 receptor. In some embodiments, orthogonal CD19 CAR-T cell comprises a first nucleic acid sequence encoding a CD19 CAR and second nucleic acid sequence encoding an orthogonalAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT hCD122 receptor. In some embodiments, the first nucleic acid sequence encoding the CD19 CAR having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 61 (CD19 CAR without signal peptide) and the second nucleic acid encodes an orthogonal hCD122 receptor having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 39 (hoCD122 without signal peptide), SEQ ID NO: 47 (chimeric orthogonal IL4 receptor), SEQ ID NO: 50 (chimeric orthogonal IL7 receptor), SEQ ID NO: 53 (chimeric orthogonal IL9 receptor) and SEQ ID NO: 56 (chimeric orthogonal IL21 receptor). In some embodiments, orthogonal CD19 CAR-T cell comprises a first nucleic acid sequence encoding the CD19 CAR is a nucleic acid sequence having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 62 (DNA encoding CAR without signal peptide) and the second nucleic acid encoding the orthogonal hCD122 receptor is a nucleic acid sequence having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 103 (DNA encoding ho CD122 without signal peptide).

[0182] In some embodiments, orthogonal CD19 CAR-T cell comprises a nucleic acid sequence first nucleic acid sequence encoding a CD19 CAR receptor with a signal peptide, the CD19 CAR having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 61 (CD19 CAR without signal peptide), and a second nucleic acid encoding an orthogonal receptor with a signal peptide, the orthogonal CD122 receptor having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 39 (hoCD122 without signal peptide), SEQ ID NO: 47 (chimeric orthogonal IL4 receptor), SEQ IDAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT NO: 50 (chimeric orthogonal IL7 receptor), SEQ ID NO: 53 (chimeric orthogonal IL9 receptor) and SEQ ID NO: 56 (chimeric orthogonal IL21 receptor).

[0183] In some embodiments, orthogonal CD19 CAR-T cell comprises a nucleic acid sequence having ^ 90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 64 (DNA sequence encoding both hoCD122 receptor and CD19 CAR with 2A peptide).

[0184] The disclosure further provides a method preparing a population of orthogonal CAR- T cells, the method comprising contacting the orthogonal CAR-T cells ex vivo in the presence of an a / b biased IL2 mutein. a / b biased IL2 muteins are described in PCT International Publication No WO 2019 / 104092 A1 published May 31, 2019 and PCT International Publication No WO 2021 / 1464S1 Al published January 14, 2020, the entire teachings of which are herein incorporated by reference.

[0185] The orthogonal CAR-T cells prepared in accordance with the foregoing form of a cell therapy product comprising at least one (alternatively 2, 3, 4 or more) species of orthogonal CAR-T cells wherein the fraction of orthogonal CAR-T cells in the cell therapy product comprises at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, or alternatively at least 90% of the total number of cells in the cell therapy product. Orthogonal IL2 Ligand

[0186] The contact of immune cells recombinantly modified to express the orthogonal IL2 receptor with an orthogonal IL2 ligand that selectively binds to orthogonal CD122 ECD (“IL2 ortholog”) provides intracellular signal that results in activation and / or proliferation of the recombinantly modified immune cells expressing the orthogonal IL2 receptor.

[0187] In some embodiments, the orthogonal hIL2 ligand exhibits significantly reduced binding to the extracellular domain of wild type hCD122 compared to wild type hIL2. In some embodiments the orthogonal hIL2 ligand exhibits less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, alternatively less than about 0.5% of the level of binding of wild type hIL2 to the extracellular domain of wild type CD122. Similarly, the ECD of an orthogonal receptor exhibits significantly reduced binding to wild-type hIL2 if the ECD of the orthogonal receptor binds to wild type hIL2 with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less thanAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, alternatively less than about 0.5% of the affinity of wild type hCD122.

[0188] In some embodiments, the orthogonal IL2 ligand exhibits reduced IL2 activity compared to wild type IL2. In some embodiments, the orthogonal human IL2 ligand exhibits reduced IL2 activity compared to wild type human IL2. The reference specific activity of recombinant human IL-2 is approximately 2.1 x 104IU / mg, which is calibrated against recombinant human IL-2 WHO International Standard (NIBSC code: 86 / 500). An orthogonal human IL2 ligand may have less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, alternatively less than about 0.5% of the activity of WHO International Standard (NIBSC code: 86 / 500) human IL2 polypeptide in a comparable assay. In some embodiments the assay measures the phosphorylation of intracellular STAT5.

[0189] In some embodiments, the orthogonal IL2 ligand comprises a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO: 12, further comprising amino acid substitutions at one or more of E15, H16, L19, D20, Q22, and M23 numbered in accordance with the mature human IL2 polypeptide (SEQ ID NO: 12), optionally further comprising: (a) one or more amino acid substitutions selected from the group consisting of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P, T3C N30E; K32E; N33D; P34G; T37I, R38W, R38G, M39Q, R39L, R39V, F42K, F42Y, F44Y, P47G, T51I, E52K, L53N, H55Y, Q57E, V91R, K97E M104A, T113N C125S, and C125A; and / or (b) a deletion of one or more N- terminal amino acids, the deletion of positions 1-9, alternatively positions 1-8, alternatively positions 1-7 alternatively positions 1-6, alternatively positions 1-5, alternatively positions 1-4, alternatively positions 1-3, alternatively positions 1-2, or alternatively position 1 numbered in accordance with SEQ ID NO: 12.

[0190] In some embodiments, the orthogonal IL2 ligand exhibits significantly reduced binding to the extracellular domain of wild type CD122 compared to wild type hIL2. In some embodiments the orthogonal IL2 ligand exhibits less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, alternatively less than about 0.5% of the level of binding of wild type IL2 to the extracellular domain of wild type CD122. Similarly, an ECD of an orthogonal receptor exhibits significantly reduced binding to wild-type IL2 if the ECD of the orthogonal receptor binds to wild type IL2 with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than aboutAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, alternatively less than about 0.5% of the affinity of wild type CD122.

[0191] In some embodiments, the one or more of amino acid substitutions at positions E15, H16, L19, D20 Q22, and M23 are the amino acid substitutions E15S, H16Q, L19V, D20L, Q22K, M23A numbered in accordance with the mature human IL2 polypeptide (SEQ ID NO: 12).

[0192] In some embodiments, the one or more of amino acid substitutions at positions E15, H16, L19, D20 Q22, and M23 is a set of amino acid substitutions selected from the group consisting of: [E15S-H16Q-L19V-D20L-Q22K-M23A], [E15S-H16Q-L19V-D20L-Q22K]; [H16N, L19V, D20N, Q22T, M23H, G27K]; [E15D, H16N, L19V, D20L, Q22T, M23H]; [E15D, H16N, L19V, D20L, Q22T, M23A]; [E15D, H16N, L19V, D20L, Q22K, M23A]; [E15S; H16Q; L19V, D20T; Q22K, M23L]; [E15S; H16Q; L19V, D20T; Q22K, M23S]; [E15S; H16Q; L19V, D20S; Q22K, M23S]; [E15S; H16Q; L19I, D20S; Q22K; M23L]; [E15S; L19V; D20M; Q22K; M23S]; [E15T; H16Q; L19V; D20S; M23S]; [E15Q; L19V; D20M; Q22K; M23S]; [E15Q; H16Q; L19V; D20T; Q22K; M23V]; [E15H; H16Q; L19I; D20S; Q22K; M23L]; [E15H; H16Q; L19I; D20L; Q22K; M23T]; and [L19V; D20M; Q22N; M23S], numbered in accordance with the mature human IL2 polypeptide (SEQ ID NO: 12).

[0193] In some embodiments, the present disclosure provides orthogonal hIL2 ligands to facilitate recombinant expression in bacterial cells by eliminating the unpaired cysteine residue at position 125 and / or elimination of the N-terminal Met of the directly expressed IL2 polypeptide. Such mutations are typically used to avoid misfolding of the protein when expressed recombinantly in bacteria and isolated from inclusion bodies. In some embodiments, the orthogonal IL2 ligand comprises an amino acid substitution at C125 selected from C125A or C125S.

[0194] In addition to the foregoing modifications that contribute to the activity and selectivity of the orthogonal IL2 ligand for an orthogonal receptor, the orthogonal IL2 ligand may comprise one or more modifications to its primary structure that do not substantially diminish the activity of the orthogonal IL2 ligand including but not limited to the substitutions: N30E; K32E; N33D; P34G; T37I, M39Q, F42Y, F44Y, P47G, T51I, E52K, L53N, Q57E, M104A.

[0195] In some embodiments, the orthogonal IL2 ligand comprises a substitution of the T3 residue to eliminate the O-glycosylation site at position T3 to facilitate the production of an aglycosylated orthogonal IL2 ligand when the orthogonal IL2 ligand is recombinantly expressed in mammalian cells such as CHO or HEK cells. Exemplary amino acid substitutions of T3Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P which removes the glycosylation site at position 3 without eliminating biological activity (see U.S. Pat. No.5,116,943; Weiger et al., (1989) Eur. J. Biochem., 180:295-300). In one embodiment, the amino acid substitution at T3 is T3A. In some embodiments, the substitution of the T3 residue is a T3C substitution which, when combined with a deletion of the deletion of the first two N-terminal amino acids (desAla1- desPro2) provides an orthogonal IL2 ligand with an N-terminal cysteine residue to facilitate for selective N-terminal modification, especially PEGylation of the sulfhydryl group of the cysteine (See, e.g. Katre, et al. United States Patent No 5,206,344 issued April 27, 1993) or conjugation to fatty acid molecules (acylation).

[0196] In some embodiments, the orthogonal IL2 ligand comprises a substitution of position M104 with an alanine residue (M104A). The M104A substitution provides an oxidation resistant orthogonal IL2 ligand polypeptide (See e.g., Koths, et al. United States patent 4,752,585 issued June 21, 1988).

[0197] In some embodiments, the orthogonal IL2 ligand polypeptide comprises a deletion of one or more N-terminal amino acids, the deletion of positions 1-9, alternatively positions 1-8, alternatively positions 1-7 alternatively positions 1-6, alternatively positions 1-5, alternatively positions 1-4, alternatively positions 1-3, alternatively positions 1-2, or alternatively position 1 numbered in accordance with SEQ ID NO: 12.

[0198] In some embodiments, the orthogonal IL2 ligand polypeptide comprises amino acid substitutions to avoid vascular leak syndrome, a substantial negative and dose limiting side effect of the use of IL2 therapy in human beings without out substantial loss of efficacy. See, Epstein, et al., United States Patent No 7,514,073B2 issued April 7, 2009. In some embodiments, the orthogonal IL2 ligand comprises amino acid substitutions include one or more amino acid substitutions selected from the group consisting of R38W, R38G, R39L, R39V, F42K, and H55Y.

[0199] The orthogonal IL2 ligand polypeptide may optionally comprise one or more modifications (e.g. PEGylation, acylation, Fc conjugation) that result in an orthogonal IL2 ligand exhibiting an extended lifetime in vivo and / or extended duration of action in a subject. In addition to, or in the alternative to, such modifications, the orthogonal IL2 ligand may comprise amino acid substitutions that result in prolonged in vivo lifetime. Examples of amino acid substitutions that result in extended stability and activity include V91R, K97E and T113N, Dakshinamurthi, et al. (2009) International Journal of Bioinformatics Research 1(2):4-13) In some embodiments, the orthogonal IL2 ligands of the present disclosure comprise one, two or all three of the V91R, K97E and T113N modifications.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0200] In some embodiments, the orthogonal IL2 ligand comprises a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO: 41. In some embodiments, the orthogonal IL2 ligand comprises a polypeptide a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO: 43. In some embodiments, the orthogonal IL2 ligand comprises a polypeptide a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO: 44. Modifications of the Orthogonal IL2 Ligand to Provide Extended Duration of Action:

[0201] In some embodiments, the orthogonal IL2 ligand comprises one or more modifications to extend the duration of action of the molecule in the mammalian subject following administration of the commonly referred to as half-life extension. In some embodiments, the orthogonal IL2 ligand modified to provide an extended half-life and / or duration of action in vivo exhibits a half-life in a human subject of greater than 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, or 30 days. Conjugation to Carrier

[0202] In some embodiments, the one or more modifications to extend the duration of action of the orthogonal IL2 ligand is covalent attachment of the orthogonal IL2 ligand to a carrier molecule. As used herein, the term “carrier molecules” refers to large, slowly metabolized macromolecules. Examples such slowly metabolized macromolecules carriers include proteins, polysaccharides; fatty acid molecule molecules (acylation), polymeric amino acids such as polyglutamic acid, or polylysine; amino acid copolymers.

[0203] Examples of protein carrier molecules which may be covalently linked to the orthogonal IL2 ligand to provide an extended duration of action in vivo include, but are not limited to albumins, antibodies, and antibody fragments such and Fc domains of IgG molecules. In many embodiments, the modification to prolong half-life is an albumin or an albumin fragment, an antibody, an antibody fragment, an antibody or an antibody fragment that minds to albumin, one or more PEG molecules, one or more fatty acid molecules, or any combination thereof. Water-Soluble PolymersAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0204] In some embodiments, half-life extension of the orthogonal IL2 ligand in a mammalian subject may be achieved by covalent linkage of orthogonal IL2 ligand to one or more water-soluble polymers. Examples of water-soluble polymers that may be conjugated to the orthogonal IL2 ligand include polyethylene glycol (PEG), poly-propylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyolefinic alcohol, polysaccharides, poly-alpha-hydroxy acid, polyvinyl alcohol (PVA), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), XTEN polymers, or a combination thereof. Polyethylene Glycol (PEG):

[0205] In some embodiments, half-life extension of the orthogonal IL2 ligand in a mammalian subject may be achieved by covalent linkage of the orthogonal IL2 ligand to one or more polyethylene glycol molecules (“PEGylation”), wherein the covalent linkage may optionally include a linker molecule between the PEG and the orthogonal IL2 ligand. PEGs suitable for covalent linkage with the orthogonal IL2 ligand have the general formula R(O-CH2- CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons. The PEG covalently linked to the polypeptide sequence can be linear or branched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs are contemplated by the present disclosure.

[0206] PEGs useful in the present disclosure is not restricted to any particular molecular weight or architecture. In some embodiments, the PEG is a linear PEG, branched PEG, star-PEG, or multi-armed PEG having an average molecular mass from about 2 kDa to about 80 kDa, alternatively from about 2 kDa to about 70 kDa, alternatively from about 5 kDa to about 50 kDa, alternatively from about 5 kDa to about 50 kDa, alternatively from about 20 kDa to about 50 kDa, alternatively from about 30 kDa to about 50 kDa, alternatively from about 20 kDa to about 40 kDa, alternatively from about 5 kDa to about 10 kDa, alternatively from about 5 kDa to about 15 kDa, alternatively from about 5 kDa to about 20 kDa, from about 10 kDa to about 15 kDa, alternatively from about 10 kDa to about 20 kDa, alternatively from about 10 kDa to about 25 kDa or alternatively from about 10 kDa to about 30 kDa alternatively from about 30 kDa to about 40 kDa, alternatively about 5 kDa, alternatively about 10 kDa, alternatively about 15 kDa, alternatively about 20 kDa, alternatively about 25 kDa, alternatively about 30 kDa, alternatively about 35 kDa, alternatively about 40 kDa, alternatively about 45 kDa, alternatively about 50 kDa, alternatively about 55 kDa, alternatively about 60 kDa, alternatively about 65 kDa, alternatively about 70 kDa, alternatively about 75 kDa, or alternatively about 80 kDa. InAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT some embodiments, the PEG is a 40 kDa linear PEG. In some embodiments, the PEG is a branched chain PEG having an average molecular weight of about 40 kDa. In some embodiments, the PEG is a branched PEG having an average molecular weight of about 40 kDa comprising two 20 kDa arms.

[0207] PEGs useful in the practice of the present disclosure include a 10 kDa linear PEG- aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10 kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), a 20 kDa linear PEG-aldehyde (e.g. Sunbright® ME-200AL, NOF, a 20kDa linear PEG- NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME- 200HS, NOF), a 20 kDa 2-arm branched PEG-aldehyde the 20 kDa PEG-aldehyde comprising two 10kDA linear PEG molecules (e.g., Sunbright® GL2-200AL3, NOF), a 20 kDa 2-arm branched PEG-NHS ester the 20 kDa PEG-NHS ester comprising two 10kDA linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), a 40 kDa 2-arm branched PEG-aldehyde the 40 kDa PEG-aldehyde comprising two 20 kDa linear PEG molecules (e.g., Sunbright® GL2-400AL3), a 40 kDa 2-arm branched PEG-NHS ester the 40 kDa PEG-NHS ester comprising two 20 kDa linear PEG molecules (e.g., Sunbright® GL2- 400AL3, Sunbright® GL2-400GS2, NOF), a linear 30 kDa PEG-aldehyde (e.g., Sunbright® ME-300AL) and a linear 30 kDa PEG-NHS ester.

[0208] In some embodiments, the PEG is an activated monomethoxy PEGs (mPEGs) are succinimdyl 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 react preferentially with lysine residues to form a carbamate linkage but are also known to react with histidine and tyrosine residues. Use of a PEG-aldehyde linker targets a single site on the N-terminus of a polypeptide through reductive amination.

[0209] In some embodiments, the PEG is conjugated to the N-terminal amino acid of the orthogonal IL2 polypeptide (including where the N-terminal amino acid is proline which lacks an amino group (e.g., SEQ ID: 41), alternatively the PEG is conjugated to the epsilon amino group on the side chain of lysine residues, alternatively the PEG is conjugated to the imidazole group of histidine residues. Since most recombinant polypeptides possess a single alpha and a number of epsilon amino and imidazole groups, numerous positional isomers can be generated depending on the linker chemistry. In some embodiments, the orthogonal IL2 ligand comprises multiple (e.g., 2, 3 or 4) PEGs.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0210] In some embodiments, the orthogonal IL2 ligand may be modified to incorporate one or more amino acid substitutions to facilitate conjugation of the PEG to a particular location on the molecule (herein referred to as "site specific PEGylation"). Site specific PEGylation may be achieved by substitution of one or more amino acids of the orthogonal IL2 ligand with an amino acid selected from the group consisting of naturally occurring amino acids or non-natural amino acids wherein the side chain of such substituted amino acid facilitates PEGylation. One example of an amino acid substitution that facilitates PEGylation is the incorporation of a cysteine residue to facilitate PEGylation via the sulfhydryl side chain of cysteine. Alternatively, the amino acid substitution to facilitate PEGylation non-natural amino acids having side chains to facilitate selective PEG covalently linkage. For example, the α / ^-orthogonal IL2 ligand may comprise a substitution of a cysteine may be for the threonine at position 3 (3TC) to facilitate N-terminal PEGylation using particular chemistries. Incorporation of non-natural amino acids having side chains to facilitate selective PEG conjugation chemistries as described Ptacin, et al., (PCT International Application No. PCT / US2018 / 045257 filed August 3, 2018, and published February 7, 2019 as International Publication Number WO 2019 / 028419Al.

[0211] Conversely, site-specific conjugation of the PEG to an orthogonal IL2 ligand to may be used to generate an orthogonal IL2 ligand by incorporating non-natural amino acids having a PEGylatable specific moiety at those sequences or residues of hIL2 identified as interacting with hCD132 including amino acids such as residues 18, 22, 109, 126, and 119-133. In some embodiments, the non-natural amino acid is N6-azidoethoxy-L-lysine (AzK), N6- propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8- oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L- phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p- propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L- phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4- propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3 -(benzyl oxy)-3- oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine.

[0212] The PEG may be attached directly to the orthogonal IL2 ligand or via a linker molecule. Suitable linkers include “flexible linkers” which are generally of sufficient length toAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT permit some movement between the modified polypeptide sequences and the linked components and molecules. n some embodiments, the linker molecule is about 6-50 atoms long. In some embodiments, the linker molecule is selected from the group consisting of aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. In some embodiments, the polypeptide linker is a GSA linker.

[0213] In one embodiment of the disclosure, the orthogonal IL2 ligand is a compound of the structure: (PEG)-(linker)n-(MAPTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 44). wherein n = 0 (absent) or 1 (present).

[0214] In one embodiment of the disclosure, the orthogonal IL2 ligand is a compound of the structure: (PEG)-(linker)n-(APTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 43). wherein n = 0 (absent) or 1 (present).

[0215] In one embodiment of the disclosure, the orthogonal IL2 ligand is a compound of the structure: (PEG)-(linker)n-(PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 41). wherein n = 1 (present),

[0216] In some embodiments, the 40kDa-PEG of the above structures is a linear 40kDa PEG. In some embodiments, the 40kDa-PEG of the above structures is a branched 40kDa PEG In some embodiments, the 40kDa-PEG of the above structures is a branched 40kDa PEG comprising two 20kDa PEG arms.

[0217] In some embodiments, the orthogonal IL2 ligand is a compound of the structure: (40 kDa-PEG)-(linker)n-(MAPTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKL TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 44). wherein n = 1 (present), wherein the 40kDa-PEG is a branched 40 kDa-PEG comprising two 20kDa arms.

[0218] In some embodiments, the orthogonal IL2 ligand is a compound of the structure:Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT (40 kDa-PEG)-(linker)n-(APTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKL TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 43), wherein n = 1 (present), wherein the 40kDa-PEG is a branched 40 kDa-PEG comprising two 20kDa arms.

[0219] In some embodiments, the orthogonal IL2 ligand is a compound of the structure: (40 kDa-PEG)-(linker)n-(PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKL TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 41), wherein n = 1 (present), wherein the 40kdA-PEG is a branched 40 kDa-PEG comprising two 20kDa arms. Xten Polymers:

[0220] In some embodiments, the half-life extended orthogonal IL2 ligand comprises the orthogonal IL2 ligand covalently linked to an XTEN polypeptide carrier molecule. The XTEN polypeptide may be covalently linked (either by chemical conjugation or as a fusion protein) to the orthogonal IL2 ligand to provide extended duration of action of the orthogonal IL2 ligand. The XTEN-linked orthogonal IL2 ligand may be produced as a recombinant fusion protein in E. coli. XTEN polymers suitable for use in conjunction with the hIL2 muteins of the present disclosure are provided in Podust, et al. (2016) “Extension of in vivo half-life of biologically active molecules by XTEN protein polymers”, J Controlled Release 240:52-66 and Haeckel et al. (2016) “XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic” PLOS ONE | DOI:10.1371 / journal.pone.0157193 June 13, 2016. The XTEN polymer fusion protein may incorporate a protease sensitive cleavage site between the XTEN polypeptide and the hIL2 mutein such as an MMP-2 cleavage site. Albumin

[0221] In some embodiments, half-life extension of the orthogonal IL2 ligand in a mammalian subject may be achieved by covalent linkage to the orthogonal IL2 ligand to an albumin molecule or an albumin fragment. The term “albumin” includes albumins such as human serum albumin (HSA), cyno serum albumin, and bovine serum albumin (BSA). In some embodiments, the HSA the HSA comprises a C34S or K573P amino acid substitution relative to the wild-type HSA sequence. The albumin molecule can be covalently linked to an orthogonal IL2 ligand at the carboxyl terminus, the amino terminus, or both the carboxyl and amino termini, and internally (see, e.g., U.S. Patent Nos.5,876,969 and 7,056,701). In some embodiments, theAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT orthogonal IL2 ligand is covalently linked to albumin via chemical conjugation. In some embodiments, the half-life extended orthogonal IL2 ligand is a fusion protein comprising: (1) the polypeptide sequence of an albumin (including but not limited to human serum albumin) and (2) the polypeptide sequence of an orthogonal IL2 ligand, wherein (1) and (2) are optionally covalently bonded via a polypeptide linker including but not limited to GSA linkers. Anti-albumin antibodies

[0222] In some embodiments, half-life extended orthogonal IL2 ligand comprises the orthogonal IL2 ligand is linked to an antibody that binds to albumin. In some embodiments, the antibody that binds to albumin is selected from the group consisting of anti-albumin IgG1 antibodies, anti-albumin IgG2 antibodies, anti-albumin IgG3 antibodies, anti-albumin IgG4, anti-albumin and antibody fragments thereof. In some embodiments, the anti-albumin antibody is a single chain antibody including but not limited to scFvs derived from anti-albumin antibodies. In some embodiments, the anti-albumin antibody is a VHH derived from a camelid antibody. In some embodiments, the antibody, scFv, or VHH is humanized. In some embodiments, the antibody is a human antibody. In some embodiments, the half-life extended orthogonal IL2 ligand is a fusion protein comprising: (1) the polypeptide sequence of an anti- albumin single chain antibody and (2) the polypeptide sequence of an orthogonal IL2 ligand, wherein (1) and (2) are optionally covalently bonded via a polypeptide linker. Examples of VHHs that may be used in the preparation of half-life extended orthogonal IL2 ligand polypeptides useful in the practice of the present disclosure are disclosed in Ferrante, et al. WO2022 / 169757A published August 11, 2022. Fc Domains

[0223] In some embodiments, the half-life extended orthogonal IL2 ligand is an orthogonal IL2 ligand covalently linked to the Fc domain derived from a mammalian (preferably human) immunoglobulin such as but not limited to an IgG1, IgG2, IgG3 or IgG4 molecule. Engineered Fc domains have been extensively investigated in the context of therapeutic antibodies, particularly bi-specific antibodies, with numerous Fc engineered antibodies being developed and commercialized. See, e.g., Czajkowsky, et al. (2012) EMBO Mol Med 4:1015-1028. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells that line the blood vessels, and, upon binding, the Fc fusion molecule is protected from degradation and re-released into the circulation, keeping the molecule in circulation longer. In some embodiments, the Fc domain of the Fc half-life extended orthogonal IL2 ligand is an Fc dimer comprising a first Fc polypeptide and a second Fc polypeptide wherein the first Fc polypeptide and a second Fc polypeptide are the same (a homodimeric Fc) or different (a heterodimeric Fc).Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0224] In some embodiments, the first and / or second Fc polypeptides comprising the Fc dimer comprise one or more modifications to the naturally occurring Fc sequence including but not limited to modified hinge regions, modifications to reduce effector function of the Fc, modifications to facilitate disulfide linkages between the Fc subunits as well as incorporation of amino acid substitutions in Fc monomers to provide geometrically complementary structures enabling consistent 1:1 association of heterodimeric Fc dimers.

[0225] The upper hinge region of the Fc domain typically contains an unpaired cysteine residue at position 220 (EU numbering) that, in a complete immunoglobulin molecule, binds to a cysteine on a light chain. However, when only the Fc domain is used comprising the hinge domain, the unpaired cysteine in the hinge domain creates the potential of the formation of improper disulfide bonds. Consequently, in some embodiments the cysteine at position 220 (C220, numbered in accordance with EU numbering) of the one or both of the Fc polypeptides of the Fc dimer is substituted with an amino acid that does not promote disulfide bonding including but not limited to C220S, C220G and C220A.

[0226] In some embodiments, one or both of the Fc polypeptides of the Fc dimer comprise the amino acid substitutions selected from the group consisting of M428L and N434S (LS modification); M252Y, S254T and T256E (YTE modification); T250 and M428 (QL modification); N434A; N434H; H433K and N434F; T307A, E380A, and N434A (AAA modification); or V259I, V308F, and M428L (IFL modification) (all amino acid substitutions are numbered in accordance with EU numbering). See, e.g., Zalevsky, et al. (2010) Nature Biotechnology 28:157-159; and Rath, Timo et al. “Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics.” Critical reviews in biotechnology vol.35,2 (2015): 235-54. doi:10.3109 / 07388551.2013.834293. Engineered Fc domains useful in the preparation of extended duration orthogonal IL2 ligand s may optionally comprise a mutation that inhibits complement fixation and Fc receptor binding.

[0227] In some embodiments, the Fc domain comprises a first Fc polypeptide and the second Fc polypeptide wherein the first and second Fc polypeptides are different, i.e., the dimeric Fc is a heterodimer. In some embodiments, the first Fc polypeptide and the second Fc polypeptide of the heterodimer contain one or more amino acid substitutions to promote heterodimerization. A variety of techniques are established for the promotion of heterodimerization of Fc domains. See, e.g., U.S. Patent No.11,087,249. In some embodiments, the modifications to promote heterodimerization of the first and second Fc monomer polypeptides are the HF-TA mutations and the HA-TF mutations as described in Moore, et al (2011) mAbs 3(6):546-557. The HF-TA method employs the S364H / T394F substitutions on one Fc monomer and the Y349T / F405AAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT substitutions on the complementary Fc monomer. The HA-TF method employs the S364H / F405A substitutions on one Fc monomer and the Y349T / T394F substitutions on the complementary Fc monomer. Alternatively, the first Fc polypeptide and the second Fc polypeptide of the dimeric Fc are modified to promote heterodimerization by the ZW1 heterodimerization method which employs the T350V / L351Y / F405A / Y407V substitutions on one Fc monomer and the T350V / T366L / K392L / T394W substitutions on the complementary Fc monomer. Von Kreudenstein, et al (2013) mAbs, 5(5):646-654. Alternatively, the first Fc polypeptide and the second Fc polypeptide of the dimeric Fc are modified to promote heterodimerization by the EW-RVT heterodimerization method which employs the K360E / K409W substitutions on one Fc monomer and the Q347R / D399V / F405T substitutions on the complementary Fc monomer. Choi , et al (2015) Molecular Immunology 65(2):377–83. In some embodiments, the first Fc polypeptide and the second Fc polypeptide of the dimeric Fc comprise “knob-into-hole” (KiH) modifications to promote heterodimerization. The KiH modification comprises one or more amino acid substitutions in a first Fc monomer polypeptide that creates a bulky “knob” domain on the first Fc monomer polypeptide, and one or more amino acid substitutions in a second Fc monomer polypeptide that creates a complementary pocket or “hole” to receive the “knob” of the first Fc monomer. Examples of “KiH” Fc modifications useful in the practice of the present disclosure are described in Ridgeway et al. (1996) Protein Eng.9617–621, and U.S. Patent Nos.5,731,168 and 11,087,249. Engineered Fc domains useful in the preparation of extended duration orthogonal IL2 ligand s may optionally be modified by the introduction of cysteine residues at positions S354 and Y349 which results in a stabilizing disulfide bridge between the two antibody heavy chains in the Fe region (Carter, et al. (2001) Immunol Methods 248, 7-15). In some embodiments, the KiH modification comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one Fc polypeptide and the amino acid substitutions T366S, L368A, Y407V, and optionally Y349C in the other Fc polypeptide.

[0228] In some embodiments, first and second Fc monomer polypeptides are linked by at least one disulfide bond. In some embodiments, the incorporation of a disulfide bond between the first and second Fc monomer polypeptides is achieved by cysteine substitutions at particular points within the Fc1 and Fc2 monomers. In some embodiments, a first Fc monomer polypeptide derived from the Fc domain of hIgG1 comprises the an amino acid substitution S354C (EU numbering) and the second Fc monomer polypeptides is derived from the Fc domain of hIgG1 comprising an amino acid substitution Y349C (EU numbering). AcylationAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0229] In some embodiments, half-life extension of the orthogonal IL2 ligand in a mammalian subject may be achieved by covalent linkage of the orthogonal IL2 ligand to a fatty acid molecule. Acylation of polypeptides to achieve an extended in vivo duration of action is described in Resh (2016) Progress in Lipid Research 63: 120–131. In some embodiments, the fatty acid molecule is a medium chain fatty acid (MCFA) comprising an aliphatic tail 6, 7, 8, 9, 10, 11 or 12 carbons. In some embodiments, the fatty acid molecule is a long chain fatty acid (LCFA) comprising an aliphatic tail of 13, 14, 15, 16, 17, 18, 19, 20, of 21 carbons. In some embodiments, the fatty acid molecule is a very long chain fatty acid (VLCFA) comprising an aliphatic tail of 22, 23, 24, 15, 26, 27, 28, or more carbons. Examples of fatty acids that may be covalently linked include myristate, palmitate and palmitoleic acid. Myristoylate is typically linked to an N-terminal glycine but lysines may also be myristoylated. Palmitoylation is typically achieved by enzymatic modification of free cysteine -SH groups such as DHHC proteins catalyze S-palmitoylation. Palmitoylation of serine and threonine residues is typically achieved enzymatically using PORCN enzymes. In some embodiments, the orthogonal IL2 ligand is acetylated at the N-terminus by enzymatic reaction with N-terminal acetyltransferase and, for example, acetyl CoA. Alternatively, or in addition to N-terminal acetylation, the orthogonal IL2 ligand is acetylated at one or more lysine residues, e.g., by enzymatic reaction with a lysine acetyltransferase. See, for example Choudhary, et al. (2009) Science 325 (5942):834L2 ortho840. In some embodiments, the fatty acid molecule is covalently linked to the sulfhydryl side chain of a cysteine residue of the orthogonal IL2 ligand. In some embodiments, the polypeptide sequence comprising a cystine residue with a free sulfhydryl group is added to the N- or C-terminus of the orthogonal IL2 ligand to provide a covalent attachment point for the fatty acid molecule. In some embodiments, the orthogonal ligand comprises a deletion of the first two amino acids (DesAla1-DesPro2) and a T3C amino acid substitution to facilitate acylation at the C3 residue. Amino Acid Substitutions:

[0230] As previously noted, the orthogonal IL2 ligand may optionally comprise amino acid substitutions that provide an extended half-life and / or duration of action in vivo. Examples of amino acid substitutions associated with prolonging the half-life and / or providing an extended duration action in vivo of include the amino acid substitutions V91R, K97E and T113. In some embodiments, the orthogonal IL2 ligand comprises one, two or all three of the amino acid substitutions V91R, K97E and T113N. Targeted Orthogonal IL2 Ligands:Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0231] In some embodiments, the orthogonal IL2 ligand is covalently linked to a targeting moiety. As used herein the term "targeting moiety" refers to a molecule exhibits selective binding to a molecule associated with the surface of a particular cell type or tissue. In some embodiments, the orthogonal IL2 ligand is covalently linked to a targeting moiety via a chemical linker. In some embodiments, the orthogonal IL2 ligand is covalently linked to a targeting moiety via a polypeptide linker molecule of from 1-40 (alternatively 2-20, alternatively 5-20, alternatively 10-20) amino acids between the orthogonal IL2 ligand sequence and the sequence of the targeting moiety of the fusion protein. In some embodiments, the polypeptide linker is a GSA linker. In some embodiments, wherein the targeting domain, the orthogonal IL2 ligand is a fusion protein comprising a targeting moiety and an orthogonal IL2 ligand polypeptide, optionally wherein the targeting moiety and orthogonal IL2 ligand polypeptide are covalently bonded via a polypeptide linker, optionally a GSA linker.

[0232] In some embodiments, the targeting moiety of a targeted orthogonal IL2 ligand is an antibody, an antigen-binding portion of an antibody, an antibody fragment (e.g., ), an scFv, and / or VHH that specifically binds to molecule on the surface of a cell. Methods of generating cytokine-antibody chimeric polypeptides are described, for example, in U.S. Pat. No.6,617,135. In one embodiment, the targeting moiety of a targeted orthogonal IL2 ligand specifically binds to a B-cell antigen. In some embodiments, the targeting moiety of a targeted orthogonal IL2 ligand specifically binds to a B cell antigen selected from the group consisting of CD19, CD20, CD22, BAFF, TACI and BCMA.

[0233] In some embodiments, the targeting moiety of a targeted orthogonal IL2 ligand is an scFv that specifically binds to a B cell antigen selected from the group consisting of CD19, CD20, CD22, BAFF, TACI and BCMA. VHH As Targeting Moiety:

[0234] In some embodiments, the targeting moiety of the targeted orthogonal ligand is a VHH that that exhibits specific binding to at one least cell surface antigen. The term VHH refers to a fragment of a camelid heavy-chain antibody that exhibits specific binding to the antigen associated with the parent heavy chain antibody. In some embodiments, the VHH is a humanized VHH. In some embodiments, targeting moiety of the targeted orthogonal ligand comprises a VHH that specifically binds to a B-cell antigen, wherein the B-cell antigen selected from the group consisting of the extracellular domains of hCD19, hCD20, hCD22, BAFF, TACI and hBCMA. scFvs As Targeting Moiety

[0235] In some embodiments, targeting moiety of the targeted orthogonal ligand that exhibitsAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT specific binding to at one least cell surface antigen is a single chain Fv (ScFv). An ScFv is a polypeptide comprised of the variable regions of the immunoglobulin heavy and light chain of an antibody covalently connected by a peptide linker (Bird, et al. (1988) Science 242:423-426; Huston, et al. (1988) PNAS(USA) 85:5879-5883; S-z Hu, et al. (1996) Cancer Research, 56, 3055-3061; Ladner, United States Patent No 4946778 issued August 7, 1990). In some embodiments, the scFv is a humanized scFv. In some embodiments, targeting moiety of the targeted orthogonal ligand comprises a scFv that specifically binds to a B-cell antigen, wherein the B-cell antigen selected from the group consisting of the extracellular domains of hCD19, hCD20, hCD22, BAFF, TACI and hBCMA.

[0236] In some embodiments the linker that joins the heavy and light chain domains of the scFv is a GSA linker of 4-20, alternatively 12-16, amino acids. In some embodiments the linker that joins the heavy and light chain domains of the scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. CD19ScFv As Targeting Moiety

[0237] In some embodiments, the targeting moiety of the targeted orthogonal ligand comprises an scFv that binds to CD19 (an "anti-CD19 scFv" or "CD19 scFv"). Examples of anti-CD19 scFvs that may be incorporated into the ABD of the CAR include but are not limited to FMC63, 1D3, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b (Kang, et al (2020) Int J Mol Sci 21(23):9163.

[0238] In some embodiments, anti-CD19 scFv comprises a light chain polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 26, a linker that joins the heavy and light chain domains of the scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 and a heavy chain sequence having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98%Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 27. In some embodiments, the anti-CD19 scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 35.

[0239] In some embodiments, an anti-CD19 scFv comprises a light chain polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 28, a linker that joins the heavy and light chain domains of the scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 and a heavy chain sequence having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 29. In some embodiments, the anti-CD19 scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 36.

[0240] In some embodiments, the targeting moiety of the targeted orthogonal ligand comprises an scFv that binds to, the orthogonal IL2 ligand is provided as a fusion protein with a BCMA targeting moiety, such as antibody or scFv comprising the CDRs of anti-BMCA antibodies as described in in Kalled, et al. (United States Patent 9,034,324 issued May 9, 2015) or antibodies or scFvs comprising the CDRs as described in Brogdon, et al., (United States Patent No 10,174,095 issued January 8, 2019). Synthesis of Orthogonal IL2 Ligands

[0241] The orthogonal IL2 ligands of the present disclosure may be produced by conventional methodology for the construction of polypeptides including but not limited recombinant production or solid phase synthesis.

[0242] In some embodiments, orthogonal IL2 ligands of the present disclosure are produced by chemical synthesis The chemical synthesis of the orthogonal IL2 ligand of may proceed viaAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS) allows the incorporation of unnatural amino acids and / or peptide / protein backbone modification. Various forms of SPPS are available for synthesizing orthogonal IL2 ligand of the present disclosure are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem.6:3-10; and Camarero J.A. et al., (2005) Protein Pept Lett.12:723-8). In the course of chemical synthesis, the alpha functions and any reactive side chains may be protected with acid-labile or base-labile groups that are stable under the conditions for linking amide bonds but can readily be cleaved without impairing the peptide chain that has formed.

[0243] In one embodiment, the orthogonal IL2 ligand of the present disclosure are produced by recombinant DNA technology. The practice of recombinant production of polypeptides requires: (a) preparation of an isolated nucleic acid sequence encoding the orthogonal IL2 ligand; (b) incorporation of the isolated nucleic acid encoding the orthogonal IL2 ligand into an expression vector suitable for the host cell in which expression will be accomplished, wherein the nucleic acid sequence is inserted into the expression vector so as to be in operable association one or more expression control sequences of the expression vector; (c) introducing the expression vector into a compatible host cell; (d) culturing the host cell under conditions that facilitate transcription and translation of the nucleic acid sequence such that the host cell produces the orthogonal IL2 ligand; and (e) isolating the orthogonal IL2 ligand from the host cell culture.

[0244] In some embodiments, the orthogonal IL2 ligand is produced by recombinant methods using a nucleic acid sequence encoding the orthogonal IL2 ligand (or fusion protein comprising the orthogonal IL2 ligand). Due to the known degeneracy of the genetic code, multiple nucleic acid sequences encode the same the orthogonal IL2 ligand. The nucleic acid molecules encoding the orthogonal IL2 ligand (and fusions thereof) may contain naturally occurring sequences or sequences that differ from those that occur naturally. A nucleic acid sequence encoding the orthogonal IL2 ligand may be synthesized by conventional means using an oligonucleotide synthesizer. In some embodiments, the nucleic acid sequence encoding the orthogonal IL2 ligand is codon optimized for expression in the host cell of employed for recombinant production. Techniques for codon optimization in a wide variety of expression systems, including mammalian, yeast and bacterial host cells, are well known in the and there are online tools to provide for a codon optimized sequences for expression in a variety of host cell types. See e.g. Hawash, et al., (2017) 9:46-53 and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols, edited by David Hacker (Human Press New York). Additionally, there are a variety of software tools that may be used in theAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT preparation of codon optimized nucleic acid sequences. Services providing synthesis of nucleic acid sequences encoding the orthogonal IL2 ligand may be obtained from various commercial sources that provide custom made nucleic acid sequences.

[0245] An orthogonal IL2 ligand of the present disclosure may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus or C-terminus of the mature orthogonal IL2 ligand. In general, the signal sequence may be a component of the vector, or it may be a part of the coding sequence that is inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In some embodiments, the signal sequence is the signal sequence that is natively associated with the orthogonal IL2 ligand (i.e. the human IL2 signal sequence). The inclusion of a signal sequence depends on whether it is desired to secrete the orthogonal IL2 ligand from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be encoded and most preferably that the wild type IL2 signal sequence be used. Alternatively, heterologous mammalian signal sequences may be suitable, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex gD signal. When the recombinant host cell is a yeast cell such as Saccharomyces cerevisiae, the alpha mating factor secretion signal sequence may be employed to achieve extracellular secretion of the orthogonal IL2 ligand into the culture medium as described in Singh, United States Patent No.7,198,919 B1 issued April 3, 2007.

[0246] In the event the orthogonal IL2 ligand to be expressed is to be expressed as a chimera (e.g., a fusion protein comprising an orthogonal IL2 ligand and a heterologous polypeptide sequence such as a targeting domain or carrier protein), the chimeric protein can be encoded by a hybrid nucleic acid molecule comprising a first sequence that encodes all or part of the orthogonal IL2 ligand and a second sequence that encodes all or part of the heterologous polypeptide, optionally further comprising a nucleic acid sequence encoding a linker between the nucleic acid sequence encoding the orthogonal IL2 ligand the nucleic acid sequence encoding the heterologous peptide. By first and second, it should not be understood as limiting to the orientation of the elements of the fusion protein and a heterologous polypeptide can be linked at either the N-terminus and / or C-terminus of the orthogonal IL2 ligand.

[0247] In some embodiments, the heterologous polypeptide sequence is a polypeptide that facilitates purification of the recombinantly produced protein. In some embodiments, theAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT heterologous polypeptide sequence that facilitates purification of the recombinantly produced protein is a chelating peptide (e.g. a histidine polymer comprising 4, 5, 6, 7, 8, 9 or 10 histidine residues) from to purification by immobilized metal affinity chromatography (IMAC). In some embodiments, the heterologous polypeptide sequence that facilitates purification of the recombinantly produced protein is a to a hemagglutinin tag.

[0248] The complete amino acid sequence of the polypeptide (or fusion / chimera) to be expressed can be used to construct a back-translated gene. A DNA oligomer containing a nucleotide sequence coding for orthogonal IL2 ligand can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0249] In some embodiments, the nucleic acid sequence encoding the orthogonal IL2 ligand encodes a polypeptide having the amino acid sequence of an orthogonal IL2 ligand selected from the group consisting of SEQ ID NO: 41, SEQ ID NO:43 and SEQ ID NO:44. In some embodiments, the nucleic acid sequence encoding the orthogonal IL2 ligand encodes a polypeptide having the amino acid sequence SEQ ID NO: 41. In some embodiments, the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 42.

[0250] For recombinant production, the nucleic acid sequence encoding the orthogonal IL2 ligand is inserted into an expression vector wherein the nucleic acid sequence encoding the orthogonal IL2 polypeptide sequence to be expressed is operably linked to transcriptional and translational regulatory control sequences that are functional in the chosen expression host. Expression vectors for orthogonal IL2 ligand of the present disclosure contain a regulatory sequence that is recognized by the host organism and is operably linked to nucleic acid sequence encoding the orthogonal IL2 ligand. The terms “regulatory control sequence,” “regulatory sequence” or “expression control sequence” are used interchangeably herein to refer to one or more genetic components that facilitate transcription of the nucleic acid sequence encoding the orthogonal IL2 polypeptide including but not limited promoters, enhancers, and other control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego CA USA Regulatory sequences include those that direct constitute expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. In selecting an expressionAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT control sequence, a variety of factors understood by one of skill in the art are to be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject orthogonal IL2 ligand, particularly as regards potential secondary structures. A variety of expression vectors for uses in various host cells are available and are typically selected based on the host cell for expression. An expression vector typically includes, but is not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, and the like. Plasmids are examples of non-viral vectors.

[0251] The expression vector may optionally encode a nucleic acid sequence encoding a selectable marker. Typical selection markers are polypeptides that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, (c) supply critical nutrients not available from complex media, or (d) cell surface expressed proteins that may be readily identifiable through the use of antibodies, including fluorescently tagged antibodies. Examples of selectable markers include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT).

[0252] Vectors suitable for eukaryotic host cells include insect cells (examples of Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al. (1987) EMBO J.6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corporation, San Diego, Calif.)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0253] Host cells useful in the recombinant production of orthogonal IL2 ligands include procaryotic and eucarytoic host cells. In some embodiments the host cell used for the recombinant production of the orthogonal IL2 ligand is a eucaryotic cell. In some embodiments, the eucaryotic cell is a yeast cell. Examples of yeast cells useful for the recombinant production of orthogonal IL2 ligands include but are not limited to Saccharomyces cerevisiae and Pichia pastoris.

[0254] In some embodiments, the eucaryotic cell used for the recombinant production of the orthogonal IL2 ligand is a mammalian cell. Examples of useful mammalian host cells useful in the practice of the present disclosure include but are not limited to mouse L cells (L-M[TK-], ATCC#CRL-2648), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or HEK293 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-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Suitable host cells are available from commercial sources, including the American Type Culture Collection (Manassas, Va.).

[0255] The expression vector encoding the orthogonal IL2 ligand is introduced into host cells to thereby produce the orthogonal IL2 ligand disclosed herein or to produce biologically active muteins thereof. In order to facilitate transfection of the target cells with expression vector encoding the orthogonal IL2 ligand, the target cell may be exposed directly with the non-viral vector may under conditions that facilitate uptake of the non-viral vector. Examples of conditions which facilitate uptake of foreign nucleic acid by mammalian cells are well known in the art and include but are not limited to chemical means (such as Lipofectamine®, Thermo- Fisher Scientific), high salt, and magnetic fields (electroporation).

[0256] Host cells containing the expression vector encoding the orthogonal IL2 ligand may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Mammalian host cells may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (suchAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.

[0257] The recombinatly produced orthogonal IL2 ligand is recovered from the host cell culture. Recombinantly produced orthogonal IL2 ligand polypeptides can be recovered from the culture medium as a secreted polypeptide if a secretion leader sequence is employed. Alternatively, the orthogonal IL2 ligand polypeptides can also be recovered from host cell lysates. The recombinatly produced orthogonal IL2 ligand is may isolated from the culture medium or host cell lysate using conventional chromatographic techniques. The orthogonal IL2 ligand produced by the transformed host can be purified according to any suitable method. Various methods are known for purifying IL2. See, e.g. Current Protocols in Protein Science, Vol 2. Eds: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, John Wiley and Sons, Inc. Examples of chromatographic techniques useful in the isolation of the orthogonal IL2 ligand from the culture medium or host cell lysate include affinity chromatography, size-exclusion chromatography (SEC), gel filtration, reversed phase liquid chromatography, cation exchange chromatography, anion exchange chromatography, high-pressure liquid chromatography.

[0258] In some embodiments, when the orthogonal IL2 ligand is isolated from inclusion bodies generated in E. coli by solubilization of the inclusion bodies followed by chromatographic separation using one or more chromatographic procedures including but not limited to include affinity chromatography, size-exclusion chromatography (SEC), gel filtration, reversed phase liquid chromatography, cation exchange chromatography, anion exchange chromatography, high-pressure liquid chromatography.

[0259] When wt hIL2 is expressed endogenously in mammalian cells, it is expressed as a pre- protein comprising a signal peptide which is efficiently cleaved in mammalian cells resulting in the N-terminal amino acid of the mature wild type hIL2 polypeptide being a serine residue (Ala1). In an alternative to recombinant expression in mammalian cells, the orthogonal IL2 ligands may also be recombinantly expressed in bacterial cells. Direct expression (i.e., not as a N-terminal fusion protein) of an orthogonal IL2 ligand results in the addition of a N-terminal methionine residue to the orthogonal IL2 ligand (i.e., an N-terminal sequence of the beginningAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT with Met-Ala-Pro-…). If the Ala1 characteristic of the native N-terminal sequence of the wt IL2 sequence is retained at the N-terminus of the orthogonal IL2 ligand polypeptide, this will result in a proline (P) residue at the +2 position relative to N-terminal methionine of the orthogonal IL2 ligand. When a proline is present at the +2 position relative to the N-terminal methionine of a polypeptide, the endogenous bacterial methionyl amino peptidase (MAP) of the bacterial host cell frequently does not efficiently cleave the N terminal methionine. (See e.g., FIG.4B of Frottin, et al. (2019) The Proteomics of N-terminal Methionine Cleavage, Molecular & Cellular Proteomics 5(12):2336-2349). Consequently, bacterial direct expression of the orthogonal IL2 ligands comprising the native N-terminal sequence may result in a mixture of orthogonal IL2 ligand species, one fraction having an N-terminal methionine residue and another species lacking the N-terminal methionine. Such a mixture of orthogonal IL2 ligand species may be difficult to resolve by typical manufacturing procedures which may result in increased processing, loss of product and other difficulties when attempting to conjugate the molecules to N-terminus of the orthogonal IL2 ligand such as a targeting molecules or carrier molecules such as a PEG molecule. However, by deleting Ala1 (des-Ala1) from the orthogonal IL2 ligand, the residue in the +2 position relative to the N-terminal methionine is a glycine residue (G3) which provides for efficient cleavage of the N-terminal methionine and facilitates bacterial production of the orthogonal IL2 ligand and provides a more uniform orthogonal IL2 ligand product. In some embodiments, the present disclosure, provides orthogonal IL2 ligand comprising a deletion of the serine at position 1 (des-Ala1), numbered in accordance with hIL2) of the orthogonal IL2 ligand. Alternatively, it has been shown that deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the N-terminus of the hIL2 molecule results in a hIL2 molecule substantially retaining the activity of hIL2. In some embodiments, the hIL2A of the orthogonal IL2 ligand comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the N-terminus of the hIL2 of the orthogonal IL2 ligand. Administration of the Orthogonal IL2 ligand:

[0260] In some embodiments, the orthogonal IL2 ligand is administered to the mammalian subject suffering from lupus by intramuscular, intraperitoneal, intra-cerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, inhalation routes, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Administration of the orthogonal IL2 ligand to the subject may be administered as single administration, multiple administrations or by continuous infusion over a period of time. Devices useful in the administration of the orthogonal IL2 ligand to the subject include but are not limited to syringes, autoinjector devices, and infusion pumps. In some embodiments, the delivery of the orthogonalAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT IL2 ligand from the infusion pump is manually activated or electronically activated wherein the electronic activation mechanism is operably linked to a programmable computer using software to control the frequency and / or quantity of the administration of the orthogonal IL2 ligand. Administration of Nucleic Acids Encoding the Orthogonal IL2 Ligand:

[0261] In some embodiments, the administration of the orthogonal IL2 ligand includes the administration of a nucleic acid encoding the orthogonal IL2 ligand. Administration of a nucleic acid encoding the orthogonal IL2 ligand provides for extended delivery of the orthogonal IL2 ligand to the subject and prolonged activation of the corresponding cells engineered to express the cognate orthogonal receptor associated with such orthogonal IL2 ligand. In some embodiments of the method of the present disclosure, the nucleic acid encoding the orthogonal IL2 ligand is administered to the subject by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol.20: 1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm.53: 151-160, 1996, erratum at Am. J. Health Syst. Pharm.53:325, 1996

[0262] In one embodiment, the nucleic acid encoding the orthogonal IL2 ligand orthogonal IL2 ligand is administered to the subject in a viral vector or non-viral delivery system. Non-viral delivery systems are typically complexes to facilitate transduction of the target cell with a nucleic acid wherein the nucleic acid is complexed with agents such as cationic lipids (DOTAP, DOTMA), surfactants, biologicals (gelatin, chitosan), metals (gold, magnetic iron) and synthetic polymers (PLG, PEI, PAMAM). Numerous embodiments of non-viral delivery systems are well known in the art including lipidic vector systems (Lee et al. (1997) Critical Reviews of Therapeutic Drug Carrier Systems 14:173-206); polymer coated liposomes (Marin et al., U.S. Pat. No.5,213,804, issued May 25, 1993; Woodle, et al., U.S. Pat. No.5,013,556, issued May 7, 1991); cationic liposomes (Epand et al., U.S. Pat. No.5,283,185, issued Feb.1, 1994; Jessee, J. A., U.S. Pat. No.5,578,475, issued Nov.26, 1996; Rose et al, U.S. Pat. No.5,279,833, issued Jan.18, 1994; Gebeyehu et al., U.S. Pat. No.5,334,761, issued Aug.2, 1994). In one embodiment, the nucleic acid sequence in the non-viral vector system encoding the IL2 receptor is operably linked to one or more expression control sequences operable in a mammalian cell.

[0263] In some embodiments, orthogonal IL2 ligand is administered to a subject by administration of a viral vector encoding the orthogonal IL2 ligand. The terms “viral vector” and “virus” are used interchangeably herein to refer to any of the obligate intracellular parasites having no protein-synthesizing or energy-generating mechanism. The viral genome may be RNA or DNA contained with a coated structure of protein of a lipid membrane. The terms virus(es)Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT and viral vector(s) are used interchangeably herein. The viruses useful in the practice of the present invention include recombinantly modified enveloped or nonenveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesviridiae, poxviridae, or adenoviridiae. In some embodiments the viral vector is a recombinant adeno- associated virus (rAAV) or recombinant adenovirus (rAd), for example in some embodiments, a replication deficient adenovirus derived from human adenovirus serotypes 3 and / or 5. In some embodiments, the replication deficient adenovirus has one or more modifications to the E1 region which interfere with the ability of the virus to initiate the cell cycle and / or apoptotic pathways. The replication deficient adenoviral vector may optionally comprise deletions in the E3 domain. In some embodiments the adenovirus is a replication competent adenovirus. The viruses are modified by recombinant DNA techniques to include expression of exogenous transgenes (e.g. a nucleic acid sequence encoding the orthogonal IL2 ligand) and may be engineered to be replication deficient, conditionally replicating or replication competent. Minimal vector systems in which the viral backbone contains only the sequences need for packaging of the viral vector and may optionally include a transgene expression cassette may also be employed. The term “replication deficient” refers to vectors that are highly attenuated for replication in a wild type mammalian cell. In order to produce such vectors in quantity, a producer cell line is generally created by co-transfection with a helper virus or genomically modified to complement the missing functions. The term “replication competent viral vectors” refers to a viral vector that is capable of infection, DNA replication, packaging, and lysis of an infected cell. The term “conditionally replicating viral vectors” is used herein to refer to replication competent vectors that are designed to achieve selective expression in particular cell types. Such conditional replication may be achieved by operably linking tissue specific or cell type specific or other selectively induced regulatory control sequences to early genes (e.g., the E1 gene of adenoviral vectors). Infection of the subject with the recombinant virus or non-viral vector can provide for long term expression of the orthogonal IL2 ligand in the subject and provide continuous selective maintenance of the orthogonal CAR-T cells expressing the CD122 orthogonal receptor. In one embodiment, the nucleic acid sequence in the viral vector system encoding the IL2 receptor is under control of a regulatable promoter, inducible promoter, tissue specific, or temporally regulated promoter. Pharmaceutical Formulations Comprising the Orthogonal IL2 Ligand:

[0264] In some embodiments, the administration of the orthogonal IL2 ligand includes the administration of a pharmaceutical formulation comprising the orthogonal IL2 ligand or a nucleic acid encoding the orthogonal IL2 ligand. Such pharmaceutically acceptableAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT formulations comprise orthogonal IL2 ligand (or nucleic acid encoding the orthogonal IL2 ligand) and one or more pharmaceutically acceptable carriers, buffers, stabilizers, diluents, dispersants, solubilizers that are compatible with (i.e., do not promote degradation, structural modification or chemical modification of) the orthogonal IL2 ligand. Pharmaceutical formulations for parenteral administration include sterile aqueous solutions (where water soluble). The pharmaceutical formulation comprising the orthogonal IL2 ligand (and / or nucleic acids encoding the orthogonal IL2 ligand) is provided in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In one embodiment, the pharmaceutical formulation comprising the orthogonal IL2 ligand (and / or nucleic acids encoding the orthogonal IL2 ligand) is provided in a prefilled syringe. In one embodiment, the pharmaceutical formulation comprising the orthogonal IL2 ligand (and / or nucleic acids encoding the orthogonal IL2 ligand) is provided in cartridge adapted for use in auto injector format, the cartridge containing a sufficient quantity the orthogonal IL2 ligand (and / or nucleic acids encoding the orthogonal IL2 ligand) to provide multiple dosing.

[0265] In some embodiments, the pharmaceutical formulation comprising the orthogonal IL2 ligand (or nucleic acid encoding the orthogonal IL2 ligand) is an extended-release formulation. An extended-release formulation is a formulation that delivers the active agent to the circulation of the subject over a prolonged period of time (e.g. at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days). In some embodiments, the controlled release formulation comprises one or more biodegradable biocompatible polymers including but not limited to ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811. Methods of Treatment

[0266] In some embodiments, the present disclosure provides methods of treating a mammalian subject suffering from lupus, the method comprising administering to the subject an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand. In some embodiments, the mammalian subject is a human being. In some embodiments, the mammalian subject is a female human being. In some embodiments, the mammalian subject is a male human being. In some embodiments, the present disclosure provides methods of treating a mammalian subjectAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT suffering from lupus, the method comprising administering to the subject an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand wherein the subject has not been treated with a lymphodepleting treatment regimen prior to the initiation of treatment with orthogonal CAR-T cell in combination with an orthogonal IL2 ligand.

[0267] As used herein the term "lupus" includes but is not limited to systemic lupus erythramatosis, acute cutaneous lupus erythematosus (ACLE), subacute cutaneous lupus erythematosus (SCLE), chronic cutaneous lupus erythematosus (CCLE) including but not limited to discoid lupus erythematosus (DLE), hypertrophic / verrucous, lupus panniculitis / profundus, lupus tumidus, chilblains lupus, mucosal discoid lupus, lupus nephritis and lichenoid discoid lupus.

[0268] In some embodiments, the lupus is lupus nephritis (LN). In some embodiments, the lupus nephritis is selected from the group consisting of Class I (minimal mesangial) lupus nephritis, Class II (mesangial proliferative) lupus nephritis, Class III (focal) lupus nephritis, Class IV (diffuse segmental or diffuse global) lupus nephritis; Class V (membranous) lupus nephritis; Class VI (advanced sclerosing) lupus nephritis. Classification of LN may be determined by histological evaluation of renal biopsy tissue.

[0269] The presence of elevated levels of protein in urine (proteinurea) is indicative of impaired renal function. A study was performed to evaluate the effects of the administration of the orthogonal CAR-T cell alone and in combination with an orthogonal ligand in the NZB / W mouse model of lupus as more fully described in Example 8 and the results of which are provided in Figure 7. As illustrated in Figure 7, the majority of (4 of 5) NZB / W mice treated with either PBS (p) or the CAR-T cell alone (Ä) exhibited elevated levels of urine protein suggesting a substantial (approximately 80%) risk of developing impaired kidney function associated with lupus nephritis when untreated (PBS) or treated with CD19 CAR-T cells alone. In contrast, the mice treated with the orthogonal CD19 CAR-T cell in combination with the orthogonal ligand at a dose of either 1.25 x106(s) or 4x106orthogonal CD19 CAR-T cells (¡) exhibited no or trace amounts of urine protein. These data in an established mammalian model of lupus demonstrate that the administration of an orthogonal CAR-T cell (e.g., an orthogonal CD19 CAR-T cell) in combination with an orthogonal ligand (e.g., an orthogonal IL2 ligand) is effective in the treatment and / or prevention of lupus nephritis in a mammalian subject, in particular without lymphodepleting preconditioning with chemotherapy or irradiation. Suffering from Lupus:

[0270] The determination of whether a subject is "suffering from lupus" is made by a physician in accordance with the skill in the art. In some embodiments, a "subject suffering fromAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT lupus" exhibits one or more clinical symptoms of lupus. Clinical symptoms of lupus include but are not limited to fatigue, malaise, fever, anorexia, weight loss, skin lesions, butterfly (malar) facial rash, joint pain, widespread maculopapular or macular rash in a photosensitive pattern, a photosensitive nonscarring, nonindurated rash, discoid lesions, oral ulcers, nasal ulcers, arthritis, anemia, headaches, seizures, aseptic meningitis, demyelinating syndrome, movement disorders, pericarditis esophageal dysmotility, mesenteric vasculitis, lupus enteritis, peritonitis and ascites, protein-losing enteropathy, pancreatitis, and lupoid hepatitis, keratoconjunctivitis sicca, retinal vasculitis, optic neuritis, uveitis, scleritis, peripheral ulcerative keratitis, episcleritis, spontaneous abortions, pre-eclampsia, and maternal thrombosis.

[0271] In some embodiments, the human subject suffering from lupus is diagnosed in accordance with one or more of the following clinical measures of lupus disease activity: EULAR; the SLE Disease Activity Index (SLEDAI) (Bombardier (1992) Arthritis Rheum 35:630-40); the SLE Disease Activity Index 2000 (SLEDAI-2K) (Gladman, et al. (2002)The Journal of Rheumatology 2002; 29(2):288-291); the Systemic Lupus Activity Measure (SLAM) (Liang, et al. (1988) Arthritis & Rheumatism 31(7):817–25); the British Isles Lupus Assessment Group (BILAG) Activity Index (Symmons (1988) Q J Med 1988; 69:927-932); BILAG 2004 (Isenberg, et al. (2005) Rheumatology, 44(7):902–906) and Safety of Estrogens in Systemic National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA- SLEDAI)] Petri, et al. (2005) N Engl J Med 353:2550–2558.

[0272] In some embodiments, the mammalian subject is a human being having a clinical diagnosis of SLE according to the 2019 European League Against Rheumatism (EULAR) / ACR classification criteria and positive for at least one of anti-dsDNA antibodies (above the upper limit of normal [ULN]); anti-Smith antibodies (above the ULN); or anti-chromatin antibodies (above the ULN). In some embodiments, the mammalian subject is a human being having a clinical diagnosis of SLE and a clinical diagnosis of active severe disease as defined by presence of ≥ 1 major organ system with BILAG A score in the mucocutaneous, musculoskeletal, cardiorespiratory, gastrointestinal or renal categories at screening. In some embodiments, the mammalian subject is a human being having a clinical diagnosis of SLE and insufficient response to ≥ 2 standard immunosuppressive / biologic therapies, with at least 1 being an alkylating or biologic therapy (including but not limited to belimumab, anitfrolumab, rituximab) for ≥ 3 months of treatment. The term "insufficient response" includes lack of response, inadequate response, lack of sustained response at appropriate doses to treatment. In some embodiments, the mammalian subject is a human being having a clinical diagnosis of SLE and a serum creatinine clearance (Cockcroft-Gault formula) > 30 mL / min.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0273] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus, the method comprising administering to the subject an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand wherein the administering results an improvement in one or more clinical symptoms of lupus in the subject. In some embodiments, the improvement in one or more symptoms of lupus occurs within 14 days, optionally within 21 days, alternatively within 30 days, alternatively within 45 days, alternatively within 60 days, alternatively within 90 days, alternatively within 180 days, alternatively within 270 days, alternatively within one year, alternatively within 18 months, alternatively within two years from the date of the administration of the orthogonal CAR-T cell.

[0274] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus, the method comprising administering to the subject an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand wherein the administering results an improvement in one or more lupus efficacy criteria in the subject compared to the subject prior to the onset of treatment.

[0275] As used herein the term "lupus efficacy criteria" refers to: (a) remission of lupus per definition of remission in SLE (DORIS) criteria (van Vollenhoven, et al. (2021) Lupus Sci Med. 8(1):e000538); (b) lupus low disease activity state (LLDAS) attainment rate (Franklyn (2016) Ann Rheum Dis.75(9):1615-21.; (c) improvement in SLEDAI-2K scores; (d) improvement in BILAG individual system scores; (e) improvement in urine protein creatinine ratio (UPCR); (f) a decrease of ≥ 50% of Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) activity score (Albrecht, et al. (2005) J Invest Dermatol 125(5): 889- 894) in subjects with baseline activity score ≥ 10 prior to initiation of treatment; (g) complete renal response (CRR) defined as a UPCR < 0.5 and eGFR ≥ 60 mL / min per 1.73 m2or no confirmed eGFR decrease > 20%) in subjects with having a UPCR >1.0 mg / mg prior to the initiation of treatment; (h) decrease in total tender and swollen joint counts in subjects with ≥ 4 tender and swollen joints; (i) time to occurrence of a moderate or severe BILAG flare; (j) time to occurrence of a mild / moderate or severe flare as defined by SELENA-SLEDAI Flare index; (k) reduction in levels of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies; (l) reduction in levels of anti-nuclear antibodies (ANAs); (m) an ANA titer less than 1:80; (n) reduction in levels of anti-Smith antibodies; (o) reduction in level of complement factor C3, (p) reduction in level of complement factor C3C4) ; (q) improvement in HAQ-DI, SF-36; (r) improvement in FACIT- Fatigue Scale version 4 (Tennant, K. (2015) Supportive Care in Cancer 23(5):1355-1364); (s) reduction in disease activity as assessed by the UCSF / JHU Lupus Activity Index (LAI); reduction in disease activity as assessed by the SLE Disease Activity Index (SLEDAI); reductionAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT in disease activity as assessed by the Systemic Lupus Activity Measure (SLAM) (Liang, et al (1989) Arthritis Rheum.32(9):1107-18) or SLAM-R (Bae, et al (2001) Lupus 2001; 10: 405-9) and the reduction in disease activity as assessed by the British Isles Lupus Assessment Group (BILAG) Activity Index. Efficacy may be measured at any time following administration of the CAR-T cells. In some embodiments, evaluation of efficacy criteria is performed at 14 days, 30 days, 60 days, 90 days, 12 weeks, alternatively within 24 weeks following administration of the orthogonal CAR-T cells, alternatively with 48 weeks following administration of the orthogonal CAR-T cells, or alternatively within 96 weeks following administration of the orthogonal CAR- T cells.

[0276] In some embodiments, the present disclosure provides a method of treating a subject suffering from lupus by the administration of an orthogonal ligand expressing CAR-Ts in the absence of lymphodepletion prior to administration of the orthogonal ligand CAR-Ts.

[0277] The typical clinical practice with respect to CAR-T cell therapies is to administer to the subject one or more lymphodepletion treatment regimens. Studies have correlated lymphodepletion of the subject prior to the administration of CD19-CAR-T cell therapies with improved therapeutic outcomes in the treatment of hematological malignancies such that prior lymphodepletion has become the standard of clinical practice. In the clinic, human subjects suffering from B-cell malignancies treated with commercially available CD-19 CAR-T cell therapies (e.g. Kymriah® and Yescarta®) and are typically subjected to a lymphodepletion treatment regimen prior to administration of the CAR-T cells, the lymphodepletion treatment regimen typically involving the administration of cyclophosphamide in combination with fludarabine (commonly referred to as "Flu / Cy chemotherapy").

[0278] However, lymphodepletion treatment regimens are associated with significant toxicity. Lymphodepletion regimens employing cytotoxic chemotherapeutics such as Fly / Cy chemotherapy are associated with significant, potentially life-threatening, toxicities such as cytopenias, cytokine release syndrome (CRS) and immune effector cell neurotoxicity syndrome (ICANS). Bechman & Maher (2021) Expert Opinion on Biological Therapy, 21:5, 627-637. lymphodepletion significantly compromises the human subject by leaving them vulnerable to environmental factors. Consequently, the practice of the methods of the present disclosure in the absence of prior lymphodepletion regimens provide a significant benefit to the subject.

[0279] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus, the method comprising administering to the subject an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand wherein the subject suffering from lupus has not been treated with a lymphodepletion treatment regimen within 90Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day, prior to administering of the orthogonal CAR-T cell.

[0280] As used herein, the terms "lymphodepleting treatment regimen", "lymphodepletion treatment regimen" and "lymphodepletion regimen" are used interchangeably to refer to the administration of one or more agents selected from the group consisting of fludarabine (typically provided in a dosage range of from 75mg / m2to 120mg / m2), cyclophosphamide (typically provided in a dosage range of from 750mg / m2to 2000mg / m2), bendamustine (typically provided in a dosage range of from 70mg / m2to 90mg / m2), busulfan, etoposide, cytarabine, dexamethasone, alemtuzumab, oxaliplatin, clofarabine and radiation. Treatment of Cytopenic Lupus Subjects:

[0281] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus and one or more cytopenias, the method comprising administering to the subject an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand. In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus and one or more cytopenias, the method comprising administering to the subject an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand wherein the subject suffering from lupus has not been treated with a lymphodepletion treatment regimen within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day, prior to administering of the orthogonal CAR-T cell. As used herein the term "cytopenia" means a condition existing in a subject having an abnormally low number of blood cells. The term cytopenia includes anemia, thrombocytopenia, and leukopenia, lymphopenia, neutropenia. Cytopenia may arise as a result of naturally occurring abnormalities or may be drug induced. As used herein the term cytopenia includes drug induced cytopenia.

[0282] A significant fraction of human subjects suffering from lupus also suffer from one or more cytopenias. Thrombocytopenia is observed in approximately 19% of SLE patients, neutropenia is observed in approximately 30% of SLE patients, leukopenia is observed in approximately 32% of SLE patients, and lymphopenia is observed in approximately 49% of SLEAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT patients. Linda, et al. (2015) Seminars in Arthritis and Rheumatism 45(2):190-194; Jung, et al. (2016) Medicine (Baltimore).2016 Feb; 95(6): e2818; Hepburn, et al. (2010) 49(12): 2243– 2254. Lymphodepleting regimens are associated with inducing cytopenias such that lymphodepletion would exacerbate cytopenias in subjects suffering from lupus and cytopenia.

[0283] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus and one or more cytopenias, the method comprising administering to the subject an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand, wherein the subject has not been treated with a lymphodepletion treatment regimen within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day, prior to the administering of the orthogonal CAR-T cell.

[0284] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus, the method comprising administering to the subject an immune cell recombinantly modified to express an orthogonal receptor and a chimeric antigen receptor, wherein the extracellular domain of the CAR selectively binds to a B-cell antigen, in combination with an orthogonal IL2 ligand, wherein the subject is an adult human being and the orthogonal CAR-T cell is administered to the subject at a dose of from 50x106to150x106cells, alternatively from 70 x106to 120x106cells, alternatively from 90x106to 120x106cells, alternatively 90 x106cells, alternatively 100 x106cells, alternatively 110x106cells, alternatively 120x106cells, alternatively 120x106cells, alternatively 120x106cells, alternatively 120x106cells, alternatively 130x106cells, alternatively 140x106cells, or alternatively 150x106cells; and wherein the orthogonal IL2 ligand is administered to the subject in one or more doses of approximately 0.05mg / kg to 1 mg / kg; alternatively from approximately 0.05mg / kg to 0.7 mg / kg; alternatively of approximately 0.05mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1mg / kg to 0.3 mg / kg wherein the orthogonal wherein is administered daily, twice weekly, thrice weekly, weekly, about every 10 days, or about every 14 days over a period of at least one month, optionally at least 2 months, optionally at least 3 months following the administration of the orthogonal CAR-T cells. In one embodiment, the orthogonal IL2 ligand is administered weekly for a period of at least 10, alternatively at least 11, alternatively at least 12 weeks following the intravenous infusion of the of the orthogonal CAR-T cells.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT

[0285] In some embodiments, the present disclosure provides a method of treating a mammalian subject suffering from lupus, the method comprising administering to the subject an orthogonal CAR-T cell, wherein the subject is an adult human being and the orthogonal CAR-T cell is administered to the subject at a dose of from 0.5x106to 3x106cells per kg of bodyweight, alternatively from 1.0x106to 2x106cells per kg of bodyweight, alternatively from 1.5x106to 2x106cells per kg of bodyweight, alternatively 1x106cells per kg of bodyweight, alternatively 1.1x106cells per kg of bodyweight, alternatively 1.2 x106cells per kg of bodyweight, alternatively 1.3x106cells per kg of bodyweight, alternatively 1.4 x106cells per kg of bodyweight, alternatively 1.5x106cells per alternatively 1.6 x106cells per kg of bodyweight, alternatively 1.7x106cells per kg of bodyweight, alternatively 1.8 x106cells per kg of bodyweight, alternatively 1.9x106cells per kg of bodyweight, alternatively 2x106cells per kg of bodyweight, alternatively 2.1x106cells per kg of bodyweight, alternatively 2.2x106cells per kg of bodyweight, alternatively 2.3x106cells per kg of bodyweight, alternatively 2.4x106cells per kg of bodyweight, alternatively 2.5x106cells per kg of bodyweight, or alternatively 3.0x106c...

Claims

Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT CLAIMS 1. A method of treating a mammalian subject suffering from lupus the method comprising, administering to said subject a therapeutically effective amount of an orthogonal chimeric antigen receptor (CAR)-T cell in combination with an orthogonal IL2 ligand, wherein the CAR-T cell comprises a CAR, wherein the CAR comprises an antigen binding domain (ABD), a transmembrane domain (TMD) and an intercellular domain (ICD), wherein the antigen binding domain (ABD) of the CAR of the orthogonal CAR-T cell selectively binds to at least one human B cell antigen, and wherein CAR-T cell comprises an orthogonal receptor, wherein the orthogonal receptor comprises an extracellular domain (ECD), a transmembrane domain (TMD) and an intercellular domain (ICD), wherein the ECD comprises an orthogonal CD122 ECD.

2. The method of claim 1 wherein the lupus is systemic lupus erythramatosis (SLE).

3. The method of claim 1 wherein the lupus is lupus nephritis.

4. The method of any one of claims 1-3 wherein the subject is also suffering from cytopenia.

5. The method of any one of claims 1-4 wherein the subject has not received a lymphodepletion treatment regimen, optionally within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day, prior to administering of the orthogonal CAR-T cell.

6. The method of any one of claims 1-5 wherein the ABD specifically binds to a B- cell antigen.

7. The method of claim 6 wherein the B-cell antigen is selected from the group consisting of CD19, CD20, CD22 and BCMA.

8. The method of claim 7 wherein the B-cell antigen is CD19.

9. The method of claim 8 wherein the ABD of the CAR comprises an anti-CD19 scFv.

10. The method of claim 9 wherein the anti-CD19 scFv comprises a light chain polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity,Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 26, a linker that joins the heavy and light chain domains of the scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 and a heavy chain sequence having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO:

27.

11. The method of claim 10 wherein the anti-CD19 scFv is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO:

35.

12. The method of any one of claims 1-11, wherein the CAR further comprises a CD3z stimulatory domain and a CD28 costimulatory domain.

13. The method of claim 12 wherein the CAR polypeptide the orthogonal CAR-T cell is an orthogonal CD19 CAR-T cell wherein the CAR of the orthogonal CD19 CAR-T is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO:

61.

14. The method of any one of claims 1-13 wherein the orthogonal CAR-T comprises an orthogonal hCD122 ECD.

15. The method of claim 14 wherein the orthogonal receptor is selected from the group consisting of an orthogonal hIL2 receptor, a chimeric orthogonal IL4R receptor, a chimeric orthogonal IL7R receptor, a chimeric orthogonal IL9 receptor, a chimeric orthogonal IL21 receptor.

16. The method of claim 15 wherein the orthogonal receptor comprises an orthogonal hCD122 ECD.

17. The method of claim 16 wherein the orthogonal hCD122 ECD comprises a variant polypeptide derived from the ECD of a human CD122 wherein the polypeptide has at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at leastAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising one or more amino acid substitutions or deletions of amino acid residues selected from the group consisting of R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and / or Q214 (numbered in accordance with SEQ ID NO:3).

18. The method of claim 17 wherein the hCD122 ECD having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to the ECD of wild type hCD122 (SEQ ID NO:3), the polypeptide further comprising one or more amino acid substitutions or deletions of amino acid residues selected from the group consisting of H133 and Y134 (numbered in accordance with SEQ ID NO:3).

19. The method of claim 18 wherein the one or more amino acid substitutions or deletions of amino acid residues selected from the group consisting of H133 and Y134 are amino acid substitutions selected from the group consisting of H133D, H133E, H133K, Y134F, Y134E, and Y134R (numbered in accordance with SEQ ID NO:3).

20. The method of claim 19 wherein the amino acid substitution at position H133 is selected from the group consisting of H133D, H133E, and H133K, and the amino acid substitution at position H134 is selected from the group consisting of Y134F, Y134E, and Y134R (numbered in accordance with SEQ ID NO:3).

21. The method of claim 20 wherein the amino acid substitution at position H133 is H133D and the amino acid substitution at position H134 is Y134F (numbered in accordance with SEQ ID NO:3).

22. The method of claim 21 wherein the hCD122 ECD is a polypeptide having the amino acid sequence: AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPV SQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLM APISLQVVHVETHRCNISWEISQASDFFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT (SEQ ID NO:40) 23. The method of any one of claims 16-22 wherein the orthogonal hCD122 receptor has the amino acid sequence:Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPV SQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLM APISLQVVHVETHRCNISWEISQASDFFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWL GHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGD VQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLT SCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLS GEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDW DPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEF RALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO:39).

24. The method of any one of claims 1-23 wherein the orthogonal IL2 ligand comprises a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO: 12, further comprising amino acid substitutions at one or more of E15, H16, L19, D20, Q22, and M23 numbered in accordance with the mature human IL2 polypeptide (SEQ ID NO: 12), optionally further comprising: (a) one or more amino acid substitutions selected from the group consisting of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P, T3C N30E; K32E; N33D; P34G; T37I, R38W, R38G, M39Q, R39L, R39V, F42K, F42Y, F44Y, P47G, T51I, E52K, L53N, H55Y, Q57E, V91R, K97E M104A, T113N C125S, and C125A; and / or (b) a deletion of one or more N-terminal amino acids, the deletion of positions 1-9, alternatively positions 1-8, alternatively positions 1-7 alternatively positions 1-6, alternatively positions 1-5, alternatively positions 1-4, alternatively positions 1-3, alternatively positions 1-2, or alternatively position 1 numbered in accordance with SEQ ID NO:

12.

25. The method of claim 24, wherein the one or more of amino acid substitutions at positions E15, H16, L19, D20 Q22, and M23 is a set of amino acid substitutions selected from the group consisting of: [E15S-H16Q-L19V-D20L-Q22K-M23A], [E15S-H16Q-L19V-D20L- Q22K]; [H16N, L19V, D20N, Q22T, M23H, G27K]; [E15D, H16N, L19V, D20L, Q22T, M23H]; [E15D, H16N, L19V, D20L, Q22T, M23A]; [E15D, H16N, L19V, D20L, Q22K, M23A]; [E15S; H16Q; L19V, D20T; Q22K, M23L]; [E15S; H16Q; L19V, D20T; Q22K, M23S]; [E15S; H16Q; L19V, D20S; Q22K, M23S]; [E15S; H16Q; L19I, D20S; Q22K; M23L]; [E15S; L19V; D20M; Q22K; M23S]; [E15T; H16Q; L19V; D20S; M23S]; [E15Q; L19V; D20M; Q22K; M23S]; [E15Q; H16Q; L19V; D20T; Q22K; M23V]; [E15H; H16Q; L19I; D20S; Q22K; M23L]; [E15H; H16Q; L19I; D20L; Q22K; M23T]; and [L19V; D20M; Q22N; M23S], numbered in accordance with the mature human IL2 polypeptide (SEQ ID NO: 12).Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT 26. The method of claim 25 wherein the set of amino acids is [E15S-H16Q-L19V- D20L-Q22K-M23A] numbered in accordance with the mature human IL2 polypeptide (SEQ ID NO: 12).

27. The method of claim 26 wherein the orthogonal IL2 ligand comprises a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO:

43.

28. The method of claim 26 wherein the orthogonal IL2 ligand comprises a polypeptide a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO:

43.

29. The method of claim 26 wherein the orthogonal IL2 ligand comprises a polypeptide a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100%, sequence identity to SEQ ID NO:

41.

30. The method of any one of claims 24-29 wherein the orthogonal IL2 ligand comprises one or more modifications to extend the duration of action of the molecule in the mammalian subject.

31. The method of claim 30 wherein the one or more modifications to extend the duration of action of the molecule covalently linkage to one or more molecules selected from the group consisting of a carrier molecule, albumin, an Xten polymer, an anti-albumin antibody, an Fc domain, a fatty acid molecule.

32. The method of claim 31 wherein the carrier molecule is a water-soluble polymer.

33. The method of claim 32 wherein the water-soluble polymer is polyethylene glycol (PEG).

34. The method of claim 33 wherein the PEG is a linear PEG, branched PEG, star- PEG, or multi-armed PEG having an average molecular mass from about 2 kDa to about 80 kDa, alternatively from about 2 kDa to about 70 kDa, alternatively from about 5 kDa to about 50 kDa, alternatively from about 5 kDa to about 50 kDa, alternatively from about 20 kDa to about 50 kDa, alternatively from about 30 kDa to about 50 kDa, alternatively from about 20 kDa to about 40 kDa, alternatively from about 5 kDa to about 10 kDa, alternatively from about 5 kDa to about 15 kDa, alternatively from about 5 kDa to about 20 kDa, from about 10 kDa to about 15 kDa,Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT alternatively from about 10 kDa to about 20 kDa, alternatively from about 10 kDa to about 25 kDa or alternatively from about 10 kDa to about 30 kDa alternatively from about 30 kDa to about 40 kDa, alternatively about 5 kDa, alternatively about 10 kDa, alternatively about 15 kDa, alternatively about 20 kDa, alternatively about 25 kDa, alternatively about 30 kDa, alternatively about 35 kDa, alternatively about 40 kDa, alternatively about 45 kDa, alternatively about 50 kDa, alternatively about 55 kDa, alternatively about 60 kDa, alternatively about 65 kDa, alternatively about 70 kDa, alternatively about 75 kDa, or alternatively about 80 kDa.

35. The method of claim 34 wherein the PEG is a 40 kDa linear PEG.

36. The method of claim 34 wherein the PEG is a branched PEG having an average molecular weight of about 40 kDa comprising two 20 kDa arms.

37. The method of any one of claims 33-36 wherein the PEG is covalently attached to the orthogonal IL2 ligand via a linker.

38. The method of any one of claims 33-36 wherein the orthogonal IL2 ligand is a compound of the structure: (PEG)-(linker)n-(PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 41). wherein n = 0 (absent) or 1 (present) and PEG is 40kDa-PEG.

39. The method of claim 38 wherein the PEG is a branched 40kDa PEG comprising two 20kDa PEG arms.

40. The method of claim 38 or 39 wherein n=1.

41. The method of any one of claims 1-40 wherein the subject is a human being and the orthogonal CAR-T cell is administered to the subject at a dose from 50x106to150x106cells, alternatively from 70 x106to 120x106cells, alternatively from 90x106to 120x106cells, alternatively 90 x106cells, alternatively 100 x106cells, alternatively 110x106cells, alternatively 120x106cells, alternatively 120x106cells, alternatively 120x106cells, alternatively 120x106cells, alternatively 130x106cells, alternatively 140x106cells, or alternatively 150x106cells.

42. The method of any one of claims 1-40 wherein the subject is a human being and the orthogonal CAR-T cell is administered to the subject at a dose from 0.5x106to 3x106cells per kg of bodyweight, alternatively from 1.0x106to 2x106cells per kg of bodyweight, alternatively from 1.5x106to 2x106cells per kg of bodyweight, alternatively 1x106cells per kg of bodyweight, alternatively 1.1x106cells per kg of bodyweight, alternatively 1.2 x106cells perAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT kg of bodyweight, alternatively 1.3x106cells per kg of bodyweight, alternatively 1.4 x106cells per kg of bodyweight, alternatively 1.5x106cells per alternatively 1.6 x106cells per kg of bodyweight, alternatively 1.7x106cells per kg of bodyweight, alternatively 1.8 x106cells per kg of bodyweight, alternatively 1.9x106cells per kg of bodyweight, alternatively 2x106cells per kg of bodyweight, alternatively 2.1x106cells per kg of bodyweight, alternatively 2.2x106cells per kg of bodyweight, alternatively 2.3x106cells per kg of bodyweight, alternatively 2.4x106cells per kg of bodyweight, alternatively 2.5x106cells per kg of bodyweight, or alternatively 3.0x106cells per kg of bodyweight.

43. The method of claim 41 or 42 wherein the orthogonal IL2 ligand is administered to the subject in one or more doses of approximately 0.05 mg / kg to 1 mg / kg; alternatively from approximately 0.05 mg / kg to 0.7 mg / kg; alternatively of approximately 0.05 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.3 mg / kg wherein the orthogonal wherein is administered daily, twice weekly, thrice weekly, weekly, about every 10 days, or about every 14 days over a period of at least one month, optionally at least 2 months, optionally at least 3 months.

44. The method of claim 43 wherein the orthogonal CAR-T cell is an orthogonal CD19 CAR-T cell wherein the CAR of the orthogonal CD19 CAR-T is a polypeptide having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: 61 and the orthogonal receptor is an orthogonal hIL2 receptor has the amino acid sequence having at least 95% sequence identity, alternatively at least 96% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively 100% sequence identity to SEQ ID NO: SEQ ID NO:

39.

45. The method of claim 44 wherein the orthogonal IL2 ligand is a compound of the structure: (PEG)-(linker)n-(PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 41). wherein n = 0 (absent) or 1 (present) and PEG is 40kDa-PEG.

46. The method of claim 45 wherein the orthogonal IL2 ligand wherein is administered daily, twice weekly, thrice weekly, weekly, about every 10 days, or about every 14 days.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT 47. The method of claim 46 wherein the orthogonal IL2 ligand is administered weekly for a period at least 10 weeks.

48. The method of claim 45 wherein the orthogonal IL2 ligand is administered at an initial dose of approximately 0.05 mg / kg to 0.5 mg / kg for a period of approximately 1 week, alternatively approximately 2 weeks followed by a maintenance dose 0.02 mg / kg to 0.3 mg / kg for a period of at least 2 weeks, alternatively at least 3 weeks, alternatively at least one month, alternatively at least at least two months, alternatively at least three months.

49. The method of claim 48 wherein the method the orthogonal IL2 ligand is administered to the subject at a dose and frequency sufficient to maintain a therapeutically effective quantity of orthogonal CAR-T cells in the subject wherein the therapeutically effective quantity of orthogonal CAR-T cells is from about 1x105cells per kg of bodyweight to about 3x106cells per kg of bodyweight, alternatively from about 2x105to about 3x106cells per kg of bodyweight, alternatively from about 2x105to about 2x106cells per kg of bodyweight, alternatively from about 3x105to about 2x106cells per kg of bodyweight, alternatively from about 5x105to about 1.5x106cells per kg of bodyweight, alternatively from about 5x105to about 1x106cells per kg of bodyweight, alternatively from about 1x106to about 2x106cells per kg of bodyweight, alternatively from about 1x106to about 1.3x106cells per kg of bodyweight, alternatively from about 1x106to about 1.5x106cells per kg of bodyweight, alternatively from about 1.3x106to about 1.7x106cells per kg of bodyweight, or alternatively from about 1.5x106to about 2x106cells per kg of bodyweight, alternatively about 1.2x106cells per kg of bodyweight, alternatively about 1.3x106cells per kg of bodyweight, alternatively about 1.4x106cells per kg of bodyweight, alternatively about 1.5x106cells per kg of bodyweight, alternatively about 1.6x106cells per kg of bodyweight, alternatively about 1.7x106cells per kg of bodyweight, alternatively about 1.8x106cells per kg of bodyweight, alternatively about 1.9x106cells per kg of bodyweight, alternatively about 2.0x106cells per kg of bodyweight, wherein the orthogonal IL2 ligand is administered to the subject in one or more doses of approximately 0.05 mg / kg to 1 mg / kg; alternatively from approximately 0.05 mg / kg to 0.7 mg / kg; alternatively of approximately 0.05 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.3 mg / kg wherein the orthogonal wherein is administered daily, twice weekly, thrice weekly, weekly, about every 10 days, or about every 14 days over a period of at least one month, optionally at least 2 months, optionally at least 3 months.

50. The method of any one of claims 41-49 wherein the orthogonal CAR-T cell is an autologous orthogonal CAR-T cell.Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT 51. The method of any one of claims 1-50 wherein the subject exhibits an improvement in one or more lupus efficacy criteria in the subject compared to the subject prior to the initiation of treatment, wherein the lupus efficacy criteria are selected from the group consisting of : (a) remission of lupus per definition of remission in SLE (DORIS) criteria; (b) lupus low disease activity state (LLDAS) attainment rate; (c) improvement in SLEDAI-2K scores; (d) improvement in BILAG individual system scores; (e) improvement in urine protein creatinine ratio (UPCR); (f) a decrease of ≥ 50% of Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) activity score in subjects with baseline activity score ≥ 10 prior to initiation of treatment; (g) complete renal response (CRR) defined as a UPCR < 0.5 and eGFR ≥ 60 mL / min per 1.73 m2or no confirmed eGFR decrease > 20%) in subjects with having a UPCR >1.0 mg / mg prior to the initiation of treatment; (h) decrease in total tender and swollen joint counts in subjects with ≥ 4 tender and swollen joints; (i) time to occurrence of a moderate or severe BILAG flare; (j) time to occurrence of a mild / moderate or severe flare as defined by SELENA-SLEDAI Flare index; (k) reduction in levels of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies; (l) reduction in levels of anti-nuclear antibodies (ANAs); (m) an ANA titer less than 1:80; (n) reduction in levels of anti-Smith antibodies; (o) reduction in level of complement factor C3, (p) reduction in level of complement factor C3C4) ; (q) improvement in HAQ-DI, SF-36; (r) improvement in FACIT-Fatigue Scale version 4 (; (s) reduction in disease activity as assessed by the UCSF / JHU Lupus Activity Index (LAI); reduction in disease activity as assessed by the SLE Disease Activity Index (SLEDAI); reduction in disease activity as assessed by the Systemic Lupus Activity Measure (SLAM) or SLAM-R and the reduction in disease activity as assessed by the British Isles Lupus Assessment Group (BILAG) Activity Index, wherein the improvement in one more efficacy criteria occurs within 14 days, 30 days, 60 days, 90 days, 12 weeks, alternatively within 24 weeks following administration of the orthogonal CAR-T cells, alternatively with 48 weeks following administration of the orthogonal CAR-T cells, or alternatively within 96 weeks following administration of the orthogonal CAR-T cells.

52. The method of any one of claims 1-51 wherein the lupus is lupus nephritis.

53. The method of any one of claims 1-52 wherein the subject is not treated with a lymphodepleting regimen within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3Attorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT days, alternatively within 2 days, or alternatively within 1 day prior to administering of the orthogonal CAR-T cell.

54. The method of any one of claims 1-52 wherein the mammalian subject suffering from lupus is suffering from one or more cytopenias, the method comprising administering to the subject an orthogonal CAR-T cell in combination with an orthogonal IL2 ligand, wherein the subject has not been treated with a lymphodepleting regimen within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day, prior to the administering of the orthogonal CAR-T cell.

55. The method of any one of claims 1-53 wherein: (a) the orthogonal CAR T cell autologous orthogonal CD19 CAR-T cell wherein the CD19 CART cell expresses a CD19 CAR having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to SEQ ID NO: 61 and an orthogonal hCD122 receptor having ^90%, alternatively ^91%, alternatively ^92%, alternatively ^93%, alternatively ^94%, alternatively ^95%, alternatively ^96%, alternatively ^ 97%, alternatively ^98%, alternatively ^99%, or alternatively 100% sequence identity to the amino acid sequence of SEQ ID NO: 39; (b) the orthogonal ligand is a compound of the structure: (PEG)-(linker)n-(PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT, SEQ ID NO: 41), wherein n = 0 (absent) or 1 (present) and PEG is 40kDa-PEG; (c) CD19 CAR-T cell is administered at a dose of from 1x105cells per kg of bodyweight to about 3x106cells per kg of bodyweight, alternatively from about 2x105to about 3x106cells per kg of bodyweight, alternatively from about 2x105to about 2x106cells per kg of bodyweight, alternatively from about 3x105to about 2x106cells per kg of bodyweight, alternatively from about 5x105to about 1.5x106cells per kg of bodyweight, alternatively from about 5x105to about 1x106cells per kg of bodyweight, alternatively from about 1x106to about 2x106cells per kg of bodyweight, alternatively from about 1x106to about 1.3x106cells per kg ofAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT bodyweight, alternatively from about 1x106to about 1.5x106cells per kg of bodyweight, alternatively from about 1.3x106to about 1.7x106cells per kg of bodyweight, or alternatively from about 1.5x106to about 2x106cells per kg of bodyweight, alternatively about 1.2x106cells per kg of bodyweight, alternatively about 1.3x106cells per kg of bodyweight, alternatively about 1.4x106cells per kg of bodyweight, alternatively about 1.5x106cells per kg of bodyweight, alternatively about 1.6x106cells per kg of bodyweight, alternatively about 1.7x106cells per kg of bodyweight, alternatively about 1.8x106cells per kg of bodyweight, alternatively about 1.9x106cells per kg of bodyweight, alternatively about 2.0x106cells per kg of bodyweight; (d) the orthogonal IL2 ligand is administered to the subject in one or more doses of approximately 0.05mg / kg to 1 mg / kg; alternatively from approximately 0.05 mg / kg to 0.7 mg / kg; alternatively of approximately 0.05mg / kg to 0.5 mg / kg; alternatively of approximately 0.1 mg / kg to 0.5 mg / kg; alternatively of approximately 0.1mg / kg to 0.3 mg / kg wherein the orthogonal wherein is administered daily, twice weekly, thrice weekly, weekly, about every 10 days, or about every 14 days over a period of at least one month, optionally at least 2 months, optionally at least 3 months; (e) the subject has not been treated with a lymphodepleting regimen within 90 days, alternatively within 60 days, alternatively within 30 days, alternatively within 21 days, alternatively within 14 days, alternatively within 10 days, alternatively within 9 days, alternatively within 8 days, alternatively within 7 days, alternatively within 6 days, alternatively within 5 days, alternatively within 4 days, alternatively within 3 days, alternatively within 2 days, or alternatively within 1 day, prior to the administering of the orthogonal CAR-T cell; and (f) the subject exhibits an improvement in one or more lupus efficacy criteria in the subject compared to the subject prior to the initiation of treatment, wherein the lupus efficacy criteria are selected from the group consisting of : (a) remission of lupus per definition of remission in SLE (DORIS) criteria; (b) lupus low disease activity state (LLDAS) attainment rate; (c) improvement in SLEDAI-2K scores; (d) improvement in BILAG individual system scores; (e) improvement in urine protein creatinine ratio (UPCR); (f) a decrease of ≥ 50% of Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) activity score in subjects with baseline activity score ≥ 10 prior to initiation of treatment; (g) complete renal response (CRR) defined as a UPCR < 0.5 and eGFR ≥ 60 mL / min per 1.73 m2or no confirmed eGFR decrease > 20%) in subjects with having a UPCR >1.0 mg / mg prior to the initiation of treatment; (h) decrease in total tender and swollen joint counts in subjects with ≥ 4 tender and swollen joints; (i) time to occurrence of a moderate or severe BILAG flare; (j) time to occurrence of a mild / moderate or severe flare as defined by SELENA-SLEDAI Flare index; (k) reduction inAttorney Docket No.106249-1487045(010720PC) Synthekine Ref. No. SYN-107PCT levels of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies; (l) reduction in levels of anti-nuclear antibodies (ANAs); (m) an ANA titer less than 1:80; (n) reduction in levels of anti-Smith antibodies; (o) reduction in level of complement factor C3, (p) reduction in level of complement factor C3C4) ; (q) improvement in HAQ-DI, SF-36; (r) improvement in FACIT- Fatigue Scale version 4; (s) reduction in disease activity as assessed by the UCSF / JHU Lupus Activity Index (LAI); reduction in disease activity as assessed by the SLE Disease Activity Index (SLEDAI); reduction in disease activity as assessed by the Systemic Lupus Activity Measure (SLAM) or SLAM-R and the reduction in disease activity as assessed by the British Isles Lupus Assessment Group (BILAG) Activity Index wherein the improvement in the one more efficacy criteria occurs within 14 days, 30 days, 60 days, 90 days, 12 weeks, alternatively within 24 weeks following administration of the orthogonal CAR-T cells, alternatively with 48 weeks following administration of the orthogonal CAR-T cells, or alternatively within 96 weeks following administration of the orthogonal CAR-T cells.

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