Il-2 orthologs and methods of use thereof

Abstract

This invention relates to hIL2 orthogonal ligands (“IL2 orthogonal homologs”) that specifically and selectively bind to transmembrane peptides comprising an extracellular domain (ECD) of a modified hCD122 polypeptide. The binding of the hIL2 orthogonal homolog to the modified hCD122 polypeptide participates in intracellular signal transduction pathways, resulting in biological activity associated with the native intracellular signal transduction pattern of hIL2 binding to intermediate- or high-affinity hIL2 receptors, but exhibiting selectivity for engineered hCD122 orthogonal receptors. Compared to its binding to the extracellular domain of wild-type hCD122, the hIL2 orthogonal homologs of this invention exhibit significantly reduced binding, either alone or when hCD122 is present in the form of an endogenous high- or intermediate-affinity hIL2 receptor.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 948066, filed on December 13, 2019.

[0003] Statement regarding government funding

[0004] No funds from the U.S. government were used in conceiving the subject matter of this invention or in putting it into practice. Background Technology

[0005] Controlled manipulation of cell differentiation, development, and proliferation, particularly engineered immune cells, has significant clinical implications. T cells have been engineered for therapeutic applications, such as recognizing and killing cancer cells, intracellular pathogens, and cells involved in autoimmunity. Selectively activating and expanding engineered T cells that provide specific functions and guiding them to selectively attack cancer cells facilitates the application of engineered cell therapy in cancer treatment. In some examples of adoptive immunotherapy, T cells are isolated from the subject's blood, processed ex vivo, and re-infused into the subject. Therefore, there is a need for compositions and methods capable of selectively activating targeted engineered cell populations.

[0006] The challenge in manufacturing cell therapy products is that these “living drugs” require a closely controlled environment to maintain their viability and function. In practice, isolated cells, whether from a patient (autologous) or a single donor (allogeneic), begin to rapidly lose function after leaving the target or controlled culture conditions. Successfully maintaining the health and function of isolated cells upon leaving the target or controlled culture conditions enables them to regain function and be reintegrated into the cell product manufacturing workflow or into the patient.

[0007] Furthermore, a challenge in the clinical application of engineered T-cell therapy is the selective stimulation of these engineered cells to maximize their therapeutic efficacy. A typical approach to providing sustained activation of engineered T-cell products is systemic administration of cytokines, such as IL-2. However, systemic administration of IL-2 is associated with non-specific stimulation outside the engineered cell population, particularly at high doses, and is linked to significant toxicity in human subjects. Additionally, IL-2 has a short lifespan in vivo, requiring frequent administration to maintain the engineered T-cells in an activated state. While initially administered engineered cells from the initial population can be detected months or even years after administration of the engineered cell product, a significant proportion of these cells become quiescent and require reactivation to demonstrate significant therapeutic effects. Therefore, the challenge of cell-based therapies is to endow transferred cells with the desired modifiable behavior, unaffected by endogenous signaling pathways, without impacting non-targeted endogenous cells, and selectively controllable after administration of the engineered cell population to the subject.

[0008] CD122 is a component of intermediate- and high-affinity IL2 receptor complexes. Sockolosky et al. (Science (2018) 359:1037–1042) and Garcia et al. (US Patent Application Publication US2018 / 0228841A1, published August 16, 2018) described an orthogonal IL2 / CD122 ligand / receptor system to facilitate selective stimulation of cells engineered to express the orthogonal CD122 receptor. Mutant IL2 proteins that are homologous ligands of the orthogonal receptor are also described. Engineered T cells expressing orthogonal CD122 are contacted with the orthogonal ligand corresponding to this orthogonal CD122 (“IL2 ortholog”), enabling specific activation of these engineered T cells. In particular, this orthogonal IL2 receptor ligand complex provides a mixed population of cells, especially a mixed population of T cells, for selective expansion of cells engineered to express the orthogonal receptor.

[0009] IL-2 orthologs with attenuated affinity for non-engineered intermediate-affinity (CD122 / CD132) or high-affinity (CD25 / CD122 / CD132) IL-2 receptor complexes are also used to selectively target the activity of ortholog IL-2 to cells exhibiting high CD25 expression, for example, in the treatment of autoimmune diseases. IL-2 orthologs with significantly attenuated affinity for the extracellular domain (ECD) of natural wild-type hCD122, but retaining binding to the CD25 ECD, can also be used as competitive antagonists of wild-type IL-2 by interfering with the formation of high-affinity IL-2 receptor complexes, and thus can be used in the treatment of autoimmune diseases or graft-versus-host disease (GVH).

[0010] This invention relates to ligands that interact with orthogonal hCD122 receptors. Specifically, an hIL-2 orthogonal ligand (hIL2 orthogonal homolog) is provided that provides selective binding and signal transduction via a receptor comprising the extracellular domain of the hCD122 orthogonal receptor, particularly the extracellular domain of human CD122 containing amino acid substitutions for H133D and Y134F. The IL2 activity of this hIL2 orthogonal homolog is significantly reduced in cells expressing wild-type hCD122 compared to the activity of the hIL2 orthogonal homolog present on cells expressing orthogonal hCD122. Therefore, engineered cells expressing the extracellular domain of orthogonal hIL2 are provided using the hIL-2 orthogonal homolog in engineered cell populations for selective activation and / or amplification. Summary of the Invention

[0011] This invention relates to a human IL2 orthogonal ligand (“hIL2 orthogonal homolog”) that specifically and selectively binds to a transmembrane polypeptide comprising a modified hCD122 polypeptide having modifications at positions 133 and / or 134 of the ECD of the orthogonal hCD122 polypeptide. In some embodiments, the orthogonal hCD122 polypeptide comprises amino acid substitutions for H133D and Y134F. The binding of the hIL2 orthogonal homolog to the modified hCD122 polypeptide participates in intracellular signal transduction pathways, resulting in biological activity associated with native intracellular signal transduction patterns of IL2 binding to intermediate- or high-affinity IL2 receptors, but exhibiting selectivity for engineered expression of the hCD122 orthogonal receptor. In some embodiments, the hIL2 orthographic homolog is a variant of hIL2 of Formula 1 described herein. The hIL2 orthographic homolog of the present invention exhibits significantly reduced binding to the extracellular domain of wild-type hCD122, either alone or in the presence of hCD122, for binding of the hIL2 orthographic homolog to the hCD122 orthogonal receptor in the form of an endogenous high- or moderate-affinity hIL2 receptor. In some embodiments, the affinity of the hIL2 orthographic homolog to the extracellular domain of orthogonal hCD122 is comparable to that of wild-type hIL2 to wild-type hCD122. In some embodiments, the affinity of the hIL2 orthographic homolog to the extracellular domain (“ECD”) of orthogonal hCD122 is greater than that of wild-type hIL2 to the extracellular domain of wild-type hCD122. In some embodiments, the affinity of the hIL2 orthogonal homolog for the extracellular domain of orthogonal hCD122 is less than the affinity of wild-type hIL2 for the extracellular domain of wild-type hCD122. In one embodiment, the ECD of the orthogonal hCD122 receptor comprises a human hCD122 ECD polypeptide (numbered according to wild-type hCD122) containing modifications substituted for H133D and Y134F, wherein the amino acid sequence of the ECD of the orthogonal hCD122 receptor comprises a 214-amino acid polypeptide having the following sequence:

[0012] AVNGTSQFTC FYNSRANISC VWSQDGALQD TSCQVHAWPD

[0013] RRRWNQTCEL

[0014] LPVSQASWAC NLILGAPDSQ KLTTVDIVTL RVLCREGVRW

[0015] RVMAIQDFKP

[0016] FENLRLMAPI SLQVVHVETH RCNISWEISQ ASDFFERHLE

[0017] FEARTLSPGH

[0018] TWEEAPLLTL KQKQEWICLE TLTPDTQYEF QVRVKPLQGE

[0019] FTTWSPWSQP

[0020] LAFRTKPAAL GKDT(SEQ ID NO:1)

[0021] In one embodiment, the orthogonal hCD122 receptor is a modified human CD122 having an amino acid sequence (minus the signal peptide) comprising the amino acid sequence of the ECD of hCD122 substituted with H133D and Y134F (SEQ ID NO:1) and the transmembrane (TM) and intracellular (ICD) domains of the wild-type hCD122 molecule. The orthogonal hCD122 receptor (hoRb) has the following amino acid sequence:

[0022] AVNGTSQFTC FYNSRANISC VWSQDGALQD TSCQVHAWPD

[0023] RRRWNQTCEL

[0024] LPVSQASWAC NLILGAPDSQ KLTTVDIVTL RVLCREGVRW

[0025] RVMAIQDFKP

[0026] FENLRLMAPI SLQVVHVETH RCNISWEISQ ASDFFERHLE

[0027] FEARTLSPGH

[0028] TWEEAPLLTL KQKQEWICLE TLTPDTQYEF QVRVKPLQGE

[0029] FTTWSPWSQP

[0030] LAFRTKPAAL GKDTIPWLGH LLVGLSGAFG FIILVYLLIN

[0031] CRNTGPWLKK

[0032] VLKCNTPDPS KFFSQLSSEH GGDVQKWLSS PFPSSSFSPG

[0033] GLAPEISPLE

[0034] VLERDKVTQL LLQQDKVPEP ASLSSNHSLT SCFTNQGYFF

[0035] FHLPDALEIE

[0036] ACQVYFTYDP YSEEDPDEGV AGAPTGSSPQ PLQPLSGEDD

[0037] AYCTFPSRDD

[0038] LLLFSPSLLG GPSPPSTAPG GSGAGEERMP PSLQERVPRD

[0039] WDPQPLGPPT

[0040] PGVPDLVDFQ PPPELVLREA GEEVPDAGPR EGVSFPWSRP

[0041] PGQGEFRALN

[0042] ARLPLNTDAY LSLQELQGQD PTHL(SEQ ID NO:2)

[0043] In one embodiment, the present invention provides an hIL2 orthogonal homolog whose amino acid sequence has at least 80% identity with the polypeptide of formula #1:

[0044]

[0045] in:

[0046] AA1 is A (wild type) or absent;

[0047] AA2 is P (wild type) or absent;

[0048] AA3 is T (wild type), C, A, G, Q, E, N, D, R, K, P or missing;

[0049] AA4 is S (wild type) or absent;

[0050] AA5 is S (wild type) or absent;

[0051] AA6 is either S (wild type) or absent;

[0052] AA7 is T (wild type) or absent;

[0053] AA8 is K (wild type) or absent;

[0054] AA9 is K (wild type) or absent;

[0055] AA13 is Q (wild type), W, or absent;

[0056] AA14 is L (wild type), M, W, or absent;

[0057] AA15 is E (wild-type), K, D, T, A, S, Q, H or absent;

[0058] AA16 can be H (wild type), N, Q, or absent;

[0059] AA18 is L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T;

[0060] AA19 is L (wild type), A, V, I, or absent;

[0061] AA20 is D (wild-type), T, SML, or absent;

[0062] AA22 is Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, F or missing;

[0063] AA23 is M (wild type), A, W, H, Y, F, Q, S, V, L, T or absent;

[0064] AA27 is G (wild type), K, S, or absent;

[0065] AA38 is R (wild type), W, or G;

[0066] AA39 is M (wild type), L, or V;

[0067] AA42 is either F (wild type) or K;

[0068] AA51 is T (wild type), I, or absent.

[0069] AA55 is either H (wild type) or Y;

[0070] AA74 is Q (wild type), N, H, S;

[0071] AA80 is L (wild type), F, or V;

[0072] AA81 is R (wild-type), I, D, Y, T, or absent.

[0073] AA85 is either L (wild type) or V;

[0074] AA86 is either I (wild type) or V;

[0075] AA88 is N (wild type), E or Q, or missing;

[0076] AA89 is either I (wild type) or V;

[0077] AA91 is V (wild type), R, or K;

[0078] AA92 is either I (wild type) or F;

[0079] AA97 is either K (wild type) or Q;

[0080] AA104 is either M (wild type) or A;

[0081] AA109 is a non-natural amino acid, either D (wild-type), C, or with an activated side chain;

[0082] AA113 is either T (wild type) or N;

[0083] AA125 is C (wild type), A, or S;

[0084] AA126 is Q (wild type) or H, M, K, C, D, E, G, I, R, S or T;

[0085] and / or

[0086] AA130 is S (wild type), T, or R.

[0087] In some embodiments of the present invention, hIL2 orthogonal homologs comprising IL2 variant peptides are provided, which contain amino acid modifications at the following positions: E15, L16, L19, D20, and M23, and optionally Q22. In some embodiments, the hIL2 orthogonal homologs comprise IL2 variant peptides containing amino acid substitutions: L12, Q13, H16, L19, D20, M23, R81, D84, S87, N88, V91, I92, and E95. In some embodiments of the present invention, hIL2 orthogonal homologs comprising IL2 variant peptides are provided, which contain amino acid modifications at the following positions: Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I92F. In some embodiments, the present invention provides hIL2 orthogonal homologs, including IL2 variant peptides comprising amino acid modifications selected from the group consisting of:

[0088] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-L85V-I86V-I92F];

[0089] [E15S-H16Q-L19V-D20L-Q22K-L80F-R81D-L85V-I86V-I92F];

[0090] [E15S-H16Q-L19V-D20L-Q22K-M23A

[0091] L80F-R81D-L85V-I86V-I92F];

[0092] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-L85V-I86V-I92F-Q1

[0093] 26H];

[0094] [E15S-H16Q-L19V-D20L-Q22K-L80F-R81D-L85V-I86V-I92F-Q1

[0095] 26H];

[0096] [E15S-H16Q-L19V-D20L-Q22K-M23A-L80F-R81D-L85V-I86V-I

[0097] 92F-Q126H];

[0098] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-L85V-I86V-I92F-Q1

[0099] 26M];

[0100] [E15S-H16Q-L19V-D20L-Q22K-L80F-R81D-L85V-I86V-I92F-Q1

[0101] 26M]; or

[0102] [E15S-H16Q-L19V-D20L-Q22K-M23A-L80F-R81D-L85V-I86V-I

[0103] 92F-Q126M].

[0104] In some embodiments, the present invention provides hIL2 orthogonal homologs, including IL2 variant polypeptides comprising amino acid substitutions at the following positions: S4, K8, K9, T10, Q11, Q13, N26, N29, N30, N30, Y31, K35, T37, R38, T41, F42, K43, F44, Y45, M46, K48, K49, K54, E61, E62, K64, P65, E67, G, E68, V69, N71, L72, Q74, S75, K76, H79, I89, N90, I92, S99, T101, F103, Y107, I114, I128, and T133. In some embodiments, the present invention provides hIL2 orthogonal homologs, including IL2 variant peptides comprising one or more amino acid substitutions: S4P, K8R, K9T, T10A, Q11R, Q13R, N26D, N29S, N30S, N30D, N30T, Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K4 9R, K49E, K54R, E61D, K64R, E67G, E68D, V69A, N71T, N71A, N71R, A73V, Q74PS75P, K76E, K76R, H79R, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, T133A, and T133N. In some embodiments, the present invention provides IL2 variant peptides comprising amino acid modifications selected from the following amino acid substitution group:

[0105] [R38A-F42A-Y45A-E62A]; [F42A-Y45A-L72G]; [V69A, Q74P];

[0106] [V69A, Q74, T101A]; [V69A, Q74P, I128T]; [N30D, V69A, Q74P, F103S]; [K49E, V69A, A73V, K76E], [V69A, Q74P, T101A, T133N];

[0107] [N30S, V69A, Q74P, I128A]; [N30S, V69A, Q74P, I128T]; [K9T,

[0108] Q11R, K35R, V69A, Q74P], [A1T, M46L, K49R, E61D, V69A,

[0109] H79R]; [K48E, E68D, N71T, N90H, F103S, I114V]; [S4P T10A,

[0110] Q11R, V69A, Q74P, T133A]; [N30S, Y31H, K35R, K48E, V69A,

[0111] Q74P, I92T]; [N30S, E68D, V69A, N71A, Q74P, S75P, K76R, N90H]; [N30S, Y31C, T37A, V69A, A73V, Q74P.H79R, I128T],

[0112] [N26D, N29S, N30S, K54R, E67G, V69A, Q74P, I92T]; [K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, I92T]; [N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, I39V] and [T41P-T51P].

[0113] In some embodiments of the present invention, hIL2 orthogonal homologs comprising IL2 variant peptides are provided, which contain amino acid modifications at the following positions: Q11, L18, Q22, E110, N119, T123, Q126, S127, Q126, S127, I129, S130, and T133. In some embodiments, the present invention provides hIL2 orthogonal homologs comprising IL2 variant peptides having one or more amino acid substitutions selected from the group consisting of: L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18T, Q22E, Q22E, Q22E, Q22E, Q22E, Q2 2G, Q22E, Q22A, Q22L, Q22M, Q22F, Q22W, Q22K, Q22S, Q22V, Q22I, Q22Y, Q22H, Q22R, Q22N, Q22D, Q22T, Q22F, Q126H, Q126M, Q126K, Q126C, Q126D, Q126E, Q126G, Q126I, Q126R, Q126S, or Q126T. In some embodiments, the present invention provides hIL2 orthogonal homologs comprising IL2 variant peptides having an amino acid substitution group selected from the following amino acid substitution group:

[0114] [E15S-H16Q-L18R-L19V-D20L-Q22E-M23A];

[0115] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A];

[0116] [E15S-H16Q-L18R-L19V-D20L-M23A];

[0117] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A];

[0118] [E15S-H16Q-L18R-L19V-D20L-M23A-Q126H];

[0119] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126H];

[0120] [E15S-H16Q-L18R-L19V-D20L-M23A-Q126K];

[0121] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126K];

[0122] [E15S-H16Q-L19V-D20L-M23A-Q126H];

[0123] [E15S-H16Q-L19V-D20L-Q22K-M23A-Q126H];

[0124] [E15S-H16Q-L19V-D20L-M23A-Q126K];

[0125] [E15S-H16Q-L19V-D20L-Q22K-M23A-Q126K];

[0126] [E15S-H16Q-L18R-L19V-D20L-M23A-Q126H];

[0127] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126H];

[0128] [E15S-H16Q-L18R-L19V-D20L-M23A-Q126K];

[0129] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126K];

[0130] [E15S-H16Q-L19V-D20L-Q22K-Q126H];

[0131] [E15S-H16Q-L19V-D20L-M23A-Q126M];

[0132] [E15S-H16Q-L19V-D20L-Q22K-M23A-Q126M];

[0133] [E15S-H16Q-L19V-D20L-Q22K-Q126M];

[0134] [desAla1-E15S-H16Q-L19V-D20L-Q126M];

[0135] [desAla1-E15S-H16Q-L19V-D20L-Q22K-Q126M];

[0136] [desAla1-E15S-H16Q-L19V-D20L-M23A-Q126M];

[0137] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-Q126M];

[0138] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-Q126M];

[0139] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-I86V-I92F-Q126H];

[0140] [E15S-H16Q-L19V-D20L-Q22K-L80F-R81D-I86V-I92F-Q126H];

[0141] [E15S-H16Q-L19V-D20L-Q22K-M23A-L80F-R81D-I86V-I92F-Q12

[0142] 6H];

[0143] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-I86V-I92F-Q126M];

[0144] [E15S-H16Q-L19V-D20L-Q22K-M23A-L80F-R81D-I86V-I92F-Q12

[0145] 6M];

[0146] [E15S-H16Q-L19V-D20L-M23A-L85V-Q126H];

[0147] [E15S-H16Q-L19V-D20L-Q22K-L85V-Q126H];

[0148] [E15S-H16Q-L19V-D20L-Q22K-M23A-L85V-Q126H];

[0149] [E15S-H16Q-L19V-D20L-M23A-L85V-Q126M];

[0150] [E15S-H16Q-L19V-D20L-Q22K-L85V-Q126H]; or

[0151] [E15S-H16Q-L19V-D20L-Q22K-M23A-L85V-Q126M].

[0152] In some embodiments, the present invention provides hIL2 orthogonal homologs, including IL2 variant peptides selected from SEQ ID NO:5-138.

[0153] In some embodiments, the present invention provides an hIL2 orthogonal homolog operably linked to at least one carrier molecule. In some embodiments, the present invention provides an hIL2 orthogonal homolog comprising at least one polyethylene glycol (PEG) molecule.

[0154] In some embodiments, the present invention provides hIL2 orthogonal homologs comprising the following structures:

[0155] [PEG]-[Connector] n -[hoIL2]

[0156] Where n = 0 or 1 and hoIL2 is a human orthogonal IL2 polypeptide variant of Formula 1. In some embodiments, the PEG molecular weight is between 5 kDa and 80 kDa. In some embodiments, the PEG molecular weight is approximately 40 kDa. In some embodiments, the present invention provides an hIL2 orthogonal homolog of the above structure, wherein hoIL2 is an IL2 polypeptide variant comprising an amino acid substituent group.

[0157] -[desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A]. In some embodiments, the present invention provides an hIL2 orthogonal homolog of the above structure, wherein hoIL2 is an IL2 polypeptide variant comprising the amino acid sequence:

[0158] PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRM

[0159] LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK

[0160] NFHLRPRDLSNINVIVLELKGSETTFMCEYADETATIV

[0161] EFLNRWITFCQSIISTLT(SEQ ID NO:5).

[0162] In some embodiments, the present invention provides a nucleic acid sequence encoding an hIL2 orthogonal homolog polypeptide of formula #1.

[0163] In some embodiments, the present invention provides a recombinant vector encoding a nucleic acid sequence of an hIL2 orthogonal homolog polypeptide of formula #1A, including a recombinant vector of the nucleic acid sequence of claim 22.

[0164] In some embodiments, the present invention provides a method for treating a disease, disorder, or symptom suffered by a subject by administering to the subject:

[0165] a. An engineered mammalian cell comprising a nucleic acid sequence encoding a transmembrane receptor molecule containing an extracellular domain (ECD) of orthogonal hCD122, the nucleic acid sequence being operatively linked to one or more expression control elements capable of acting on the expression and surface presentation of the ECD of the transmembrane receptor molecule; and

[0166] b. Administer to the patient a therapeutically effective dose of the hIL2 ortholog of Formula #1.

[0167] In some embodiments, the present invention provides a method for preparing an engineered T cell product comprising at least 20% hoCD122 T cells, the method comprising the steps of:

[0168] a. Isolating T cell populations from mammalian subjects;

[0169] b. Contact the isolated T cell population with a recombinant vector comprising a nucleic acid sequence encoding hoCD122 operatively linked to one or more expression control sequences such that expression in mammalian T cells is promoted while allowing the recombinant vector to be taken up by T cells.

[0170] c. Contact the isolated T cell population with an effective amount of the hIL2 ortholog as described in claim 1.

[0171] In some embodiments, the present invention provides a cell population of at least 20% engineered hoCD122 T cells.

[0172] The present invention further provides a method for preparing the hIL2 orthogonal homolog of the present invention. In particular, the present invention provides a recombinant expression vector comprising a nucleic acid sequence encoding an hIL2 orthogonal homolog operatively linked to a control element to provide expression of the nucleic acid sequence encoding the hIL2 orthogonal homolog in a host cell.

[0173] The present invention further provides a composition comprising a mixed cell population containing at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70% of T cells (e.g., T cells, CD8+ T cells, Tregs, TILs, NK cells, TCR-modified cells, CAR-T cells, etc.), wherein the T cells have been recombinantly modified to express an orthogonal hCD122 receptor polypeptide. The present invention also provides a method for producing a pharmaceutically acceptable dosage form of an engineered cell therapy product comprising a T cell population substantially enriched with one or more engineered T cell species expressing a receptor containing the extracellular domain of the hCD122 orthogonal polypeptide, the method comprising, in the presence of the hIL2 orthogonal homolog of the present invention, in vitro culturing the T cell population comprising the engineered T cells expressing a receptor containing the extracellular domain of the hCD122 orthogonal polypeptide for a time sufficient to enrich the cell population with one or more such engineered T cells.

[0174] In some embodiments, the present invention provides a recombinant vector comprising a nucleic acid sequence encoding an hIL2 orthogonal homolog operatively linked to a control element herein to promote the expression and secretion of the hIL2 orthogonal homolog from mammalian cells, administered to a subject to provide in situ expression of the hIL2 orthogonal homolog. In some embodiments, the recombinant vector is administered intratumorally to a cancerous subject. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) or a 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 virus's ability to initiate cell circulation and / or apoptosis pathways. The replication-deficient adenovirus vector may optionally include a deletion in the E3 domain. In some embodiments, the adenovirus is a replication-capable adenovirus. In some embodiments, the adenovirus is a replication-capable recombinant virus engineered to selectively replicate in tumor cells.

[0175] The present invention further provides a method for preparing a pharmaceutically acceptable dosage form of a cell therapy product comprising at least one (or two, three, four or more) engineered T cells expressing a transmembrane receptor protein, wherein the extracellular domain of such transmembrane receptor protein includes the extracellular domain of an hCD122 orthogonal polypeptide, wherein in the cell therapy product, the engineered cell portion constitutes at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the total number of cells in the cell therapy product.

[0176] In some embodiments, a therapeutic method is provided, comprising introducing an engineered cell population into a subject suffering from a disease, disorder, or symptom. The engineered cell population comprises a nucleic acid sequence encoding a transmembrane orthogonal receptor polypeptide of the ECD of SEQ ID NO: 1, a transmembrane domain, and an intracellular signal transduction domain, which reacts with an hIL2 orthogonal homolog ligand to the ECD of the transmembrane orthogonal receptor polypeptide to generate an intracellular signal. The nucleic acid sequence is operatively linked to an expression control element to promote transcription and translation of the ECD of the transmembrane polypeptide and cell surface presentation, in combination with the hCD122 orthogonal homolog of the present invention. Such a cell population may comprise cells that have been in vitro modified and are autologous or allogeneic to the subject. In some embodiments, the therapeutic method includes: (1) ex vivo contact of an engineered cell population with an amount of a homologous hIL2 orthogonal homolog at a concentration and duration sufficient to activate the engineered cells, the engineered cells expressing a receptor comprising an hIL2 orthogonal CD122 ECD; and (2) administration of the cell population to a subject; and (3) administration of the homologous hIL2 orthogonal homolog to the subject in combination with the engineered cells. In some embodiments, the subject to which the engineered hIL2 orthogonal CD122 ECD receptor cell population and hIL2 orthogonal homolog are administered has a neoplastic disease. In some embodiments, the orthogonal receptor and ligand are administered in combination with at least one other / complementary therapeutic or prophylactic agent.

[0177] Brief description of the attached figures

[0178] The invention will be better understood through the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, by convention, the various features in the drawings are not to scale. Instead, the dimensions of the various features are arbitrarily enlarged or reduced for clear display. The accompanying drawings include the following figures.

[0179] The attached diagram Figure 1 293 NKL cells transfected with supernatant were provided for use in the experiments described in more detail in Example 7 of this document. Values. For NKL cells receiving each supernatant of a specified dilution, indicated in bold, with duplicates... The values ​​are displayed in side-by-side columns.

[0180] The attached diagram Figure 2 NKL cells (“NKL hoRB cells”) were provided that were recombinantly modified to express the hCD122 orthogonal receptor SEQ ID NO2. Values ​​were obtained by processing 293 transfection supernatants from the experiments described in Example 7. NKL hoRB cells receiving each specified dilution of supernatant are indicated in bold, with replicates... The values ​​are displayed in side-by-side columns. Detailed Implementation

[0181] To facilitate understanding of this invention, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be interpreted based on the knowledge of someone skilled in the art.

[0182] Before describing the methods and compositions of the present invention, it should be understood that the invention is not limited to the methods or compositions described herein, as they may, of course, vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be limiting.

[0183] When providing a numerical range, it should also be considered as specifically disclosing intermediate values ​​between the upper and lower limits of the range, spaced one-tenth of the lower limit unit, unless the context explicitly states otherwise. This invention also includes smaller ranges between any set value or intermediate value within the set range and any other set value or intermediate value within the set range. Depending on any explicitly excluded limit value within the set range, the range may independently include or exclude the upper and lower limits of these smaller ranges. This invention also includes ranges that do not contain a limit value, or that contain one or both limit values. When a set range contains one or two limit values, this invention also includes ranges that exclude one or both of those limit values.

[0184] Unless otherwise stated, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used to practice or test the invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0185] It should be noted that the singular forms "a," "an," and "the" used herein and in the appended claims include the plural meaning unless the context clearly indicates otherwise. Thus, for example, reference to "cell" includes a plurality of such cells, and reference to "the peptide" includes one or more peptides and their equivalents known to those skilled in the art, such as polypeptides, etc.

[0186] The publications discussed herein refer only to their disclosures prior to the filing date of this application. Nothing herein should be construed as an admission that the invention does not precede these publications by virtue of a prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may require separate verification.

[0187] Unless otherwise specified, parts are by weight, molecular weight is by weight-average molecular weight, temperature is in degrees Celsius (°C), and pressure is atm or near atm. Standard abbreviations are used, including the following: bp = base pair; kb = kilobase; pl = picolitrogen; s or sec = second; min = minute; h or hr = hour; aa = amino acid; kb = kilobase; nt = nucleotide; pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar; mM = millimole; M = mole; kDa = kilodalton; im = intramuscular (via intramuscular); ip = Intraperitoneal (transperitoneal); SC or SQ = subcutaneous (transcutaneous); QD = once daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = no statistical significance; PBS = phosphate buffered saline; PCR = polymerase chain reaction; NHS = N-hydroxysuccinimide; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Darwin's modified Irwin medium; GC = genome copy; EDTA = ethylenediaminetetraacetic acid.

[0188] It should be understood that amino acids are referred to by single-letter or three-letter codes in this disclosure. For the convenience of readers, the single-letter and three-letter amino acid codes are provided in Table 1 below:

[0189]

[0190] The standard methods of molecular biology are described in the scientific literature (see, for example, Sambrook and Russell (2001), Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; and Ausubel, et al. (2001)).

[0191] *Current Protocols in Molecular Biology*, Volumes 1-4, John Wiley and Sons, Inc., NY, describes cloning and site-directed mutagenesis in bacterial cells (Volume 1), cloning in mammalian cells and yeast (Volume 2), glycoconjugates and protein expression (Volume 3), and bioinformatics (Volume 4). The scientific literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, fusion protein production, and protein glycosylation (see, for example, Coligan et al. (2000), *Current Protocols in Protein Science*, Volumes 1-2, John Wiley and Sons, Inc., NY).

[0192] Unless otherwise stated, the following terms are intended to have the following meanings. Other terms are defined throughout this specification.

[0193] activation The term “activation” as used herein refers to a receptor or receptor complex to reflect biological effects that arise directly and / or through participation in a multi-component signal transduction cascade, which is a ligand-binding response produced by the binding of an agonist ligand to the receptor. For example, it is alleged that the binding of an IL2 agonist to its homologous IL2 receptor “activates” receptor signal transduction to produce one or more intracellular biological effects (e.g., phosphorylation of STAT5).

[0194] active As used herein, the term "activity" refers to a molecule and describes its properties in relation to a test system or biological function, such as the extent to which the molecule binds to another molecule. Examples of such biological functions include, but are not limited to, the catalytic activity of a biopharmaceutical, its ability to stimulate intracellular signal transduction, gene expression, cell proliferation, and its ability to regulate immunological activities (such as inflammatory responses). "Activity" is often expressed as the biological activity of an administered reagent per unit, such as [catalytic activity] / [mg protein], [immunological activity] / [mg protein], international units of activity (IU), [STAT5 phosphorylation] / [mg protein], [T-cell proliferation] / [mg protein], plaque-forming units (PFU), etc. The term "proliferative activity" encompasses the activity that promotes cell division, including dysregulated cell division observed in neoplastic diseases, inflammatory diseases, fibrosis, developmental abnormalities, cell transformation, metastasis, and angiogenesis.

[0195] Dosage / Administration The terms “giving” and “application” are used interchangeably herein and refer to the act of contacting a subject, including contact of a reagent with cells, tissues, organs, or biological fluids of the subject, whether in vitro, in vivo, or ex vivo (e.g., IL-2 orthologs, CAR-T cells, chemotherapeutic agents, antibodies, or modulators, or pharmaceutical preparations comprising one or more of the foregoing). The administration of a reagent can be achieved by any of a variety of methods recognized in the art, including but not limited to local, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, percutaneous, transmucosal, iontophoretic delivery, intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhaler, intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), etc. The term “giving” includes contact of a reagent with cells, tissues, or organs, as well as contact of a reagent with a liquid, wherein the liquid contacts cells.

[0196] Adverse events As used herein, the term "adverse event" refers to any unintended experience associated with the use of a therapeutic or prophylactic agent in a subject. Adverse events are not necessarily caused by the administration of a therapeutic or prophylactic agent (e.g., an IL-2 ortholog), and may also be caused by unrelated circumstances. Adverse events are generally classified as mild, moderate, or severe. The classification of adverse events used herein conforms to the Common Terminology Standard for Adverse Events v4.03 (CTCAE), published by the U.S. Department of Health and Human Services, the National Institutes of Health, and the National Cancer Institute on June 14, 2010.

[0197] Affinity As used in this article, “affinity” refers to the degree to which a first molecule (e.g., a ligand) specifically binds to a second molecule (e.g., a receptor), and through K... d The binding kinetics measure is expressed as the dissociation constant (K) between the molecule and its target. off ) and the association constant between the molecule and its target (K on The ratio of ).

[0198] agonistsAs used herein, the term "agonist" refers to a first agent that specifically binds to a second agent ("target") and interacts with the target to induce or promote an increase in target activation. In some cases, an agonist is an activator of a receptor protein that can modulate, enhance, sensitize cells to activation by a second agent, or upregulate the expression of one or more genes, proteins, ligands, receptors, or biological pathways that may lead to cell proliferation or cell cycle arrest, or cell death (e.g., through apoptosis). In some embodiments, an agonist is an agent that binds to a receptor and alters the receptor state to induce a biological response that mimics the action of the receptor's endogenous ligands. The term "agonist" includes partial agonists, complete agonists, and superagonists. An agonist may be described as a "complete agonist" when it induces a substantially complete biological response (i.e., a response related to a naturally occurring ligand / receptor binding interaction) in the receptor under study; alternatively, an agonist may also be described as a partial agonist. "Super agonists" are agonists capable of producing a maximal response greater than that of the endogenous agonist to the target receptor, thus possessing more than 100% of the activity of the natural ligand. Super agonists are typically synthetic molecules that, when evaluated at similar concentrations in comparative studies, exhibit a response greater than 110%, or greater than 120%, or greater than 130%, or greater than 140%, or greater than 150%, or greater than 160%, or greater than 170% in assessable quantitative or qualitative parameters of the naturally occurring form of the molecule. It should be noted that the biological effects associated with full agonists may differ in degree and / or type from those of partial or super agonists. In contrast to agonists, antagonists can specifically bind to the receptor but do not cause the signaling cascade normally initiated by the receptor and can alter the action of the agonist on that receptor. Inverse agonists are agents that produce a pharmacological response in the opposite direction to that of the agonist.

[0199] Antagonist As used herein, the terms “antagonist” or “inhibitor” refer to a molecule that has one or more effects in contrast to an agonist. Antagonists can prevent, reduce, inhibit, or neutralize the activity of an agonist, and they can also prevent, inhibit, or reduce the constitutive activity of a target (such as a target receptor), even without an identified agonist. Inhibitors are molecules that reduce, block, prevent, delay activation, inactivate, desensitize, or downregulate, for example, genes, proteins, ligands, receptors, biological pathways (including immune checkpoint pathways), or cellular molecules.

[0200] AntibodyAs used herein, the term “antibody” collectively refers to: (a) glycosylated and non-glycosylated immunoglobulins (including, but not limited to, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4) that specifically bind to target molecules, and (b) immunoglobulin derivatives, including but not limited to IgG(1-4)ΔC H 2, F(ab')2, Fab, ScFv, V H V L Antibodies include tetraclonal antibodies, triclonal antibodies, biclonal antibodies, dsFv, F(ab')3, scFv-Fc, and (scFv)2, which compete with their derived immunoglobulins for binding to target molecules. The term antibody is not limited to immunoglobulins, antibodies, or human antibodies from any particular mammalian species (including mice, humans, horses, and camels). The term antibody includes so-called "heavy chain antibodies" or "VHH" or... Immunizations are typically obtained from camelids (including camels, llamas, and alpacas) (see, for example, Hamers-Casterman, et al. (1993) Nature 363:446-448). Antibodies with a given specificity can also be derived from non-mammal sources, such as VHHs obtained from immunizations of cartilaginous fish (including but not limited to sharks). The term "antibody" includes antibodies that can be isolated from animals from natural sources or after immunization with an antigen, as well as engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted, veneered, or deimmunized (e.g., T-cell epitope removed) antibodies, camel-derived (in the case of VHHs), or molecules containing antibody-binding domains (such as CDRs) within a non-immunoglobulin scaffold. The term "antibody" should be interpreted as not limited to any particular synthetic method, including naturally occurring antibodies that can be isolated from natural sources, and engineered antibody molecules prepared by "recombinant" means, including antibodies isolated from transgenic animals carrying human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with nucleic acid constructs that lead to antibody expression, and antibodies isolated from combinatorial antibody libraries (including phage display libraries). In one embodiment, an "antibody" is a mammalian immunoglobulin. In some embodiments, the antibody is a "full-length antibody" that includes variable and constant domains that provide binding and effector functions. As used herein, the term "single-domain antibody" (sdAb) refers to an antibody fragment composed of monomeric variable antibody domains capable of selectively binding to an antigen and competitively binding to its derived parental antibody. As used herein, the term "VHH" refers to a single-domain antibody derived from camelid antibodies, typically obtained from immunizations of camels (including camels, llamas, and alpacas) (see, for example, Hamers-Casterman et al. (1993) Nature 363:446-448). VHHs may also be referred to as heavy chain antibodies or... Single-domain antibodies can also be derived from non-mammal sources, such as VHH obtained from immunization with IgNAR antibodies from cartilaginous fish (including but not limited to sharks). The term antibody includes antibody-drug conjugates, which include modifications to prolong the duration of action, such as fusion proteins or conjugated to polymers (e.g., PEGylated), described in more detail below.

[0201] biological samplesAs used herein, the term "biological sample" or "sample" refers to a sample obtained from or derived from a subject. For example, a biological sample includes materials selected from the group consisting of: body fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluids (such as vitreous fluid, aqueous humor), lymph, lymph node tissue, spleen tissue, bone marrow, and immunoglobulin-enriched portions of one or more of these tissues. In some embodiments, the sample is obtained from a subject who has been exposed to a treatment regimen comprising a pharmaceutical formulation including an hhIL2 ortholog, e.g., repeated exposure to the same drug. In other embodiments, the sample is obtained from a subject who has not recently been exposed to an hIL2 ortholog, or from the subject prior to planned administration of an hIL2 ortholog.

[0202] "CAR" or "Chimeric Antigen Receptor" As used herein, the terms "chimeric antigen receptor" and "CAR" are used interchangeably and refer to a chimeric polypeptide comprising multiple functional domains, the sequence of which, from the amino terminus to the carboxyl terminus, is arranged as follows: (a) an antigen-binding domain (ABD); (b) a transmembrane domain (TD); and (c) one or more cytoplasmic signal transduction domains (CSD), wherein the aforementioned domains may optionally be linked by one or more spacer subdomains. The CAR may further comprise a signal peptide sequence, which is typically removed during post-translational processing and presented on the surface of cells transformed with an expression vector containing a nucleic acid sequence encoding the CAR. CARs useful in the practice of this invention can be prepared according to principles well known in the art. See, for example, Eshhaar et al., U.S. Patent No. 7,741,465B1, issued June 22, 2010; Sadelain et al. (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross et al. (1989) PNAS (USA) 86(24):10024-10028; Curran et al. (2012) J Gene Med 14(6):405-15. Examples of commercially available CAR-T cell products that can be modified to incorporate the orthogonal receptor of the present invention include axicabtagene ciloleucel (from Gilead Pharmaceuticals under the name...). Commercially available) and tisagenlecleucel (from Novartis under the name) Commercially available.

[0203] CAR-T cells As used herein, the terms “chimeric antigen receptor T-cell” and “CAR-T cell” are used interchangeably and refer to T cells that have been recombinantly modified to express a chimeric antigen receptor. As used herein, CAR-T cells can be engineered to express the hCD122 orthogonal peptide.

[0204] CD-122 Orthogonal: As used herein, the terms "CD122 ortho" or "hoCD122" or "hoIL2Rb" are used interchangeably to refer to a variant of the hCD122 polypeptide containing amino acid substitutions at histidine (H133) at position 133 and tyrosine (Y134) at position 134 of the ECD of the hCD122 polypeptide. In some embodiments, the CD-122 ortho includes an amino acid substitution at position 133 from histidine to aspartic acid (H133D), glutamic acid (H133E), or lysine (H133K) and / or at position 134 from tyrosine to phenylalanine (Y134F), glutamic acid (Y134E), or arginine (Y134R). In a preferred embodiment, the hCD122 ortho receptor is an hCD122 molecule having amino acid substitutions H133D and Y134F. In one embodiment, the hCD122 ortho receptor is a polypeptide having the amino acid sequence of SEQ ID NO:2.

[0205] CDR. As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site found within the variable region of a heavy-chain or light-chain immunoglobulin polypeptide. CDRs have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977); Kabat et al., (1991) US Department of Health and Human Services, “Sequences of Proteins of Immunological Interest” (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes overlap or subsets of amino acid residues when compared to one another. However, the definition of CDR for antibodies or transplanted antibodies or variants thereof is intended to fall within the scope of the definitions or terms used herein. In the context of this invention, the CDR position numbering is provided according to the Kabat numbering method.

[0206] QuiteAs used herein, the term "comparable" is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, two measurements are considered "comparable" when a first measurement of an evaluable quantitative parameter (e.g., IL-2 activity level determined by a CTLL-2 proliferation or phosphorylation-STAT5 assay) and a second measurement of the same evaluable parameter do not deviate from a range that a person skilled in the art would consider not to present a statistically significant difference between the two results. In some cases, a measurement may be considered "comparable" if one measurement deviates from another measurement by less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 7%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%. In a particular embodiment, a measurement is considered comparable to a reference standard if it deviates from a reference standard by less than 15%, or less than 10%, or less than 5%.

[0207] Source As used herein, the term "derived from" in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence "derived from" an IL-2 polypeptide) means that the polypeptide or nucleic acid has a sequence based on a reference polypeptide or nucleic acid (e.g., a naturally occurring IL-2 polypeptide or a nucleic acid encoding IL-2), and is not intended to be limited to a source or method for preparing a protein or nucleic acid. For example, the term "derived from" includes homologs or variants of the reference amino acid or DNA sequence.

[0208] Effective concentration (EC) As used herein, the term "effective concentration" or its abbreviation "EC" is used interchangeably to indicate the concentration of a reagent sufficient to cause a change in a given parameter in the test system. The abbreviation "E" refers to the magnitude of a given biological effect observed in the test system when it is exposed to the test reagent. The abbreviation "EC" is used when the magnitude of the reaction is expressed as a factor of the concentration ("C") of the test reagent. In the context of biological systems, the term E... 最大 This refers to the maximum order of magnitude of a given biological effect observed at a saturation concentration of the activating assay reagent. When provided with the abbreviation EC (e.g., EC30), it is indicated by a subscript. 40 EC 50 When (etc.), the subscript refers to the E value at which a biological response was observed. 最大值 The percentage. For example, in a testing system, the reaction of this test reagent is sufficient to cause a reduction in the concentration of a measurable biological parameter by 30% of the maximum level of that measurable biological parameter, which is referred to as the "EC" of the test reagent for that biological parameter. 30 Similarly, the term "EC" 100"Effective concentration" is used to indicate the effective concentration of a reagent, which results in a maximum (100%) response to the measurable parameter. Similarly, the term EC... 50 (Used in pharmacokinetics) refers to the concentration of a reagent sufficient to cause a half-maximum (50%) change in a measurable parameter. The term "saturation concentration" refers to the maximum possible amount of a test reagent that can dissolve in a standard volume of a specific solvent (e.g., water) under standard temperature and pressure conditions. In pharmacokinetics, the saturation concentration of a drug is often used to express the concentration of the drug sufficient to occupy all available receptors, while EC... 50 It is the drug concentration at which the half-maximal effect is achieved.

[0209] enriched As used herein, the term “enriched” means that a sample has been subjected to non-natural manipulation so that the molecule of interest is present in a concentration that is (a) higher than that in the starting sample (e.g., at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or at least 1000-fold) or (b) higher than that in the environment in which the molecule was prepared (e.g., in recombinantly modified bacterial or mammalian cells).

[0210] extracellular domain As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of cell surface proteins (e.g., cell surface receptors) located outside the cytoplasmic membrane. ECDs can include the entire extracellular portion of transmembrane proteins, cell surface or membrane-associated proteins, secretory proteins, and cell surface targeting proteins.

[0211] hCD-122 As used herein, the term "hCD122" refers to the naturally occurring human CD122 polypeptide, including its naturally occurring variants. The amino acid sequence of a naturally occurring hCD122 variant is:

[0212] AVNGTSQFTC FYNSRANISC VWSQDGALQD TSCQVHAWPD

[0213] RRRWNQTCEL

[0214] LPVSQASWAC NLILGAPDSQ KLTTVDIVTL RVLCREGVRW

[0215] RVMAIQDFKP

[0216] FENLRLMAPI SLQVVHVETH RCNISWEISQ ASHYFERHLE

[0217] FEARTLSPGH

[0218] TWEEAPLLTL KQKQEWICLE TLTPDTQYEF QVRVKPLQGE

[0219] FTTWSPWSQP

[0220] LAFRTKPAAL GKDTIPWLGH LLVGLSGAFG FIILVYLLIN

[0221] CRNTGPWLKK

[0222] VLKCNTPDPS KFFSQLSSEH GGDVQKWLSS PFPSSSFSPG

[0223] GLAPEISPLE

[0224] VLERDKVTQL LLQQDKVPEP ASLSSNHSLT SCFTNQGYFF

[0225] FHLPDALEIE

[0226] ACQVYFTYDP YSEEDPDEGV AGAPTGSSPQ PLQPLSGEDD

[0227] AYCTFPSRDD

[0228] LLLFSPSLLG GPSPPSTAPG GSGAGEERMP PSLQERVPRD

[0229] WDPQPLGPPT

[0230] PGVPDLVDFQ PPPELVLREA GEEVPDAGPR EGVSFPWSRP

[0231] PGQGEFRALN

[0232] ARLPLNTDAY LSLQELQGQD PTHL(SEQ ID NO:3)

[0233] identityFor polypeptide or DNA sequences, the term "identity" as used herein refers to the subunit sequence identity between two molecules. Two molecules are identical at that position when the subunit position is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide). The similarity between two amino acid or two nucleotide sequences is a direct function of the amount of identical position. Typically, sequences are aligned to obtain the highest-order match. If necessary, identity can be calculated using publicly available techniques and widely used computer programs, such as the GCS package (Devereux et al., (1984) Nucleic Acids Res. 12:387), BLASTP, BLASTN, and FASTA (Atschul et al., (1990) J. Molecular Biol. 215:403-410). Suitable algorithms for determining sequence identity percentages and sequence similarity percentages are BLAST and BLAST 2.0, described by Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1977) Nucleic Acids Res.25:3389-3402. Software for BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. These HSPs match or satisfy a positive threshold score “T” when aligned with words of the same length in the database sequence. T is called the adjacent word score threshold (Altschul et al., ibid.). These initial adjacent word hits are used as seeds to initiate a search for longer HSPs containing them. Then, the word hits are extended in both directions along each sequence until the cumulative alignment score is increased. For nucleotide sequences, a cumulative score is calculated using parameters "M" (reward score for a pair of matching residues; always >0) and "N" (penalty score for mismatched residues; always <0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. Word matching is halted in all directions if: (a) the cumulative alignment score decreases by X from its maximum gain; the cumulative score becomes zero or below due to the accumulation of one or more negatively scored residues; 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) is similar but uses the following default values: word length ("W") 28, expected value ("E") 10, M = 1, N = -2, and compares two strands.For the amino acid sequence, the BLASTP program uses the following default values: word length (W) of 3, expected value (E) of 10, and BLOSUM62 score matrix (see Henikoff and Henikoff, (1989) PNAS (USA) 89:10915-10919).

[0234] IL-2: As used herein, the term "interleukin-2" or "IL-2" refers to a naturally occurring IL-2 polypeptide having IL-2 activity. In some embodiments, IL-2 refers to mature wild-type human IL-2. Mature wild-type human IL-2 (hIL2) is a 133-amino acid polypeptide (minus the signal peptide consisting of an additional 20 N-terminal amino acids), as described by Fujita, et al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of a naturally occurring variant of mature wild-type human IL-2 (hIL2) is:

[0235] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML

[0236] TFKFYMPKKA

[0237] TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN

[0238] VIVLELKGSE

[0239] TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT(SEQ ID NO:4)

[0240] As used herein, residue numbering is based on the hIL2 sequence UniProtID P60568, excluding the same signal peptide as SEQ ID NO:4.

[0241] IL2 activity The term "IL2 activity" refers to one or more biological effects on cells in response to contact with an effective amount of the IL2 peptide. IL2 activity can be measured, for example, in cell proliferation assays using CTLL-2 mouse cytotoxic T cells; see Gearing, AJH, and CBBird (1987) in *Lymphokines and Interferons, A Practical Approach*, Clemens, MJ, et al. (eds.): IRL Press, 295. The specific activity of recombinant human IL-2 is approximately 2.1 x 10⁻⁶.4 IU / μg, calibrated against the WHO international standard (NIBSC code: 86 / 500) for recombinant human IL-2. In some embodiments, IL2 activity can be assessed in human cells (e.g., YT cells) that do not require CD25 for signal transduction via the IL2 receptor but are capable of signal transduction via the intermediate-affinity CD122 / CD132 receptor, for example, when the orthogonal human IL-2 of interest exhibits (or is engineered to have) a weakened affinity for CD25. When evaluated at similar concentrations in comparative experiments, the orthogonal human IL-2 of the present invention can have less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5% of the activity of wild-type mature human IL-2 according to the WHO international standard (NIBSC code: 86 / 500).

[0242] IL-2 orthologs As used herein, the term "IL-2 orthogonal homolog" refers to a variant of hIL2 derived from the IL-2 parent polypeptide that specifically binds to an orthogonal hCD122 ECD and exhibits significantly reduced binding to the extracellular domain of wild-type hCD122. In some embodiments, the hIL2 orthogonal homolog exhibits specific binding to a receptor containing an orthogonal hCD122 ECD and contacts cells expressing a transmembrane receptor containing an ECD of the orthogonal hCD122 polypeptide in an amount sufficient to alter the signal signature of the signal generated by the intracellular domain (ICD) of the transmembrane receptor. When the transmembrane receptor contains both an orthogonal hCD122 ECD and an hCD122 ICD, the binding of the hIL2 orthogonal homolog to this receptor produces the activation of the intracellular signal signature of the Cd25 / CD122 / CD132 high affinity hIL2 receptor, characterized by moderate CD122 / CD132 affinity. IL-2 orthogonal homologs exhibit significantly reduced binding to wild-type hCD122. The term hIL2 orthogonal homologs include orthogonal variants of IL-2 and modified hIL2 orthogonal homologs. In some embodiments, hIL2 orthogonal homologs are derived from naturally occurring variants of human IL2, which may be referred to as “hoCD122” or “hoRb”. Certain modified IL-2 peptides are provided in Garcia et al. (U.S. Patent Application Publication US2018 / 0228842A1, published August 16, 2018). As used herein, the term hIL2 orthogonal homologs does not include modified hIL2 peptides described in Garcia et al.'s U.S. Patent Application Publication US2018 / 0228842A1, published August 16, 2018.

[0243] Quantities sufficient to produce change:As used herein, the phrase "amount sufficient to produce a change" refers to an amount of test reagent sufficient to produce a detectable difference between a level of the indicator measured before (e.g., baseline level) and after administration of the test reagent, such as in cell-based assays assessing the biological function of a response to an amount of test reagent administered. "Amount sufficient to produce a change" can be an amount sufficient to be therapeutically effective, but it may be more or less than a therapeutically effective amount.

[0244] Needs treatment The term "in need of treatment" as used in this article refers to a physician's or other caregiver's judgment about a subject and whether the subject needs or is likely to benefit from treatment. This judgment is based on a number of factors within the physician's or caregiver's area of ​​expertise.

[0245] Preventable The term "in need of prevention" as used in this article refers to a physician's or other caregiver's judgment that the subject needs or is likely to benefit from preventive care. This judgment is based on a number of factors within the physician's or caregiver's area of ​​expertise.

[0246] Inhibitors As used herein, an inhibitor is a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes, or downregulates, such as genes, proteins, ligands, receptors, or cells. Inhibitors can also be defined as molecules that reduce, block, or inactivate the constitutive activity of cells or organisms.

[0247] Intracellular domains: As used herein, the term "intracellular domain" or its abbreviation "ICD" refers to the portion of a cell surface protein (such as a cell surface receptor) within the cell membrane. An ICD may contain the entire intracellular portion of a transmembrane protein or a membrane-associated protein, or an intracellular protein.

[0248] Separate As used herein, the term "isolated" refers to a polypeptide of interest that, if naturally occurring, exists in an environment different from that in which it may naturally occur. "Isolated" means a polypeptide included in a sample that is substantially enriched with the polypeptide of interest and / or that the polypeptide of interest has been partially or substantially purified. If the polypeptide is not naturally occurring, "isolated" means that the polypeptide has been isolated from the environment in which it was prepared by synthetic or recombinant means.

[0249] Intracellular domain of orthogonal receptorAs used herein, the term "intracellular domain of orthogonal receptor" or "ICD-OR" refers to a portion of a transmembrane orthogonal receptor located within the plasma membrane of a cell expressing such a transmembrane orthogonal receptor. ICD-OR may include one or more "proliferation signal transduction domains" or "PSDs," which are protein domains that signal the cell to enter mitosis and initiate cell growth. Examples include Janus kinases, including but not limited to JAK1, JAK2, JAK3, Tyk2, Ptk-2, homologous members of the Janus kinase family from other mammalian or eukaryotic species, the β and / or γ chains of the IL-2 receptor, and other subunits from the cytokine receptor superfamily proteins, which may interact with proteins of the Janus kinase family to transduce signals, or portions, modifications, or combinations thereof. Examples of signals include phosphorylation of one or more STAT molecules, including but not limited to one or more of STAT1, STAT3, STAT5a, and / or STAT5b.

[0250] Kabat ID: As used herein, the term "Kabat numbering" is a recognized term in the art, referring to a system for numbering amino acid residues (e.g., hypervariable) that are more variable than other amino acid residues in the heavy and light chain regions of immunoglobulins (Kabat, et al., (1971) Ann. NY Acad. Sci. 190:382-93; Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the purposes of this disclosure, the location of CDRs in variable regions of antibodies follows the Kabat numbering or is simply referred to as "Kabat".

[0251] ligands As used herein, the term "ligand" refers to a molecule that specifically binds to a receptor and causes changes in the receptor, resulting in altered receptor activity or a response in cells expressing the receptor. In one embodiment, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor. As used herein, the term "ligand" includes both natural and synthetic ligands. "Ligand" also includes peptide mimics of small molecules, cytokines, and antibodies. A complex of a ligand and a receptor is referred to as a "ligand-receptor complex." A ligand may include a domain of a multiprotein or fusion protein (e.g., a domain of an antibody / ligand fusion protein).

[0252] transfer As used in this article, the term "metastasis" describes the spread of cancer cells from the primary cancer to surrounding tissues and distant organs.

[0253] Modified IL-2 orthologs: As used herein, the term "modified IL-2 orthogonal homolog" refers to an IL-2 orthogonal homolog having one or more modifications, such as PEGylation, glycosylation (N- and O-linking), acylation, or polysialylation, or by coupling with other peptide carrier molecules (whether chemical or fusion proteins), including but not limited to albumin fusion peptides containing serum albumin (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)) or Fc-fusion proteins, or having a targeting moiety (e.g., containing hhIL2 orthogonal polysaccharides). IgG-like peptide fusion proteins, targeting IL-2 orthogonal peptides, such as ScFv-hIL2 orthogonal peptide fusion protein and VHH-IL-2 orthogonal peptide fusion protein. Modified hIL2 orthogonal homologs can be prepared to enhance one or more properties, such as modulating immunogenicity; increasing water solubility, bioavailability, serum half-life and / or therapeutic half-life; and / or modulating biological activity. Certain modifications can also be used, for example, to enhance antibodies used in detection assays (e.g., epitope tags) and to facilitate protein purification.

[0254] adjust As used herein, the terms "modulation" and "modulation" refer to the ability of a test reagent to affect a reaction in a system (including biological systems or biochemical pathways), which can be positive or negative, direct or indirect. The term modulator includes agonists and antagonists.

[0255] Tumors As used herein, the term “tumor disease” refers to a disorder or symptom in a subject caused by excessive or uncontrolled (or disordered) cell replication. The term “tumor disease” refers to a disorder in a subject caused by the presence of a tumor. Tumors can be classified as: (1) benign; (2) precancerous (or “precancerous”); and (3) malignant (or “cancerous”). The term “tumor disease” includes tumor-related disorders, disorders, and symptoms that are directly or indirectly related to a tumor disease, including, for example, angiogenesis and precancerous conditions such as dysplasia or slowly progressive multiple myeloma. Examples of benign disorders caused by cell replication disorders include hypertrophic scars, such as keloidscars. N-terminusAs used in the context of polypeptide structure, “N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the polar amino and carboxyl terms of the polypeptide, respectively, while the terms “N-terminal” and “C-terminal” refer to the relative positions of the amino acid sequences of the polypeptide toward the N-terminus and C-terminus, respectively, and may include N-terminal and C-terminal residues, respectively. “Near-neighbor N-terminus” or “near-neighbor C-terminus” refers to the position of the first amino acid residue relative to the second amino acid residue, wherein the first and second amino acid residues are covalently linked to provide a continuous amino acid sequence.

[0256] Nucleic acid The terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably in this document and refer to any polymer of nucleotides of any length, namely 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, etc.

[0257] According to hIL2 number As used herein, "according to hIL2 number" means determining the position of a specific amino acid based on its position in the sequence of mature wild-type hIL2 (SEQ ID NO:4). For example, for hIL2, "R81" refers to arginine at the 81st amino acid (numbered from the N-terminus) in the sequence of mature wild-type hIL2. It should be noted that the amino acid sequences of IL2 molecules vary in number and sequence among different mammalian species. Therefore, referring to residues according to this convention is helpful in identifying the IL2 species under discussion.

[0258] According to hCD122 number As used herein, "based on hCD122 number" means determining the position of a specific amino acid based on the position of an amino acid typically present in the sequence of the mature wild-type hCD122 molecule, in one embodiment, hCD122 of SEQ ID NO. 3. For example, for human CD122, H133 refers to histidine at the 133rd amino acid (numbered from the N-terminus) in the sequence of mature wild-type hCD122.

[0259] Based on the extracellular domain numbering of hCD122As used herein, "based on hCD122 extracellular domain number" or "based on hCD122 ECD number" refers to determining the position of a specific amino acid based on the location of an amino acid typically present in the extracellular domain (ECD) sequence (SEQ ID NO. 3) of a mature wild-type hCD122 molecule. For example, for human CD122 ECD, H133 refers to histidine at the 133rd amino acid (numbered from the N-terminus) of the sequence of mature wild-type hCD122 ECD.

[0260] Operable connection In this paper, the term "operably linked" refers to a relationship between molecules, typically peptides or nucleic acids, arranged in a construct such that the functions of the individual component molecules are preserved, even though an operably linked construct may result in positive or negative modulation of the activity of the individual components within the construct. For example, an operably linked polyethylene glycol (PEG) molecule may result in a decrease in the biological activity of that protein in the construct relative to the wild-type molecule, but the two are still considered operably linked. The term "operably linked" is also used to refer to the relationship between multiple nucleic acid sequences encoding different functions when combined into a single nucleic acid molecule, for example, when introduced into cells using recombinant technology, providing nucleic acids capable of enabling transcription and / or translation of a specific nucleic acid sequence within the cell. For example, if it leads to the expression of a preprotein, a nucleic acid sequence encoding a signal sequence may be considered operably linked to DNA encoding a peptide, thereby the signal peptide promoting peptide secretion; if it affects the transcription of a sequence, a promoter or enhancer may be considered operably linked to the coding sequence; or if it is localized to promote translation, a ribosome binding site may be considered operably linked to the coding sequence. Generally, in the context of nucleic acid molecules, the term "operably linked" means that the linked nucleic acid sequences are continuous; in the context of secretory leaders or associated subdomains of molecules, they are continuous and in the reading segment. However, some genetic elements, such as enhancers, can function at a distance and do not require the sequences that interact with them to be continuous, but can still be considered operably linked.

[0261] orthogonal hCD122 As used herein, the terms “orthogonal hCD122” or “CD122 orthogonal receptor” are used interchangeably to refer to an hCD122 polypeptide variant containing amino acid substitutions that result in specific binding to the hhIL2 orthogonal homolog but not specific binding to hIL2. In one embodiment, hCD122 is hCD122 having amino acid modifications at positions 133 and 134 of SEQ ID NO:4 (naturally occurring hCD122). In some embodiments, orthogonal hCD122 includes an hCD122 molecule (SEQ ID NO:2) having amino acid substitutions H133D and Y134F.

[0262] Orthogonal receptor As used herein, the term "orthogonal receptor" refers to a variant of the receptor that includes modifications to the amino acid sequence such that the orthogonal receptor exhibits significantly reduced binding to its homologous ligand, but specifically binds to an orthogonal ligand engineered to interact with the orthogonal receptor. In some embodiments, the orthogonal receptor may include an extracellular domain that exhibits significantly reduced binding to its homologous natural ligand, while the orthogonal receptor exhibits significantly reduced binding to the ECD of one or more homologous natural receptors. In some embodiments, the orthogonal ligand exhibits an affinity for the homologous orthogonal receptor comparable to that of the natural ligand to the natural receptor, for example, having at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, and possibly higher, of the affinity for the natural cytokine receptor, for example, 2, 3, 4, 5, 10, or more times that of the natural cytokine affinity for the natural receptor. Orthogonal receptors can be named after their derived parent molecules (e.g., orthogonal hCD122) or after their homologous ligands derived from orthogonal receptors (e.g., orthogonal hIL2 receptor).

[0263] Orthogonal homologs As used herein, "orthogonal homolog" refers to the ligand component of an orthogonal ligand / receptor pair, meaning a modified polypeptide incorporated into its primary structure to provide a polypeptide variant exhibiting: (a) a significantly reduced affinity for its native homologous receptor (i.e., the native receptor of the parent polypeptide from which the orthogonal homolog is derived); and (b) specific binding to an engineered orthogonal receptor, which is a variant of the homologous receptor of the orthogonal homolog. Upon binding of the orthogonal homolog to the orthogonal receptor (expressed on the cell surface by recombinant DNA technology to incorporate a nucleic acid sequence encoding the orthogonal receptor, operatively linked to control elements to enable expression of the orthogonal receptor in recombinantly modified cells), activated orthogonal receptor initiation signal transduction, transduced through native cellular elements, provides biological activity mimicking the native response of the homolog but specific to recombinantly modified cell populations expressing the orthogonal receptor. In some embodiments of the invention, the ortholog exhibits significant selectivity for the orthogonal receptor relative to the homologous receptor, and optionally, significantly reduced potency relative to the homologous receptor. Selectivity is typically assessed by activity measurements in characterization assays of the activity induced by the ligand / receptor binding response. In some embodiments, the ortholog exhibits at least a 5-fold, or at least a 10-fold, or at least a 20-fold, or at least a 30-fold, or at least a 40-fold, or at least a 50-fold, or at least a 100-fold, or at least a 200-fold difference in affinity compared to the EC50 increase of the orthogonal receptor as measured in the same assay.

[0264] Parental polypeptidesAs used herein, the terms "parental polypeptide" or "parental protein" are used interchangeably to indicate the source of a second polypeptide (e.g., a derivative or variant) that is modified relative to a first "parental" polypeptide. In some cases, the parental polypeptide is the wild-type or naturally occurring form of the protein. In other cases, the parental polypeptide may be modified to form a further modified naturally occurring protein. The term "parental polypeptide" may refer to the polypeptide itself or a composition containing the parental polypeptide (e.g., a glycosylated or PEGylated form and / or a fusion protein containing the parental polypeptide).

[0265] Partial agonists As used herein, the term "partial agonist" refers to a molecule that specifically binds to and activates a given receptor, but only partially activates the receptor as opposed to a full agonist. Partial agonists can exhibit both agonist and antagonist effects. For example, when a full agonist and a partial agonist are present, the partial agonist acts as a competitive antagonist, competing with the full agonist for receptor binding, resulting in a net reduction in receptor activation compared to the receptor's contact with the full agonist in its absence. When the amount of endogenous ligands present is insufficient, partial agonists in the subject can be used to activate the receptor to provide the desired submaximal response, or when the amount of endogenous ligands is excessive, they can reduce overstimulation of the receptor. The maximal response (E) produced by partial agonists... 最大 This is referred to as its intrinsic activity, which can be expressed as a percentage when a full agonist produces a 100% response. When evaluated at similar concentrations in a given test system, a partial agonist may have greater than 10% but less than 100%, or greater than 20% but less than 100%, or greater than 30% but less than 100%, or greater than 40% but less than 100%, or greater than 50% but less than 100%, or greater than 60% but less than 100%, or greater than 70% but less than 100%, or greater than 80% but less than 100%, or greater than 90% but less than 100%.

[0266] PEG-hIL2 orthogonal homologs:As used herein, the term "PEG-IL2 ortholog" refers to an hIL2 ortholog covalently bound to at least one polyethylene glycol (PEG) molecule, wherein at least one PEG molecule is covalently attached to at least one amino acid residue of the IL-2 ortholog. PEGylated peptides may also be referred to as mono-PEGylated, di-PEGylated, tri-PEGylated (etc.), to indicate PEG-hIL2 orthologs comprising one, two, three (or more) PEG moieties attached to an IL-2 ortholog, respectively. In some embodiments, PEG may be directly covalently attached to the IL-2 ortholog (e.g., via a lysine side chain, a thiol group of cysteine, or an N-terminal amine) or optionally a linker may be used between the PEG and the IL-2 ortholog. In some embodiments, the PEG-hIL2 ortholog comprises more than one PEG molecule, each attached to a different amino acid residue. In some embodiments, the PEG-hIL2 ortholog is derived from SEQ ID NO:4.

[0267] polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to a polymer of amino acids of any length, which may include genetically encoded or non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified polypeptide backbone. These terms include fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences; fusion proteins having heterologous and homologous leader sequences; fusion proteins with or without N-terminal methionine residues; fusion proteins having immunotagged proteins; fusion proteins of immunologically active proteins (e.g., fragments of antigenic diphtheria or tetanus toxin), etc.

[0268] prevent The terms "prevention" and "avoidance" as used in this article refer to actions initiated before the onset of a disease, disorder, condition, or its symptoms, thereby temporarily or permanently preventing, mitigating, suppressing, or reducing the risk of the subject developing a certain disease, disorder, condition, or similar illness (e.g., determined by the absence of clinical symptoms), or delaying its onset. This generally applies when the subject is susceptible to a particular disease, disorder, or condition due to genetic, experiential, or environmental factors. In some cases, the terms "prevention" and "avoidance" are also used to refer to slowing the progression of a disease, disorder, or condition from its current state to a more harmful state.

[0269] receptorAs used herein, the term "receptor" refers to a polypeptide having a ligand-binding domain that binds to a ligand, the binding of which results in a change in at least one biological property of the polypeptide. In some embodiments, the receptor is a cell surface receptor comprising an extracellular domain (ECD) and a membrane-associated domain for anchoring the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide comprising an intracellular domain (ICD) and an extracellular domain (ECD) linked by a transmembrane spanning domain commonly referred to as a transmembrane domain (TM). Homologous ligand binding to the receptor results in a conformational change in the receptor, thereby producing a measurable biological effect. In some cases, if the receptor is a transmembrane polypeptide comprising an ECD, a TM, and an ICD, ligand binding to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to ligand binding to the ECD. In some embodiments, the receptor is a component of a multi-component complex to facilitate intracellular signal transduction. For example, ligands can bind to cell surface receptors that are not associated with any intracellular signal transduction on their own, but which, upon binding, promote the formation of heteropolymer (including heterodimers, heterotrimers, etc.) or homopolymer (e.g., homodimers, homotrimers, homotetramers, etc.) complexes, leading to measurable biological effects in the cell, such as activation of intracellular signal transduction cascades (e.g., the Jak / STAT pathway). For example, a ligand can bind to a cell surface molecule that is not associated with any intracellular signal transduction on its own, but, upon ligand binding, promotes the formation of heterodimers (including heterodimers (e.g., the intermediate-affinity hCD122 / CD132 hIL2 receptor), heterotrimers (e.g., the high-affinity CD25 / CD122 / CD132 hIL2 receptor), or homodimers (homodemers, holotrimers, holotetramers) complexes, leading to activation of intracellular signal transduction cascades (e.g., the Jak / STAT pathway). In some embodiments, the receptor is a transmembrane single-chain polypeptide comprising ECD, TM, and ICD domains, wherein the ECD, TM, and ICD domains are derived from the same or different naturally occurring receptor variants or their synthetic functional equivalents.

[0270] ReorganizedAs used herein, the term “recombinant” is used as an adjective to refer to the method of modifying polypeptides, nucleic acids, or cells using recombinant DNA technology. “Recombinant protein” refers to a protein produced using recombinant DNA technology, usually abbreviated with a lowercase “r” before the protein name to indicate the method of production (e.g., recombinant human growth hormone is usually abbreviated as “rhGH”). Similarly, if cells are modified by incorporating (e.g., transfection, transduction, infection) exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, granules, etc.) using recombinant DNA technology, the cells are called “recombinant cells.” The techniques and protocols used for recombinant DNA technology are well-known in the art and can be found, for example, in Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY), and other standard molecular biology laboratory manuals.

[0271] reaction The term "response" refers to, for example, the response of a cell, tissue, organ, or organism, including quantitative or qualitative changes in assessable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, rate of energy consumption, differentiation level, or state) that are associated with activation, stimulation, or treatment, or with exposure to exogenous reagents or internal mechanisms (such as genetic programming). In some cases, the terms "activation," "stimulation," etc., refer to cell activation regulated by internal mechanisms and external or environmental factors; while the terms "inhibition," "downregulation," etc., refer to the opposite effect. A "response" can be assessed in vitro, for example, using assay systems, surface plasmon resonance, enzyme activity, mass spectrometry, amino acid, or protein sequencing techniques. A "response" can be assessed quantitatively in vivo, qualitatively by assessing objective physiological parameters such as body temperature, weight, tumor volume, blood pressure, X-ray, or other imaging techniques, or by changes in reported subjective feelings (happiness, depression, anxiety, or pain). In some implementations, the proliferation level of CD3-activated primary human T cells can be assessed in a bioluminescent assay that produces a luminescent signal proportional to the amount of ATP present, which in turn is proportional to the amount of cells present in the culture, as described by Crouch et al. (1993) J. Immunol. Methods 160:81–8, or by using commercially available assays, such as 2.0 Cell viability assay or The 3D cell viability kit, commercially available from Promega Corporation, Madison WI 53711, catalog numbers G9241 and G9681, was used substantially according to the manufacturer's instructions. In some embodiments, the activation level of T cells reacting with the test reagent can be determined by the flow cytometry method described above, such as by determining the phosphorylation level of STAT (e.g., STAT1, STAT3, STAT5), according to methods well known in the art. For example, STAT5 phosphorylation can be measured using flow cytometry techniques, as described above by Horta et al., Garcia et al., or by using a commercially available kit, such as the phosphate-STAT5 (Tyr694) kit (commercially available from Perkin-Elmer, Waltham, MA, part number 64AT5PEG), substantially according to the manufacturer's instructions.

[0272] Significantly reduced binding: The term "exhibiting significantly reduced binding" refers to a variant of a first molecule (e.g., a ligand) exhibiting significantly reduced affinity for a second molecule (e.g., a receptor) relative to its parental form. As used herein, the term "exhibiting significantly reduced binding" refers to the binding affinity of an orthogonal ligand to an orthogonal receptor relative to the binding of the orthogonal ligand to its native form. An orthogonal ligand exhibits significantly reduced binding relative to the native form of the receptor if the binding of the orthogonal ligand to the native form of the receptor is less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5% of the native form of the ligand. Similarly, if the binding of the native form of the ligand to the orthogonal form of the receptor is less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5% of the native receptor, then the orthogonal receptor exhibits a significantly reduced binding relative to the native form of the ligand.

[0273] One or more small molecules The term "small molecule" refers to chemical compounds (usually pharmaceutically active compounds) with a molecular weight of less than about 10 kDa, less than about 2 kDa, or less than about 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, and synthetic molecules. The term "small molecule" is a term familiar to those skilled in the pharmaceutical industry and is commonly used to distinguish between organic chemical compounds and biological agents.

[0274] Specific binding:As used herein, the term "specific binding" refers to the degree of selectivity or affinity of one molecule to another. In the context of binding pairs (such as ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pairs), a molecule is said to specifically bind to a second molecule when the first molecule of the binding pair does not bind to other components present in the sample in a significant amount. A molecule is said to specifically bind to a second molecule when the affinity of the first molecule to the second molecule is at least 2, 5, 10, 20, or 100 times greater than the affinity of the first molecule to other components present in the sample. In a specific embodiment where the first molecule in the binding pair is an antibody, the equilibrium dissociation constant between the antibody and the second molecule of the binding pair is greater than about 10. 6 M, or greater than approximately 10 8 M, or greater than approximately 10 10 M, or greater than approximately 10 11 M, or greater than approximately 10 10 M, or greater than approximately 10 12 M, for example, as determined by Scatchard analysis, indicates that the antibody specifically binds to the second molecule (e.g., protein, antigen, ligand, or receptor) in the binding pair (Munsen, et al. 1980 Analyt. Biochem. 107:220-239). In one embodiment, the ligand is an hIL2 orthogonal homolog and the receptor comprises an orthogonal hCD122 ECD if the equilibrium dissociation constant of the hIL2 orthogonal homolog / orthogonal hCD122 ECD is greater than about 10. 5 M, or greater than approximately 10 6 M, or greater than approximately 10 7 M, or greater than approximately 10 8 M, or greater than approximately 10 9 M, or greater than approximately 10 10 M, or greater than approximately 10 11M, then the hIL2 orthogonal homolog specifically binds. Specific binding can be assessed using techniques known in the art, including but not limited to competitive ELISA, radioligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting procedures, and competitive binding assays); non-radioligand 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., multi-well plate assays, bead-on-bead ligand binding assays, column-on-column ligand binding assays, and filtration assays) and surface plasmon resonance assays (see, for example, Drescher et al. (2009), Methods Mol Biol 493:323-343, using commercially available instruments such as Biacore 8+, Biacore S200, and Biacore T200 (GE Healthcare Life Sciences, 100 Results Way, Marlborough, MA 01752)).

[0275] object The terms “recipient,” “individual,” “object,” and “patient” are used interchangeably herein and refer to any mammalian object requiring diagnosis, treatment, or therapy, particularly a human. For therapeutic purposes, “mammal” means any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sporting, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0276] Suffering from As used herein, the term "having" refers to a physician's judgment, based on existing information recognized in the field for identifying a disease, disorder, or condition (including but not limited to X-rays, CT scans, routine laboratory diagnostic tests (e.g., blood cell counts), genomic data, protein expression data, and immunohistochemistry), that a person needs or will benefit from treatment. The term "having" is often used in conjunction with a specific disease state; for example, "having a neoplastic disease" means a patient has been diagnosed as carrying a tumor.

[0277] Basically pure As used herein, the term "substantially pure" means that the components of the composition constitute more than about 50%, or more than about 60%, or more than about 70%, or more than about 80%, or more than about 90%, or more than about 95% of the total content of the composition. "Substantially pure" means that the proteins constitute more than about 50%, or more than about 60%, or more than about 70%, or more than about 80%, or more than about 90%, or more than about 95% of the total content of the composition.

[0278] T cells: As used herein, the term "T-cell" or "T cell" in its conventional sense refers to a lymphocyte differentiated in the thymus that possesses specific cell surface antigen receptors, including some that control the initiation or suppression of cell-mediated immunity and humoral immunity, as well as the lysis of antigen-carrying cells. In some embodiments, T cells include, but are not limited to, naïve CD8 cells. + T cells, cytotoxic CD8 + T cells, immature CD4 + T cells, helper T cells, such as T cells H 1. T H 2. T H 9. T H 11. T H 22. T FH Regulatory T cells, such as T cells R 1. Treg cells, induced Treg cells; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TILs), and engineered variants of these T cells, including but not limited to CAR-T cells, recombinant modified TILs, and TCR-engineered cells.

[0279] End / Terminal: As used in the context of polypeptide structure, “N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the polar amino and carboxyl terms of the polypeptide, respectively. The terms “N-terminal” and “C-terminal” refer to the relative positions of the amino acid residues of the polypeptide toward the N-terminus and C-terminus, respectively, and may include N-terminal and C-terminal residues, respectively. “Nearest N-terminus” refers to the position of the first amino acid residue relative to the second amino acid residue in a continuous polypeptide sequence, with the first amino acid being closer to the N-terminus of the polypeptide. “Nearest C-terminus” refers to the position of the first amino acid residue relative to the second amino acid residue in a continuous polypeptide sequence, with the first amino acid being closer to the C-terminus of the polypeptide.

[0280] Therapeutic effective doseThe term "therapeutic effective dose" as used herein refers to the amount of an agent administered to a subject, alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a series of doses, to produce any detectable, positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition. Therapeutic effective doses can be determined by measuring the associated physiological effects and can be adjusted in conjunction with the dosing regimen and diagnostic analysis of the subject's condition. The assessment parameters used to determine the therapeutic effective dose of an agent are determined by a physician using recognized diagnostic criteria, including but not limited to indicators such as age, weight, sex, general health status, ECOG score, observable physiological parameters, blood concentrations, blood pressure, electrocardiogram, computed tomography, X-ray, etc. Alternatively, other parameters typically assessed in clinical settings can be monitored to determine whether the subject has received a therapeutically effective dose of the agent, such as normalization of body temperature, heart rate, blood chemistry, blood pressure, cholesterol levels, or any symptoms, aspects, or characteristics of the disease, disorder, or condition; reductions in biomarkers (such as inflammatory cytokines, IFN-γ, granzymes, etc.); reductions in serum tumor markers; improvements in the Responsive Response Standard for Solid Tumors (RECIST); improvements in the Immune Related Response Standard (irRC); prolonged survival; prolonged progression-free survival; prolonged time to progression; prolonged time to treatment failure; prolonged event-free survival; prolonged time to next treatment; improved objective response rate; improved duration of response; reduced tumor burden; complete remission; partial remission; stable disease, etc. Clinicians in this field assess improvements in the subject's response to the administered agent based on these parameters. As used herein, the terms “complete response (CR),” “partial response (PR),” “stable disease (SD),” and “progressive disease (PD)” relating to target lesions, and the terms “complete response (CR),” “incomplete response / stable disease (SD),” and “progressive disease (PD)” relating to non-target lesions, should be understood as defined in the RECIST criteria. The terms “immune-related complete response (irCR),” “immune-related partial response (irPR),” “immune-related progressive disease (irPD),” and “immune-related stable disease (irSD)” as used herein are as defined according to the immune-related response criteria (irRC).As used herein, the term “immune-related response criteria (irRC)” refers to a system used to assess the response to immunotherapy, as described in Wolchok et al. (2009), Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-Related Response Criteria, Clinical Cancer Research 15(23):7412-7420. During the course of treatment, the therapeutically effective dose may be adjusted based on the dosing regimen and / or assessment of the patient’s condition and changes in the aforementioned factors. In one embodiment, the therapeutically effective dose is the amount of reagent that, when used alone or in combination with another reagent, does not cause irreversible serious adverse events during administration to mammalian subjects.

[0281] Transmembrane domain The term "transmembrane domain" or "TM" refers to a domain of a transmembrane polypeptide (e.g., a transmembrane receptor) that, when associated with the cell membrane, is embedded within the cell membrane and linked by peptidyl linkage to both the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide. The transmembrane domain can be homologous (naturally associated) or heterologous (not naturally associated) to one or both of the extracellular and / or intracellular domains. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with the ECD domain of a homologous receptor of a derived orthogonal receptor. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with the ICD domain of a homologous receptor of a derived orthogonal receptor. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with a proliferation signal transduction domain. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with a different protein. Alternatively, the transmembrane domain of an orthogonal receptor can be an artificial amino acid sequence that crosses the plasma membrane. In some implementations, the transmembrane domain of the orthogonal receptor is the transmembrane domain that is typically associated with the ICD of the homologous receptor from which the orthogonal receptor is derived.

[0282] treatThe terms "treatment," "therapy," "treatment," etc., refer to an action process initiated for a subject in response to a diagnosis of a disease, disorder, or condition or its symptoms (e.g., exposing the subject to hIL-2 orthogolog, hoRb T cells, hoCAR-T cells, or a pharmaceutical composition containing them, alone or in combination with a supplement) to temporarily or permanently eliminate, reduce, suppress, alleviate, or improve at least one of the following: (a) the underlying cause of the disease, disorder, or condition troubling the subject; and / or (b) at least one symptom associated with such disease, disorder, or condition. In some embodiments, treatment includes an action process taken on a subject suffering from a disease, wherein the action process results in suppression of the subject's disease (e.g., preventing the development of the disease, disorder, or condition or improving one or more symptoms associated therewith).

[0283] Treg cells or regulatory T cells As used in this article, "regulatory T cells" or "Treg cells" refers to CD4+ cells. + T cells are types of T cells that can suppress the responses of other T cells, including but not limited to effector T cells (Teff). Treg cells are characterized by the expression of CD4, the α subunit of the IL-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004). "Conventional CD4 + "T cells" refers to CD4 cells other than regulatory T cells. + T cells.

[0284] variants The terms "variant," "protein variant," "variant protein," or "variant polypeptide" are used interchangeably herein to refer to a polypeptide that differs from a parent polypeptide due to at least one amino acid modification, substitution, or deletion. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide or a modified form of a WT polypeptide. The term "variant polypeptide" may refer to the polypeptide itself, a composition comprising the polypeptide, or the nucleic acid sequence encoding it. In some embodiments, the variant polypeptide comprises, relative to the parent polypeptide, about 1 to about 10, or about 1 to about 8, or about 1 to about 7, or about 1 to about 5, or about 1 to about 4, or about 1 to about 3, or 1 to 2 amino acid modifications, substitutions, or deletions, or a single amino acid modification, substitution, or deletion. The variant may have at least about 99% identity with the parent polypeptide from which it is derived, or at least about 98% identity, or at least about 97% identity, or at least about 95% identity, or at least about 90% identity.

[0285] wild type:"Wild-type," "WT," or "natural" in this article refers to an amino acid or nucleotide sequence that exists in nature, including allelic variations. WT proteins, peptides, antibodies, immunoglobulins, IgG, etc., have amino acid or nucleotide sequences that are not artificially modified.

[0286] hIL2 ortholog

[0287] Naming conventions:

[0288] This invention provides multiple polypeptide ligands for hIL2 receptor polypeptide variants. The following nomenclature is used herein to indicate substitutions, deletions, or insertions. Residues herein may be designated by a one-letter or three-letter amino acid code of a naturally occurring amino acid found in the wild-type molecule, followed by the position of the hIL2 amino acid in the mature hIL2 molecule; for example, “Cys125” or “C125” refers to the cysteine ​​residue at position 125 of the wild-type hIL2 molecule. For hIL2 orthologs, substitutions are specified herein by a one-letter amino acid code followed by the position of the hIL2 amino acid, followed by the one-letter amino acid code of the substitution. The hIL2 orthologs of this invention are numbered according to the hIL2 number. For example, an hIL2 ortholog with the “K35A” modification refers to the lysine (K) residue at position 35 of the wild-type hIL2 sequence being replaced by an alanine (A) residue. Deletions of amino acid residues are referred to as “des”, followed by the amino acid residue and its position in SEQ ID NO:4. For example, the terms “des-Ala1” or “ΔA1” refer to the deletion of alanine at position 1 of the polypeptide in the wild-type hIL2 sequence. Similarly, for amino acid substitutions in orthogonal hCD122, this document specifies amino acid substitutions as a one-letter amino acid code of the naturally occurring amino acid, followed by its position number in the wild-type hIL2 sequence, followed by the one-letter amino acid code of the substituted amino acid at that position. hCD122 modifications to hCD122 incorporating orthogonal receptors are based on hCD122 numbering. For example, an hCD122 orthogonal receptor having a phenylalanine substitution for a tyrosine residue at position 134 is abbreviated as “Y134F”. The abbreviation hoRb is used synonymously with hoCD122, referring to human CD122 containing the orthogonal hCD122 receptor. Similarly, references to “hoRb cells” (e.g., hoRb T cells or hoRbNKL cells) refer to cells expressing the orthogonal hCD122 receptor. As used in this article, SQVLKA refers to the hIL2 ortholog, which contains amino acid substitutions: E15S, H16Q, L19V, D20L, Q22K, and M23A.

[0289] hIL2 ortholog

[0290] In some embodiments, the present invention provides hIL2 orthogonal homologs and methods of using the same, wherein the hIL2 orthogonal homologs are homologous ligands of orthogonal receptors comprising modified hCD122 ECDs. In some embodiments, the term hIL2 orthogonal homolog refers to an hIL2 variant that is a ligand of a receptor comprising the extracellular domain of human orthogonal hCD122 containing amino acid substitutions at positions H133 and / or Y134. In some embodiments, hIL2 orthogonal homologs refer to ligands of orthogonal receptors comprising the extracellular domain of human hCD122 containing amino acid substitutions at positions H133 and / or Y134. In some embodiments, hIL2 orthogonal homologs refer to ligands of transmembrane receptors comprising the extracellular domain of human CD122 containing amino acid substitutions at positions H133 and / or Y134, wherein the ICD contains one or more STAT3 binding motifs. In some embodiments, hIL2 orthogonal homologs refer to ligands of orthogonal human CD122 containing amino acid substitutions at positions H133 and Y134. In some embodiments, hIL2 orthogonal homologs refer to ligands of orthogonal hCD122 containing amino acid substitutions at H133D and Y134F.

[0291] In various embodiments, the compositions and methods of the present invention include the use of an hIL2 ortholog polypeptide having at least 80%, 85%, 90%, 95%, 97%, or 99% identity with a polypeptide of formula #1.

[0292]

[0293] in:

[0294] AA1 is A (wild type) or absent;

[0295] AA2 is P (wild type) or absent;

[0296] AA3 is T (wild type), C, A, G, Q, E, N, D, R, K, P or missing;

[0297] AA4 is S (wild type) or absent;

[0298] AA5 is S (wild type) or absent;

[0299] AA6 is either S (wild type) or absent;

[0300] AA7 is T (wild type) or absent;

[0301] AA8 is K (wild type) or absent;

[0302] AA9 is K (wild type) or absent;

[0303] AA13 is Q (wild type), W, or absent;

[0304] AA14 is L (wild type), M, W, or absent;

[0305] AA15 is E (wild-type), K, D, T, A, S, Q, H or absent;

[0306] AA16 can be H (wild type), N, Q, or absent;

[0307] AA18 is L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T;

[0308] AA19 is L (wild type), A, V, I, or absent;

[0309] AA20 is D (wild-type), T, SML, or absent;

[0310] AA22 is Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, F or missing;

[0311] AA23 is M (wild type), A, W, H, Y, F, Q, S, V, L, T or absent;

[0312] AA27 is G (wild type), K, S, or absent;

[0313] AA38 is R (wild type), W, or G;

[0314] AA39 is M (wild type), L, or V;

[0315] AA42 is either F (wild type) or K;

[0316] AA51 is T (wild type), I, or absent.

[0317] AA55 is either H (wild type) or Y;

[0318] AA74 is Q (wild type), N, H, S;

[0319] AA80 is L (wild type), F, or V;

[0320] AA81 is R (wild-type), I, D, Y, T, or absent.

[0321] AA85 is either L (wild type) or V;

[0322] AA86 is either I (wild type) or V;

[0323] AA88 is N (wild type), E or Q, or missing;

[0324] AA89 is either I (wild type) or V;

[0325] AA91 is V (wild type), R, or K;

[0326] AA92 is either I (wild type) or F;

[0327] AA97 is either K (wild type) or Q;

[0328] AA104 is either M (wild type) or A;

[0329] AA109 is a non-natural amino acid, either D (wild-type), C, or with an activated side chain;

[0330] AA113 is either T (wild type) or N;

[0331] AA125 is C (wild type), A, or S;

[0332] AA126 is Q (wild type) or H, M, K, C, D, E, G, I, R, S or T;

[0333] and / or

[0334] AA130 is S (wild type), T, or R.

[0335] According to the embodiments, the series of hIL2 orthogonal homologs of Formula 1 (mature protein sequences expressed with an additional N-terminal signal peptide of sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO. 421) were prepared as shown in Table 2 below, and their ability to selectively activate NKL cells relative to NKL cells was evaluated. These homologs were engineered to express orthogonal CD122 of SEQ ID NO: 2, expressing T cells containing a substituted orthogonal IL2 receptor.

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365] The activity of the aforementioned hIL2 ortholog was assessed for its ability to activate NKL and hoRb NKL cells, largely based on the teachings of this paper in the following dilutions. The results of these experiments are shown in the attached figures. Figure 1 and 2 And provided in Table 3 below:

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380] The activity of the IL2 ortholog in CD4+ human T cell clone 3F8 cells was evaluated, and is described in more detail in Example 8. The data obtained from these experiments are shown in Table 4 below.

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396] As the data above show, the hIL2 ortholog of Formula 1 selectively activates hoCD122 human T cells relative to human T cells (NKL cells) that do not express orthogonal receptors.

[0397] Conservative amino acid substitution

[0398] In some embodiments, the hIL2 ortholog of Formula 1 may optionally contain one or more conserved amino acid substitutions. Such conserved substitutions include those described by Dayhoff in *The Atlas of Protein Sequence and Structure 5* (1978) and by Argos in *EMBO J., 8:779-785* (1989). Conserved substitutions are typically made according to those shown in Table 4 below:

[0399]

[0400]

[0401] Substantial alterations in functional or immunological properties can be made by selecting less conserved amino acid substitutions than those shown in Table 4. For example, substitutions that more significantly affect the polypeptide backbone structure or disrupt secondary or tertiary elements can be made, including replacing amino acids with small, uncharged side chains (e.g., glycine) with large, highly charged side chains (asparagine). In particular, substitutions of hIL2 residues that participate in the interaction with one or more of CD25, CD122, and / or CD123 can be observed in the hIL2 crystal structure associated with the receptor described in Wang et al. (2005) Science 310:1159-1163. The modifications to the primary structure provided above may optionally include further modifications, including but not limited to substitutions of: N30E; K32E; N33D; P34G; T37I; M39Q; F42Y; F44Y; P47G; T51I; E52K; L53N; Q57E; M104A (see U.S. Patent No. 5,206,344).

[0402] Cys125:

[0403] In some embodiments, the present invention provides an hIL2 orthogonal homolog of Formula 1, comprising modifications that promote recombinant expression in bacterial cells by eliminating an unpaired cysteine ​​residue at position 125 through substitution of C125A or C125S. In some embodiments, the hIL2 orthogonal homolog of the present invention comprises one of the following amino acid modification groups:

[0404] [E15S-H16Q-L19V-D20L-Q22K-M23A-C125S];

[0405] [E15S-H16Q-L19V-D20L-Q22K-C125S];

[0406] [E15S-H16Q-L19V-D20L-M23A-C125S];

[0407] [E15S-H16Q-L19V-D20L-C125S];

[0408] [E15S-H16Q-L19V-D20L-Q22K-M23A-C125A];

[0409] [E15S-H16Q-L19V-D20L-M23A-C125A];

[0410] [E15S-H16Q-L19V-D20L-Q22K-C125A];

[0411] [E15S-H16Q-L19V-D20L-C125A];

[0412] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125S];

[0413] [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125S];

[0414] [desAla1-E15S-H16Q-L19V-D20L-C125S];

[0415] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125A];

[0416] [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125A];

[0417] [desAla1-E15S-H16Q-L19V-D20L-C125A];

[0418] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A];

[0419] [desAla1-E15S-H16Q-L19V-D20L-M23A];

[0420] [desAla1-E15S-H16Q-L19V-D20L-Q22K]; or

[0421] [desAla1-E15S-H16Q-L19V-D20L].

[0422] Mutations that enhance hCD122 affinity

[0423] In some embodiments, the hIL-2 ortholog of Formula 1 contains one or more mutations at positions in the hIL-2 sequence that either contact hCD122 or alter the orientation of other positions contacting hCD122, thereby modulating the binding affinity of the hIL-2 ortholog to hCD122. hIL-2 residues identified as involved in hIL2 binding to hCD122 include L12, Q13, H16, L19, D20, M23, R81, D84, S87, N88, V91, I92, and E95. In some embodiments, the hIL-2 ortholog comprises one or more amino acid substitutions: Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I92F, or combinations thereof. In some embodiments, the hIL2 orthogonal homolog comprises one or more amino acid substitutions: L80F, R81D, L85V, I86V, and I92F. In some embodiments, the hIL2 orthogonal homolog comprises one or more amino acid substitutions: N74Q, L80F, R81D, L85V, I86V, I89V, and I92F. In some embodiments, the hIL2 orthogonal homolog comprises one or more amino acid substitutions: Q74N, L80V, R81T, L85V, I86V, and I92F. In some embodiments, the hIL2 orthogonal homolog comprises one or more amino acid substitutions: Q74H, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the hIL2 orthogonal homolog comprises one or more amino acid substitutions: Q74S, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the hIL2 orthogonal homolog comprises one or more amino acid substitutions: Q74N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the hIL2 orthogonal homolog comprises one or more amino acid substitutions: Q74S, R81T, L85V, and I92F. In some embodiments, the hIL2 orthogonal homolog includes the mutant group [L80F-R81D-L85V-I86V-I92F], which has been identified as increasing the affinity of hIL2 for hCD122. In some embodiments, the present invention provides an hIL2 orthogonal homolog, which is an hIL2 polypeptide comprising one of the following groups of amino acid modifications:

[0424] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-L85V-I86V-I92F];

[0425] [E15S-H16Q-L19V-D20L-Q22K-L80F-R81D-L85V-I86V-I92F];

[0426] [E15S-H16Q-L19V-D20L-Q22K-M23A L80F-R81D-L85V-I86V-I92F];

[0427] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-L85V-I86V-I92F-Q126H];

[0428] [E15S-H16Q-L19V-D20L-Q22K-L80F-R81D-L85V-I86V-I92F-Q126H];

[0429] [E15S-H16Q-L19V-D20L-Q22K-M23A-L80F-R81D-L85V-I86V-I92F-

[0430] Q126H];

[0431] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-L85V-I86V-I92F-Q126M];

[0432] [E15S-H16Q-L19V-D20L-Q22K-L80F-R81D-L85V-I86V-I92F-Q126M]

[0433] ;or

[0434] [E15S-H16Q-L19V-D20L-Q22K-M23A-L80F-R81D-L85V-I86V-I92F-

[0435] Q126M].

[0436] In some embodiments, the hIL-2 orthogonal homolog of Formula 1 comprises a substituted L85V identified as increasing the affinity of hIL2 for hCD122. In some embodiments, the present invention provides an hIL2 orthogonal homolog that is an hIL2 polypeptide comprising one of the following groups of amino acid modifications:

[0437] [E15S-H16Q-L19V-D20L-M23A-L85V];

[0438] [E15S-H16Q-L19V-D20L-Q22K-M23A-L85V];

[0439] [E15S-H16Q-L19V-D20L-M23A-L85V];

[0440] [E15S-H16Q-L19V-D20L-Q22K-M23A-L85V];

[0441] [E15S-H16Q-L19V-D20L-M23A-L85V-Q126H];

[0442] [E15S-H16Q-L19V-D20L-Q22K-M23A-L85V-Q126H];

[0443] [E15S-H16Q-L19V-D20L-M23A-L85V-Q126M]; or

[0444] [E15S-H16Q-L19V-D20L-Q22K-M23A-L85V-Q126M].

[0445] Modifications that adjust CD25 affinity

[0446] In some embodiments, the hIL-2 orthogonal homolog of Formula 1 contains one or more mutations at the position of the hIL-2 orthogonal homolog that modulates the binding affinity to hCD25. Based on the crystal structure of IL2 associated with the IL2 receptor (Wang et al. (2005) Science 310:1159), as part of the trimer IL2 receptor complex, the mutated region of hIL2 is adjacent to hCD25. In some embodiments, the hIL-2 ortholog of Formula 1 includes modifications located at one or more positions selected from the group consisting of: S4, K8, K9, T10, Q11, Q13, N26, N29, N30, N30, Y31, K35, T37, R38, T41, F42, K43, F44, Y45, M46, K48, K49, K54, E61, E62, K64, P65, E67G, E68, V69, N71, L72, Q74, S75, K76, H79, I89, N90, I92, S99, T101, F103, Y107, I114, I128, and T133. Examples of amino acid substitutions that can be included in hIL2 ortholog sequences include one or more substitutions selected from the group consisting of: S4P, K8R, K9T, T10A, Q11R, Q13R, N26D, N29S, N30S, N30D, N30T, Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K49R, K49E, K54R, E61D, K64R, E67G, E68D, V69A, N71T, N71A, N71R, A73V, Q74P S75P, K76E, K76R, H79R, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, T133A and T133N.In some embodiments, the hIL2 orthologs of the present invention comprise one or more point mutations: S4P, K8R, K9T, T10A, Q11R, Q13R, N26D, N29S, N30S, N30D, N30T, Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K49R, K49E, K54R, E61D, K64R, E67G, E68D, V69A, N71T, N71A, N71R, A73V, Q74P, S75P, K76E, K76R, H79R, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, T133A and T133N (Wittrup, aka ibid.), R38A, F41A and / or F42A (Suave, et al. (1991) PNAS (USA) 88:4636-4640); P65L (Chen et al. Cell Death and Disease (2018) 9:989).

[0447] F42A / G / S / T / Q / E / N / R / K, Y45A / G / S / T / Q / E / N / D / R / K and / or

[0448] L72G / A / S / T / Q / E / N / D / R / K (Ast, U.S. Patent Application Publication 2012 / 0244112A1, published on September 27, 2012; U.S. Patent No. 9,266,938B2, granted on February 23, 2016).

[0449] In addition to point mutations, the combination of the above modifications can be used to regulate the binding of the hIL-2 ortholog of Formula 1 to CD25. In some embodiments, the hIL2 orthologs of the present invention comprise one or more of the following amino acid substitution groups: [R38A-F42A-Y45A-E62A](Carmenate, et al. (2013) J Immunol 190:6230-6238); [F42A-Y45A-L72G](Roche RG7461 (RO6874281); [V69A,Q74P]; [V69A,Q74,T101A]; [V69A,Q74P,I128T]; [N30D,V69A,Q74P,F103S]; [K49E,V69A,A73V,K76E]; [V69A,Q74P,T101A,T133N];

[0450] [N30S,V69A,Q74P,I128A];[N30S,V69A,Q74P,I128T];[K9T,Q11R,K35R,V69A ,Q74P],[A1T,M46L,K49R,E61D,V69A,H79R];[K48E,E68D,N71T,N90H,F103S,I 114V]; [S4PT10A, Q11R, V69A, Q74P, T133A]; [N30S, Y31H, K35R, K48E, V69A, Q7 4P,I92T];[N30S,E68D,V69A,N71A,Q74P,S75P,K76R,N90H];[N30S,Y31C,T37A [V69A,A73V,Q74P,H79R,I128T],[N26D,N29S,N30S,K54R,E67G,V69A,Q74P,I92T];[K8R,Q13R,N26D,N30T,K35R,T37R,V69A,Q74P,I92T] and [N29S,Y31H,K35R,T37A,K48E,V69A,N71R,Q74P,I39V] (Wittrup et al., U.S. Patent No. 7,569,215, granted August 4, 2009); and / or [T41P-T51P] (Chang et al. (1995) Molecular Pharmacology 47:206-211). In some embodiments, the present invention provides an hIL-2 orthogonal homolog of Formula 1, which comprises one of the following groups of amino acid modifications:

[0451] [E15S-H16Q-L19V-D20L-M23A-R38A-F42A-Y45A-E62A];

[0452] [E15S-H16Q-L19V-D20L-M23A-R38A-F42A-Y45A-E62A];

[0453] [E15S-H16Q-L19V-D20L-Q22K-M23A-R38A-F42A-Y45A-E62A];

[0454] [E15S-H16Q-L19V-D20L-M23A-R38A-F42A-Y45A-E62A-Q126H];

[0455] [E15S-H16Q-L19V-D20L-M23A-R38A-F42A-Y45A-E62A-Q126H];

[0456] [E15S-H16Q-L19V-D20L-Q22K-M23A-R38A-F42A-Y45A-E62A-Q126

[0457] H];

[0458] [E15S-H16Q-L19V-D20L-M23A-V69A];

[0459] [E15S-H16Q-L19V-D20L-Q22K-M23A-V69A];

[0460] [E15S-H16Q-L19V-D20L-M23A-Q74P];

[0461] [E15S-H16Q-L19V-D20L-Q22K-M23A-Q74P];

[0462] [E15S-H16Q-L19V-D20L-M23A-R38A-F42A-Y45A-E62A-Q126M]; or

[0463] [E15S-H16Q-L19V-D20L-M23A-R38A-F42A-Y45A-E62A-Q126M].

[0464] Modification to adjust CD132 affinity

[0465] In some embodiments of the present invention, the hIL-2 orthogonal homolog of the present invention comprises one or more mutations that regulate the binding of the hIL-2 orthogonal homolog to CD132. Exemplary hIL-2 orthogonal homologs contain one or more mutations at positions in the hIL-2 sequence that either contact CD132 or alter the orientation of other positions contacting hCD122, resulting in altered binding to CD132. hIL-2 residues identified as regulating the affinity of hIL2 for CD132 include Q11, L18 (e.g., L18R), Q22 (e.g., Q22E), E110, N119, T123, Q126 (e.g., Q126K / H), S127, I129, S130, and T133. In some embodiments, the present invention provides an hIL2 orthogonal homolog that is an hIL2 polypeptide comprising one of the following groups of amino acid modifications:

[0466] [E15S-H16Q-L18R-L19V-D20L-Q22E-M23A];

[0467] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A];

[0468] [E15S-H16Q-L18R-L19V-D20L-M23A];

[0469] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A];

[0470] [E15S-H16Q-L18R-L19V-D20L-M23A-Q126H];

[0471] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126H];

[0472] [E15S-H16Q-L18R-L19V-D20L-M23A-Q126K];

[0473] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126K];

[0474] [E15S-H16Q-L19V-D20L-M23A-Q126H];

[0475] [E15S-H16Q-L19V-D20L-Q22K-M23A-Q126H];

[0476] [E15S-H16Q-L19V-D20L-M23A-Q126K];

[0477] [E15S-H16Q-L19V-D20L-Q22K-M23A-Q126K];

[0478] [E15S-H16Q-L18R-L19V-D20L-M23A-Q126H];

[0479] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126H];

[0480] [E15S-H16Q-L18R-L19V-D20L-M23A-Q126K];

[0481] [E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126K];

[0482] [E15S-H16Q-L19V-D20L-Q22K-Q126H];

[0483] [E15S-H16Q-L19V-D20L-M23A-Q126M];

[0484] [E15S-H16Q-L19V-D20L-Q22K-M23A-Q126M];

[0485] [E15S-H16Q-L19V-D20L-Q22K-Q126M];

[0486] [desAla1-E15S-H16Q-L19V-D20L-Q126M];

[0487] [desAla1-E15S-H16Q-L19V-D20L-Q22K-Q126M];

[0488] [desAla1-E15S-H16Q-L19V-D20L-M23A-Q126M];

[0489] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-Q126M];

[0490] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-Q126M];

[0491] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-I86V-I92F-Q126H];

[0492] [E15S-H16Q-L19V-D20L-Q22K-L80F-R81D-I86V-I92F-Q126H];

[0493] [E15S-H16Q-L19V-D20L-Q22K-M23A-L80F-R81D-I86V-I92F-Q12

[0494] 6H];

[0495] [E15S-H16Q-L19V-D20L-M23A-L80F-R81D-I86V-I92F-Q126M];

[0496] [E15S-H16Q-L19V-D20L-Q22K-M23A-L80F-R81D-I86V-I92F-Q12

[0497] 6M];

[0498] [E15S-H16Q-L19V-D20L-M23A-L85V-Q126H];

[0499] [E15S-H16Q-L19V-D20L-Q22K-L85V-Q126H];

[0500] [E15S-H16Q-L19V-D20L-Q22K-M23A-L85V-Q126H];

[0501] [E15S-H16Q-L19V-D20L-M23A-L85V-Q126M];

[0502] [E15S-H16Q-L19V-D20L-Q22K-L85V-Q126H]; or

[0503] [E15S-H16Q-L19V-D20L-Q22K-M23A-L85V-Q126M].

[0504] Removal of glycosylation sites

[0505] The hIL2 orthotic homologs of the present invention may also provide, or optionally provide, the elimination of the O-glycosylation site at Thr3 to promote the generation of non-glycosylated hIL2 orthotic homolog variants when the orthotic homologs are expressed in mammalian cells (e.g., CHO or HEK cells). Therefore, in some embodiments, the hIL2 orthotic homologs further comprise a modification that eliminates the O-glycosylation site at the 3-position of human IL-2. In some embodiments, the modification that eliminates the O-glycosylation site at the 3-position of human IL-2 is an amino acid modification. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P, which remove the glycosylation site at position 3 without eliminating biological activity (see U.S. Patent No. 5,116,943; Weiger et al., (1989) Eur. J. Biochem., 180:295-300). In certain embodiments, the modification is an amino acid substitution for T3A. In some embodiments, the present invention provides an hIL2 orthogonal homolog, which is an hIL2 polypeptide comprising one of the following groups of amino acid modifications:

[0506] [T3A-E15S-H16Q-L19V-D20L-Q22K-M23A-C125S];

[0507] [T3A-E15S-H16Q-L19V-D20L-Q22K-C125S];

[0508] [T3A-E15S-H16Q-L19V-D20L-M23A-C125S];

[0509] [T3A-E15S-H16Q-L19V-D20L-C125S];

[0510] [T3A-E15S-H16Q-L19V-D20L-Q22K-M23A-C125A];

[0511] [T3A-E15S-H16Q-L19V-D20L-M23A-C125A];

[0512] [T3A-E15S-H16Q-L19V-D20L-Q22K-C125A];

[0513] [T3A-E15S-H16Q-L19V-D20L-C125A];

[0514] [T3A-E15S-H16Q-L19V-D20L-Q22K-M23A];

[0515] [T3A-E15S-H16Q-L19V-D20L-M23A];

[0516] [T3A-E15S-H16Q-L19V-D20L-Q22K];

[0517] [T3A-E15S-H16Q-L19V-D20L];

[0518] [desAla1-T3A-E15S-H16Q-L19V-D20L-Q22K-M23A-C125S];

[0519] [desAla1-T3A-E15S-H16Q-L19V-D20L-M23A-C125S];

[0520] [desAla1-T3A-E15S-H16Q-L19V-D20L-Q22K-C125S];

[0521] [desAla1-T3A-E15S-H16Q-L19V-D20L-C125S];

[0522] [desAla1-T3A-E15S-H16Q-L19V-D20L-Q22K-M23A-C125A];

[0523] [desAla1-T3A-E15S-H16Q-L19V-D20L-M23A-C125A];

[0524] [desAla1-T3A-E15S-H16Q-L19V-D20L-Q22K-C125A];

[0525] [desAla1-T3A-E15S-H16Q-L19V-D20L-C125A];

[0526] [desAla1-T3A-E15S-H16Q-L19V-D20L-Q22K-M23A];

[0527] [desAla1-T3A-E15S-H16Q-L19V-D20L-M23A];

[0528] [desAla1-T3A-E15S-H16Q-L19V-D20L-Q22K]; or

[0529] [desAla1-T3A-E15S-H16Q-L19V-D20L].

[0530] N-terminal deletion:

[0531] The IL-2 orthogonal homolog may further comprise the elimination of one or more N-terminal amino acids at positions 1-9, or positions 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2, while retaining the hIL2 orthogonal homolog activity. In some embodiments, the present invention provides an hIL2 orthogonal homolog, which is an hIL2 polypeptide comprising one of the following groups of amino acid modifications:

[0532] [desAla1-E15S-H16Q-L18R-L19V-D20L-Q22E-M23A];

[0533] [desAla1-E15S-H16Q-L18R-L19V-D20L-Q22K-M23A];

[0534] [desAla1-E15S-H16Q-L18R-L19V-D20L-M23A];

[0535] [desAla1-E15S-H16Q-L18R-L19V-D20L-Q22K-M23A];

[0536] [desAla1-E15S-H16Q-L18R-L19V-D20L-M23A-Q126H];

[0537] [desAla1-E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126H];

[0538] [desAla1-E15S-H16Q-L18R-L19V-D20L-M23A-Q126K];

[0539] [desAla1-E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126K];

[0540] [desAla1-E15S-H16Q-L19V-D20L-M23A-Q126H];

[0541] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-Q126H];

[0542] [desAla1-E15S-H16Q-L19V-D20L-M23A-Q126K];

[0543] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-Q126K];

[0544] [desAla1-E15S-H16Q-L18R-L19V-D20L-M23A-Q126H];

[0545] [desAla1-E15S-H16Q-L18R-L19V-D20L-Q22K-M23A-Q126H];

[0546] [desAla1-E15S-H16Q-L18R-L19V-D20L-M23A-Q126K];

[0547] [desAla1-desPro2-E15S-H16Q-L19V-D20L-Q22K-M23A];

[0548] [desAla1-desPro2-E15S-H16Q-L19V-D20L-Q22K];

[0549] [desAla1-desPro2-E15S-H16Q-L19V-D20L-M23A];

[0550] [desAla1-desPro2-E15S-H16Q-L19V-D20L];

[0551] [desAla1-desPro2-desThr3-E15S-H16Q-L19V-D20L-Q22K-M23A];

[0552] [desAla1-desPro2-desThr3-E15S-H16Q-L19V-D20L-Q22K];

[0553] [desAla1-desPro2-desThr3-E15S-H16Q-L19V-D20L-M23A];

[0554] [desAla1-desPro2-desThr3-E15S-H16Q-L19V-D20L];

[0555] [desAla1-desPro2-desThr3-desSer4-E15S-H16Q-L19V-D20L-Q22K-M2

[0556] 3A];

[0557] [desAla1-desPro2-desThr3-desSer4-E15S-H16Q-L19V-D20L-Q22K];

[0558] [desAla1-desPro2-desThr3-desSer4-E15S-H16Q-L19V-D20L-M23A];

[0559] [desAla1-desPro2-desThr3-desSer4-E15S-H16Q-L19V-D20L];

[0560] [desAla1-desPro2-desThr3-desSer4-desSer5-E15S-H16Q-L19V-D20L-Q

[0561] 22K-M23A];

[0562] [desAla1-desPro2-desThr3-desSer4-desSer5-E15S-H16Q-L19V-D20L-Q

[0563] 22K];

[0564] [desAla1-desPro2-desThr3-desSer4-desSer5-E15S-H16Q-L19V-D20L-

[0565] M23A];

[0566] [desAla1-desPro2-desThr3-desSer4-desSer5-E15S-H16Q-L19V-D20L];

[0567] [desAla1-desPro2-desThr3-desSer4-desSer5-desSer6-E15S-H16Q-L19V-

[0568] [D20L-Q22K-M23A];

[0569] [desAla1-desPro2-desThr3-desSer4-desSer5-desSer6-E15S-H16Q-L19V-

[0570] [D20L-Q22K];

[0571] [desAla1-desPro2-desThr3-desSer4-desSer5-desSer6-E15S-H16Q-L19V-

[0572] [D20L-M23A]; or

[0573] [desAla1-desPro2-desThr3-desSer4-desSer5-desSer6-E15S-H16Q-L19V-

[0574] D20L).

[0575] In some embodiments, the hIL2 orthogonal homolog may comprise the deletion of the first two amino acids (desAla1-desPro2), and Thr3 glycosylation with cysteine ​​residues to promote selective N-terminal modification, particularly PEGylation of the thiol group of cysteine ​​(see, for example, U.S. Patent No. 5,206,344, Katre et al., issued April 27, 1993). In some embodiments, the present invention provides an hIL2 orthogonal homolog that is an hIL2 polypeptide comprising one of the following groups of amino acid modifications:

[0576] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-Q22K-M23A-C125S];

[0577] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-Q22K-C125S];

[0578] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-M23A-C125S];

[0579] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-C125S];

[0580] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-Q22K-M23A-C125A];

[0581] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-Q22K-C125A];

[0582] [desAla1-desPro2-T3C E15S-H16Q-L19V-D20L-M23A-C125A];

[0583] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-C125A];

[0584] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-Q22K-M23A];

[0585] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-Q22K];

[0586] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L-M23A]; or

[0587] [desAla1-desPro2-T3C-E15S-H16Q-L19V-D20L].

[0588] In some embodiments, the hIL2 orthogonal homologs of the present invention comprise a deleting (“desAla1”) hIL2 orthogonal homologs. In some embodiments, the present invention provides des-Ala1-hIL2 orthogonal homologs comprising one of the following groups of amino acid modifications:

[0589] [desAla1-E15S-H16Q-L19V-D20L];

[0590] [desAla1-E15S-H16Q-L19V-D20L-Q22K];

[0591] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A];

[0592] [desAla1-E15S-H16Q-L19V-D20L-Q126H];

[0593] [desAla1-E15S-H16Q-L19V-D20L-Q22K-Q126H];

[0594] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-Q126H];

[0595] [desAla1-E15S-H16Q-L19V-D20L-Q126M];

[0596] [desAla1-E15S-H16Q-L19V-D20L-Q22K-Q126H];

[0597] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-Q126H];

[0598] [desAla1-E15S-H16Q-L19V-D20L-C125A];

[0599] [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125A];

[0600] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125A];

[0601] [desAla1-E15S-H16Q-L19V-D20L-C125A-Q126H];

[0602] [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125A-Q126H];

[0603] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125A-Q126H];

[0604] [desAla1-E15S-H16Q-L19V-D20L-C125A-Q126M];

[0605] [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125A-Q126H];

[0606] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125A-Q126H];

[0607] [desAla1-E15S-H16Q-L19V-D20L-C125S];

[0608] [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125S];

[0609] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125S];

[0610] [desAla1-E15S-H16Q-L19V-D20L-C125S-Q126H];

[0611] [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125S-Q126H];

[0612] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125S-Q126H];

[0613] [desAla1-E15S-H16Q-L19V-D20L-C125S-Q126M];

[0614] [desAla1-E15S-H16Q-L19V-D20L-Q22K-C125S-Q126M]; or

[0615] [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A-C125S-Q126M].

[0616] Modifications to minimize vascular leakage syndrome

[0617] In some embodiments of the invention, the hIL2 orthographic homolog contains amino acid substitutions that prevent vascular leakage syndrome. Epstein, et al., U.S. Patent No. 7,514,073B2, issued April 7, 2009. Examples of such modifications incorporated into the hIL2 orthographic homolog of the invention include one or more of R38W, R38G, R39L, R39V, F42K, and / or H55Y.

[0618] Oxidation-stabilized M104A:

[0619] In some embodiments of the invention, the hIL2 orthographic homolog may include a modification at position M104. In one embodiment, methionine 104 is replaced with an alanine residue (M104A) to provide a more antioxidant orthographic homolog (see Koths, et al., U.S. Patent 4,752,585, issued June 21, 1988).

[0620] Mature affinity:

[0621] In some embodiments, the hIL2 orthogonal homologs of the present invention may undergo affinity maturation to enhance their activity toward orthogonal hCD122. An “affinity-matured” polypeptide is a polypeptide with one or more alterations at one or more residues, resulting in improved affinity of the orthogonal polypeptide for its homologous orthogonal receptor, or vice versa, compared to a parental polypeptide that does not possess these alterations. Affinity maturation can increase the binding affinity of the hIL2 orthogonal homolog by at least about 10%, or at least about 50%, or at least about 100%, or at least about 150%, or 1-5 times compared to the “parental” polypeptide. When assessed by ELISA and / or FACS analysis using sufficient amounts of the molecule under suitable experimental conditions, the engineered hIL2 orthogonal homologs of the present invention activate their homologous orthogonal receptors as described above, but significantly reduce binding and activation toward the native receptor.

[0622] Modifications that prolong the duration of action in vivo

[0623] In some embodiments, the hIL-2 orthogonal homologs of the present invention may include modifications to provide extended in vivo lifetime and / or extended duration of action in a subject. In some embodiments, the hIL-2 orthogonal homologs of the present invention have a plasma half-life in human subjects 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. Such extended-life hIL-2 orthogonal homologs are achieved through primary sequence modification and / or conjugation to a carrier molecule.

[0624] First-order modifications that prolong the duration of action

[0625] The hIL-2 orthographic homologs of the present invention may contain amino acid substitutions that result in extended lifespan in vivo. Examples of the positions of amino acid substitutions that can be incorporated into hIL-2 orthographic homologs to provide extended lifespan in vivo include one or more of V111, R117, and / or T133. In some embodiments, the hIL2 orthographic homologs of the present invention contain one or more modifications of V111R, R117K, and / or T133N. Dakshinamurthi, et al. (2009) International Journal of Bioinformatics Research 1(2):4-13.

[0626] Transport molecules

[0627] In some embodiments, the hIL2 orthographic homolog is modified to provide an extended duration of action in the target, which can be achieved by coupling to a carrier molecule to provide desired pharmacological properties (e.g., extended half-life). In one embodiment, the hIL2 orthographic homolog may comprise a functional domain of a chimeric peptide. In some embodiments, the hIL2 orthographic homolog may be covalently linked to the Fc domain of IgG, albumin, or other molecules to extend their half-life, for example, through PEGylation, glycosylation, etc., as known in the art.

[0628] Fc Fusion

[0629] In some embodiments, the hIL2 orthogonal homolog of the present invention is operatively linked to the functional domain of an Fc-fusion chimeric polypeptide molecule. Fc fusion conjugates have been shown to increase the systemic half-life of biologics, thus reducing the need for frequent dosing. Fc binds to nascent Fc receptors (FcRn) lining the endothelial cells of blood vessels. Upon binding, the Fc fusion molecule is protected from degradation and re-release into circulation, allowing the molecule to remain in circulation for a longer period. This Fc binding is considered a mechanism by which endogenous IgG maintains its long plasma half-life. Recent Fc fusion technologies link a single copy of a biologic to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biologic compared to conventional Fc fusion conjugates. The "Fc region" used to prepare the Fc fusion can be a naturally occurring or synthetic polypeptide homologous to the C-terminal domain of IgG produced by papain digestion of IgG. The molecular weight of the IgG Fc is approximately 50 kDa. hIL2 orthologs can provide the entire Fc region or a smaller portion that retains the ability to extend the cycling half-life of the chimeric polypeptide to which it is part. Furthermore, the full-length or fragmented Fc region can be a variant of the wild-type molecule. In a typical presentation, each monomer of the dimer Fc carries a heteropeptide, which may be identical or different.

[0630] In some implementations, when hIL2 orthologs are to be administered as Fc fusion bodies, particularly when the polypeptide chains coupled to the various subunits of the Fc dimer are different, the Fc fusion bodies can be engineered to have a “knob-into-hole modification.” Knob-into-hole modification is described more fully in Ridgway et al. (1996), Protein Engineering, 9(7):617-621, and in U.S. Patent No. 5,731,168, issued March 24, 1998. The mortar modification refers to the modification of the interface between two immunoglobulin heavy chains in the CH3 domain, wherein: i) in the CH3 domain of the first heavy chain, amino acid residues are replaced with amino acid residues having larger side chains (e.g., tyrosine or tryptophan), creating a protrusion (“mortar”) from the surface; and ii) in the CH3 domain of the second heavy chain, amino acid residues are replaced with amino acid residues having smaller side chains (e.g., alanine or threonine), thereby creating a cavity (“mortar”) within the interface of the second CH3 domain, wherein the protruding side chain (“mortar”) of the first CH3 domain is received by the cavity in the second CH3 domain. In one embodiment, the “mortar modification” includes amino acid substitution T366W and optionally amino acid substitution S354C in one antibody heavy chain, and amino acid substitution T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. Furthermore, the Fc domain can be modified by introducing cysteine ​​residues at the S354 and Y349 positions, thereby generating stable disulfide bonds between the two antibody heavy chains in the Fe region (Carter et al. (2001) Immunol Methods 248, 7-15). The mortar and pestle configuration is used to promote the expression of the first polypeptide (such as the hIL2 ortholog) on ​​the first Fc monomer with the mortar modification and the expression of the second polypeptide on the second Fc monomer with the mortar modification, thereby promoting the expression of heterodimeric polypeptide conjugates.

[0631] The Fc region can be "soluble" or "insoluble," but is usually insoluble. Insoluble Fc regions typically lack both high-affinity Fc receptor binding sites and Clq binding sites. The high-affinity Fc receptor binding site of mouse IgG Fc includes the Leu residue at position 235. Therefore, the Fc receptor binding site can be inhibited by mutation or deletion of Leu 235. For example, replacing Leu 235 with Glu inhibits the ability of the Fc region to bind to the high-affinity Fc receptor. The mouse Clq binding site can be functionally disrupted by mutation or deletion of Glu 318, Lys 320, and Lys 322 residues in IgG. For example, replacing Glu 318, Lys 320, and Lys 322 with Ala residues prevents IgG1 Fc from guiding antibody-dependent complement cleavage. In contrast, soluble IgG Fc regions possess both high-affinity Fc receptor binding sites and Clq binding sites. The high-affinity Fc receptor binding site includes the Leu residue at position 235 of IgG Fc, while the Clq binding site includes Glu 318, Lys 320, and Lys 322 residues of IgG 1. Soluble IgG Fc has wild-type residues or conserved amino acid substitutions at these sites. Soluble IgG Fc can induce antibody-dependent cytotoxicity or complement-directed cytolysis (CDC) against cells. Appropriate mutations in human IgG are also known (see, for example, Morrison et al., The Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2:125, 1994). In some embodiments, the Fc domain monomer includes at least one mutation relative to the wild-type human IgG1, IgG2, or IgG4 Fc region, as described in U.S. Patent No. US10,259,859B2, the entire teaching of which is incorporated herein by reference. In some embodiments, the polypeptide exhibits reduced phagocytosis in phagocytosis assays compared to polypeptides having a wild-type human IgG Fc region. In some embodiments, an Fc domain monomer is linked to a second polypeptide containing a second Fc domain monomer to form an Fc domain dimer.

[0632] PEGylation:

[0633] In some embodiments, the hIL2 ortholog of the present invention can be coupled to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of the present invention include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polymers of polyvinylpyrrolidone, ethylene glycol and propylene glycol, poly(oxyethylated polyols), polyolefinic alcohols), polysaccharides, poly-α-hydroxy acids, polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0634] In some embodiments, the hIL2 orthographic homolog is operatively linked to one or more polyethylene glycol molecules or "PEGylated". Although the methods or sites of PEG attachment to the hIL2 orthographic homolog may differ, in some embodiments PEGylation does not alter or only minimally alters the activity of the hIL2 orthographic homolog.

[0635] In some embodiments, selective PEGylation of hIL2 orthologs (e.g., by incorporating non-natural amino acids with side chains to facilitate selective PEG coupling chemistry, as described in Ptacin et al. (PCT International Application No. PCT / US2018 / 045257 filed August 3, 2018, and published as International Publication No. WO on February 7, 2019) (Disclosed in 2019 / 028419A1) can be used to generate hIL2 orthologs with reduced affinity for one or more subunits (such as CD25, CD132) of the hIL2 receptor complex. For example, an hIL2 ortholog wherein a non-natural amino acid with a PEGylated specific moiety is incorporated at those sequences or residues of hIL2 identified as interacting with CD25 (including amino acids 34-45, 61-72, and 105-109) provides an hIL2 ortholog with regulated CD25 binding. Similarly, an hIL2 ortholog wherein a non-natural amino acid with a PEGylated specific moiety is incorporated at those sequences or residues of hIL2 identified as interacting with hCD132 (including, but not limited to, amino acids 18, 22, 109, 126, and / or 133) provides an hIL2 ortholog with regulated hCD132 binding affinity.

[0636] In some implementations, the increase in half-life outweighs any decrease in biological activity. PEGs suitable for coupling to peptide sequences are generally water-soluble at room temperature and have the general formula...

[0637] R(O-CH2-CH2) nOR, where R is hydrogen or a protecting group, such as alkyl or alkylol, where n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons. PEG coupled to the polypeptide sequence can be linear or branched. This invention considers branched PEG derivatives, “star-PEG” and multi-arm PEG.

[0638] The molecular weight of PEG used in the context of the hIL2 ortholog of the present invention is not limited to any particular range. The PEG component of the PEG-IL2 ortholog may have a molecular weight greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. The molecular weight of linear or branched PEG molecules ranges from about 2,000 to about 80,000 Daltons, or about 2,000 to about 70,000 Daltons, or about 5,000 to about 50,000 Daltons, or about 10,000 to about 50,000 Daltons, or about 20,000 to about 50,000 Daltons, or about 30,000 to about 50,000 Daltons, or about 20,000 to about 40,000 Daltons, or about 30,000 to about 40,000 Daltons. In one embodiment of the invention, the PEG is a 40kD branched PEG comprising two 20kD arms.

[0639] This disclosure also considers compositions of conjugates in which PEG has different n values, thus allowing for a variety of different PEGs at specific ratios. For example, some compositions comprise mixtures of conjugates where n = 1, 2, 3, and 4. In some compositions, the percentage of conjugate with n = 1 is 18-25%, the percentage of conjugate with n = 2 is 50-66%, the percentage of conjugate with n = 3 is 12-16%, and the percentage of conjugate with n = 4 is up to 5%. Such compositions can be produced using reaction conditions and purification methods known in the art. Chromatography can be used to distinguish the fractions of the conjugates, and then identify the fractions containing, for example, conjugates with the desired amount of PEG attached, purified from unmodified protein sequences and conjugates with other amounts of PEG attached.

[0640] PEGs suitable for coupling to IL2 linear conjugates are typically water-soluble at room temperature and have the general formula R(O-CH2-CH2). nOR, where R is a hydrogen or protecting group, such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons.

[0641] Two widely used first-generation activated monomethoxy PEGs (mPEGs) are succinimide carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotönol. Appl. Biochem 15:100-114) and benzotriazole methyl PEG (BTC-PEG; see, e.g., Dolence, et al., U.S. Patent No. 5,650,234), which preferably react with lysine residues to form a carbamate link, but are also known to react with histidine and tyrosine residues. PEG-aldehyde linkers are used to target a single N-terminal site of the polypeptide via reductive amination.

[0642] Polyglycolation can occur at the N-terminus of a polypeptide, at the α-amino group of a lysine residue side chain, and at the imidazole group of a histidine residue side chain. Since most recombinant polypeptides possess a single α-group and several ε- and imidazole groups, numerous positional isomers can be generated depending on the chemical nature of the linker. General polyethylene glycolation strategies known in the art are applicable herein.

[0643] PEG can be bound to the hIL2 orthologs of the present invention via a terminal reactive group (“spacer”) that mediates the binding between a free amino or carboxyl group of one or more polypeptide sequences and polyethylene glycol. PEG having a spacer that can bind to a free amino group comprises N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating a succinate of polyethylene glycol with N-hydroxysuccinimide.

[0644] In some embodiments, the PEGylation of hIL2 orthologs is facilitated by incorporating non-natural amino acids with unique side chains to promote site-specific PEGylation. Incorporating non-natural amino acids into peptides to provide a functional moiety for site-specific PEGylation of such peptides is known in the art. See, for example, Ptacin et al., (PCT International Application No. PCT / US2018 / 045257, filed August 3, 2018 and published February 7, 2019, International Publication No. WO).

[0645] 2019 / 028419A1. In one embodiment, the hIL2 orthogonal homolog of the present invention incorporates a non-natural amino acid at the D109 position of the hIL2 orthogonal homolog. In one embodiment of the present invention, the hIL2 orthogonal homolog is PEGylated at the 109 position of the hIL2 orthogonal homolog, and the PEG molecule has a molecular weight of about 20 kD, or about 30 kD, or about 40 kD.

[0646] PEG conjugated to a polypeptide sequence can be linear or branched. This invention considers branched PEG derivatives, “star-PEG”, and multi-arm PEG. Specific embodiments useful in the practice of this invention include PEG comprising 10 kDa linear PEG-aldehydes (e.g., ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA, 10kDa linear PEG-NHS ester (e.g., ME-100CS, ME-100AS, ME-100GS, ME-100HS, NOF), 20kDa linear PEG-aldehyde (e.g.) ME-200AL, NOF, 20kDa linear PEG-NHS ester (e.g., ME-200CS, ME-200AS, ME-200GS, ME-200HS, NOF), 20kDa 2-arm branched PEG-aldehyde, the 20kDA PEG-aldehyde comprising two 10kDA straight-chain PEG molecules (e.g., ME-200HS, NOF), 20kDa 2-arm branched PEG-aldehyde, which contains two 10kDA straight-chain PEG molecules (e.g., ME-200HS, NOF), 20kDa 2-arm branched PEG-aldehyde, GL2-200AL3,NOF), 20kDa 2-arm branched PEG-NHS ester, which contains two 10kDa straight-chain PEG molecules (e.g., GL2-200TS, GL200GS2,NOF), a 40kDa 2-arm branched PEG-aldehyde, which contains two 20kDa straight-chain PEG molecules (e.g., GL200GS2,NOF), and a 40kDa PEG-aldehyde containing two 20kDa straight-chain PEG molecules (e.g., GL2-400AL3), 40kDa 2-arm branched PEG-NHS ester, which contains two 20kDa straight-chain PEG molecules (e.g., GL2-400AL3, GL2-400GS2, NOF), linear 30kDa PEG-aldehyde (e.g., ME-300AL) and linear 30kDa PEG-NHS ester.

[0647] In one embodiment, the hIL2 orthogonal homolog of the present invention includes the following structure:

[0648] [PEG]-[Connector] n -[hoIL2]

[0649] Where n = 0 or 1.

[0650] In an embodiment, the hIL2 orthogonal homolog of the present invention includes the following structure:

[0651] [PEG]-[Connector] n -[desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A]

[0652] Where n = 0 or 1.

[0653] In one embodiment, the hIL2 orthogonal homolog of the present invention includes the following structure:

[0654] [40kDa-PEG]-[Connector] n -[hoIL2],

[0655] Where n = 0 or 1.

[0656] In an embodiment, the hIL2 orthogonal homolog of the present invention includes the following structure:

[0657] [40kDa-PEG]-[connect]

[0658] head] n -[desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A]

[0659] Where n = 0 or 1.

[0660] In one embodiment, the hIL2 orthogonal homolog of the present invention includes the following structure:

[0661] [40kDa-branched PEG]-[Connector] n -[hoIL2],

[0662] Where n = 0 or 1.

[0663] In an embodiment, the hIL2 orthogonal homolog of the present invention includes the following structure:

[0664] [40KD-branched PEG]-[connection]

[0665] head] n -[desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A],

[0666] Where n = 0 or 1.

[0667] In another embodiment, the hIL2 orthogonal homolog includes the structure:

[0668] [PEG]-(connected)

[0669] head) n -[PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRM LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLI

[0670] SNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0671] (SEQ ID NO:5)],

[0672] Where n = 0 or 1.

[0673] In another embodiment, the hIL2 orthogonal homolog includes the structure:

[0674] [40kDa-PEG]-(Connector) n -[PTSSSTKKTQLQLSQLLVLLKAILNGINNYK NPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKN

[0675] FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQUENCE IDNO:5)],

[0676] Where n = 0 or 1.

[0677] Suitable linkers include “flexible linkers” whose length is generally sufficient to allow some movement between the modified polypeptide sequence and the linked component and molecule. Linker molecules are typically about 6–50 atoms long. Linkers can also be, for example, arylaceyne, ethylene glycol oligomers containing 2–10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable linkers can be readily chosen and can be of any suitable length, such as 1 amino acid (e.g., glycine), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10–20, 20–30, 30–50, or more than 50 amino acids.

[0678] Examples of flexible joints include glycine polymers (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., GmSo)n, (GSGG)n, etc.

[0679] (GmSoGm)n, (GmSoGmSoGm)n, (GSGGSm)n, (GSGSmG)n, and (GGGGSm)n and combinations thereof, wherein m, n, and o are each independently selected from at least 1 to 10 integers, such as 1-18, 2-16, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or other flexible connectors. Glycine and glycine-serine polymers are relatively unstructured and therefore can be used as neutral tethers between components, including but not limited to GGGSG (SEQ ID NO: 139), GGSGG (SEQ ID NO: 140), GGSG (SEQ ID NO: 141), GGSGG (SEQ ID NO: 142), and GGGGSG (SEQ ID NO: 143). Other examples of flexible joints include glycine polymers (G)n or glycine-serine polymers (e.g., GS)n, GSGGS)n, GGGS)n, and GGGGS)n where n = 1-50, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50G joints. Exemplary flexible joints include, but are not limited to, GGGGS (SEQ ID NO: 144), GGGGS (SEQ ID NO: 145), GGSG (SEQ ID NO: 146), GGSGG (SEQ ID NO: 147), GGSG (SEQ ID NO: 148), GGSGG (SEQ ID NO: 149), GGGGSG (SEQ ID NO: 150), and GSSSG (SEQ ID NO: 151). Polymers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) can be linked together to provide a flexible linker for coupling heterologous amino acid sequences to the peptide or PEG molecules disclosed herein. Alternatively, peptide linkers may be chemical linkers, such as PEG-aldehyde linkers.

[0680] Acetylated:

[0681] In some embodiments, the IL-2 orthogonal homolog is acetylated at the N-terminus by an enzymatic reaction with an N-terminal acetyltransferase, for example, acetyl-CoA. Alternatively, in addition to N-terminal acetylation, the IL-2 orthogonal homolog may be acetylated at one or more lysine residues, for example, by an enzymatic reaction with a lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325(5942):834-840.

[0682] Flag tag

[0683] In some embodiments, the IL-2 orthogonal homolog is modified to include other polypeptide sequences that function as antigen tags, such as the FLAG sequence. As described herein, the FLAG sequence can be recognized by biotinylated, highly specific anti-FLAG antibodies (see, for example, Blanar et al. (1992) Science 256:1014 and LeClair et al. (1992) PNAS-USA 89:8145). In some embodiments, the IL-2 orthogonal homolog polypeptide further includes a C-terminal c-myc epitope tag.

[0684] Albumin fusion:

[0685] In some embodiments, an IL-2 orthogonal homolog is coupled to albumin, referred to herein as an "IL2 orthogonal homolog albumin fusion". When the term "albumin" is used in the context of an hIL2 orthogonal homolog albumin fusion, it includes albumin such as human serum albumin (HSA), cynomolgus monkey serum albumin, and bovine serum albumin (BSA). In some embodiments, the HSA contains a C34S or K573P amino acid substitution relative to the wild-type HSA sequence. According to the invention, albumin can be coupled to the hIL2 orthogonal homolog at the carboxyl terminus, amino terminus, carboxyl and amino terminus, and interior (see, for example, USP 5,876,969 and USP 7,056,701). Various forms of albumin can be used in the HSA-hIL2 orthogonal homolog polypeptide conjugates contemplated in this disclosure, such as albumin secretion pre-sequences and their variants, fragments and variants thereof, and HSA variants. This form typically possesses one or more desired albumin activities. In other embodiments, the present invention relates to fusion proteins comprising hIL2 orthogonal homolog peptides directly or indirectly fused to albumin, albumin fragments, and albumin variants, wherein the fusion protein exhibits higher plasma stability than the unfused drug molecule and / or the fusion protein retains the therapeutic activity of the unfused drug molecule. In some embodiments, direct fusion is achieved via a linker, such as a peptide linker or a modified version thereof, as discussed more fully below.

[0686] Alternatively, the hIL2 orthogonal homolog albumin fusion protein comprises an hIL2 orthogonal homolog, which is a fusion protein containing an albumin-binding domain (ABD) polypeptide sequence and an hIL2 orthogonal homolog polypeptide. As described above, the fusion protein containing the albumin-binding domain (ABD) polypeptide sequence and the hIL2 orthogonal homolog polypeptide can, for example, be genetically manipulated to bind a nucleic acid encoding HSA or a fragment thereof to a nucleic acid encoding one or more hIL2 orthogonal homolog sequences. In some embodiments, the albumin-binding peptide comprises the amino acid sequence: DICLPRWGCLW (SEQ ID NO: 152).

[0687] His tags

[0688] In some embodiments, the hIL2 orthogonal homologs of the present invention (including fusion proteins of such IL-2 orthogonal homologs) are expressed as fusion proteins having one or more transition metal chelating polypeptide sequences. The inclusion of such transition metal chelating domains facilitates purification by immobilized metal affinity chromatography (IMAC), as described in U.S. Patent No. 4,569,794, issued February 11, 1986, by Smith et al. Examples of transition metal chelating polypeptides useful in the practice of the present invention are described in U.S. Patent No. 5,320,663, issued May 10, 1995, by Smith et al. (ibid.) and Dobeli et al., the entire teachings of which are incorporated herein by reference. Particularly useful transition metal chelating polypeptides in the practice of the present invention are peptides comprising 3-6 consecutive histidine residues, such as 6-histidine peptide (His)6, often referred to in the art as “His-tags”.

[0689] The aforementioned fusion protein can be readily produced using recombinant DNA methods with techniques known in the art, by constructing a recombinant vector containing a nucleic acid sequence including a nucleic acid sequence encoding an hIL2 orthogonal homolog that is framed with a nucleic acid sequence encoding an N-terminal or C-terminal fusion mate encoding an hIL2 orthogonal homolog, the sequence optionally further including a nucleic acid sequence encoding a linker or spacer polypeptide within the frame.

[0690] Targeted hIL2 orthogonal homolog molecules

[0691] In some embodiments, the hIL2 orthogonal homolog is provided as a fusion protein having a polypeptide sequence (“targeting domain”) to facilitate selective binding to specific cell types or tissues expressing cell surface molecules that specifically bind to such targeting domains, optionally with an adapter molecule incorporated between the hIL2 orthogonal homolog and the targeting domain sequence of the fusion protein. In one embodiment, the targeting region of the hIL2 orthogonal homolog fusion protein specifically binds to cell surface molecules of cell types targeted by CAR-T cells expressing orthogonal hCD122. For example, in cases where orthogonal hCD122 CAR-T cells contain a CAR with an ECD that specifically binds to CD-19, the targeting domain of the hIL2 orthogonal homolog fusion protein may also bind to CD-19. Expression of the targeting domain may include a ligand for a cell surface receptor or an antibody that specifically binds to a molecule. In one embodiment, the hIL2 orthogonal homolog fusion protein includes a specific binding to the same cell type targeted as engineered cells expressing orthogonal ligands (e.g., hoRb CAR-T cells). In one embodiment, where the ECD of the CAR in hoRb CAR-T cells specifically binds to CD-19, an IL-2 orthogonal homolog may be provided as a fusion protein having a CD-19 targeting moiety. For example, in one embodiment, where the ECD of the CAR in hoRb CAR-T cells is an scFv molecule that specifically binds to CD-19, an IL-2 orthogonal homolog is provided as a fusion protein with a CD-19 targeting moiety (e.g., a single-chain antibody (e.g., scFv or VHH) that specifically binds to CD-19). In one embodiment, the fusion protein comprises an IL-10 orthogonal homolog and anti-CD19 scFv FMC63 (Nicholson, et al. (1997) Mol Immunol 34:1157–1165). Similarly, in some embodiments, the ECD of the CAR from hoRb CAR-T cells specifically binds to BCMA, and an IL-2 orthogonal homolog is provided as a fusion protein with a BCMA targeting moiety, such as an antibody comprising, for example, the CDR of an anti-BMCA antibody as described by Kalled et al. (US Patent No. 9,034,324, granted May 9, 2015) or an antibody comprising the CDR of an anti-BMCA antibody as described by Brogdon et al. (US Patent No. 10,174,095, granted January 8, 2019).In some embodiments, the ECD of the CAR in hoRb CAR-T cells specifically binds to GD2, and an IL-2 orthogonal homolog is provided as a fusion protein with a GD2 targeting moiety, which includes, for example, the CDR described by Cheung et al. (US Patent No. 9,315,585, granted April 19, 2016), or an antibody derived from a CDR of ME36.1 (Thurin et al., (1987) Cancer Research 47:1229-1233), 14G2a, 3F8 (Cheung et al., 1985 Cancer Research 45:2642-2649), hu14.18, 8B6, 2E12, or ic9.

[0692] In some implementations, the targeting portion of the hIL2 orthogonal homolog fusion protein is the same as or different from that provided by CAR-T cells expressing orthogonal hCD122, particularly as a substitute antigen directed to the tumor cell type targeted by the CAR. For example, in the case of orthogonal hCD122scfv 14G2a GD2 targeting CAR-T cells, the hIL2 orthogonal homolog can be provided in a targeting fusion construct containing a specific binding domain of another GD2 tumor antigen.

[0693] In alternative embodiments, the hIL2-targeting orthographic homolog of the present invention can be administered in combination with CAR-T cell therapy to provide targeted delivery of the hIL2 orthographic homolog to CAR-T cells via extracellular receptors based on CAR-T cells, for example, by combining it with an anti-FMC63 antibody to target hIL2 activity to CAR-T cells and regenerate depleted CAR-T cells in vivo. Therefore, embodiments of this disclosure include targeted delivery of the hIL2 orthographic homolog by conjugating such an hIL2 orthographic homolog to an antibody or ligand designed to interact with specific cell surface molecules of CAR-T cells. An example of such a molecule could be an anti-FMC63-hIL2 orthographic homolog.

[0694] In other embodiments, the chimeric polypeptide includes a mutant IL-2 polypeptide and a heterologous polypeptide that functions to enhance the expression of the mutant IL-2 polypeptide or guide cell localization, such as the Aga2p lectin subunit (see, for example, Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0695] Protein transduction domain fusion protein:

[0696] In some embodiments, the hIL2 orthogonal homolog may optionally include a "protein transduction domain" or "PTD". A PTD is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic molecule that facilitates crossing lipid bilayers, micelles, cell membranes, organelle membranes, or vesicle membranes. Incorporating a PTD into the hIL2 orthogonal homolog facilitates membrane crossing. In some embodiments, the PTD is covalently linked to the amino or carboxyl terminus of the hIL2 orthogonal homolog. In some embodiments, the PTD is incorporated as part of a PTD-IL2 orthogonal homolog fusion protein at the N-terminus or C-terminus of the molecule.

[0697] Exemplary protein transduction structures include, but are not limited to, a minimal decapeptide transduction domain (corresponding to residues 47-57 of HIV-1 TAT); a polyarginine sequence comprising a sufficient number of arginine residues to guide cell entry (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginine residues); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); and a polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA). 97:13003-13008), transport proteins (as described in Wierzbicki, et al., (2014) Folio Histomchemica et Cytobiologica 52(4):270-280 and Pooga, et al., (1998) FASEB J12(1)67-77 and available commercially from AnaSpec, catalog number AS-61256); KALA (as described in Wyman et al., (1997) Biochemistry 36(10)3008-3017 and available commercially from AnaSpec, catalog number AS-65459); tentacledopod mutant peptides (as described in Pietersz et al., (2001) Vaccine19:1397 and available commercially from AnaSpec, catalog number AS-61032); TAT 47-57 (available commercially from AnaSpec, catalog number AS-60023).

[0698] Preparation of hIL2 direct homologs

[0699] hIL2 orthologs can be produced by any conventional method, including recombination or solid-phase synthesis.

[0700] Solid-phase chemical synthesis:

[0701] Besides generating mutant peptides through the expression of nucleic acid molecules that have been altered using recombinant molecular biotechnology, the host hIL2 ortholog can be chemically synthesized. Chemically synthesized peptides can be routinely produced by those skilled in the art. Chemical synthesis includes the direct synthesis of peptides encoding protein sequences that exhibit the aforementioned properties of hIL2 orthologs via chemical means.

[0702] In some embodiments, the hIL2 orthologs of the present invention can be prepared by chemical synthesis. The chemical synthesis of hIL2 orthologs can be carried out in a liquid or solid phase. Solid-phase peptide synthesis (SPPS) allows for the inclusion of non-natural amino acids and / or peptide / protein backbone modifications. Various forms of SPPS that can be used to synthesize the hIL2 orthologs of the present invention are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero et al., (2005) Protein Pept Lett. 12:723-8). During chemical synthesis, α-functional groups and any reactive side chains can be protected with acid-insecure or base-insecure groups that are stable under conditions of amide bond linkage but readily cleaved without impairing the formed peptide chain.

[0703] In solid-phase synthesis, N-terminal or C-terminal amino acids can be coupled to a suitable support material. Suitable support materials are those that are inert to the kit reaction conditions of stepwise condensation and cleavage reactions during synthesis and are insoluble in the reaction medium being used. Examples of commercially available support materials include styrene / divinylbenzene copolymers modified with active groups and / or polyethylene glycol; chloromethylated styrene / divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene / divinylbenzene copolymers, etc. Continuous coupling of the protected amino acids can be performed according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer.

[0704] At the end of solid-phase synthesis, the peptide is cleaved from the support material, along with the side-chain protecting groups. The obtained peptide can be purified by various chromatographic methods, including but not limited to hydrophobic adsorption chromatography, ion exchange chromatography, partition chromatography, high-performance liquid chromatography, and reversed-phase HPLC.

[0705] Reorganization of production:

[0706] In some embodiments, hIL2 orthogonal homologs (or fusion proteins containing hIL2 orthogonal homologs) are produced via recombinant methods. A nucleic acid sequence encoding the desired hIL2 orthogonal homolog polypeptide (optionally including a secretory leader sequence or signal peptide) is introduced into an expression vector into the cells to be engineered. The nucleic acid sequence is operatively linked to one or more expression control sequences encoded by the vector and functioning in the target host cell. If a secretory leader sequence (signal peptide) is incorporated into the polypeptide, the recombinant hIL2 orthogonal homolog can be recovered by disrupting the host cell or from the cell medium. The recombinant hIL2 orthogonal homolog can be purified and concentrated for further uses, including incorporation. The recombinant production process of hIL2 peptides is known in the art and described in Fernandes and Taforo, U.S. Patent No. 4,604,377, issued August 5, 1986, and hIL2 orthologs described in Mark et al., U.S. Patent No. 4,512,584, issued May 21, 1985, and Gillis, U.S. Patent No. 4,401,756, issued August 30, 1983, the entire teachings of which are incorporated herein by reference.

[0707] DNA encoding hIL2 orthologs can be obtained from a variety of sources as designed during the engineering process. As described herein, amino acid sequence variants of the hIL2 polypeptide that produce the hIL2 orthologs of the present invention are prepared by introducing appropriate nucleotide changes into the coding sequence. Such variants represent insertions, substitutions, and / or specific deletions of the indicated residues. Any combination of insertions, substitutions, and / or specific deletions can yield the final construct, provided that the final construct possesses the desired biological activity as defined herein.

[0708] Construction of nucleic acid sequences encoding hIL2 orthogonal homologs

[0709] In some implementations, hIL2 orthogonal homologs are produced via recombination methods using nucleic acid sequences encoding hIL2 orthogonal homologs (or fusion proteins containing hIL2 orthogonal homologs). The nucleic acid sequences encoding the desired hIL2 orthogonal homologs can be chemically synthesized using an oligonucleotide synthesizer.

[0710] Nucleic acid sequences are not limited to sequences encoding polypeptides; they may also include partial or complete non-coding sequences located upstream or downstream of a coding sequence (e.g., the coding sequence for hIL2). Those skilled in molecular biology are familiar with the routine procedures used to isolate nucleic acid molecules. For example, they can be produced by treating genomic DNA with restriction endonucleases or by performing a polymerase chain reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0711] Nucleic acid molecules (and their fusions) encoding hIL2 orthologs can contain naturally occurring sequences or sequences different from naturally occurring sequences, but due to the degeneracy of the genetic code, they encode the same polypeptide. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis) or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, nucleic acid molecules can be double-stranded or single-stranded (i.e., sense or antisense strands).

[0712] The nucleic acid sequences encoding hIL2 orthologs can be obtained from a variety of commercially available sources that provide customized nucleic acid sequences. Amino acid sequence variants of the hIL polypeptides producing the hIL2 orthologs of this invention are prepared by incorporating suitable nucleotide changes into the coding sequence based on genetic codes well known in the art. Such variants represent insertions, substitutions, and / or specific deletions of the indicated residues. Any combination of insertions, substitutions, and / or specific deletions can yield the final construct, provided that the final construct possesses the desired biological activity as defined herein.

[0713] Methods for constructing DNA sequences encoding hIL2 orthogonal homologs and expressing these sequences in suitable transformed hosts include, but are not limited to, the use of PCR-assisted mutagenesis. Mutations consisting of deletions or additions of amino acid residues can also be created in the hIL2 polypeptide using standard recombinant techniques. If deletions or additions are made, the nucleic acid molecule encoding hIL2 is optionally digested with a suitable restriction endonuclease. The resulting fragment can be expressed directly or further manipulated, for example, by ligation to a second fragment. Ligation can be facilitated if the two ends of the nucleic acid molecule contain overlapping complementary nucleotides, but blunt-ended fragments can also be ligated. PCR-generated nucleic acids can also be used to generate a variety of mutant sequences.

[0714] The hIL2 orthogonal homolog of the present invention can be produced not only through direct recombinant production 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 a mature hIL2 orthogonal homolog). Typically, the signal sequence can be a component of a vector or a portion of a coding sequence inserted into the vector. The selected heterologous signal sequence is preferably a sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In some embodiments, the signal sequence is a signal sequence naturally associated with the hIL2 orthogonal homolog (i.e., a human IL2 signal sequence). The inclusion of the signal sequence depends on whether it is required to secrete the hIL2 orthogonal homolog from the prepared recombinant cells. If the selected cells are prokaryotic cells, it is generally preferred that the DNA sequence does not encode the signal sequence. If the selected cells are eukaryotic cells, it is generally preferred that a signal sequence is encoded, with wild-type IL2 signal sequences being most preferred. Alternatively, heterologous mammalian signal sequences are also suitable, such as signal sequences of secretory polypeptides from the same or related species, and viral secretion leader sequences, such as the herpes simplex gD signal. When the recombinant host cell is a yeast cell, such as Saccharomyces cerevisiae, an alpha mating factor secretion signal sequence can be used to achieve the extracellular secretion of IL2 orthologs into the culture medium, as described in Singh, U.S. Patent No. 7,198,919B1, issued April 3, 2007.

[0715] Codon optimization:

[0716] In some implementations, the nucleic acid sequence encoding the recombinant protein (IL2 orthogonal homolog, orthogonal hCD122, or CAR) can be "codon-optimized" to facilitate expression in a specific host cell type. Techniques for codon optimization in various expression systems, including mammalian, yeast, and bacterial host cells, are well-known in the art, and online tools are available to provide codon-optimized sequences for expression in multiple host cell types. See, for example, Hawash et al. (2017) 9:46-53 and Mauro and Chappell in... Recombinant Protein Expression in Mammalian Cells: Methods and Protocols

[0717] (RecombinantProteinExpressioninMammalianCells:Methods andProtocols), Edited by David Hacker (Human Press, New York). In addition, several web-based online software programs are available free of charge to assist in the preparation of codon-optimized nucleic acid sequences.

[0718] Vehicle for expression:

[0719] After assembly (by synthesis, site-directed mutagenesis, or another method), the nucleic acid sequence encoding the hIL2 orthogonal homolog is inserted into an expression vector. Multiple expression vectors for various host cells can be used, typically based on the host cell chosen for expression. Vector components typically include, but are not limited to, one or more of the following: origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Vectors include viral vectors, plasmid vectors, integrative vectors, etc. Plasmids are examples of non-viral vectors. To promote transfection of target cells, target cells can be directly exposed to non-viral vectors under conditions favorable to their uptake. Examples of conditions that promote the uptake of exogenous nucleic acids by mammalian cells are well known in the art, including but not limited to chemical methods (e.g., Thermo-Fisher Scientific, high salt and magnetic field (electroporation).

[0720] hIL2 orthogonal homologs can be produced not only through direct recombinant synthesis but also as fusion peptides with heterologous peptides (e.g., signal sequences or other peptides with specific cleavage sites at the N-terminus of mature proteins or peptides). Typically, the signal sequence can be a component of the vector or a portion of the coding sequence inserted into the vector. The selected heterologous signal sequence is preferably a sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In mammalian cell expression, native signal sequences or other mammalian signal sequences are suitable, such as signal sequences of secretory peptides from the same or related species, and viral secretion leaders, such as the herpes simplex gD signal.

[0721] Selectable markers

[0722] Expression vectors typically contain a select gene, also known as a selectable marker. This gene encodes a protein essential for the survival or growth of transformed host cells in selective media. Host cells not transformed with a vector containing a select gene cannot survive in the medium. A typical select gene encodes a protein that will (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for auxotrophic deficiencies; or (c) provide a key nutrient that is not available from a complex culture medium.

[0723] Regulatory sequence:

[0724] The expression vector for the hIL2 orthogonal homolog used in this invention comprises a regulatory sequence that is recognized by the host organism and operatively linked to a nucleic acid sequence encoding the hIL2 orthogonal homolog. The terms “regulatory sequence,” “regulatory sequence,” or “expression control sequence” are used interchangeably herein to refer to promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990), *Gene Expression Technology: Methods in Enzymology*, 185 (Academic Press, San Diego, California, USA). Regulatory sequences include nucleotide sequences that guide constitutive expression in many types of host cells and nucleotide sequences that guide expression only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of expression vectors may depend on factors such as, for example, the choice of host cells to be transformed, the desired protein expression level, etc. In the selection of expression control sequences, those skilled in the art will understand that a variety of factors will be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the host hIL2 ortholog, particularly regarding potential secondary structures.

[0725] promoter

[0726] In some implementations, the regulatory sequence is a promoter, the selection of which is based, for example, on the cell type in which expression is sought. The expression vector will contain a promoter recognized by the host organism and operatively linked to an orthogonal protein-coding sequence. A promoter is a non-translated sequence (typically within about 100 to 1000 bp) located upstream (5') of the start codon of a structural gene that controls the transcription and translation of a specific nucleic acid sequence operatively linked to it. Such promoters are generally classified into two categories: inducible and constitutive. Inducible promoters are promoters that respond to certain changes in culture conditions (such as the presence or absence of nutrients or changes in temperature), increasing the level of transcription from the DNA they control. A large number of promoters recognized by a variety of potential host cells are well known. For example, the T7 promoter can be used in bacteria, the polyhedrosis protein promoter can be used in insect cells, and the cytomegalovirus or metallothionein promoter can be used in mammalian cells. In addition, tissue-specific and cell-specific promoters are widely used in the case of higher eukaryotic cells. These promoters are named for their ability to direct the expression of nucleic acid molecules in a given in vivo tissue or cell type. Technicians are familiar with many promoters and other regulatory elements that can be used to direct the expression of nucleic acids.

[0727] Transcription of vectors in mammalian host cells can be controlled by promoters, for example, derived from viral genomes such as polyomavirus, vaccinia virus, adenovirus (e.g., human adenovirus serotype 5), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses (e.g., mouse stem cell virus), hepatitis B virus, and most ideally simian virus 40 (SV40); promoters from heterologous mammals such as actin promoters, PGK (phosphoglycerate kinase) or immunoglobulin promoters; and promoters from heat shock, provided these promoters are compatible with the host cell system. Early and late promoters of SV40 virus are readily available as SV40 restriction fragments, which also contain the SV40 virus's origin of replication.

[0728] enhancer

[0729] In higher eukaryotes, transcription is typically enhanced by inserting enhancer sequences into a vector and operatively linking them to a nucleic acid sequence encoding a direct homolog of hIL2. Enhancers are cis-acting elements of DNA, typically 10 to 300 bp in length, that act on the promoter to increase its transcription. The orientation and location of enhancers are relatively independent, found at the 5' and 3' of the transcription unit, within introns, and within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic viruses are commonly used. Examples include the SV40 enhancer post-OMI, the cytomegalovirus early promoter enhancer, the polyoma enhancer post-OMI, and the adenovirus enhancer. Enhancers can be spliced ​​into the 5' or 3' position of the coding sequence in the expression vector, but are preferably located 5' from the promoter. Expression vectors for eukaryotic host cells will also contain sequences necessary for terminating transcription and for stabilizing mRNA. These sequences can typically be obtained from the 5', and occasionally 3', untranslated regions of eukaryotic or viral DNA or cDNA. The construction of suitable vectors containing one or more of the above components employs standard techniques.

[0730] In addition to sequences that promote transcription of the inserted nucleic acid molecule, the vector may contain genes encoding origins of replication and other optional markers. For example, the neomycin resistance (neoR) gene confers resistance to G418 on its expressing cells, thus allowing phenotypic selection of transfected cells. Other examples of markers or reporter genes include β-lactamases, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-β-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine-guanine phosphoribosyltransferase (XGPRT). Those skilled in the art can readily determine whether a given regulatory element or optional marker is suitable or unsuitable for a particular expression environment.

[0731] The correct assembly of expression vectors can be confirmed by nucleic acid sequencing, restriction mapping, and expression of bioactive peptides in a suitable host.

[0732] Host cells used to produce hIL2 orthologs

[0733] In one embodiment, the invention further includes recombinant cells comprising a nucleic acid sequence encoding an hIL2 orthogonal homolog. The cells may be prokaryotic or eukaryotic. The cells of the invention are transfected cells, i.e., cells in which nucleic acid molecules have been introduced, for example, by introducing nucleic acid molecules encoding a mutant hIL2 polypeptide using recombinant DNA technology. Progeny of such cells are also considered to be within the scope of the invention.

[0734] Host cells for expressing hIL2 orthologs are typically selected based on their compatibility with the chosen expression vector, the toxicity of the product encoded by the DNA sequence of this invention, their secretory properties, their ability to correctly fold polypeptides, their fermentation or culture requirements, and the ease of purification of the product encoded by the DNA sequence. Suitable host cells for cloning or expressing the DNA in the vectors described herein are the aforementioned prokaryotic cells, yeast, or higher eukaryotic cells.

[0735] In some implementations, the host cell used for the recombinant production of hIL2 orthologs is a eukaryotic cell, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (e.g., baculovirus vectors for protein expression in cultured insect cells, such as 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 (e.g., vectors for expression in Saccharomyces cerevisiae (including pYepSecl) (Baldari et al. (1987) EMBOJ. 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, California) and pPicZ (Ingenie, San Diego, California)); or mammalian cells (mammalian expression vectors including pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.6:187:195)).

[0736] Examples of useful mammalian host cell lines include: mouse L cells (LM[TK-], ATCC CRL-2648); monkey kidney CV1 cell line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cell subclones, grown in suspension culture); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); and human lung cells (W138, ATCC CCL 1442). 75); human hepatocytes (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2).

[0737] In some implementations, the resulting hIL2 ortholog will be glycosylated or unglycosylated, depending on the host organism used to produce the mutant protein. If bacteria are chosen as the host, the produced hIL2 ortholog will be unglycosylated. On the other hand, eukaryotic cells will glycosylate the hIL2 ortholog, perhaps in a different manner than native IL2 glycosylation.

[0738] For other expression systems used in prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY). See also Goeddel (1990), *Gene Expression Technology: Methods in Enzymology*, p. 185 (Academic Press, San Diego, CA).

[0739] Transfection:

[0740] The nucleic acid expression construct encoding the hIL2 orthogonal homolog was introduced into host cells to produce the hIL2 orthogonal homolog disclosed herein, or to produce its biologically active mutant protein. The vector DNA can be introduced into prokaryotic or eukaryotic cells using 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* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY), and other standard molecular biology laboratory manuals.

[0741] To promote the transfection of target cells, the target cells can be directly exposed to a non-viral vector under conditions that favor the uptake of the non-viral vector. Examples of conditions that promote the uptake of exogenous nucleic acids by mammalian cells are well known in the art, including but not limited to chemical methods (e.g., Thermo-Fisher Scientific, high salt and magnetic field (electroporation).

[0742] Cell culture:

[0743] Cells can be cultured in conventional nutrient media modified to be suitable for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. Mammalian host cells can 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 DMEM (DSigma) are suitable for culturing host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or equivalent energy sources. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used for selecting expression host cells and are obvious to those skilled in the art.

[0744] Recombinant protein recovery:

[0745] If a secretory leader sequence is used, the recombinantly produced hIL2 ortholog peptide can be recovered from the culture medium as a secreted peptide. Alternatively, the hIL2 ortholog peptide can also be recovered from host cell lysates. Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), can be used in the recovery stage from cell lysates to inhibit protease degradation during purification and may include antibiotics to prevent the growth of exogenous contaminants. In some embodiments, the hIL2 ortholog is produced in *E. coli*, wherein overexpression of the hIL2 ortholog is isolated in inclusion bodies. Techniques for isolating and lysing inclusion bodies and recovering the active protein are well known in the art.

[0746] Various purification steps are known in the art and are available, such as affinity chromatography. Affinity chromatography utilizes highly specific binding sites commonly found in biological macromolecules to separate them based on their ability to bind to specific ligands. Covalent bonds attach ligands to an insoluble porous support medium, making the ligands readily visible on the protein sample, thus utilizing the natural specific binding of one type of molecule to separate and purify a second type from the mixture. Antibodies are commonly used in affinity chromatography. Size selection steps can also be used, for example, gel filtration chromatography (also known as size exclusion or molecular sieve chromatography) for separating proteins based on their size. In gel filtration, a protein solution is passed through a column filled with a semi-permeable porous resin. The semi-permeable resin has a range of pore sizes that determines the size of proteins that can be separated using the column.

[0747] Orthogonal hIL2 orthohomogeneous receptors can be concentrated, filtered, dialyzed, etc., using methods known in the art. For therapeutic applications, hIL2 orthohomogeneous receptors can be administered to mammals containing suitable engineered orthogonal receptors. Administration can be intravenous, by bolus injection or by continuous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes. Orthogonal hIL2 orthohomogeneous receptors can also be suitably administered intratumorally, adjacent to, within, or around the lesion, or via the lymphatic system to exert local and systemic therapeutic effects.

[0748] The pathway for the administration of hIL2 orthologs:

[0749] In embodiments of the therapeutic method of the present invention, administration includes to a subject requiring treatment a pharmaceutical preparation comprising an hIL2 orthogonal homolog (and / or a nucleic acid encoding an hIL2 orthogonal homolog). Administration may be administered intravenously, either by bolus injection or by continuous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes. The hIL2 orthogonal homolog may also be suitably administered intratumorally, adjacent to, within the lesion, intranodally, or perilesionally, or via the lymphatic system to exert local and systemic therapeutic effects.

[0750] In some embodiments, the main hIL2 orthogonal homolog (and / or the nucleic acid encoding the hIL2 orthogonal homolog) may be incorporated into the composition, including a pharmaceutical composition. Such compositions typically comprise a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. The pharmaceutical composition is prepared to be compatible with the intended route of administration and to therapeutic use for administering the hIL2 orthogonal homolog to a subject requiring treatment or prevention.

[0751] formulations of hIL2 direct homologs

[0752] In some embodiments, the present invention provides pharmaceutically acceptable formulations of hIL2 orthologs. Preferred formulations depend on the intended route of administration and therapeutic application. Pharmaceutically acceptable formulations of hIL2 orthologs include physiologically acceptable carriers that are non-toxic and non-therapeutic in themselves. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers such as phosphates, sucrose, glycine, sorbic acid, potassium sorbate, partial glycerol ester mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, and PEG. Local or gel-based carriers of peptides include polysaccharides such as sodium carboxymethyl cellulose or methyl cellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block copolymers, PEG, polyamino acids, amino acid copolymers, and lipid aggregates (e.g., oily droplets or liposomes).

[0753] The formulation may also include pharmaceutically acceptable, non-toxic carriers, excipients, stabilizers, or diluents, defined as carriers commonly used in formulating pharmaceutical compositions for administration to animals or humans. Diluents are selected to not affect the biological activity of the combination. Acceptable carriers, excipients, or stabilizers, at the doses and concentrations used, are non-toxic to the recipient and include: buffers such as phosphates, citrates, and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexahydroquinone quaternary ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butyl or benzyl alcohol; alkyl p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). Polypeptides (containing 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Alternatively, polyethylene glycol (PEG). Formulations intended for in vivo administration are typically sterile. Sterilization of the compositions of this invention can be readily achieved through sterile membrane filtration.

[0754] Typically, formulations are prepared as injectable, liquid solutions or suspensions; they can also be prepared in solid forms suitable for placement in a liquid carrier prior to injection. The preparation can also be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolic acid, or copolymers, to enhance adjuvant effects, as described above. (Langer, Science (1990) 249:1527 and Hanes, Advanced Drug Delivery Reviews (1997) 28:97-119). The reagents of the present invention can be administered in the form of depot injections or implantable formulations, formulated to allow for sustained or pulsatile release of the active ingredient. Pharmaceutical compositions are typically formulated as sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practices (GMP) regulations of the U.S. Food and Drug Administration.

[0755] In some embodiments, the method of the present invention includes parenteral administration of hIL2 directly to its homolog. Examples of parenteral routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), mucosal, and rectal administration. Parenteral formulations include solutions or suspensions for parenteral application and may include carriers and buffers. Pharmaceutical formulations suitable for parenteral administration include sterile aqueous solutions (when water-soluble) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In one embodiment, the formulation is provided in a pre-filled syringe for parenteral administration.

[0756] Oral formulations typically include one or more inert diluents and / or edible carriers. For oral therapeutic purposes, hIL2 orthologs can be incorporated into excipients and administered in tablet, lozenge, or capsule form, such as gelatin capsules. Oral compositions can also be prepared using liquid carriers for use as mouthwashes. Pharmaceutically compatible binders and / or adjuvants may be included as part of the formulation. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginate, carboxymethyl starch (Primogel™), or corn starch; lubricants such as magnesium stearate or fully hydrogenated vegetable oil (Sterotes™); gliding agents such as silica gel; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0757] In the case of administration by inhalation, the main hIL2 direct homolog or the nucleic acid encoding it is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant (e.g., carbon dioxide gas) or a spray. Such methods include those described in U.S. Patent No. 6,468,798.

[0758] The host hIL2 or nucleic acid can also be administered systemically via mucosal or transdermal formulations. For mucosal or transdermal administration, a permeabilizer suitable for the permeation barrier is used in the formulation. Such permeabilizers are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used for mucosal administration. Mucosal administration can be performed by preparing the compound for rectal delivery using nasal sprays or suppositories (e.g., using conventional suppository bases such as cocoa butter or other glycerides) or retention enemas. For transdermal administration, the active compound is formulated as an ointment, ointment, gel, or cream, as is generally known in the art, and may incorporate a penetration enhancer such as ethanol or lanolin.

[0759] In some embodiments, the hIL2 ortholog formulation is an extended-release formulation to provide prolonged delivery of the hIL2 ortholog reagent over a period of hours or days. Examples of extended-release formulations of injectable compositions can be achieved by including agents with delayed absorption (such as aluminum monostearate and gelatin) in the composition. In one embodiment, the host hIL2 ortholog or nucleic acid is prepared using a carrier that will protect the mutated IL-2 peptide from rapid clearance by the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. Liposome suspensions can also be used as pharmaceutically acceptable carriers.

[0760] Engineered hoCD122 cells:

[0761] In the practice of this invention, the preparation of useful recombinant cells is achieved by transformation and isolation using an expression vector containing a nucleic acid sequence encoding the hCD122 orthogonal receptor. The hIL2 orthogonal homolog of this invention can be applied to methods for selectively amplifying such engineered hoRb cells (e.g., human T cells) engineered to express the corresponding orthogonal hCD122 receptor. T cells used for engineering with the constructs described herein include naive T cells, central memory T cells, effector memory T cells, or combinations thereof. The aforementioned T cells for engineering are collected from a subject or donor and can be isolated from a cell mixture using techniques for enriching the desired cells, or can be engineered and cultured without isolation. Alternatively, the T cells for engineering can be isolated from other cells. Techniques providing precise isolation include fluorescence-activated cell sorting. Dead cells can be selected using a dye associated with dead cells (e.g., propidium iodide). The isolated cells can be collected in any suitable culture medium to maintain cell viability, typically with a serum pad at the bottom of the collection tube. A variety of commercially available culture media can be used depending on the cell type, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., usually supplemented with fetal bovine serum (FCS). Collected and optionally enriched cell populations can be used immediately for genetic modification or frozen and stored at liquid nitrogen temperature, and can be reused after thawing. Cells are typically stored in 10% DMSO, 50% FCS, and 40% RPMI 1640 medium.

[0762] In some implementations, the engineered cells comprise a complex mixture of immune cells, such as tumor-infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, for example, Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer”; Feldman et al. (2015) Seminars in Oncol. 42(4):626-39, “Adoptive Cell Therapy - Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01174121, “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641-645, "Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer".

[0763] CAR-T cells

[0764] In one embodiment of the invention, the hoRb cell is a T-cell (e.g., a human T-cell) modified to express a chimeric antigen receptor (“hoCAR-T cell”) on its surface. As used herein, the term antigen-binding domain (ABD) refers to a polypeptide that specifically binds to an antigen expressed on the surface of a target cell. The ABD can be any polypeptide that can specifically bind to one or more antigens expressed on the surface of a target cell. The CAR further includes a transmembrane domain that attaches the ABD (or a linker, if used) to the intracellular cytoplasmic domain of the CAR. The transmembrane domain consists of any polypeptide sequence that is thermodynamically stable in the eukaryotic cell membrane. The transmembrane domain can be derived from the transmembrane domain of a naturally occurring transmembrane protein or it can be synthetic. When designing a synthetic transmembrane domain, amino acids that favor an α-helical structure are preferred. The transmembrane domain used to construct the CAR consists of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, or 24 amino acids that favor the formation of an α-helical secondary structure. Amino acids with a favorable α-helical conformation are well known in the art. See, for example, Pace et al. (1998) Biophysical Journal 75:422-427. Amino acids particularly favorable for the α-helical conformation include methionine, alanine, leucine, glutamic acid, and lysine. In some embodiments, the CAR transmembrane domain may be derived from the transmembrane domain of a type I transmembrane protein, such as CD3ζ, CD4, CD8, CD28, etc.

[0765] The cytoplasmic domain (ICD) of a CAR peptide includes one or more intracellular signaling domains. In one embodiment, the intracellular signaling domain includes the cytoplasmic sequence of a T-cell receptor (TCR) and a co-receptor that initiate signal transduction upon antigen-receptor binding, as well as their functional derivatives and subfractions. Cytoplasmic signaling domains, such as those derived from the ζ-chain of the T-cell receptor, are used as part of the CAR to generate signals that stimulate T lymphocyte proliferation and effector function upon binding of the chimeric receptor to the target antigen. Examples of cytoplasmic signal transduction domains include, but are not limited to, the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also known as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also known as ICOS), the p110α, β, or δ catalytic subunits of PI3 kinase, the human CD3 ζ-chain, the cytoplasmic domain of CD134 (also known as OX40 and TNFRSF4), the FcεR1γ and β chains, the MB1 (Igα) chain, the 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.

[0766] In some implementations, the CAR also provides a co-stimulatory domain. The term "co-stimulatory domain" refers to the stimulatory domain of the CAR, typically an intracellular domain, which provides a secondary nonspecific activation mechanism through which primary specific stimulation is propagated. A co-stimulatory domain refers to a portion of the CAR that enhances the proliferation, survival, or development of memory cells. Examples of co-stimulation include antigen-nonspecific T cell co-stimulation following antigen-specific signal transduction via T cell receptors, and antigen-nonspecific B cell co-stimulation following signal transduction via B cell receptors. Co-stimulation, such as T cell co-stimulation, and the factors involved have been described in Chen and Flies. (2013) Nat RevImmunol 13(4):227-42. In some embodiments of the present invention, CSD includes one or more TNFR superfamily members, CD28, CD137(4-1BB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1(CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or combinations thereof.

[0767] CARs are commonly referred to as first-generation, second-generation, third-generation, or fourth-generation. The term first-generation CAR refers to a CAR in which the cytoplasmic domain transmits signals from antigen binding via a single signal transduction domain, such as the signal transduction domain of a high-affinity receptor from the CD3ζ chain or IgEFcεR1γ. This domain contains one or three immune receptor tyrosine-based activation motifs (ITAMs) for antigen-dependent T cell activation. ITAM-based activation signals confer the ability of T cells to lyse target tumor cells and secrete cytokines in response to antigen binding. Second-generation CARs, in addition to CD3ζ signaling, include co-stimulatory signals. The co-delivery of these co-stimulatory signals enhances cytokine secretion and CAR-transduced T cell-induced antitumor activity. The co-stimulatory domain is typically located proximal to the membrane relative to the CD3ζ domain. Third-generation CARs include trigonometric signal transduction domains, such as those comprising CD28, CD3ζ, OX40, or 4-1BB signal transduction regions. In the fourth generation, or "armored car" CAR T cells, they are further modified to express or block molecules and / or receptors to enhance immune activity, such as expressing IL-12, IL-18, IL-7 and / or IL-10; 4-1BB ligand, CD-40 ligand.

[0768] Examples of intracellular signal transduction domains that can be incorporated into hoCAR-T cells in the practice of this invention include (amino to carboxyl): CD3ζ; CD28–41BB-CD3ζ; CD28–OX40–CD3ζ; CD28–41BB–CD3ζ; 41BB–CD-28–CD3ζ and 41BB–CD3ζ.

[0769] The term CAR also includes CAR variants, including but not limited to splitting CAR, on-off CAR, bispecific or tandem CAR, repressive CAR (iCAR) and induced pluripotent stem (iPS) CAR-T cells.

[0770] The term "split CAR" refers to a CAR in which the extracellular portion, ABD, and cytoplasmic signal transduction domains of the CAR reside on two separate molecules. CAR variants also include on-switch CARs, which are conditionally activatable CARs, such as split CARs, in which the conditional heterodimers of the two parts of the split CAR are pharmacologically controlled. CAR molecules and their derivatives (i.e., CAR variants) have been described, for example, in PCT applications US2014 / 016527, US1996 / 017060, US2013 / 063083; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla and Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. CancerDiscov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; their published contents are included in this article by citation.

[0771] The term "bispecific or tandem CAR" refers to a CAR that contains a secondary CAR-binding domain that can amplify or inhibit the activity of the primary CAR.

[0772] The terms “inhibitory chimeric antigen receptor” or “iCAR” are used interchangeably herein and refer to a CAR in which binding to an iCAR utilizes dual antigen targeting. By binding to a second inhibitory receptor equipped with an inhibitory signal transduction domain of the secondary CAR binding domain, activation of the active CAR is shut down, resulting in inhibition of primary CAR activation. Inhibitory CARs (iCARs) are designed to modulate CAR-T cell activity through activation of an inhibitory receptor signal transduction module. This approach combines the activity of two CARs, one of which generates a dominant negative signal that limits the response of CAR-T cells activated by the activating receptor. When an iCAR binds to a specific antigen expressed only by normal tissue, it can shut down the antagonist CAR response. In this way, iCAR-T cells can distinguish between cancer cells and healthy cells and reversibly block the function of transduced T cells in an antigen-selective manner. The CTLA-4 or PD-1 intracellular domain in the iCAR triggers inhibitory signaling on T lymphocytes, leading to reduced cytokine production, decreased target cell lysis efficiency, and altered lymphocyte motility.

[0773] The term "tandem CAR" or "TanCAR" refers to a CAR that mediates bispecific activation of T cells through the conjugation of two chimeric receptors designed to deliver stimulatory or co-stimulatory signals in response to independent conjugation of two different tumor-associated antigens.

[0774] Typically, chimeric antigen receptor T-cells (CAR-T cells) are recombinant modified T cells that are transduced with an expression vector encoding CAR, based on the teachings above.

[0775] In some implementations, the hoCAR-T cells are allogeneic to the individual being treated. Graham et al. (2018) Cell 7(10) E155. In some implementations, the allogeneic engineered T cells are perfectly HLA matched. However, not all patients have perfectly matched donors, and HLA-type-independent cell products suitable for all patients offer an alternative.

[0776] Because hoCAR-T cells are derived from the recipient's own T cells, the cell population to be administered to the recipient will inevitably be variable, and the response to this agent may vary. Therefore, continuous monitoring and management of treatment-related toxicities are necessary, including management through pharmacological immunosuppression or B-cell depletion courses prior to administration of the hoCAR-T cell product. Typically, at least 1x10-1 6 1 x 10 CAR-T cells / kg, at least 1 x 10 7 1 x 10 CAR-T cells / kg, at least 1 x 10 8 1 x 10 CAR-T cells / kg, at least 1 x 10 91 x 10 CAR-T cells / kg, at least 1 x 10 10 The number of hoCAR-T cells per kg or more is typically limited by the number of T cells obtained during collection. Engineered hoCAR-T cells can be infused into the subject in any physiologically acceptable medium via any convenient route of administration, usually intravascular, although they can also be introduced via other routes where the cells can find suitable sites for growth.

[0777] If the hoCAR-T cells are allogeneic T cells, these cells can be modified to reduce graft-versus-host disease. For example, the engineered cells of this invention can be TCRαβ receptor knockout achieved through gene editing technology. TCRαβ is a heterodimer, and both the α and β chains are required for expression. One gene encodes the α chain (TRAC), and two genes encode the β chain; therefore, the TRAC locus is deleted for this purpose. Many different methods have been used to accomplish this deletion, such as CRISPR / Cas9; meganuclease; engineered I-CreI homing endonuclease, etc. See, for example, Eyquem et al. (2017) Nature 543:113–117, where the TRAC coding sequence is replaced by the CAR coding sequence; and Georgiadis et al. (2018) Mol. Ther. 26:1215–1227, which linked CAR expression with TRAC disruption through regularly clustered, spaced short palindromic repeats (CRISPR) / Cas9, rather than directly incorporating CAR into the TRAC locus. Alternative strategies to prevent GVHD include modifying T cells to express inhibitors of TCRαβ signaling, such as using a truncated form of CD3ζ as a TCR repressor molecule.

[0778] In some embodiments, the present invention provides a method for selectively expanding a population of engineered cells expressing the orthogonal hCD122 receptor from a mixed cell population, the method comprising contacting the mixed cell population with the hIL2 orthogonal homolog of the present invention under conditions that promote the expansion of the engineered cells. In one embodiment, when CAR-T cells expressing the orthogonal hCD122 receptor are used, the orthogonal receptor-expressing CAR-T cells are also selectively expanded from a background or mixed transduced and non-transduced cell population by using the hIL2 orthogonal homolog described herein. Expansion of T cells for therapeutic applications typically involves culturing cells and surface contacting them with a reagent that provides a signal associated with stimulating the CD3 TCR complex and a reagent that stimulates co-stimulatory molecules on the surface of T cells. In routine practice, engineered T cells are stimulated prior to administration of cell therapy products by contacting them with CD3 / D28, particularly in the preparation of CAR-T cells for clinical applications. A variety of or commercially available products can be used to promote bead-based T cell activation, including but not limited to… CTS CD3 / 28 (Life Technologies, Inc., Carl Pasteur, California) or Miltenyi GMPExpAct Treg beads, or Miltenyi MACS GMP TransAct TM CD3 / 28 beads (Miltenyi Biotec, Inc.). Suitable conditions for T cell culture are well known in the art. Lin, et al. (2009) Cytotherapy 11(7):912-922; Smith, et al. (2015) Clinical & Translational Immunology 4:e31, published online January 16, 2015. Target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2), wherein a mixed cell population containing engineered T cells expressing the hCD122 orthogonal receptor is cultured in the presence of a certain concentration of hIL2 orthogonal homolog. In some embodiments, the cells are cultured for at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 6 hours, or at least 8 hours, or at least 12 hours, or at least 24 hours, or at least 48 hours, or at least 72 hours, or more. In this in vitro setting, the concentration of hIL2 orthogonal homologs is sufficient to induce cell proliferation in this cell population. T cell proliferation can be readily assessed using microscopic methods, and determining the optimal concentration of hIL2 orthogonal homologs will depend on the relative activity of the hIL2 orthogonal homologs against the orthogonal hCD122 receptor.

[0779] In one embodiment, the present invention provides a method for preparing an engineered cell product substantially enriched with engineered cells expressing hoCD122, the method comprising the steps of: (a) obtaining a biological sample containing T cells; (b) contacting the biological sample with a recombinant vector encoding the hoCD122 receptor; and (c) contacting the biological sample with an hIL2 orthogonal homolog for a period of time sufficient to amplify the engineered cells expressing hoCD122. The duration of contact and culture can be varied depending on the degree of population enrichment.

[0780] When cells are exposed to an hIL2 orthogonal homolog in vitro, the hIL2 orthogonal homolog is added to engineered cells at a dose and duration sufficient to activate signal transduction from the hoCD122 receptor, which can utilize natural cellular mechanisms such as accessory proteins, co-receptors, etc. Any suitable culture medium can be used. Cells thus activated can be used for any desired purpose, including experimental purposes related to determining antigen specificity, cytokine profiling, and in vivo delivery.

[0781] When administered in vivo, an effective dose of engineered cells, including but not limited to CAR-T cells modified to express the orthogonal hoCD122 receptor, is combined with orthogonal cytokines (e.g., IL-2) administered to the recipient or infused prior to administration, allowing contact with T cells in their native environment, such as in lymph nodes. Dosage and frequency may vary depending on the reagent; route of administration; and the nature of the hIL2 orthogonal homolog. Those skilled in the art will understand that this guideline will be adapted to individual circumstances. Dosage may also vary depending on the route of administration, such as intramuscular, intraperitoneal, intradermal, subcutaneous, or intravenous injection. Typically, at least approximately 10 [units of measurement are needed]. 4 One engineered cell / kg, at least approximately 10 5 One engineered cell / kg, at least approximately 10 6 One engineered cell / kg, at least approximately 10 7 One engineered cell / kg, or more.

[0782] When the engineered cells modified to express hoCD122 are T cells, the enhanced immune response can manifest as an increased T cell cytolysis response to target cells present in the recipient, such as the elimination of tumor cells, infected cells; a reduction in symptoms of autoimmune diseases; and so on. In some embodiments, when the engineered T cell population is administered to the subject, an immunosuppressive regimen is provided to the subject before or in combination with the administration of the engineered T cell population. Examples of such immunosuppressive regimens include, but are not limited to, systemic corticosteroids (e.g., methylprednisolone). Treatment for B cell depletion includes intravenous immunoglobulin (IVIG) according to established clinical dosing guidelines to restore normal serum immunoglobulin levels. In some embodiments, the subject optionally undergoes a lymphatic drainage regimen before receiving the CAR-T cell therapy of the present invention. An example of such a lymphatic drainage regimen consists of administering fludarabine (30 mg / m² daily) to the subject. 2 Administered intravenously for 4 days) and cyclophosphamide (starting with the first dose of fludarabine, 500 mg / m² daily). 2 IV, lasting 2 days).

[0783] Engineered hoCD122 cells can be provided in pharmaceutical compositions suitable for therapeutic use (e.g., for human treatment). Therapeutic formulations containing these cells can be frozen or prepared for administration using physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. Ed. (1980)) in the form of an aqueous solution. hoCD122 cells will be formulated, dosed, and administered in accordance with good medical practice. Considerations in this regard include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, etiology, site of reagent delivery, method of administration, administration schedule, and other factors known to the medical professional.

[0784] hoCD122 cells can be administered via any suitable route, typically parenteral. Parenteral infusion includes intramuscular, intravenous (bolus or slow infusion), intra-arterial, intraperitoneal, intrathecal, or subcutaneous administration. In typical practice, engineered T cells can be infused into the subject in a physiologically acceptable medium, usually intravascular, although they can also be introduced at any other convenient site where the cells can find a suitable growth site. Typically, at least 1 x 102 cells will be administered. 5 Cells / kg, at least 1x10 6 Cells / kg, at least 1x10 7 Cells / kg, at least 1x10 8 Cells / kg, at least 1x10 9 Cells / kg or more, typically limited by the number of T cells obtained during collection.

[0785] For example, in practice using this method, a typical dosing range for cells modified to express the orthogonal hCD122 receptor is approximately 1 x 10-1 per kg of subject body weight per treatment cycle. 5 Up to 5x10 8 One live cell. Therefore, after adjusting for weight, the typical range of live cells administered to a patient per treatment course is approximately 1 x 102. 6 To approximately 1x10 13 One living cell, or approximately 5 x 10 6 Approximately 5x10 12 One living cell, or approximately 1 x 102 7 To approximately 1x10 12 One living cell, or approximately 5 x 10 7 Up to approximately 1x10 12 One living cell, or approximately 1 x 102 8 Up to approximately 1x10 12 One living cell, or approximately 5 x 10 8 Up to approximately 1x1012 One living cell, or approximately 1 x 102 9 Up to approximately 1x10 12 10 live cells. In one embodiment, the dose of cells per treatment cycle is 2.5-5 x 10⁻⁶. 9 Within the range of living cells.

[0786] Treatment with hIL2 orthologs and / or hoCD122 cells may include a single dose or multiple doses over a period of time. In some embodiments, hoCD122 cells are administered in a single dose. In some embodiments, hoCD122 cells are administered in two or more fractionated doses over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. In such fractionated dosing regimens, the number of engineered hoCD122 cells may be the same in each dose or may be provided at different levels. Multi-day dosing regimens over a period of time may be provided by a skilled technician (e.g., a physician) who monitors cell administration, considers the subject's response to treatment, including adverse reactions and their management, as described above.

[0787] The compositions and methods of the present invention also provide a method for treating subjects with hoCD122 cell therapy (particularly CAR T cell therapy) without prior lymph node depletion. Lymph node depletion is typically performed in conjunction with CAR T cell therapy in the subject because the subsequent administration of a mixed cell population and a nonspecific agent (e.g., hIL2) to expand the engineered cell population in the subject, combined with the administration of the cell therapy product, leads to significant systemic toxicity (including cytokine release syndrome or "cytokine storm") due to the extensive proliferation and activation of immune cells caused by the administration of agents leading to widespread activation, as well as a substantial portion of the non-engineered cells in the cell therapy product itself. The methods and compositions of the present invention avoid this significant obstacle by providing a substantially purified engineered cell population, substantially free of contamination by non-engineered cells, when employing the aforementioned in vitro methods, and / or selectively activating and expanding engineered T cells with the hIL2 ortholog of the present invention, which greatly reduces the off-target effects of nonspecific proliferators such as IL2.

[0788] For example, in current clinical practice of CAR-T cell therapy, CAR-T cells are typically administered in combination with lymphatic clearance (e.g., by administration of alemtuzumab (monoclonal anti-CD52), purine analogs, etc.) to promote CAR-T cell expansion prior to host immune recovery. In some embodiments, CAR-T cells may be modified to be resistant to alemtuzumab. In one aspect of the invention, the lymphatic clearance currently associated with CAR-T therapy can be avoided or reduced by orthogonal ligands expressing the CAR-T cells of the invention. As mentioned above, lymphatic clearance is typically used to achieve CAR-T cell expansion. However, lymphatic clearance is also associated with major side effects of CAR-T cell therapy. Since orthogonal ligands provide a means of selectively expanding specific T cell populations, the need for lymphatic clearance prior to administration of CAR-T cells expressing orthogonal ligands can be reduced. The present invention enables the practice of CAR-T cell therapy to eliminate or reduce lymphatic clearance prior to administration of CAR-T cells expressing orthogonal ligands.

[0789] In one embodiment, the present invention provides a method for treating a subject suffering from a disease, disorder, or condition (e.g., cancer) that can be treated with CAR-T cell therapy by administering a CAR-T cell expressing an orthogonal ligand without prior lymphatic clearance.

[0790] In one embodiment, the present invention provides a method for treating a mammalian subject suffering from a tumor disease, the method comprising the steps of: (a) obtaining a biological sample containing T cells from the individual; (b) enriching the T cells present in the biological sample; (c) transfecting the T cells with one or more expression vectors containing a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding an orthogonal hCD122 receptor, wherein the antigen-targeting domain of the CAR is capable of binding to at least one antigen present on tumor cells; (d) ex vivo contacting a cell population containing CAR-T cells expressing the orthogonal receptor with an hIL2 orthogonal homolog, such that the cell population is enriched with CAR T cells expressing hCD122; (e) administering a pharmaceutically effective amount of the CAR-T cells expressing the orthogonal receptor to the mammal; and (f) regulating the growth of the CAR-T cells expressing the orthogonal hCD122 receptor by administering a therapeutically effective amount of the hIL2 orthogonal homolog, the hIL2 orthogonal homolog selectively binding to the orthogonal hCD122 receptor expressed on the CAR-T cells. In the above method, when the nucleic acid sequences encoding CAR and orthogonal hCD122 receptors are provided on the same vector, the sequences may optionally be provided in a polycistronic form, and the nucleic acid sequences are separated by intercalation sequences (e.g., IRES or T2A sequences). In one embodiment, the above method is associated with lymphatic clearance or immunosuppression in mammals prior to the initiation of a CAR-T cell therapy course. In another embodiment, the above method is performed without lymphatic clearance and / or immunosuppression in mammals.

[0791] Administration of viral or non-viral vectors encoding hIL2 orthologs

[0792] An alternative to administering the hIL2 orthogonal protein is to provide the subject with a nucleic acid construct encoding the hIL2 orthogonal, thereby achieving sustained exposure to the selective hIL2 orthogonal. Administration of a recombinant vector encoding the hIL2 orthogonal provides extended delivery of the hIL2 orthogonal to the subject and corresponding extended activation of cells engineered to express the homologous orthogonal receptor associated with this hIL2 orthogonal.

[0793] Non-viral vector:

[0794] In one implementation, the hIL2 orthogonal homolog can be delivered to the target in the form of a nucleic acid expression construct of the hIL2 orthogonal homolog in a non-viral vector provided in a non-viral delivery system. Non-viral delivery systems are typically complexes designed to facilitate the transduction of target cells with nucleic acid carriers, wherein the nucleic acid is complexed with a reagent, such as cationic lipids (DOTAP, DOTMA), surfactants, bioproducts (gelatin, chitosan), metals (gold, magnetite), and synthetic polymers (PLG, PEI, PAMAM). Many implementations of non-viral delivery systems are well known in the art, including lipid carrier systems (Lee et al. (1997) Critical Reviews of Therapeutic Drug Carriers). Systems (14:173-206); polymer-coated liposomes (Marin et al., U.S. Patent No. 5,213,804, granted May 25, 1993; Woodle et al., U.S. Patent No. 5,013,556, granted May 7, 1991); cationic liposomes (Epand et al., U.S. Patent No. 5,283,185, granted February 1, 1994; Jessee, JA., U.S. Patent No. 5,578,475, granted November 26, 1996; Rose et al., U.S. Patent No. 5,279,833, granted January 18, 1994; Gebeyehu et al., U.S. Patent No. 5,334,761, granted August 2, 1994). In one embodiment, the nucleic acid sequence encoding the hIL2 receptor in the non-viral vector system is under the control of an adjustable promoter, an inducible promoter, a tissue-specific or tumor-specific promoter, or a time-regulated promoter.

[0795] Viral vector:

[0796] In another embodiment, the hIL2 orthogonal homolog can be given to the target in the form of a nucleic acid expression construct in a viral vector encoding the hIL2 orthogonal homolog. The terms "viral vector" and "virus" are used interchangeably herein, referring to any obligate intracellular parasite lacking a protein synthesis or energy production mechanism. The viral genome can be RNA or DNA with a protein-coated structure containing a lipid membrane. The terms "virus" and "viral vector" are used interchangeably herein. Viruses useful in the practice of this invention include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from the families Baculoviridiae, Parvoviridae, Picorviridae, Herpesviridae, Poxviridae, or Adenoviridae. The virus is modified using DNA recombination techniques to include the expression of exogenous transgenes (e.g., nucleic acid sequences encoding the hIL2 orthogonal homolog) and can be engineered to be replication-deficient, conditionally replicative, or replication-capable. Minimal vector systems can also be used, where the viral backbone contains only the sequences required for viral vector packaging, optionally including a transgenic expression cassette. The term "replication-deficient" refers to a vector with highly reduced replication in wild-type mammalian cells. To mass-produce such vectors, the missing function is usually supplemented by co-transfection with a helper virus or through genomic modification, thereby establishing producer cell lines. The term "replication-capable viral vector" refers to a viral vector capable of infecting, replicating, packaging, and lysing infected cells. The term "conditionally replicative viral vector" as used herein refers to a vector with replication capacity designed to achieve selective expression in a specific cell type. This conditional replication can be achieved by operatively linking tissue-specific, tumor-specific, or cell-type-specific or other selectively induced regulatory sequences to an early gene (such as the E1 gene in an adenovirus vector). Infecting subjects with recombinant viruses or non-viral vectors can provide long-term expression of the hIL2 orthogonal homolog in the subject and provide continuous selective maintenance of engineered T cells expressing the hCD122 orthogonal receptor. In one implementation, the nucleic acid sequence encoding the hIL2 orthogonal homolog in the viral vector system is under the control of an adjustable promoter, an inducible promoter, a tissue-specific or tumor-specific promoter, or a time-regulated promoter.

[0797] Treatment combination:

[0798] The compositions and methods of the present invention can be combined with other therapeutic agents. For example, when the disease, disorder, or condition to be treated is a neoplastic disease (such as cancer), the methods of the present invention can be combined with conventional chemotherapy agents or other biological anticancer drugs such as checkpoint inhibitors (such as PD1 or PDL1 inhibitors) or therapeutic monoclonal antibodies (such as Avastin or Herceptin).

[0799] Examples of chemical agents identified in this art as useful for treating neoplastic diseases include, but are not limited to: abitrexate, adriamycin, adrucil, amsacrine, asparaginase, anthracyclines, azacitidine, azathioprine, bicnu, blenoxane, busulfan, bleomycin, camptosar, and others. Camptothecins, Carboplatin, Carmustine, Cerubidine, Chlorambucil, Cisplatin, Cladribine, Cosmegen, Cytarabine, Cytosar, Cyclophosphamide, Cytoxan, Dactinomycin, Docetaxel, Doxorubicin icin), daunorubicin, (ellence), asparaginase (elspar), epirubicin, etoposide, fludarabine, fluorouracil, fludara, gemcitabine, gemzar, hycamtin, hydroxyurea, hydrea, idamycin, idarubicin, ifosfamide amide), ifosfamide (IFEX), irinotecan, lanvis, leukeran, leustatin, matulane, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, mithramycin, mitomycin, myleran, mylosarNovelbine, nipent, novantrone, oncovin, oxaliplatin, paclitaxel, paraplatin, pentostatin, platinol, plicamycin, procarbazine, purinethol, ralitrexed, taxotere, taxol, teniposide, thioguanine, tomudex, topotecan, valrubicin, velban, vepesid, vinblastine, vindesine, vincristine, vinorelbine, VP-16, and vumon. ,

[0800] The compositions of the present invention may be administered in combination with one or more other therapeutic agents selected from the group consisting of tyrosine kinase inhibitors, such as imatinib mesylate (as...). Listed on the market, also known as STI-571, gefitinib ( Also known as ZD1839), erlotinib (as...) (Listed) Sorafenib Sunitinib Dasatinib Lapatinib Nilotinib and bortezomib Ruxotinib; Janus kinase inhibitors, such as tofacitinib; ALK inhibitors, such as crizotinib; Bcl-2 inhibitors, such as obatoclax, venclexta, and gossypol; FLT3 inhibitors, such as midostaurin. IDH inhibitors, such as AG-221; PARP inhibitors, such as iniparib and olaparib; PI3K inhibitors, such as perifosine; VEGF receptor 2 inhibitors, such as apatinib; AN-152 (AEZS-108) doxorubicin linked to [D-Lys(6)]-LHRH; Braf inhibitors, such as vemurafenib, dabrafenib, and LGX818; MEK inhibitors, such as trametinib; CDK inhibitors, such as PD-0332991 and LEE011; Hsp90 inhibitors, such as salinomycin; and / or small molecule drug conjugates, such as Vintafolide; serine / threonine kinase inhibitors, such as tamsirolimus. everolimus Vemurafenib Trametinib (Mekinist) and Dabrafenib

[0801] In some implementations, particularly where the tumor antigen-binding portion of the CAR targets BCMA, engineered CAR-T cells are administered in combination with a gamma-secretase inhibitor (GSI), as described in Pont et al. (2019), "γ-secretase inhibition increases efficacy of BCMA-specific chimeric antigen receptor T cells in multiple myeloma," Blood https: / / doi.org / 10.1182 / blood.2019000050.

[0802] Examples of biological agents identified in this art that are useful for treating neoplastic diseases include, but are not limited to: cytokines or cytokine antagonists, such as IL-12, INFα, or anti-epidermal growth factor receptor; radiotherapy; irinotecan; leucovorin antimetabolites, such as pemetrexed; antibodies against tumor antigens, complexes of monoclonal antibodies and toxins, T-cell adjuvants, bone marrow transplantation, or antigen-presenting cells (e.g., dendritic cell therapy); antitumor vaccines; replicative viruses; signal transduction inhibitors (e.g., or ) or immunomodulators to achieve complementary or synergistic inhibition of tumor growth, cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g.)

[0803] and Interferon-β1a and interferon-β1b And one or more combinations of the above-mentioned known chemotherapy regimens that are easily understood by skilled clinicians in the field.

[0804] Tumor-specific monoclonal antibodies that can be combined with engineered cells may include, but are not limited to, rituximab (marketed as MabThera or Rituxan), alemtuzumab, panitumumab, and ipilimumab (Yervoy).

[0805] In some embodiments, the compositions and methods of the present invention may be combined with immune checkpoint therapy. Examples of immune checkpoint therapy include inhibitors of PD1 binding to PDL1 and / or PDL2. Inhibitors of PD1 binding to PDL1 and / or PDL2 are well known in the art. Examples of commercially available monoclonal antibodies that interfere with PD1 binding to PDL1 and / or PDL2 include nivolumab (…). BMS-936558, MDX1106 (commercially purchased from Bristol Myers Squibb, Princeton, NJ), pembrolizumab ( MK-3475, lambda antibody (commercially purchased from Merck and Company, Kenniallworth, New Jersey) and atezolizumab ( Genentech / Roche, South San Francisco, California. Other examples of PD-1 inhibitory antibodies include, but are not limited to, durvalumab (MEDI4736, Medimmune / AstraZeneca), pilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, Bristol Myers Squibb), avirumab (MSB0010718C, Merck Serono / Pfizer), and SHR-1210 (Incyte). Other antibody PD1 pathway inhibitors are described in U.S. Patent No. 8,217,149 (Genentech), issued July 10, 2012; U.S. Patent No. 8,168,757 (Merck Sharp and Dohme Corp.), issued May 1, 2012; U.S. Patent No. 8,008,449 (Medarex), issued August 30, 2011; and U.S. Patent No. 7,943,743 (Medarex), issued May 17, 2011. Furthermore, small molecule PD1 to PDL1 and / or PDL2 inhibitors are well known in the art. See, for example, Sasikumar, et al. WO2016142833A1 and Sasikumar, et al. WO2016142886A2, BMS-1166 and BMS-1001 (Skalniak, et al. (2017) Oncotarget 8(42):72167–72181).

[0806] The invention has now been fully described, and it will be apparent to those skilled in the art that many changes and modifications can be made without departing from the spirit or scope of the invention.

[0807] Example

[0808] The following embodiments are presented to more fully illustrate preferred embodiments of the invention. However, they should not be construed as limiting the scope of the invention.

[0809] Example 1. Generation of human IL2 expression vector pcDNA3.1 / hygro(+)-huIL2

[0810] Human IL2 DNA ORF (Genbank NM_000586.3) was synthesized (Life Technologies GeneArt Services, Carlsbad, CA) and amplified by PCR using the Platinum SuperFiII DNA polymerase kit (catalog number 12361050, Thermo Fisher Scientific), following the manufacturer's protocol and using the following primers:

[0811] 5'TATAGTCAGCGCCACcCATGTACAGGATGCAACTCCTGTC 3'

[0812] (SEQ ID NO:153)

[0813] Its inclusion of NheI restriction sites, and

[0814] 5'TATAGGGCCCTATCAAGTCAGTGTTGAGATG 3'(SEQ ID

[0815] NO:154)

[0816] It incorporates the ApaI restriction site. The PCR fragment was visualized on a 1% agarose gel (catalog number 54803, Lonza, Rockland, Maine), excised from the gel, and purified using the QIAquick PCR Purification Kit (catalog number 28106, Qiagen, Germany), according to the manufacturer's protocol.

[0817] Purified PCR fragments and the mammalian expression vector pcDNA 3.1 / Hygro(+) (No. V87020, Thermo Fisher Scientific) were digested with NheI and ApaI restriction enzymes (Nos. R0111S and R0114L, New England Biolabs, Ipswich, MA). The expression vector was further processed using a rapid dephosphorylation kit (No. M0508L, New England Biolabs) according to the manufacturer's protocol. PCR fragments were ligated into pcDNA3.1 / Hygro(+) using a rapid DNA ligation kit (No. 11635379001, Sigma-Aldrich, St. Louis, Missouri), according to the manufacturer's protocol, transformed into One Shot TOP10 chemicompetent E. coli (No. C404006, Life Technologies, Carlsbad, CA), inoculated onto LB agar plates containing 100 μg / ml carbenicillin (No. L1010, Teknova, Hollister, CA) and grown overnight at 37°C.

[0818] The following day, a single colony was picked and used to initiate a 3 ml bacterial culture in LB broth (No. 10855-001, Life Sciences) containing 100 μg / ml ampicillin (A9626, Teknova). The culture was incubated overnight at 37°C.

[0819] The following day, *E. coli* were precipitated (6,000 rpm, 10 min, benchtop centrifuge, No. 5424, Eppendorf, Hopoorg, NY), and the DNA expression vector was isolated using the QIAprep Spin Kit (No. 27106, Qiagen). The plasmid DNA was sequence validated (MCLab, South San Francisco, CA).

[0820] Example 2. Human IL2 ORTHO expression vector

[0821] Production of pcDNA3.1 / hygro(+)-huIL2-ORTHO

[0822] The expression vector introduced six mutations into the human IL2 ORF (E35S, H36Q, L39V, D40L, Q42K, and M43A; all numbering is based on the full-length human IL2 ORF NM_000586.3 numbering), and was assembled in accordance with the instructions in Example 1 of the human IL2 expression vector in pcDNA3.1 / Hygro(+), except that the initial template DNA for PCR was synthesized containing the E35S, H36Q, L39V, D40L, Q42K, and M43A mutations.

[0823] Example 3. Introducing a mutation into pcDNA3.1 / hygro(+)-huIL2 or reverting the mutation into pcDNA3.1 / hygro (+)-huIL2IRTHO expression vector.

[0824] All mutations or reversion mutations (restoring mutations in pcDNA3.1 / hygro(+)-huIL2-ORTHO to match wild-type IL2 ORF) were introduced into the pcDNA3.1 / Hygro(+)-huIL2 or pcDNA3.1 / Hygro(+)-huIL2-ORTHO expression vectors, using the Quik Change II site-directed mutagenesis kit (No. 200524, Agilent Technologies, Santa Clara, CA) and were performed substantially according to the manufacturer’s protocol.

[0825] Table 5 lists the generated mutations, the templates used to introduce the mutations, and the primer sets used to introduce the mutations. The transformation of *E. coli* into the QuikChange PCR reaction, as well as the isolation and sequencing of the plasmid DNA, were performed using essentially the same protocol as for generating the pcDNA3.1 / Hygro-huIL2 expression vector. The template abbreviations are: Template 1 = pcDNA3.1 / hygro(+)-huIL2; Template 2 =

[0826] pcDNA3.1 / Hygro(+)-huIL2 ORTHO.

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834]

[0835]

[0836]

[0837]

[0838]

[0839]

[0840]

[0841]

[0842]

[0843]

[0844]

[0845]

[0846]

[0847]

[0848]

[0849]

[0850]

[0851]

[0852]

[0853]

[0854]

[0855]

[0856]

[0857] Example 4. Transient transfection in HEK293 cells

[0858] All expression vectors were transiently transfected into HEK293 cells (CRL-1573, ATCC, Manassas, VA). Approximately 1E6 HEK293 cells were seeded into each well of a 6-well tissue culture plate in 2 ml of DMEM (10569044, Life Sciences, Inc.) supplemented with 10% fetal bovine serum (SH30071.03, Fisher Scientific, Chicago, ILLU, USA) and grown overnight at 37°C and 5% CO2.

[0859] The next day, cells were transfected using Lipofectamine 3000 reagent (product number L3000150, Life Sciences, Inc.). Following the manufacturer's protocol, each transfection used 2.5 μg DNA, 5 μL P3000 reagent, and 7.5 μL Lipofectamine 3000. Transfected cells were grown at 37°C and 5% CO2 for 48–72 hours, and then harvested into conditioned medium.

[0860] Example 5. Protein Expression Analysis

[0861] Protein expression of several mutant proteins was measured by ELISA using the Human IL2 V-PLEX ELISA Kit (No. K151QQD-4, Mesoscale Diagnostics, Baltimore, MD), following the manufacturer's protocol (transfection medium initially diluted 1:4, then serially diluted 1:2). Plates were read using the manufacturer's preset settings for this ELISA kit on a Meso Quickplex SQ120 (Mesoscale Diagnostics). Human IL2 standards from this kit were used to calculate approximate expression levels in the conditioned medium samples. Table 6 below details the approximate expression levels of the expressed proteins.

[0862]

[0863]

[0864] Example 7. Evaluation of the activity of orthologs in cell lines expressing hoCD122 The activity of IL2 orthogonal homologs was assessed in NKL cells (Robertson, et al. (1996) Experimental Hematology 24(3):406-15). To generate a cell line expressing human orthogonal hCD122 (hoNKL hoRB), NKL cells were infected with a retrovirus encoding hoRBhCD122 and co-expressing YFP (MSCV-hoRb-IRES-YFP) according to procedures known in the art.

[0865] NKL and NKL hoRB cells were contacted with the supernatant from 293 cells transfected with hIL2 ortholog, as follows: Cells were seeded in growth medium consisting of RPMI 1640 (Thermo Fisher Scientific), 10% fetal bovine serum (Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific), and 1% glutamax (Thermo Fisher Scientific), at a density of 0.5 x 10⁻⁶. 6Cells / ml. After 2 days of culture, cells were seeded into 96-well plates (Falcon) at 100 μl of growth medium per well (25,000 cells). The supernatant from transfected 293 cells was serially diluted 2- or 5-fold in growth medium, and 100 μl of each dilution was added to plates containing NKL and NKL hoRB cells in double replicates, with a final titration range of 1:2 to 1:31,250. The plates were then transferred to a humidified incubator (Thermo Fisher Scientific) and incubated for three days at 37°C and 5% CO2.

[0866] Remove the plate from the incubator and incubate at room temperature for 30 minutes. Centrifuge the plate at 400x g for 5 minutes and discard the supernatant. Lyse the cells by adding 50 μl of Celltiterglo (Promega) in a 1:1 dilution of PBS to each well. Mix the cell lysates at 600 rpm for 2 minutes on a track shaker (VWR Scientific) and then incubate at room temperature for 10 minutes. Transfer the lysates to 96-well plates with a black clear bottom (Costar), NKL cell lysates (… Figure 1 ) and NKL hoRB cell lysate ( Figure 2 The light emitted by the tag is read in counts per second in the Envision 2103 multi-tag reader (Perkin Elmer).

[0867] To compare the effects of each IL-2 variant on the proliferation of NKL cells and NKL hoRB cells, the CelltiterGlo values ​​of cells treated with the supernatant were compared with those of control cells treated alone with growth medium, supernatant from 293 cells transfected with empty vector, supernatant from wild-type IL-2 transfection, or supernatant from human orthogonal IL-2 transfection. The data from these experiments are shown in the attached figures. Figure 1 and 2 .

[0868] Example 8. Evaluation of the activity of orthologs in cell lines expressing hoCD122

[0869] To assess the activity of IL2 orthogonal homologs in CD4+ human T cell clone 3F8 cells. CD4-positive T cell clone 3F8 was generated by activation of PBMCs from healthy donors and the EBV-transformed B cell line JY in two consecutive rounds of mixed leukocyte reactions, followed by the production of single-cell clones through limiting dilutions, as described in (Yssel and Spits (2002), Current Protocols in Immunology, 7.19.1–7.19.12). CD4-positive T cell clone 3F8 expresses CD25 and CD122 and proliferates in response to IL-2. To generate a cell line expressing human orthogonal hCD122 (ho3F8hoRB), 3F8 cells were infected with a retrovirus encoding hoRB hCD122 and co-expressing YFP (MSCV-hoRb-IRES-YFP) according to procedures known in the art.

[0870] 3F8 and 3F8 hoRb cells were contacted with the supernatant of 293 cells transfected from hIL2 orthologs, as follows: Cells were grown at 200,000 cells per ml in a growth medium consisting of Yssel's medium (Iscove's modified Dulbecco medium (Thermo Fisher Scientific), 0.25% w / v human albumin (Sigma), 1% penicillin / streptomycin (Thermo Fisher Scientific), 1% ITS-X insulin, transferrin, selenium (Gibco), 30 mg / L transferrin (Roche), 2 mg / L palmitic acid (Sigma), 1% LA-OA-albumin linoleic acid, oleic acid (Sigma), and 1% human serum (Gemini) (Yssel et al. (1984) J Immunol Methods). 72:219–227), 50 Gy irradiated JY cells were introduced at 100,000 cells per well, and 40 Gy irradiated allogeneic PBMCs were introduced at 1,000,000 cells per mL. After ten days of culture and expansion with 100 pM human IL-2, the cells were washed and seeded into 96-well plates (Costar) with a black clear bottom, 50,000 cells per well, with 50 μl of growth medium. The supernatant of transfected 293 cells was serially diluted twofold in growth medium, and 50 μl of each dilution was added to plates of 3F8 and 3F8 hoRB cells, with a final titration range of 1:2 to 1:31250. The plates were transferred to a humidified incubator (Thermo Fisher Scientific) and incubated for three days at 37°C and 5% CO2.

[0871] Remove the plate from the incubator and keep it at room temperature for 30 minutes. Add 100 μl of Celltiterglo (Promega) to each well to lyse the cells. Mix the cell lysates at 600 rpm for two minutes on a track-controlled shaker (VWR Scientific) and then keep at room temperature for 10 minutes. The luminescence of 3F8 cell lysates and 3F8 hoRB cell lysates was read at counts per second using an Envision 2103 multi-tag reader (Perkin Elmer).

[0872] To compare the effects of each IL-2 variant on the proliferation of 3F8 and 3F8 hoRB cells, the CelltiterGlo values ​​of cells treated with the supernatant were compared with those of control cells treated with growth medium alone, 293 supernatant from empty vector transfection, wild-type IL-2 transfection, or supernatant from human orthogonal IL-2 transfection. The data obtained from these experiments are shown in Table 4.

Claims

1. An hIL2 orthogonal homolog, which is a polypeptide with the following amino acid sequence: PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRM LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHLRPRDLSNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISTLT.

2. An in vitro method for preparing an engineered T cell product, said T cell product comprising at least 20% T cells expressing orthogonal hCD122 receptor (hoCD122), said method comprising the steps of: a. Isolating T cell populations from mammalian subjects; b. Contact the isolated population of T cells with a recombinant vector comprising a nucleic acid sequence encoding hoCD122, which is operatively linked to one or more expression control sequences such that, while allowing the recombinant vector to be taken up by T cells, the expression of hoCD122 in mammalian T cells is promoted. c. Contact the isolated T cell population from step b with an effective amount of the hIL2 orthogonal homolog as described in claim 1.

3. The method of claim 2, wherein prior to step (b), the T cell population isolated in step (a) is contacted with a reagent that is associated with a signal that stimulates the CD3 TCR complex and a reagent that stimulates co-stimulatory molecules on the surface of the T cells.

4. A cell population product prepared by the method of claim 2 or 3, wherein the cell population comprises at least 20% engineered hoCD122 T cells.

5. A cell population product obtained by the method of claim 2 or 3, wherein the T cells are CAR-T cells or TILs.

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