Il-2 orthologs and methods of use

Abstract

To provide hIL2 orthogonal ligands (IL2 orthologs) that specifically and selectively bind to the extracellular domain (ECD) of a transmembrane polypeptide comprising a modified hCD122 polypeptide.SOLUTION: The binding of the hIL2 ortholog to the modified hCD122 polypeptide participates in the transduction pathway of intracellular signaling resulting in a biological activity of the native intracellular signaling patterns associated with hIL2 binding to either the intermediate or high affinity hIL2 receptor but which exhibits selectivity to an engineered cell expressing an hCD122 orthogonal receptor. The hIL2 orthologs of the present invention exhibit significantly reduced binding relative to their binding to the extracellular domain of wild type hCD122, either alone or when hCD122 is present in the form of an endogenous high or intermediate affinity hIL2 receptors.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross-reference to other applications This application claims priority to U.S. Provisional Patent Application No. 62 / 948,066, filed December 13, 2019.

[0002] Statement regarding government funding No government funds were used in the conception or practice of the subject matter of the present invention.

Background Art

[0003] Background of the invention The controlled manipulation of the differentiation, development, and proliferation of cells, particularly engineered immune cells, is of great clinical interest. T cells have been engineered for use in therapeutic applications such as the recognition and killing of cancer cells, intracellular pathogens, and cells involved in autoimmunity. The use of engineered cell therapies in the treatment of cancer is facilitated by the selective activation and expansion of engineered T cells that provide specific functions and are directed to selectively attack cancer cells. In some examples of adoptive immunotherapy, T cells are isolated from a subject's blood, processed ex vivo, and reinfused into the subject. Therefore, compositions and methods that enable the selective activation of a targeted population of engineered cells are desirable.

[0004] A challenge in the manufacture of cell therapy products is that such "living drugs" require tight control of their environment to maintain viability and functionality. In fact, isolated cells, whether patient-derived (autologous) or from a single donor source (allogeneic), rapidly begin to lose function after being removed from a subject or controlled culture conditions. The success of maintaining the health and functionality of isolated cells while outside of a subject or controlled culture conditions enables the isolated cells to regain functionality for reinsertion into a cell product manufacturing workflow or into a patient.

[0005] In addition, an issue regarding the clinical application of engineered T cell therapies is to selectively stimulate these engineered cells to maximize their therapeutic efficacy. A typical means of providing continuous maintenance of activated engineered T cell products is systemic administration of cytokines such as IL2. However, systemic administration of IL2 is accompanied by non-specific stimulatory effects that exceed the population of engineered cells, especially at high doses, and is accompanied by significant toxicity in human subjects. Furthermore, IL2 has a short in vivo lifespan, and it is necessary to frequently dose IL-2 to maintain engineered T cells in an activated state. Engineered cells from the initial population after the first administration can be detectable for months or even years after administration of the engineered cell product, but a significant proportion of these engineered cells become quiescent and require reactivation to exhibit a significant therapeutic effect. Thus, an issue in cell-based therapies is to endow the transplanted cells with a desired adjustable behavior that is protected from endogenous signaling pathways, does not affect non-target endogenous cells, and can be selectively controlled after administration to the engineered cell population.

[0006] CD122 is a component of the intermediate and high affinity IL2 receptor complexes. Sockolosky et al. (Science (2018) 359: 1037-1042 (Non-Patent Document 1)) and Garcia et al. (U.S. Patent Application Publication US2018 / 0228841A1 (Patent Document 1), published on August 16, 2018) describe an orthogonal IL2 / CD122 ligand / receptor system to facilitate selective stimulation of cells engineered to express an orthogonal CD122 receptor. Also described is an IL2 mutein, which is a cognate ligand for the orthogonal receptor. Contact of engineered T cells expressing orthogonal CD122 with the corresponding orthogonal ligand for such orthogonal CD122 (the "IL2 ortholog") enables specific activation of such engineered T cells. In particular, this orthogonal IL2 receptor ligand complex provides for selective expansion of cells engineered to express the orthogonal receptor in a mixed cell population, particularly a mixed T cell population.

[0007] In addition, an IL-2 ortholog with reduced affinity for an unoperated intermediate affinity (CD122 / CD132) IL-2 receptor complex or a high affinity (CD25 / CD122 / CD132) IL-2 receptor complex is useful, for example, in the treatment of autoimmune diseases, for selectively targeting the activity of the ortholog IL-2 to cells exhibiting high CD25 expression. Also, an IL-2 ortholog that has a significantly reduced affinity for the extracellular domain (ECD) of native wild-type hCD122 but retains binding to the ECD of CD25 can be used as a competitive antagonist of wild-type IL-2 by interfering with the formation of the high affinity IL-2 receptor complex, and thus can be used in the treatment of autoimmune diseases or graft-versus-host (GVH) disease.

[0008] The present disclosure is directed to ligands that interact with an orthogonal hCD122 receptor. Specifically, provided are hIL-2 orthogonal ligands (hIL2 orthologs) that provide selective binding and signaling through a receptor comprising the extracellular domain of the hCD122 orthogonal receptor, particularly the extracellular domain of human CD122 containing the amino acid substitutions H133D and Y134F. The IL2 activity of the present hIL2 ortholog against cells expressing wild-type hCD122 is significantly reduced as compared to the activity of the present hIL2 ortholog against cells expressing orthogonal hCD122. Accordingly, provided is the selective activation and / or expansion of engineered cells expressing a receptor comprising the extracellular domain of orthogonal hIL2 using an hIL-2 ortholog on an engineered cell population.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Non-Patent Documents

[0010]

Non-Patent Document 1

Summary of the Invention

[0011] The present disclosure relates to a human IL2 orthogonal ligand (“hIL2 ortholog”) that specifically and selectively binds to the extracellular domain (ECD) of a transmembrane polypeptide comprising a modified hCD122 polypeptide that contains a modification at position 133 and / or 134 of the ECD of an orthogonal hCD122 polypeptide. In some embodiments, the orthogonal hCD122 polypeptide comprises the amino acid substitutions H133D and Y134F. Binding of the hIL2 ortholog to the modified hCD122 polypeptide results in a bioactivity of the native intracellular signaling pattern associated with IL2 binding to either the intermediate affinity or high affinity IL2 receptor, involving the intracellular signaling pathway, but shows selectivity for engineered cells expressing the hCD122 orthogonal receptor. In some embodiments, the hIL2 ortholog is an hIL2 variant of Formula 1 described hereinbelow. The hIL2 orthologs of the present disclosure show significantly reduced binding to the extracellular domain of wild-type hCD122, either alone or when hCD122 is present in the form of an endogenous high-affinity or intermediate-affinity hIL2 receptor, compared to the binding of the hIL2 ortholog to the hCD122 orthogonal receptor. In some embodiments, the affinity of the hIL2 ortholog for the extracellular domain of orthogonal hCD122 is equivalent to the affinity of wild-type hIL2 for wild-type hCD122. In some embodiments, the affinity of the hIL2 ortholog for the extracellular domain (“ECD”) of orthogonal hCD122 is greater than the affinity of wild-type hIL2 for the extracellular domain of wild-type hCD122. In some embodiments, the affinity of the hIL2 ortholog 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 modified human hCD122 ECD polypeptide containing the substitutions H133D and Y134F (numbered according to wild-type hCD122), where the amino acid sequence of the ECD of the orthogonal hCD122 receptor comprises a 214 amino acid polypeptide having: TIFF2025108545000002.tif22128.

[0012] In one aspect, the orthogonal hCD122 receptor is a modified human CD122 comprising the ECD of hCD122 (SEQ ID NO:1) having substitutions H133D and Y134F and the amino acid sequences of the transmembrane (TM) and intracellular domain (ICD) of the wild-type hCD122 molecule (smaller than the signal peptide), and the orthogonal hCD122 receptor (hoRb) has the amino acid sequence: Includes TIFF2025108545000003.tif50128.

[0013] In one aspect, the present disclosure provides an hIL2 ortholog, the amino acid sequence of which has at least 80% identity to the polypeptide of formula #1: TIFF2025108545000004.tif72155 wherein, AA1 is A (wild type) or a deletion; AA2 is P (wild type) or a deletion; AA3 is T (wild type), C, A, G, Q, E, N, D, R, K, P, or a deletion; AA4 is S (wild type) or a deletion; AA5 is S (wild type) or a deletion; AA6 is S (wild type) or a deletion; AA7 is T (wild type) or a deletion; AA8 is K (wild type) or a deletion; AA9 is K (wild type) or a deletion; AA13 is Q (wild type), W or a deletion; AA14 is L (wild type), M, W or a deletion; AA15 is E (wild type), K, D, T, A, S, Q, H or a deletion; AA16 is H (wild type), N or Q, or a deletion; AA18 is L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T; AA19 is L (wild type), A, V, I or a deletion; AA20 is D (wild type), T, S, M, L, or a deletion; AA22 is Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, F, or a deletion; AA23 is M (wild type), A, W, H, Y, F, Q, S, V, L, T, or a deletion; AA27 is G (wild type), K, S or a deletion; AA38 is R (wild type), W or G; AA39 is M (wild type), L or V; AA42 is F (wild type) or K; AA51 is T (wild type), I or a deletion AA55 is H (wild type) or Y; AA74 is Q (wild type), N, H, S; AA80 is L (wild type), F or V; AA81 is R (wild type), I, D, Y, T or a deletion AA85 is L (wild type) or V; AA86 is I (wild type) or V; AA88 is N (wild type), E or Q, or a deletion; AA89 is I (wild type) or V; AA91 is V (wild type), R or K; AA92 is I (wild type) or F; AA97 is K (wild type) or Q; AA104 is M (wild type) or A; AA109 is D (wild type), C or a non-natural amino acid having an activated side chain; AA113 is T (wild type) or N; AA125 is C (wild type), A or S; AA126 is Q (wild type) or H, M, K, C, D, E, G, I, R, S, or T; and / or AA130 is S (wild type), T or R.

[0014] In some embodiments, the disclosure provides an hIL2 ortholog comprising an IL2 variant polypeptide comprising amino acid modifications at positions E15, L16, L19, D20 and M23 and optionally at position Q22. In some embodiments, the hIL2 ortholog comprises an IL2 variant polypeptide comprising the amino acid substitutions L12, Q13, H16, L19, D20, M23, R81, D84, S87, N88, V91, I92 and E95. In some embodiments, the disclosure provides an hIL2 ortholog comprising an IL2 variant polypeptide comprising an amino acid modification at a position. The hIL2 ortholog comprises an IL2 variant polypeptide comprising the amino acid substitutions Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I92F. In some embodiments, the disclosure provides a set of the following amino acid substitutions: An hIL2 ortholog comprising an IL2 variant polypeptide comprising an amino acid modification selected from TIFF2025108545000005.tif66135 is provided.

[0015] In some aspects, the present disclosure provides hIL2 orthologs comprising an IL2 variant polypeptide comprising amino acid modifications at 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, E67G, E68, V69, N71, L72, Q74, S75, K76, H79, I89, N90, I92, S99, T101, F103, Y107, I114, I128 and T133. In some aspects, the present disclosure provides hIL2 orthologs comprising an IL2 variant polypeptide comprising one or more of the following amino acid substitutions: 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 aspects, the present disclosure provides the following sets of amino acid substitutions: TIFF2025108545000006.tif74147 provides an IL2 variant polypeptide comprising an amino acid modification selected from

[0016] In some embodiments, the present disclosure provides hIL2 orthologs comprising an IL2 variant polypeptide having amino acid modifications at positions Q11, L18, Q22, E110, N119, T123, Q126, S127, Q126, S127, I129, S130, and T133. In some embodiments, the present disclosure provides hIL2 orthologs comprising an IL2 variant polypeptide having one or more amino acid substitutions selected from L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18T, Q22E, Q22E, Q22E, Q22E, Q22G, 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 disclosure provides the following set of amino acid substitutions: TIFF2025108545000007.tif59128TIFF2025108545000008.tif214124, and provides hIL2 orthologs comprising an IL2 variant polypeptide having a set of amino acid substitutions selected therefrom.

[0017] In some embodiments, the present disclosure provides hIL2 orthologs comprising an IL2 variant polypeptide selected from the group consisting of SEQ ID NOs: 5-138.

[0018] In some embodiments, the present disclosure provides hIL2 orthologs functionally linked to at least one carrier molecule. In some embodiments, the present disclosure provides hIL2 orthologs comprising at least one polyethylene glycol (PEG) molecule.

[0019] In some embodiments, the present disclosure provides hIL2 orthologs having the following structure: [PEG]-[linker] n -[hoIL2] Wherein n = 0 or 1, and hoIL2 is a human orthogonal IL2 polypeptide variant of Formula 1. In some embodiments, the PEG has a molecular weight of 5 kDa to 80 kDa. In some embodiments, the PEG has a molecular weight of approximately 40 kDa. In some embodiments, the present disclosure provides an hIL2 ortholog of the above structure, wherein hoIL2 is an IL2 polypeptide variant comprising the set of amino acid substitutions [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A]. In some embodiments, the present disclosure provides an hIL2 ortholog of the above structure, wherein hoIL2 is an IL2 polypeptide variant comprising the amino acid sequence: TIFF2025108545000009.tif29128.

[0020] In some embodiments, the present disclosure provides a nucleic acid sequence encoding an hIL2 ortholog polypeptide of Formula #1.

[0021] In some embodiments, the present disclosure provides a recombinant vector encoding a nucleic acid sequence encoding an hIL2 ortholog polypeptide of Formula #1. A recombinant vector comprising the nucleic acid sequence according to claim 22.

[0022] In some embodiments, the present disclosure provides a. A nucleic acid sequence encoding a transmembrane receptor molecule comprising the extracellular domain (ECD) of orthogonal hCD122, operably linked to one or more expression control elements capable of achieving expression and surface presentation of the ECD of the transmembrane receptor molecule, administering the engineered mammalian cell comprising the same to a subject suffering from a disease, disorder or condition; and b. administering a therapeutically effective dose of an hIL2 ortholog of Formula #1 to the subject to provide a method of treating a disease, disorder or condition in a subject.

[0023] In some embodiments, the present disclosure provides a method of preparing an engineered T cell product comprising at least 20% hoCD122 T cells, comprising a. A step of isolating a population of T cells from a mammalian subject; b. A step of contacting the isolated population of T cells ex vivo with a recombinant vector comprising a nucleic acid sequence encoding hoCD122 operably linked to one or more expression control sequences to facilitate expression in mammalian T cells, under conditions that allow uptake of the recombinant vector by the T cells; c. A step of contacting the isolated population of T cells with an effective amount of the hIL2 ortholog of claim 1 A method is provided that includes the above steps.

[0024] In some embodiments, the disclosure provides a cell population comprising at least 20% engineered hoCD122 T cells.

[0025] The disclosure further provides a method for producing the hIL2 ortholog of the invention. In particular, the disclosure provides a recombinant expression vector comprising a nucleic acid sequence encoding an hIL2 ortholog operably linked to a control element to provide expression of the nucleic acid sequence encoding the hIL2 ortholog in a host cell.

[0026] The present disclosure further provides a composition comprising a mixed cell population comprising 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% T cells (e.g., T cells, CD8+ T cells, Tregs, TILs, NK cells, TCR-modified cells, CAR-T cells, etc.), wherein the T cells are recombinantly modified to express an orthogonal hCD122 receptor polypeptide. The present disclosure further provides a method of making an engineered cell therapy product in a pharmaceutically acceptable dosage form, wherein the dosage form comprises a population of T cells, and in the population of T cells, one or more species of engineered T cells are substantially enriched, and the engineered T cells express a receptor comprising an extracellular domain of an hCD122 orthogonal polypeptide, the method comprising ex vivo culturing a population of T cells comprising engineered T cells that express a receptor comprising an extracellular domain of an hCD122 orthogonal polypeptide, in the presence of the hIL2 ortholog of the invention, for a period of time sufficient to enrich a cell population of one or more such engineered T cells.

[0027] In some embodiments, the disclosure provides a recombinant vector comprising a nucleic acid sequence encoding an hIL2 ortholog described herein operably linked to a control element to facilitate the expression and secretion of the hIL2 ortholog from mammalian cells, and is administered to a subject to provide in situ expression of the hIL2 ortholog. In some embodiments, the recombinant vector is administered intratumorally to a subject suffering from cancer. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) or a recombinant adenovirus (rAd), for example, in some embodiments, a replication-deficient adenovirus derived from human adenovirus serotype 3 and / or 5. In some embodiments, the replication-deficient adenovirus has one or more modifications to the E1 region that interfere with the ability of the virus to initiate the cell cycle and / or the apoptotic pathway. The replication-deficient adenovirus vector may optionally contain a deletion in the E3 domain. In some embodiments, the adenovirus is a replicable adenovirus. In some embodiments, the adenovirus is a replicable recombinant virus engineered to replicate selectively in newborn cells.

[0028] The disclosure further provides a method of preparing a cell therapy product in a pharmaceutically acceptable dosage form, comprising at least 1 (or 2, 3, 4 or more) engineered T cells that express a transmembrane receptor protein, wherein the extracellular domain of such transmembrane receptor protein comprises the extracellular domain of an hCD122 orthogonal polypeptide, and the fraction of engineered cells in the cell therapy product comprises at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the total number of cells in the cell therapy product.

[0029] In some aspects, there is provided a therapy comprising introducing into a subject suffering from a disease, disorder or condition a population of engineered cells, wherein the engineered cell population comprises a nucleic acid sequence encoding a cell membrane-passing orthogonal receptor polypeptide comprising an extracellular domain (ECD) of SEQ ID NO:1, a transmembrane domain, and an intracellular signaling domain that results in an intracellular signal in response to binding of the ECD of the cell membrane-passing orthogonal receptor polypeptide to an orthologous ligand of hIL2, and wherein the nucleic acid sequence is operably linked to expression control elements to facilitate transcription, translation and cell surface presentation of the ECD of the membrane-passing polypeptide in combination with administration of the hCD122 ortholog of the present disclosure. Such cell populations may comprise cells that have been modified ex vivo and are autologous or allogeneic with respect to the subject. In some aspects, the therapy comprises: (1) contacting, ex vivo, a population of engineered cells expressing a receptor comprising an hIL2 orthogonal CD122 ECD with an amount, concentration and duration of the cognate hIL2 ortholog sufficient to activate the engineered cells; (2) administering the cell population to the subject; and (3) administering the cognate hIL2 ortholog to the subject in combination with administration of the engineered cells to the subject. In some aspects, the subject to whom the engineered hIL2 orthogonal CD122 ECD receptor cell population and the hIL2 ortholog are administered has a neoplastic disease. In some aspects, the orthogonal receptor and ligand are administered in combination with at least one additional / adjuvant therapeutic or prophylactic agent. [Invention 1001] The hIL2 ortholog, wherein its amino acid sequence has at least 90% identity to the polypeptide of Formula #1: TIFF2025108545000010.tif72155 wherein, AA1 is A (wild type) or a deletion; AA2 is P (wild type) or a deletion; AA3 is T (wild type), C, A, G, Q, E, N, D, R, K, P, or a deletion; AA4 is S (wild type) or a deletion; AA5 is S (wild type) or a deletion; AA6 is S (wild type) or a deletion; AA7 is T (wild type) or a deletion; AA8 is K (wild type) or a deletion; AA9 is K (wild type) or a deletion; AA13 is Q (wild type), W or a deletion; AA14 is L (wild type), M, W or a deletion; AA15 is E (wild type), K, D, T, A, S, Q, H or a deletion; AA16 is H (wild type), N or Q, or a deletion; AA18 is L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T; AA19 is L (wild type), A, V, I or a deletion; AA20 is D (wild type), T, S, M, L, or a deletion; AA22 is Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, F, or a deletion; AA23 is M (wild type), A, W, H, Y, F, Q, S, V, L, T, or a deletion; AA27 is G (wild type), K, S or a deletion; AA38 is R (wild type), W or G; AA39 is M (wild type), L or V; AA42 is F (wild type) or K; AA51 is T (wild type), I or a deletion AA55 is H (wild type) or Y; AA74 is Q (wild type), N, H, S; AA80 is L (wild type), F or V; AA81 is R (wild type), I, D, Y, T or a deletion AA85 is L (wild type) or V; AA86 is either I (wild-type) or V; AA88 is either N (wild-type), E or Q, or a deletion; AA89 is either I (wild-type) or V; AA91 is either V (wild-type), R or K; AA92 is either I (wild-type) or F; AA97 is either K (wild-type) or Q; AA104 is either M (wild-type) or A; AA109 is either D (wild-type), C or a non-natural amino acid with an activated side chain; AA113 is either T (wild-type) or N; AA125 is either C (wild-type), A or S; AA126 is either Q (wild-type) or H, M, K, C, D, E, G, I, R, S, or T; and / or AA130 is either S (wild-type), T or R. [Invention 1002] The hIL2 ortholog of Invention 1001 comprising modifications at positions AA15, AA16, AA19, AA20 and AA24. [Invention 1003] The hIL2 ortholog of Invention 1002 further comprising an amino acid substitution at one or more positions selected from the group consisting of L12, Q13, H16, L19, D20, M23, R81, D84, S87, N88, V91, I92 and E95. [Invention 1004] The IL2 ortholog of Invention 1003, wherein one or more substitutions are selected from the group consisting of Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I92F. [Invention 1005] One or more amino acid substitutions are a set of the following amino acid substitutions: The IL2 ortholog of Invention 1003 selected from TIFF2025108545000011.tif67135. [Invention 1006] The hIL2 ortholog of the present invention 1002, further comprising an amino acid substitution 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. [The present invention 1007] The IL2 ortholog of the present invention 1006, wherein one or more substitutions are 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. [The present invention 1008] One or more amino acid substitutions are a set of the following amino acid substitutions: The IL2 ortholog of the present invention 1006, selected from TIFF2025108545000012.tif74147. [The present invention 1009] The IL2 ortholog of the present invention 1002, further comprising an amino acid substitution at one or more positions selected from the group consisting of Q11, L18, Q22, E110, N119, T123, Q126, S127, Q126, S127, I129, S130, and T133. [The present invention 1010] The hIL2 ortholog of the present invention 1009 further comprising an amino acid substitution at one or more positions 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, Q22G, 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. [The present invention 1011] The following set of amino acid modifications: The hIL2 ortholog of the present invention 1001 comprising one of TIFF2025108545000013.tif43128TIFF2025108545000014.tif229124. [The present invention 1012] The hIL2 ortholog of the present invention 1001 selected from the group consisting of SEQ ID NO:5 - 138. [The present invention 1013] The hIL2 ortholog of the present invention 1001 functionally linked to at least one carrier molecule. [The present invention 1014] The hIL2 ortholog of the present invention 1013, wherein the carrier molecule is independently selected from the group consisting of a water-soluble polymer, the Fc domain of IgG, a sugar, and / or albumin. [The present invention 1015] The hIL2 ortholog of the present invention 1014, wherein at least one carrier molecule is polyethylene glycol (PEG). [The present invention 1016] The hIL2 ortholog of the present invention 1014, wherein at least one carrier molecule is polyethylene glycol (PEG). [The present invention 1017] Structure: [PEG]-[Linker] n -[hoIL2] comprising wherein n = 0 or 1, and hoIL2 is a human orthogonal IL2 polypeptide variant the hIL2 ortholog of the present invention 1014 [the present invention 1018] the hIL2 ortholog of the present invention 1017, wherein PEG has a molecular weight of 5 kDa to 80 kDa [the present invention 1019] the hIL2 ortholog of the present invention 1017, wherein PEG has a molecular weight of approximately 40 kDa [the present invention 1020] the hIL2 ortholog polypeptide of the present invention 1019, wherein hoIL2 is an IL2 polypeptide variant comprising the set of amino acid substitutions [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A] [the present invention 1021] hoIL2 has the amino acid sequence: the hIL2 ortholog polypeptide of the present invention 1019, which is an IL2 polypeptide variant of TIFF2025108545000015.tif29128 [the present invention 1022] a nucleic acid sequence encoding any hIL2 ortholog polypeptide of the present invention 1001 [the present invention 1023] a recombinant vector comprising the nucleic acid sequence of the present invention 1022 [the present invention 1024] a. a nucleic acid sequence encoding a transmembrane receptor molecule comprising the extracellular domain (ECD) of orthogonal hCD122 and operably linked to one or more expression control elements capable of achieving expression and surface presentation of the ECD of the transmembrane receptor molecule administering the engineered mammalian cell comprising the same to a subject suffering from a disease, disorder or condition; and b. administering a therapeutically effective dose of the hIL2 ortholog of the present invention 1001 to the subject a method of treating the disease, disorder or condition in the subject thereby [the present invention 1025] The method of the present invention 1024, wherein the engineered mammalian cell is an engineered T cell. [The present invention 1026] The method of the present invention 1025, wherein the engineered T cell is a CAR-T cell. [The present invention 1027] The method of the present invention 1026, wherein the hIL2 ortholog is the hIL2 ortholog of the present invention 1012. [The present invention 1028] The method of the present invention 1024, wherein the hIL2 ortholog is the hIL2 ortholog of the present invention 1017. [The present invention 1029] A method for preparing an engineered T cell product comprising at least 20% hoCD122 T cells, comprising the following steps: a. Isolating a population of T cells from a mammalian subject; b. Exposing the isolated population of T cells ex vivo to a recombinant vector comprising a nucleic acid sequence encoding hoCD122 operably linked to one or more expression control sequences to facilitate expression in mammalian T cells, under conditions that allow uptake of the recombinant vector by the T cells; c. Contacting the isolated population of T cells with an effective amount of the hIL2 ortholog of the present invention 1001. [The present invention 1030] The cell population product of the method of the present invention 1029, wherein the cell population comprises at least 20% engineered hoCD122 T cells.

Brief Description of the Drawings

[0030] The present invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that the various features of the drawings are not to scale. On the contrary, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. The drawings include the following figures.

[0031] [[FIG. 1]]Figure 1 of the accompanying drawings provides the Celltiterglo® values of NKL cells treated with 293 transfection supernatants from the experiments described in more detail in Example 7 of this specification. For NKL cells that received dilutions of each supernatant indicated in bold, duplicate Celltiterglo® values are shown in parallel columns. [[FIG. 2]] Figure 2 of the accompanying drawings provides the Celltiterglo® values of NKL cells ( "NKL hoRB cells") recombinantly modified to express the hCD122 orthogonal receptor of SEQ ID NO:2, treated with 293 transfection supernatants from the experiments described in Example 7. For NKL hoRB cells that received dilutions of each supernatant indicated in bold, duplicate Celltiterglo® values are shown in parallel columns.

Mode for Carrying Out the Invention

[0032] Detailed Description To make the present disclosure more readily understandable, certain terms and phrases are defined below and throughout this specification. The definitions provided herein are non-limiting and should be read in light of the knowledge known to those skilled in the art.

[0033] Before describing the methods and compositions, it should be understood that the present invention is not limited to the specific methods or compositions described, and thus, of course, can vary. It should also be understood that the technical terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0034] When a range of values is provided, it is understood that each intervening value, to one tenth of the unit of the lower limit, between the upper and lower limits of the range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any specified value or intervening value within the specified range and any other specified value or intervening value within the specified range is included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range, and each range such that either one or both of the upper and lower limits of the smaller range are included, or neither are included, is also included within the invention, subject to any specifically excluded limit values within the specified range. When the specified range includes one or both of the upper and lower limits, ranges excluding one or both of the included upper and lower limits are also included within the invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but some promising and preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which they are cited.

[0036] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, such as polypeptides, known to those of ordinary skill in the art.

[0037] Publications discussed in this specification are provided solely for their disclosure prior to the filing date of this application. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the date of publication of a provided publication may be different from the actual publication date and may need to be independently confirmed.

[0038] Unless otherwise indicated, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric pressure. Standard abbreviations are used, including the following: bp = base pair; kb = kilobase; pl = picoliter; s or sec = second; min = minute; h or hr = hour; aa = 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 concentration; mM = millimolar concentration; M = molar concentration; kDa = kilodalton; i.m. = intramuscular (into muscle); i.p. = intraperitoneal (into the peritoneal cavity); SC or SQ = subcutaneous (under the skin); QD = daily; BID = twice daily; QW = weekly; QM = monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = not statistically significant; PBS = phosphate buffered saline; PCR = polymerase chain reaction; NHS = N-hydroxysuccinimide; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Dulbecco's modified Eagle's medium; GC = genomic copy; EDTA = ethylenediaminetetraacetic acid.

[0039] Throughout this disclosure, it will be recognized that amino acids are referred to according to either the one-letter or three-letter designations. For the convenience of the reader, the one-letter and three-letter amino acid codes are provided in Table 1 below.

[0040] (Table 1) Abbreviations for Amino Acids TIFF2025108545000016.tif108128

[0041] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describe cloning and DNA 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 protein purification (including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization), as well as methods for chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).

[0042] Unless otherwise indicated, the following terms are intended to have the meanings set forth below. Other terms are defined elsewhere throughout this specification.

[0043] Activate: As used herein, the term "activate" is used with respect to a receptor or receptor complex to refer to a biological effect that results directly and / or through involvement in a multi-component signaling cascade in response to the binding of an agonist ligand to the receptor. For example, it can be said that the binding of an IL2 agonist to its cognate IL2 receptor "activates" receptor signaling to produce one or more intracellular biological effects (e.g., phosphorylation of STAT5).

[0044] Activity : As used herein, the term "activity" is used with respect to a molecule to describe a property of the molecule related to a test system or biological function, such as the degree of binding of one molecule to another. Examples of such biological functions include, but are not limited to, catalytic activity of a biologic agent, intracellular signaling, gene expression, the ability to stimulate cell proliferation, and the ability to modulate immunological activities such as inflammatory responses. "Activity" is typically expressed as biological activity per unit of agent administered, e.g., [catalytic activity] / [mg of protein], [immunological activity] / [mg of protein], international units of activity (IU), [STAT5 phosphorylation] / [mg of protein], [T-cell proliferation] / [mg of protein], plaque forming units (pfu), etc. The term "proliferative activity" encompasses activities that promote cell division, including uncontrolled cell division as observed in neoplastic diseases, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis, and angiogenesis.

[0045] Administer / Administration: The terms "administer" and "administering" are used interchangeably herein and refer to the act of contacting a subject (including in vitro, in vivo or ex vivo cells, tissues, organs or biological fluids of the subject) with an agent (e.g., an IL-2 ortholog, CAR-T cell, chemotherapeutic agent, antibody or modulator, or a pharmaceutical formulation comprising one or more of the foregoing). Administration of the agent can be accomplished through any of a variety of methods recognized in the art, including but not limited to topical, intravascular injection (including intravenous or intraarterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal, transmucosal, iontophoretic delivery, intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., into the bladder), inhalation via a respiratory inhaler, intraocular injection, intra-abdominal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), etc. The term "administer" includes contact of the agent with cells, tissues or organs as well as contact of the agent with a fluid (wherein the fluid is in contact with cells).

[0046] Adverse event : As used herein, the term "adverse event" refers to any undesirable experience associated with the use of a therapeutic or prophylactic agent in a subject. An adverse event need not necessarily be caused by the administration of a therapeutic or prophylactic agent (e.g., an hIL2 ortholog) and may arise from unrelated circumstances. Adverse events are typically classified as mild, moderate or severe. As used herein, the classification of adverse events as used herein follows the Common Terminology Criteria for Adverse Events v4.03 (CTCAE) published on June 14, 2010 by the U.S. Department of Health and Human Services, National Institutes of Health, National Cancer Institute.

[0047] Affinity : As used herein, the term "affinity" refers to the degree of specific binding of a first molecule (e.g., a ligand) to a second molecule (e.g., a receptor), K d (the dissociation constant (K between a molecule and its target off) and the binding constant (K on ) between the molecule and its target) as measured by the binding kinetics represented by the ratio).

[0048] Agonist: As used herein, the term "agonist" refers to a first agent that specifically binds to a second agent (the "target") and interacts with the target to cause or promote an increase in the activation of the target. In some instances, an agonist modulates cell activation, enhances activation, primes a cell to be activated by a second agent, or upregulates the expression of one or more genes, proteins, ligands, receptors, biological pathways, resulting in a pathway that can lead to cell proliferation or cell death such as cell cycle arrest or apoptosis, and is an activator of a receptor protein. In some embodiments, an agonist is an agent that binds to a receptor and changes the state of the receptor, resulting in a biological response that mimics the action of the receptor's endogenous ligand. The term "agonist" includes partial agonists, full agonists and superagonists. An agonist can be described as a "full agonist" if it leads to a substantially complete biological response (i.e., a response associated with the binding interaction of a naturally occurring ligand / receptor) induced by the receptor or partial agonist under study. A "superagonist" is a type of agonist that is capable of generating a greater maximal response than the endogenous agonist to a target receptor, and thus has an activity greater than 100% of the natural ligand. A superagonist is typically a synthetic molecule that exhibits 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% of the evaluable quantitative or qualitative parameters of the molecule in its naturally occurring form when evaluated at similar concentrations in comparable assays. It should be noted that the biological effects associated with a full agonist may differ in degree and / or type from those of a partial agonist or superagonist. In contrast to an agonist, an antagonist can specifically bind to a receptor but typically does not initiate a signal cascade mediated by the receptor and can modify the action of an agonist at that receptor. An inverse agonist is an agent that produces a pharmacological response opposite in direction to that of an agonist.

[0049] Antagonist : As used herein, the terms "antagonist" or "inhibitor" refer to a molecule that counteracts the action of an agonist. An antagonist blocks, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also block, inhibit, or reduce the constitutive activity of a target, e.g., a target receptor, even in the absence of a specified agonist. An inhibitor is, for example, a molecule that reduces, blocks, inhibits, delays activation, inactivates, desensitizes, or downregulates a gene, protein, ligand, receptor, biological pathway including an immune checkpoint pathway, or cell.

[0050] Antibody : As used herein, the term "antibody" collectively refers to (a) glycosylated and non-glycosylated immunoglobulins that specifically bind to a target molecule (including, without limitation, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4) and (b) IgG(1-4) delta C that competes with immunoglobulins derived with respect to binding to the target molecule H 2, F(ab')2, Fab, ScFv, V H 、V LRefers to immunoglobulin derivatives including, without limitation, tetrabody, tribody, diabody, dsFv, F(ab')3, scFv-Fc and (scFv)2. The term antibody is not limited to immunoglobulins derived from any particular mammalian species and includes murine, human, equine, camelid antibodies, human antibodies. The term antibody includes so-called "heavy chain antibodies" or "VHH" or "Nanobodies®" typically obtained from immunization of camelids (including camels, llamas and alpacas) (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448). Antibodies with a given specificity can also be derived from non-mammalian sources, such as VHH obtained from immunization of cartilaginous fish including sharks without limitation. The term "antibody" includes antibodies isolable from natural sources or from animals after immunization with an antigen, as well as monoclonal antibodies, bispecific antibodies, trispecific ones, chimeric antibodies, humanized antibodies, human antibodies, CDR grafting, veneering or deimmunized (e.g., to remove T-cell epitopes) antibodies, camelized ones (in the case of VHH), or engineered antibodies including molecules containing the binding domain of an antibody (e.g., CDR) in a non-immunoglobulin scaffold. The term "antibody" should not be construed as limited to any particular synthetic means and includes naturally occurring antibodies isolable from natural sources, as well as engineered antibody molecules prepared by "recombinant" means including antibodies isolated from transgenic animals that are transgenic for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with nucleic acid constructs that effect the expression of an antibody, antibodies isolated from combinatorial antibody libraries including phage display libraries. In one aspect, an "antibody" is a mammalian immunoglobulin. In some aspects, an antibody is a "full-length antibody" that includes variable and constant domains that provide binding and effector functions.The term "single domain antibody" (sdAb), as used herein, refers to an antibody fragment consisting of a monomeric variable antibody domain that is capable of selectively binding to an antigen and competing with respect to binding to the parental antibody from which it is derived. As used herein, the term "VHH" refers to a single domain antibody typically derived from camelid antibodies obtained from immunization of camelids (including camels, llamas and alpacas) (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448). Single domain antibodies can also be derived from non-mammalian sources such as VHHs obtained from immunization of cartilaginous fish, including sharks (IgNAR antibodies), and thus VHHs are also referred to as heavy chain antibodies or Nanobodies®. The term antibody includes antibody conjugates that include modifications that extend the duration of action, such as conjugation (e.g., PEGylation) to fusion proteins or polymers as described in more detail below.

[0051] Biological sample : As used herein, the term "biological sample" or "sample" refers to a sample obtained from or derived from a subject. By way of example, biological samples include materials selected from the group consisting of body fluids, blood, whole blood, plasma, serum, mucosal secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluids (e.g., vitreous humor, aqueous humor), lymph fluid, lymph node tissue, spleen tissue, bone marrow, and immunoglobulin-enriched fractions derived from one or more of these tissues. In some embodiments, the sample is obtained from a subject that has been exposed to a therapeutic regimen that includes a pharmaceutical formulation of an hhIL2 ortholog, e.g., repeatedly exposed to the same drug. In other embodiments, the sample is obtained from a subject that has not been recently exposed to an hIL2 ortholog or from a subject prior to the planned administration of an hIL2 ortholog.

[0052] "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, in order from amino-terminus to carboxy-terminus: (a) an antigen-binding domain (ABD), (b) a transmembrane domain (TD); and (c) one or more cytoplasmic signaling domains (CSD), wherein the domains described above may optionally be linked by one or more spacer domains. A CAR may also further comprise a signal peptide sequence that is customarily removed during post-translational processing of the CAR and presentation on the cell surface of a cell transformed with an expression vector containing the nucleic acid sequence encoding the CAR. CARs useful in the practice of the present invention are prepared according to principles well known in the art. See, for example, U.S. Patent No. 7,741,465 B1 to Eshhaar et al., 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 may be modified to incorporate the orthogonal receptors of the present invention include axicabtagene ciloleucel (sold by Gilead Pharmaceuticals under the trade name Yescarta®) and tisagenlecleucel (sold by Novartis under the trade name Kymriah®).

[0053] CAR-T cell : As used herein, the terms "chimeric antigen receptor T-cells" and "CAR-T cells" are used interchangeably and refer to T-cells that have been recombinantly modified to express a chimeric antigen receptor. As used herein, CAR-T cells may be engineered to express the hCD122 orthopolypeptide.

[0054] CD-122 ortholog : As used herein, the terms "CD122 ortho", "hoCD122", or "hoIL2Rb" are used interchangeably and refer to variants of the hCD122 polypeptide that contain amino acid substitutions at positions histidine 133 (H133) and tyrosine 134 (Y134) in the ECD of the hCD122 polypeptide. In some embodiments, CD-122 ortho contains an amino acid substitution from histidine to aspartic acid (H133D), glutamic acid (H133E) or lysine (H133K) at position 133 and / or an amino acid substitution from tyrosine to phenylalanine (Y134F), glutamic acid (Y134E) or arginine (Y134R) at position 134. In a preferred embodiment, the hCD122 orthogonal receptor is an hCD122 molecule having the amino acid substitutions H133D and Y134F. In one embodiment, the hCD122 orthogonal receptor is a polypeptide having the amino acid sequence of SEQ ID NO:2.

[0055] CDR.As used herein, the terms "CDR" or "complementary determining region" are intended to mean the discontinuous antigen-binding sites found within the variable regions of both the heavy and light chain immunoglobulin polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlaps or subsets of amino acid residues when compared to each other. In any event, the application of any definition for referring to the CDRs of an antibody or a grafted antibody or variants thereof is intended to be within the scope of the terms defined and used herein. In the context of the present disclosure, the numbering of CDR positions is provided according to the Kabat numbering convention.

[0056] Equivalent: As used herein, the term "equivalent" is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, a first measurement result of an evaluable quantitative parameter (e.g., the level of IL-2 activity as determined by CTLL-2 proliferation or phospho-STAT5 assay) and a second measurement result of an evaluable parameter do not deviate beyond the range that one of ordinary skill in the art would recognize as not producing a statistically significant difference in effect between the two results in that context, then the two measurement results would be considered "equivalent". In some cases, if one measurement result deviates 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% from another measurement result, the measurement results may be considered "equivalent". In certain embodiments, if one measurement result deviates less than 15%, or less than 10%, or less than 5% from a reference standard, then that measurement result is equivalent to the reference standard.

[0057] Derived from : 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 a polypeptide or nucleic acid has a sequence based on that of a reference polypeptide or nucleic acid (e.g., a naturally occurring IL-2 polypeptide or a nucleic acid encoding IL-2), but is not meant to be limiting with respect to the source or method by which the protein or nucleic acid is made. By way of example, the term "derived from" includes homologs or variants of the reference amino acid or DNA sequence.

[0058] Effective concentration (EC): As used herein, the terms "effective concentration" or its abbreviation "EC" are used interchangeably and refer to the concentration of an agent in an amount sufficient to effect a change in a given parameter in a test system. The abbreviation "E" refers to the magnitude of a given biological effect observed in a test system when the test system is exposed to a test substance. The abbreviation "EC" is used when the magnitude of the response is expressed as a function of the concentration of the test substance ("C"). In the context of a biological system, the term Emax refers to the maximum value of a given biological effect observed in response to an activating test substance at a saturating concentration. When the abbreviation EC is provided with a subscript (e.g., EC 40 , EC 50 , etc.), the subscript refers to the fraction of Emax of the biological response observed at that concentration. For example, if the concentration of a test substance sufficient to effect an induction of a measurable biological parameter in a test system is 30% of the maximum level of the measurable biological parameter in response to such test substance, this is referred to as the "EC 30 " of the test substance with respect to such biological parameter. Similarly, the term "EC 100 " is used to represent the effective concentration of an agent that results in a maximal (100%) response of a measurable parameter in response to the agent. Similarly, the term EC 50 (commonly used in the field of pharmacodynamics) refers to the concentration of an agent sufficient to effect a half-maximal (50%) change in a measurable parameter. The term "saturating concentration" refers to the maximum possible amount of a test substance that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacodynamics, the saturating concentration of a drug is typically used to represent a concentration of drug sufficient such that all available receptors are occupied by the drug, and EC 50 is the drug concentration that gives a half-maximal effect.

[0059] Concentrated: As used herein, the term "concentrated" refers to a molecule of interest being present at a concentration greater than (e.g., at least 3-fold greater, or at least 5-fold greater, or at least 10-fold greater, or at least 50-fold greater, or at least 100-fold greater, or at least 1000-fold greater than) the concentration of the molecule in an starting sample such as a biological sample (e.g., a sample in which the molecule is naturally present or a sample in which the molecule is present after administration); or (b) the sample being non-naturally manipulated such that the molecule is present at a greater concentration than in the environment in which the molecule was made (e.g., as in a recombinant modified bacterium or mammalian cell).

[0060] Extracellular domain : As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is outside the plasma membrane of a cell. The ECD can include the entire extracellular portion of a transmembrane protein, a cell surface or membrane-associated protein, a secreted protein, or a cell surface targeting protein.

[0061] hCD-122 : As used herein, the term "hCD122" refers to a naturally occurring human CD122 polypeptide, including its naturally occurring variants. The amino acid sequence of one naturally occurring hCD122 variant is as follows. TIFF2025108545000017.tif50128

[0062] Identity: As used herein with respect to polypeptides or DNA sequences, the term "identity" refers to subunit sequence identity between two molecules. The molecules are identical at a position if the subunit positions in both molecules are occupied by the same monomer subunit (i.e., the same amino acid residue or nucleotide). Similarity between two amino acid or two nucleotide sequences is a function of the number of identical positions. Generally, sequences are aligned so as to obtain the highest order match. If desired, identity can be calculated using published techniques and widely available computer programs such as the GCS program package (Devereux, et al., (1984) Nucleic Acids Res. 12:387), BLASTP, BLASTN, FASTA (Atschul, et al. (1990) J. Molecular Biol. 215:403-410). Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in the query sequence that match or satisfy some positive-valued threshold score "T" when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can increase.The cumulative score for nucleotide sequences is calculated using the parameters "M" (reward score for pairs of matching residues; always >0) and "N" (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of a word hit in each direction stops when (a) the cumulative alignment score drops by an amount X from its achieved maximum value; when the cumulative score drops below zero due to the accumulation of one or more negatively scored residues in the alignment; or (b) when the end of either sequence is reached. The BLAST algorithm parameters "W", "T", and "X" determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) functions similarly, but by default has a word size ("W") of 28, an expectation value ("E") of 10, M = 1, N = -2, and uses comparison of both strands. For amino acid sequences, the BLASTP program by default uses a word size (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, (1989) PNAS (USA) 89:10915-10919).

[0063] IL-2: As used herein, the terms "interleukin-2" or "IL-2" refer to naturally occurring IL-2 polypeptides that possess IL-2 activity. In some embodiments, IL-2 refers to mature wild-type human IL-2. Mature wild-type human IL-2 (hIL2) results as a 133 amino acid polypeptide (smaller than the signal peptide, consisting of an additional 20 N-terminal amino acids), as described in Fujita, et. al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequences of naturally occurring variants of mature wild-type human IL-2 (hIL2) are TIFF2025108545000018.tif is 13128. When used herein, the number of residues excludes the signal peptide that is the same as that of SEQ ID NO:4 based on the hIL2 sequence UniProt ID P60568.

[0064] IL2 activity : The term "IL2 activity" refers to one or more biological effects on cells in response to contacting the cells with an effective amount of an IL2 polypeptide. IL2 activity can be measured, for example, in a cell proliferation assay using CTLL-2 mouse cytotoxic T cells. See Gearing, A.J.H. and C.B. Bird (1987) in Lymphokines and Interferons, A Practical Approach. Clemens, M.J. et al. (eds): IRL Press. 295. The specific activity of recombinant human IL-2 is approximately 2.1×10 4 IU / μg, which is calibrated against the recombinant human IL-2 WHO International Standard (NIBSC code: 86 / 500). In some embodiments, for example, if the hIL2 orthogonal polypeptide of interest exhibits a reduced affinity for CD25 (or is engineered to possess such), IL2 activity can be evaluated in human cells such as YT cells that do not require CD25 to effect signal transduction through the IL2 receptor but can signal through the intermediate-affinity CD122 / CD132 receptor. The orthogonal human IL-2 of the present disclosure 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 the WHO International Standard (NIBSC code: 86 / 500) wild-type mature human IL-2 when evaluated at similar concentrations in comparable assays.

[0065] IL-2 ortholog: As used herein, the term "IL-2 ortholog" refers to a variant of hIL2 derived from an IL-2 parental 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 ortholog exhibits specific binding to a receptor comprising the orthogonal hCD122 ECD, and when contacted in an amount sufficient to effect a change with a cell expressing a transmembrane receptor comprising the ECD of the orthogonal hCD122 polypeptide, a signal characteristic of the signal generated by the intracellular domain (ICD) of the transmembrane receptor is produced. When the transmembrane receptor comprises the orthogonal hCD122 ECD and hCD122 ICD, binding of the hIL2 ortholog to such a receptor produces an intracellular signal characteristic of the activation of the Cd25 / CD122 / CD132 high-affinity of the CD122 / CD132 intermediate-affinity hIL2 receptor. The IL-2 ortholog exhibits significantly reduced binding to wild-type hCD122. The term "hIL2 ortholog" includes IL-2 orthogonal variants and modified hIL2 orthologs. In some embodiments, the hIL2 ortholog is derived from a naturally occurring variant of human IL2, and such a human IL2 ortholog may also be referred to as "hoCD122" or "hoRb". Certain modified IL-2 polypeptides are provided by Garcia et al. (U.S. Patent Application Publication US2018 / 0228842A1, published August 16, 2018). As used herein, the term "hIL2 ortholog" does not include the modified hIL2 polypeptides described in Garcia et al.'s U.S. Patent Application Publication US2018 / 0228842A1, published August 16, 2018.

[0066] In an amount sufficient to effect a change: As used herein, the phrase "in an amount sufficient to effect a change" refers to an amount of a test substance that provides a detectable difference, such as a biological function evaluated in a cell-based assay, in response to administration of an amount of the test substance between the level of an indicator measured prior to application of the test substance to the system (e.g., a baseline level) and the level after application. An "amount sufficient to effect a change" may be sufficient to be a therapeutically effective amount, but an "amount sufficient to effect a change" may be more or less than a therapeutically effective amount.

[0067] Requires treatment : As used herein, the term "in need of treatment" refers to a determination made by a physician or other caregiver with respect to a subject that the subject is in need of treatment or would potentially benefit from treatment. This determination is made based on a variety of factors that are within the realm of the physician's or caregiver's expertise.

[0068] Requires prevention : As used herein, the term "in need of prophylaxis" refers to a determination made by a physician or other caregiver with respect to a subject that the subject is in need of prophylactic care or would potentially benefit from it. This determination is made based on a variety of factors that are within the realm of the physician's or caregiver's expertise.

[0069] Inhibitor : As used herein, the term "inhibitor" refers to a molecule that, for example, decreases, blocks, prevents, delays the activation of, inactivates, desensitizes, or down-regulates a gene, protein, ligand, receptor, or cell. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates the constitutive activity of a cell or organism.

[0070] Intracellular domain: As used herein, the term "intracellular domain" or its abbreviation "ICD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is inside the plasma membrane of a cell. The ICD can include the transmembrane protein or membrane-associated protein, or the entire cytoplasmic portion of an intracellular protein.

[0071] Isolated : As used herein, the term "isolated" is used with respect to a polypeptide of interest that, when it occurs in nature, is in an environment different from the environment in which it may occur in nature. "Isolated" means that the polypeptide of interest is included in a sample in which the polypeptide of interest is substantially enriched and / or the polypeptide of interest is partially or substantially purified. When the polypeptide does not occur in nature, "isolated" indicates that the polypeptide is separated from the environment in which it was made by either synthetic or recombinant means.

[0072] Intracellular domain of an orthogonal receptor : As used herein, the term "intracellular domain of an orthogonal receptor" or "ICD-OR" refers to the portion of a transmembrane orthogonal receptor that is inside the plasma membrane of a cell that expresses the transmembrane orthogonal receptor. The ICD-OR can include one or more "proliferation signaling domains" or "PSDs", which refer to protein domains that send signals to cause the cell to enter mitosis and initiate cell proliferation. Examples include Janus kinases including, without limitation, JAK1, JAK2, JAK3, Tyk2, Ptk-2, homologous members of the Janus kinase family from other mammalian or eukaryotic species, the IL-2 receptor β and / or γ chains, and other subunits, or portions, modifications or combinations thereof, from proteins of the cytokine receptor superfamily that can interact with proteins of the Janus kinase family to transmit signals. Examples of signals include phosphorylation of one or more STAT molecules including, without limitation, one or more of STAT1, STAT3, STAT5a and / or STAT5b.

[0073] Kabat numbering : As used herein, the term "Kabat numbering" refers to the numbering system of amino acid residues that are more variable (e.g., hypervariable) than other amino acid residues in the heavy and light chain regions of immunoglobulins, as recognized in the art (Kabat, et al., (1971) Ann. NY Acad. Sci. 190:382-93; Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the purposes of the present disclosure, the positioning of CDRs in the variable regions of antibodies follows Kabat numbering or simply "Kabat".

[0074] Ligand : As used herein, the term "ligand" refers to a molecule that specifically binds to a receptor and causes a change in the activity of the receptor or a change in the response in a cell expressing the receptor. In one aspect, 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" encompasses natural and synthetic ligands. "Ligand" also encompasses small molecules, peptidomimetics of cytokines, and antibodies. The complex of a ligand and a receptor is referred to as a "ligand-receptor complex". A ligand can comprise one domain of a polypeptide or fusion protein (e.g., either domain of an antibody / ligand fusion protein).

[0075] Metastasis : As used herein, the term "metastasis" describes the spread of cancer cells from a primary tumor to surrounding tissues and distant organs.

[0076] Modified IL-2 ortholog: As used herein, the term "modified IL-2 ortholog" refers to an IL-2 ortholog that has been modified by one or more modifications such as pegylation, glycosylation (N-linked and O-linked), acylation or polysialylation, or conjugation (either chemically or as a fusion protein) with another polypeptide carrier molecule including albumin fusion polypeptides (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)) or / and Fc-fusion proteins, or with a targeting moiety such as IgG including target-directed IL-2 orthogonal polypeptides such as hhIL2 orthogonal polypeptide fusion proteins, ScFv-hIL2 orthogonal polypeptide fusion proteins and VHH-IL-2 orthogonal polypeptide fusion proteins. Modified hIL2 orthologs 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 useful, for example, for generating antibodies for use in detection assays (e.g., epitope tags) and for facilitating protein purification.

[0077] Modulate : As used herein, terms such as "modulate", "modulation" refer to the ability of a test substance to have a positive or negative or either direct or indirect effect on a response in a system including a biological system or biochemical pathway. The term "modulator" includes both agonists and antagonists.

[0078] Neoplastic disease: As used herein, the term "neoplastic disease" refers to a disorder or condition in a subject resulting from the overgrowth of cells or uncontrolled (or deregulated) cell replication. The term "neoplastic disease" refers to a disorder resulting from the presence of a neoplasm in a subject. Neoplasms can be classified as (1) benign, (2) premalignant (or "precancerous"); and (3) malignant (or "cancerous"). The term "neoplastic disease" includes diseases, disorders and conditions related to neoplasms that refer to conditions directly or indirectly related to neoplastic diseases, and includes, for example, pre-cancerous conditions such as angiogenesis and dysplasia or smoldering multiple myeloma. Examples of benign disorders resulting from deregulated cell replication include hypertrophic scars such as keloid scars.

[0079] N-terminus : As used herein in the context of the structure of a polypeptide, "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxy terminus") refer to the amino and carboxyl ends, respectively, that are at the very ends of a polypeptide, while the terms "N-terminal" and "C-terminal" refer to the relative positions in the amino acid sequence of a polypeptide with respect to the N-terminus and C-terminus, respectively, and can include the residues at the N-terminus and C-terminus. "Immediately N-terminal" or "immediately C-terminal" refers to the position of the first amino acid residue with respect to the second amino acid residue when the first and second amino acid residues are covalently linked to provide a continuous amino acid sequence.

[0080] Nucleic acid : The terms "nucleic acid", "nucleic acid molecule", "polynucleotide", etc. are used interchangeably herein and refer to nucleotides in polymeric form, either deoxyribonucleotides or ribonucleotides, of any length, 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.

[0081] Numbered according to hIL2: As used herein, the term "numbered according to hIL2" refers to identifying the position of a particular amino acid based on the position where the particular amino acid normally occurs in the sequence of mature wild-type hIL2 (SEQ ID NO:4). For example, with respect to hIL2, "R81" refers to the 81st (numbered from the N-terminus) amino acid in the sequence of mature wild-type hIL2 being arginine. It should be noted that the amino acid sequences of IL2 molecules from different mammalian species have different numbers of amino acids and sequences. Thus, referring to residues according to this convention helps to identify the IL2 species in question.

[0082] Numbered according to hCD122 : As used herein, the term "numbered according to hCD122" refers to identifying the position of a particular amino acid based on the position where the particular amino acid normally occurs in the sequence of the mature wild-type hCD122 molecule, in one embodiment, the hCD122 of SEQ ID NO. 3. For example, with respect to human CD122, H133 refers to the 133rd (numbered from the N-terminus) amino acid in the sequence of mature wild-type hCD122 being histidine.

[0083] Numbered according to the extracellular domain of hCD122 : As used herein, the term "numbered according to the extracellular domain of hCD122" or "numbered according to hCD122 ECD" refers to identifying the position of a particular amino acid based on the position where the particular amino acid normally occurs in the extracellular domain (ECD) sequence of the mature wild-type hCD122 molecule (SEQ ID NO. 3). For example, with respect to human CD122 ECD, H133 refers to the 133rd (numbered from the N-terminus) amino acid in the sequence of mature wild-type hCD122 ECD being histidine.

[0084] Functionally linked: The term "functionally linked" as used herein refers to a relationship between molecules, typically between polypeptides or nucleic acids, that are arranged in a construct such that each of the functions of the constituent molecules is retained, but where functional linkage can result in modulation of the positive or negative activity of the individual components of the construct. For example, functional linkage of a polyethylene glycol (PEG) molecule to a wild-type protein can result in a construct in which the biological activity of the protein is decreased compared to the wild-type molecule, yet the two are still considered to be functionally linked. When the term "functionally linked" is applied to the relationship of multiple nucleic acid sequences encoding different functions, the multiple nucleic acid sequences are combined into a single nucleic acid molecule and provide a nucleic acid that can achieve transcription and / or translation of a particular nucleic acid sequence in a cell when introduced into the cell using, for example, recombinant techniques. For example, a nucleic acid sequence encoding a signal sequence can be considered to be functionally linked to a DNA encoding a polypeptide if it results in the expression of the preprotein, thereby facilitating the secretion of the polypeptide; a promoter or enhancer can be considered to be functionally linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site can be considered to be functionally linked to a coding sequence if it is positioned to facilitate translation. Generally, in the context of nucleic acid molecules, the term "functionally linked" means that the linked nucleic acid sequences are adjacent, and in the case of a secretion leader or a subdomain of a related molecule, adjacent and in the reading phase. However, certain genetic elements, such as enhancers, can function distally and need not be adjacent to the sequences that provide their effects, yet they can still be considered to be functionally linked.

[0085] Orthogonal hCD122: As used herein, the terms "orthogonal hCD122" or "CD122 orthogonal receptor" are used interchangeably herein and refer to an hCD122 polypeptide variant that results in specific binding to an hhIL2 ortholog but does not specifically bind to naturally occurring variants of hIL2 and includes amino acid substitutions. 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) containing the substitutions H133D and Y134.

[0086] Orthogonal receptor : As used herein, the term "orthogonal receptor" refers to a variant of a receptor, i.e., an orthogonal receptor that exhibits significantly reduced binding to its cognate ligand but exhibits specific binding to an orthogonal ligand engineered to interact therewith and includes modifications to the amino acid sequence. In some embodiments, the orthogonal receptor may include an extracellular domain that exhibits significantly reduced binding to its cognate native ligand, while the orthogonal ligand exhibits significantly reduced binding to the ECD of its cognate native receptor. In some embodiments, the affinity of the orthogonal ligand for the cognate orthogonal receptor exhibits an affinity that is equivalent to the affinity of the native ligand for the native receptor, e.g., at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100% of the native cytokine receptor pair affinity, and may also have a higher affinity, e.g., 2×, 3×, 4×, 5×, 10× or more of the affinity of the native cytokine for the native receptor. The orthogonal receptor may be indicated by the parent molecule from which it is derived (e.g., orthogonal hCD122) or by the cognate ligand from which the orthogonal ligand for the orthogonal receptor is derived (e.g., orthogonal hIL2 receptor).

[0087] Ortholog: As used herein, the term "ortholog" refers to the ligand component of an orthogonal ligand / receptor pair and also refers to a polypeptide that incorporates into its primary structure a modification that provides a polypeptide variant that exhibits: (a) a significantly reduced affinity for its native cognate receptor (i.e., the native receptor for the parent polypeptide from which the ortholog is derived); and (b) specific binding to an engineered orthogonal receptor that is a variant of the cognate receptor for the ortholog. When an ortholog binds to an orthogonal receptor (which is expressed on the surface of a cell that has been modified by recombinant DNA technology such that a nucleic acid sequence encoding the orthogonal receptor is incorporated so as to effect expression of the orthogonal receptor in the recombinant modified cell), the activated orthogonal receptor initiates signal transduction that is transmitted through native cellular elements to provide a biological activity that mimics the native response of its cognate but is specific to a population of recombinant modified cells that express the orthogonal receptor. In some embodiments of the invention, the ortholog possesses significant selectivity for the orthogonal receptor compared to the cognate receptor and optionally possesses significantly reduced potency with respect to the cognate receptor. Selectivity is typically evaluated by the activity measured in an assay specific for the activity induced in response to ligand / receptor binding. In some embodiments, the ortholog has an EC50 where the affinity for the orthogonal receptor is increased by a differential of at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 100-fold, or at least 200-fold, as compared to that measured in the same assay.

[0088] Parent polypeptide: As used herein, the terms "parent polypeptide" or "parent protein" are used interchangeably and refer to the source of a second polypeptide (e.g., a derivative or variant) that is modified with respect to a first "parent" polypeptide. In some cases, the parent polypeptide is a wild-type or naturally occurring form of the protein. In some cases, the parent polypeptide can be a modified form of a naturally occurring protein that is further modified. The term "parent polypeptide" can refer to the polypeptide itself or a composition comprising the parent polypeptide (e.g., a glycosylated or PEGylated form and / or a fusion protein comprising the parent polypeptide).

[0089] Partial agonist : As used herein, the term "partial agonist" refers to a molecule that specifically binds to a given receptor and activates that receptor, but activates the receptor only partially compared to a full agonist. A partial agonist can exhibit both agonist and antagonist effects. For example, when both a full agonist and a partial agonist are present, the partial agonist acts as a competitive antagonist by competing with the full agonist for receptor binding, resulting in a net decrease in receptor activation compared to the case where the receptor is contacted with the full agonist in the absence of the partial agonist. Partial agonists can be used to activate a receptor to give a desired submaximal response in a subject when an inappropriate amount of endogenous ligand is present, or they can reduce overstimulation of the receptor when an excess amount of endogenous ligand is present. The maximal response (E) generated by a partial agonist max) is referred to as its intrinsic activity and can be represented on a percentage scale where a full agonist produces a 100% response. A partial agonist has an activity 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% of the activity of the reference polypeptide when evaluated at similar concentrations in a given assay system.

[0090] PEG-hIL2 ortholog : As used herein, the term "PEG-hIL2 ortholog" refers to an hIL2 ortholog covalently attached 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. The PEGylated polypeptide may further be referred to as monopegylated, dipegylated, tripegylated, etc., representing a PEG-hIL2 ortholog containing 1, 2, 3 (or more) PEG moieties attached to the IL-2 ortholog, respectively. In some embodiments, the PEG may be covalently attached directly to the IL-2 ortholog (e.g., through a lysine side chain, a sulfhydryl 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 contains more than one PEG molecule, each of which is attached to a different amino acid residue. In some embodiments, the PEG-hIL2 ortholog is derived from SEQ ID NO:4.

[0091] Polypeptide: As used herein, the terms "polypeptide," "peptide," and "protein," which are used interchangeably herein, refer to any length of amino acid multimers, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified polypeptide backbone. This term includes fusion proteins having non-homologous amino acid sequences; fusion proteins having non-homologous and homologous leader sequences; fusion proteins having or not having an N-terminal methionine residue; fusion proteins having immunologically tagged proteins; fusion proteins of immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments), etc., including but not limited to fusion proteins.

[0092] Prevent : As used herein, the terms "prevent," "preventing," "prevention," etc. generally refer to a series of actions initiated with respect to a subject who has a tendency to have a particular disease, disorder, or condition due to genetic, experiential, or environmental factors, to prevent, suppress, inhibit, or reduce the risk (e.g., as determined by the absence of clinical symptoms) or the occurrence of the subject's disease, disorder, condition, etc., either temporarily or permanently, before the onset of the disease, disorder, condition, or its symptoms. In certain instances, the terms "prevent," "preventing," "prevention" are also used to refer to slowing the progression of a disease, disorder, or condition from its current state to a worse state.

[0093] Receptor: As used herein, the term "receptor" refers to a polypeptide having a domain that specifically binds a ligand, and binding of the ligand alters at least one biological property of the polypeptide. In some embodiments, the receptor is a cell surface receptor that includes an extracellular domain (ECD) and a membrane-associated domain that serves to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide that includes an intracellular domain (ICD) and an extracellular domain (ECD) linked by a transmembrane domain, typically referred to as a transmembrane domain (TM). Binding of the cognate ligand to the receptor results in a conformational change in the receptor, which in turn produces a measurable biological effect. In some cases where the receptor is a transmembrane polypeptide that includes an ECD, TM, and ICD, binding of the ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to binding of the ligand to the ECD. In some embodiments, the receptor is a component of a multi-component complex that facilitates intracellular signaling. For example, a ligand may not be associated with any intracellular signaling alone, but binding of the ligand allows for the formation of a heteromultimer (including heterodimers, heterotrimers, etc.) or homomultimer (including homodimers, homotrimers, homotetramers, etc.) complex that results in a measurable biological effect such as activation of an intracellular signaling cascade (e.g., the Jak / STAT pathway). For example, a ligand may not be associated with any intracellular signaling alone, but binding of the ligand allows for the formation of a heteromultimer (e.g., intermediate affinity hCD122 / CD132 hIL2 receptor), heterotrimer (e.g., high affinity CD25 / CD122 / CD132 hIL2 receptor) including heteromultimers or homomultimers (homodimers, homotrimers, homotetramers) that result in activation of an intracellular signaling cascade (e.g., the Jak / STAT pathway), and can bind to cell surface molecules.In some embodiments, the receptor is a transmembrane single-chain polypeptide comprising an ECD, a TM, and an ICD domain, wherein the ECD, TM, and ICD domains are derived from the same or different naturally occurring receptor variants, or functional equivalents of their synthesis.

[0094] Recombinant As used herein, the term "recombinant" is used as an adjective and refers to the manner in which a polypeptide, nucleic acid, or cell has been modified using recombinant DNA technology. A "recombinant protein" is a protein produced using recombinant DNA technology, and is often abbreviated by prefixing the protein name with the lowercase letter "r" to denote that the protein was produced by that method (e.g., recombinantly produced human growth hormone is commonly abbreviated as "rhGH"). Similarly, a cell is referred to as a "recombinant cell" if it has been modified by incorporating an exogenous nucleic acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vector, plasmid, cosmid, etc.) using recombinant DNA technology (e.g., transfection, transduction, infection). Techniques and protocols 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 (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0095] Response:For example, the term "response" in relation to a cell, tissue, organ or organism encompasses a quantitative or qualitative change in an evaluable biochemical or physiological parameter (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy consumption rate, level or state of differentiation), which change correlates with activation, stimulation or treatment by an exogenous agent or internal mechanisms such as genetic programming, or contact therewith. In certain situations, terms such as "activation", "stimulation" etc. refer to cell activation when regulated by internal mechanisms as well as by external or environmental factors; while terms such as "inhibition", "downregulation" etc. refer to the opposite effects. A "response" can be evaluated in vitro through the use of an assay system, surface plasmon resonance, enzyme activity, mass spectrometry, amino acid or protein sequencing techniques etc. A "response" can be evaluated quantitatively in vivo by assessment of objective physiological parameters such as body temperature, body weight, tumor volume, blood pressure, results of X-ray or other imaging techniques, or qualitatively through changes in reported subjective sensations of well-being, depression, mood swings or pain. In some embodiments, the level of proliferation of CD3-activated primary human T-cells generates a luminescence signal proportional to the amount of ATP present, which is proportional to the number of cells present in a culture as described in Crouch, et al. (1993) J. Immunol. Methods 160: 81-8, in a bioluminescence assay, or can be evaluated by using a commercially available assay such as the CellTiter-Glo® 2.0 Cell Viability Assay or the CellTiter-Glo® 3D Cell Viability Kit, catalog numbers G9241 and G9681 from Promega Corporation (Madison WI 53711), substantially according to the instructions provided by the manufacturer. In some embodiments, the level of activation of T-cells in response to administration of a test substance can be determined by flow cytometry methods as described, such as by determining the level of phosphorylation 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 in commercially available kits implemented substantially in accordance with the instructions provided by manufacturers such as the aforementioned Horta et al., the aforementioned Garcia et al., or the Phospho-STAT5(Tyr694) kit (commercially available from Perkin-Elmer (Waltham MA) with Part Number 64AT5PEG).

[0096] Significantly reduced binding The term "showing significantly reduced binding" is used with respect to variants of a first molecule (e.g., a ligand) that show a significant decrease in affinity for a second molecule (e.g., a receptor) compared to the parental form of the first molecule. As used herein, the term "showing significantly reduced binding" is used with respect to the affinity of the binding of an orthogonal ligand to an orthogonal receptor compared to the binding of the orthogonal ligand to the naturally occurring form of its cognate receptor. If the orthogonal ligand binds to the naturally occurring form of the receptor at less than 20%, or about less than 10%, or about less than 8%, or about less than 6%, or about less than 4%, or about less than 2%, or about less than 1%, or about less than 0.5% of the binding of the naturally occurring ligand, then the orthogonal ligand shows significantly reduced binding with respect to the naturally occurring ligand. Similarly, if the naturally occurring ligand binds to the orthogonal form of the receptor at less than 20%, or about less than 10%, or about less than 8%, or about less than 6%, or about less than 4%, or about less than 2%, or about less than 1%, or about less than 0.5% of the binding of the naturally occurring receptor, then the orthogonal receptor shows significantly reduced binding with respect to the naturally occurring ligand.

[0097] Small molecule: The term "small molecule" refers to a compound (typically a pharmaceutically active compound) having 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 well understood by those of ordinary skill in the pharmaceutical art and is typically used to distinguish organic compounds from biologic agents.

[0098] Specifically bind : As used herein, the term "specifically binds" refers to the degree of selectivity or affinity for one molecule to bind to another molecule. In the context of a binding pair (e.g., ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pair), the first molecule of the binding pair is said to specifically bind to the second molecule of the binding pair if the first molecule of the binding pair does not bind in significant amounts to other components present in the sample. The affinity of the first molecule for the second molecule is at least 2-fold greater, or at least 5-fold greater, or at least 10-fold greater, or at least 20-fold greater, or at least 100-fold greater than the affinity of the first molecule for other components present in the sample, then the first molecule of the binding pair is said to specifically bind to the second molecule of the binding pair. In certain embodiments, when the first molecule of the binding pair is an antibody, the antibody binds to the second molecule of the binding pair (e.g., a protein, antigen, ligand or receptor) with an equilibrium dissociation constant between the antibody and the second molecule of the binding pair of about 10 6 M or greater, or about 10 8 M or greater, or about 10 10 M or greater, or about 10 11 M or greater, or about 10 10 M or greater, about 10 12When it is M ultra, for example, when determined by Scatchard analysis (Munsen, et al. 1980 Analyt. Biochem. 107:220-239), it binds specifically. In one aspect where the ligand is an hIL2 ortholog and the receptor contains an orthogonal hCD122 ECD, the hIL2 ortholog has an equilibrium dissociation constant of the hIL2 ortholog / orthogonal hCD122 ECD of about 10 5 M ultra, or about 10 6 M ultra, or about 10 7 M ultra, or about 10 8 M ultra, or about 10 9 M ultra, or about 10 10 M ultra, or about 10 11 When it is M ultra, it binds specifically. Specific binding can be evaluated using techniques known in the art, including competitive ELISA assays, radioligand binding assays (e.g., saturation binding, Scatchard plots, non-linear curve fitting programs, 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., multiwell plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays)) as well as surface plasmon resonance assays (e.g., see Drescher et al., (2009) Methods Mol Biol 493:323-343, and using commercially available devices such as Biacore 8+, Biacore S200, Biacore T200, etc. (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA 01752) non-limitingly).

[0099] Subject: The terms "recipient", "individual", "subject", and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, laboratory and domestic animals, and zoo, sports, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0100] Suffering from : As used herein, the term "afflicted" refers to a determination by a physician with respect to a subject, based on available information accepted in the art for the identification of a disease, disorder, or condition, including, but not limited to, x-rays, CT scans, conventional laboratory diagnostic tests (e.g., blood cell counts, etc.), genomic data, protein expression data, immunohistochemistry, that the subject requires treatment or would benefit from treatment. The term "afflicted" is typically used in conjunction with a specific disease state; for example, "afflicted with a neoplastic disease" refers to a subject diagnosed with the presence of a neoplasm.

[0101] Substantially pure : As used herein, the term "substantially pure" indicates that the components of a 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. A "substantially pure" protein comprises 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.

[0102] T-cell: As used herein, the term "T-cell" or "T cell" is used in its conventional meaning and refers to lymphocytes that differentiate in the thymus, possess specific cell surface antigen receptors, and control the initiation or suppression of cellular and humoral immunity and lyse antigen-containing cells. In some embodiments, T cells include, without limitation, naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, such as T H 1, T H 2, T H 9, T H 11, T H 22, T FH ; regulatory T cells, such as T R 1, Treg, induced Treg; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TIL), and engineered variants of such T-cells including, without limitation, CAR-T cells, recombinant modified TIL, and TCR-engineered cells.

[0103] Terminal / terminus : As used herein in the context of the structure of a polypeptide, "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxy terminus") refer, respectively, to the amino and carboxyl ends that are at the very ends of the polypeptide, while the terms "N-terminal" and "C-terminal" refer, respectively, to the relative positions in the amino acid sequence of the polypeptide with respect to the N-terminus and C-terminus, and can include the residues at the N-terminus and C-terminus, respectively. "Immediately N-terminal" refers to the position of the first amino acid residue relative to the second amino acid residue in a contiguous polypeptide sequence, with the first amino acid being closer to the N-terminus of the polypeptide. "Immediately C-terminal" refers to the position of the first amino acid residue relative to the second amino acid residue in a contiguous polypeptide sequence, with the first amino acid being closer to the C-terminus of the polypeptide.

[0104] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of an agent that, when administered to a subject, alone or as part of a pharmaceutical composition or treatment regimen, is capable of having any detectable positive effect on any symptom, aspect, or feature of a disease, disorder, or medical condition. A therapeutically effective amount can be determined by measuring the relevant physiological effects, and the amount can be adjusted in relation to the dosing regimen and in response to, for example, a diagnostic analysis of the subject's medical condition. Parameters for evaluation to determine a therapeutically effective amount of an agent are determined by a physician using accepted diagnostic criteria in the art, including, but not limited to, evidence such as age, weight, sex, general health, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, X-ray, etc. Alternatively, or in addition, other parameters commonly evaluated in a clinical setting, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or feature of a disease, disorder, or medical condition, biomarker (e.g., inflammatory cytokine, IFN-γ, granzyme, etc.), reduction of serum tumor marker, improvement of response evaluation criteria in solid tumors (RECIST), improvement of immune-related response criteria (irRC), increase in survival period, extended progression-free survival period, extended time to progression, increase in treatment success period, extended event-free survival period, extended time to next treatment initiation, improvement of objective response rate, improvement of response duration, reduction of tumor burden, complete response, partial response, stable disease, etc. (relying on clinicians in the art for evaluation of improvement of a subject's medical condition in response to administration of an agent) can be monitored to determine whether a therapeutically effective amount of the agent has been administered to the subject. As used herein, the terms "complete response (CR)", "partial response (PR)", "stable disease (SD)", and "progressive disease (PD)" with respect to target lesions, and the terms "complete response (CR)", "incomplete response / stable disease (SD)", and "progressive disease (PD)" with respect to non-target lesions are understood to be defined by RECIST criteria.As used herein, the terms "immune-related complete response (irCR)", "immune-related partial response (irPR)", "immune-related progressive disease (irPD)", and "immune-related stable disease (irSD)" are defined according to immune-related response criteria (irRC). As used herein, the term "immune-related response criteria (irRC)" refers to a system for evaluating responses 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. The therapeutically effective amount can be adjusted during the treatment of a subject in relation to the dosing regimen and / or evaluation of the subject's condition and variations in the foregoing factors. In one aspect, the therapeutically effective amount is the amount of an agent that does not cause irreversible serious adverse events during administration to a mammalian subject when used alone or in combination with another agent.

[0105] Transmembrane domain: The term "transmembrane domain" or "TM" refers to a domain of a transmembrane polypeptide (e.g., a transmembrane receptor) that, when the transmembrane polypeptide is associated with the cell membrane, is embedded in the cell membrane and is in a peptidyl-linked state with the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide. The transmembrane domain can be homologous (naturally related) or non-homologous (not naturally related) to either or both of the extracellular domain and / or intracellular domain. In some embodiments, the transmembrane domain is a transmembrane domain that is naturally related to the ECD domain of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, the transmembrane domain is a transmembrane domain that is naturally related to the ICD domain of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, the transmembrane domain is a transmembrane domain that is naturally related to a growth signaling domain. In some embodiments, the transmembrane domain is a transmembrane domain that is naturally related to a different protein. Alternatively, the transmembrane domain of the orthogonal receptor can be an artificial amino acid sequence that traverses the plasma membrane. In some embodiments, the transmembrane domain of the orthogonal receptor is a transmembrane domain that is normally associated with the ICD of the cognate receptor from which the orthogonal receptor is derived.

[0106] Treat: The terms "treating", "treatment", "treat" and the like refer to a series of acts ((such as contacting the subject with an hIL-2 ortholog, hoRb T cell, hoCAR-T cell, or a pharmaceutical composition comprising the same) alone or in combination with an adjuvant agent) initiated with respect to a subject in accordance with a diagnosis that the subject is suffering from a disease, disorder or medical condition or a symptom thereof, and the series of acts are initiated to eliminate, reduce, suppress, alleviate or improve at least one of the following, either temporarily or permanently: (a) the root cause of such a disease, disorder or medical condition that afflicts the subject; and / or (b) at least one of the symptoms associated with such a disease, disorder or medical condition. In some embodiments, treating is a series of acts carried out with respect to a subject suffering from a disease, the series of acts including those that result in inhibition of the disease in the subject (e.g., preventing the occurrence of the disease, disorder or medical condition or improving one or more symptoms associated therewith).

[0107] Treg cell or regulatory T cell . As used herein, the term "regulatory T cell" or "Treg cell" refers to a type of CD4 T cell capable of suppressing the responses of other T cells, including, without limitation, 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" means CD4 T cells other than regulatory T cells. + T cells. 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" means CD4 T cells other than regulatory T cells. + T cells.

[0108] Variant: The terms "variant", "protein variant", "variant protein", or "variant polypeptide" are used interchangeably herein and refer to a polypeptide that differs from a parent polypeptide due to at least one amino acid modification, substitution, or deletion. The parent polypeptide can be a naturally occurring or wild-type (WT) polypeptide or a modified version of a WT polypeptide. The term "variant polypeptide" can refer to the polypeptide itself, a composition containing the polypeptide, or the nucleic acid sequence encoding it. In some embodiments, the variant polypeptide contains 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 compared to the parent polypeptide, or alternatively, an amino acid modification, substitution, or deletion of a single amino acid. The variant can be at least about 99% identical, or at least about 98% identical, or at least about 97% identical, or at least about 95% identical, or at least about 90% identical to the parent polypeptide from which the variant is derived.

[0109] Wild type : As used herein, "wild-type" or "WT" or "native" means a naturally found amino acid sequence or nucleotide sequence that includes allelic variations. WT proteins, polypeptides, antibodies, immunoglobulins, IgG, etc. have amino acid sequences or nucleotide sequences that have not been artificially modified.

[0110] hIL2 ortholog Nomenclature: The present disclosure provides various polypeptide ligands of hIL2 receptor polypeptide variants. The following nomenclature is used herein to refer to substitutions, deletions or insertions. Residues may be designated herein by the one-letter or three-letter amino acid code of the naturally occurring amino acid found in the wild-type molecule followed by the hIL2 amino acid position of the mature hIL2 molecule, for example, "Cys125" or "C125" refers to the cysteine residue being at position 125 of the wild-type hIL2 molecule. For hIL2 orthologs, substitutions are designated herein by the one-letter amino acid code, followed by the hIL2 amino acid position, followed by the one-letter amino acid code of the substituted amino acid. The numbering of the hIL2 orthologs of the present disclosure follows that of hIL2. For example, an hIL2 ortholog having the modification "K35A" refers to the lysine (K) residue at position 35 of the wild-type hIL2 sequence being substituted at this position by an alanine (A) residue. Deletion of an amino acid residue is 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", "desA1" refer to the deletion of alanine at position 1 of the polypeptide of the wild-type hIL2 sequence. Similarly, for amino acid substitutions in orthogonal hCD122, amino acid substitutions are designated herein by the one-letter amino acid code of the naturally occurring amino acid, followed by the number of its position in the wild-type hIL2 sequence, followed by the one-letter amino acid code of the amino acid substituted at that position. Modifications to hCD122 incorporated into the orthogonal receptor are numbered according to hCD122. For example, in an hCD122 orthogonal receptor in which the tyrosine residue at position 134 is substituted by a phenylalanine residue, the substitution is abbreviated as "Y134F". The abbreviation hoRb is used synonymously with hoCD122 and refers to human CD122 including the orthogonal hCD122 receptor. Similarly, reference to "hoRb cells" (e.g., hoRb T cells or hoRb NKL cells) refers to cells expressing the orthogonal hCD122 receptor.The term SQVLKA, as used herein, refers to an hIL2 ortholog comprising the amino acid substitutions: E15S, H16Q, L19V, D20L, Q22K and M23A.

[0111] hIL2 ortholog In some embodiments, the present disclosure provides an hIL2 ortholog, which is a cognate ligand of an orthogonal receptor comprising a modified hCD122 ECD, and methods of using the same. In some embodiments, the term hIL2 ortholog refers to an hIL2 variant that is a ligand for a receptor comprising the extracellular domain of human orthogonal hCD122, which comprises an amino acid substitution at position H133 and / or Y134. In some embodiments, the hIL2 ortholog is a ligand for an orthogonal receptor comprising the extracellular domain of human hCD122, which comprises an amino acid substitution at position H133 and / or Y134. In some embodiments, the hIL2 ortholog is a ligand for a transmembrane receptor comprising the extracellular domain of human CD122, which comprises an amino acid substitution at position H133 and / or Y134, and whose ICD comprises one or more STAT3 binding motifs. In some embodiments, the hIL2 ortholog is a ligand for an orthogonal human CD122, which comprises an amino acid substitution at positions H133 and Y134. In some embodiments, the hIL2 ortholog is a ligand for an orthogonal hCD122, which comprises the amino acid substitutions H133D and Y134F.

[0112] In various embodiments, the compositions and methods of the present disclosure include the use of an hIL2 ortholog polypeptide having at least 80%, or 85%, or 90%, or 95%, or 97%, or 99% identity to the polypeptide of Formula #1 below: TIFF2025108545000019.tif72156wherein, AA1 is A (wild type) or a deletion; AA2 is P (wild type) or a deletion; AA3 is T (wild type), C, A, G, Q, E, N, D, R, K, P, or a deletion; AA4 is S (wild type) or a deletion; AA5 is S (wild type) or a deletion; AA6 is S (wild type) or a deletion; AA7 is T (wild type) or a deletion; AA8 is K (wild type) or a deletion; AA9 is K (wild type) or a deletion; AA13 is Q (wild type), W or a deletion; AA14 is L (wild type), M, W or a deletion; AA15 is E (wild type), K, D, T, A, S, Q, H or a deletion; AA16 is H (wild type), N or Q, or a deletion; AA18 is L (wild type) or R, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T; AA19 is L (wild type), A, V, I or a deletion; AA20 is D (wild type), T, S, M, L, or a deletion; AA22 is Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, F, or a deletion; AA23 is M (wild type), A, W, H, Y, F, Q, S, V, L, T, or a deletion; AA27 is G (wild type), K, S or a deletion; AA38 is R (wild type), W or G; AA39 is M (wild type), L or V; AA42 is F (wild type) or K; AA51 is T (wild type), I or a deletion AA55 is H (wild type) or Y; AA74 is Q (wild type), N, H, S; AA80 is L (wild type), F or V; AA81 is R (wild type), I, D, Y, T or a deletion AA85 is L (wild type) or V; AA86 is either I (wild type) or V; AA88 is either N (wild type), E or Q, or a deletion; AA89 is either I (wild type) or V; AA91 is either V (wild type), R or K; AA92 is either I (wild type) or F; AA97 is either K (wild type) or Q; AA104 is either M (wild type) or A; AA109 is either D (wild type), C or a non-natural amino acid with an activated side chain; AA113 is either T (wild type) or N; AA125 is either C (wild type), A or S; AA126 is either Q (wild type) or H, M, K, C, D, E, G, I, R, S, or T; and / or AA130 is either S (wild type), T or R.

[0113] A series of hIL2 orthologs of Formula 1 above (represented with the signal peptide at the N-terminus of the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO. 421) added to the mature protein sequence) were prepared as provided in Table 2 below according to the examples and evaluated for their ability to selectively activate NKL cells compared to NKL cells engineered to express the orthogonal CD122 of SEQ ID NO:2. T cells expressing an orthogonal IL2 receptor containing the substitution.

[0114] (Table 2) hIL2 ortholog TIFF2025108545000020.tif187164TIFF2025108545000021.tif225163TIFF2025108545000022.tif225163TIFF2025108545000023.tif225163TIFF2025108545000024.tif225163TIFF2025108545000025.tif225163TIFF2025108545000026.tif225163TIFF2025108545000027.tif225163TIFF2025108545000028.tif225163TIFF2025108545000029.tif220163TIFF2025108545000030.tif220163TIFF2025108545000031.tif225163TIFF2025108545000032.tif110163

[0115] The activity of the aforementioned hIL2 ortholog was evaluated for its ability to activate NKL and hoRb NKL cells substantially in accordance with the teachings herein at the following dilutions. The results of these experiments are provided in FIGS. 1 and 2 of the accompanying drawings, and in Table 3 below.

[0116] (Table 3) Proliferative activity of hIL2 ortholog against hoCD122 NKL cells vs. NKL cells TIFF2025108545000033.tif226162TIFF2025108545000034.tif227162TIFF2025108545000035.tif227162TIFF2025108545000036.tif226162TIFF2025108545000037.tif226162TIFF2025108545000038.tif226162TIFF2025108545000039.tif227162TIFF2025108545000040.tif227162TIFF2025108545000041.tif227162TIFF2025108545000042.tif226162TIFF2025108545000043.tif188162

[0117] The IL2 ortholog was evaluated for its activity in CD4-positive human T cell clone 3F8 cells, as described in detail in Example 8. The data obtained from these experiments are provided in Table 4 below.

[0118] (Table 4) Proliferative activity of hIL2 ortholog on hoCD122 3F8 cells vs. 3F8 cells TIFF2025108545000044.tif222164TIFF2025108545000045.tif222164TIFF2025108545000046.tif222164TIFF2025108545000047.tif222164TIFF2025108545000048.tif222164TIFF2025108545000049.tif222164TIFF2025108545000050.tif222164TIFF2025108545000051.tif222164TIFF2025108545000052.tif222164TIFF2025108545000053.tif222164TIFF2025108545000054.tif222164TIFF2025108545000055.tif203163

[0119] As demonstrated by the above data, the hIL2 ortholog of Formula 1 selectively activates hoCD122 human T cells compared to human T cells (NKL cells) that do not express an orthogonal receptor.

[0120] Conservative amino acid substitution In some embodiments, the hIL2 ortholog of Formula 1 may optionally include one or more conservative amino acid substitutions. Such conservative substitutions include those described in The Atlas of Protein Sequence and Structure 5 (1978) by Dayhoff and in EMBO J., 8:779-785 (1989) by Argos. Conservative substitutions are generally made according to Table 4 below.

[0121] (Table 4) Conservative amino acid substitutions TIFF2025108545000056.tif117138

[0122] Substantial changes in function or immunological identity can be made by selecting amino acid substitutions that are less conservative than those shown in Table 4. For example, substitutions can be made that more significantly affect the structure of the polypeptide backbone or disrupt secondary or tertiary elements, including substitutions of amino acids with small uncharged side chains (e.g., glycine) with large bulky charged side chains (asparagine). In particular, substitutions of hIL2 residues involved in amino acids that interact with one or more of CD25, CD122, and / or CD123 can be discerned from the crystal structure of hIL2 associated with its receptor, as described in Wang, et al (2005) Science 310: 1159-1163. Modifications to the primary structure as provided above can optionally further include modifications including, without limitation, substitutions: N30E; K32E; N33D; P34G; T37I, M39Q, F42Y, F44Y, P47G, T51I, E52K, L53N, Q57E, M104A (see U.S. Patent No. 5,206,344).

[0123] Cys125: In some embodiments, the disclosure provides an hIL2 ortholog of Formula 1 containing a modification that facilitates recombinant expression in bacterial cells by eliminating the unpaired cysteine residue at position 125 by substitution with C125A or C125S. In some embodiments, the hIL2 ortholog of the invention comprises one of the following sets of amino acid modifications: including one of TIFF2025108545000057.tif136128.

[0124] Mutation to enhance hCD122 affinity In some embodiments, the hIL-2 ortholog of Formula 1 contains one or more mutations that change the orientation of the hCD122 contact position or other positions contacting hCD122 in the hIL-2 sequence, modifying the binding affinity of the hIL2 ortholog for hCD122. hIL-2 residues identified as being involved in the binding of hIL2 to hCD122 include L12, Q13, H16, L19, D20, M23, R81, D84, S87, N88, V91, I92, and E95. In some embodiments, the hIL2 ortholog contains one or more of the amino acid substitutions: Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I92F, or combinations thereof. In some embodiments, the hIL2 ortholog contains one or more of the amino acid substitutions: L80F, R81D, L85V, I86V and I92F. In some embodiments, the hIL2 ortholog contains one or more of the amino acid substitutions: N74Q, L80F, R81D, L85V, I86V, I89V, and I92F. In some embodiments, the hIL2 ortholog contains one or more of the amino acid substitutions: Q74N, L80V, R81T, L85V, I86V, and I92F. In some embodiments, the hIL2 ortholog contains one or more of the amino acid substitutions: Q74H, L80F, R81D, L85V, I86V and I92F. In some embodiments, the hIL2 ortholog contains one or more of the amino acid substitutions: Q74S, L80F, R81D, L85V, I86V and I92F. In some embodiments, the hIL2 ortholog contains one or more of the amino acid substitutions: Q74N, L80F, R81D, L85V, I86V and I92F. In some embodiments, the hIL2 ortholog contains one or more of the amino acid substitutions: Q74S, R81T, L85V, and I92F. In some embodiments, the hIL2 ortholog contains the set of mutations [L80F-R81D-L85V-I86V-I92F] identified as increasing the affinity of hIL2 for hCD122. In some embodiments, the present disclosure provides the following set of amino acid modifications: Provided is an hIL2 ortholog that is an hIL2 polypeptide comprising one of TIFF2025108545000058.tif66135.

[0125] In some embodiments, the hIL-2 ortholog of Formula 1 comprises the substitution L85V, which has been identified as increasing the affinity of hIL2 for hCD122. In some embodiments, the present disclosure provides the following set of amino acid modifications: Provided is an hIL2 ortholog that is an hIL2 polypeptide comprising one of TIFF2025108545000059.tif59128.

[0126] Modification to modulate CD25 affinity In some embodiments, the hIL-2 ortholog of Formula 1 contains one or more mutations at the position of the hIL-2 ortholog that modulate the binding affinity for hCD25. The mutated region of hIL2 is proximal to hCD25 as part of the trimeric IL2 receptor complex based on the crystal structure of IL2 associated with the IL2 receptor (Wang, et al (2005) Science 310:1159). In some embodiments, the hIL-2 ortholog of Formula 1 contains a modification at one or more positions selected from 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 incorporated into the hIL2 ortholog sequence include one or more substitutions selected from 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 ortholog of the present disclosure comprises one or more point mutations 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 (Wittrup et al., supra); 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); F42A / G / S / T / Q / E / N / R / K, Y45A / G / S / T / Q / E / N / D / R / K and / or L72G / A / S / T / Q / E / N / D / R / K (U.S. Patent Application Publication 2012 / 0244112 A1 by Ast et al. published on September 27, 2012; U.S. Patent No. 9,266,938 B2 issued on February 23, 2016).

[0127] In addition to point mutations, combinations of the aforementioned modifications may be used to modulate the binding of the hIL-2 ortholog of Formula 1 to CD25. In some embodiments, the hIL2 ortholog of the present disclosure comprises one or more of the following sets of amino acid substitutions: TIFF2025108545000060.tif90169. In some embodiments, the present disclosure comprises one of the following sets of amino acid modifications: TIFF2025108545000061.tif90128 provides an hIL-2 ortholog of Formula 1 comprising one of the following.

[0128] Modification to modulate CD132 affinity In some aspects of the present invention, the hIL-2 orthologs of the present disclosure contain one or more mutations that modulate the binding of the hIL-2 ortholog to CD132. Exemplary hIL-2 orthologs contain one or more mutations at positions in the hIL-2 sequence that change the orientation of other positions that contact CD132 or contact hCD122, resulting in a change in binding to CD132. hIL-2 residues that have been identified as modulating 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 aspects, the present disclosure provides hIL2 orthologs that are hIL2 polypeptides comprising one of the following sets of amino acid modifications: TIFF2025108545000062.tif35128TIFF2025108545000063.tif237124, and provides an hIL2 ortholog that is an hIL2 polypeptide comprising one of them.

[0129] Removal of glycosylation site The hIL2 orthologs of the present disclosure may further or optionally provide for the elimination of the O-glycosylation site at position Thr3 to facilitate the production of glycosylated hIL2 ortholog variants when the ortholog is expressed in mammalian cells such as CHO or HEK cells. Thus, in certain aspects, the hIL2 ortholog further comprises a modification that eliminates the O-glycosylation site of IL-2 at the position corresponding to residue 3 of human IL-2. In one aspect, the modification that eliminates the O-glycosylation site of IL-2 at the position corresponding to residue 3 of human IL-2 is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P, which remove the glycosylation site at position 3 without eliminating biological activity (see U.S. Patent No. 5,116,943; Weiger et al., (1989) Eur. J. Biochem., 180:295-300). In certain aspects, the modification is the amino acid substitution T3A. In some aspects, the present disclosure provides the following sets of amino acid modifications: Provided is an hIL2 ortholog that is an hIL2 polypeptide comprising one of 184128 of TIFF2025108545000064.tif.

[0130] N-terminal deletion: The IL-2 ortholog may further include elimination of one or more N-terminal amino acids at positions 1-9, or positions 1-8, or positions 1-7, or positions 1-6, or positions 1-5, or positions 1-4, or des1-3, or positions 1-2 while retaining hIL2 ortholog activity. In some embodiments, the present disclosure is a set of the following amino acid modifications: Provided is an hIL2 ortholog that is an hIL2 polypeptide comprising one of 90128 of TIFF2025108545000065.tif and 191146 of TIFF2025108545000066.tif.

[0131] In some embodiments, the hIL2 ortholog may include deletion of the first two amino acids (desAla1-desPro2), as well as a selective N-terminal modification, particularly substitution of Thr3 glycosylation with a cysteine residue that facilitates PEGylation of the sulfhydryl group of the cysteine (see, for example, U.S. Patent No. 5,206,344 to Katre et al., issued April 27, 1993). In some embodiments, the present disclosure is a set of the following amino acid modifications: Provided is an hIL2 ortholog that is an hIL2 polypeptide comprising one of 90128 of TIFF2025108545000067.tif.

[0132] In some embodiments, the hIL2 ortholog of the present disclosure includes deletion of alanine at position 1 ("desAla1" hIL2 ortholog). In some embodiments, the present disclosure is a set of the following amino acid modifications: Provided is a des-Ala1-hIL2 ortholog that is one of 207111 of TIFF2025108545000068.tif.

[0133] Modification to minimize vascular leak syndrome In some aspects of the present disclosure, the hIL2 orthologs contain amino acid substitutions to avoid vascular leak syndrome. U.S. Patent No. 7,514,073B2 to Epstein et al., issued April 7, 2009. Examples of such modifications that can be incorporated into the hIL2 orthologs of the present disclosure include one or more of R38W, R38G, R39L, R39V, F42K, and / or H55Y.

[0134] Oxidation-stabilized M104A: In some aspects of the present disclosure, the hIL2 orthologs can contain a modification at position M104. In one aspect, substitution of the methionine 104 with an alanine residue (M104A) provides a more oxidation-resistant ortholog (U.S. Patent 4,752,585 to Koths et al., issued June 21, 1988).

[0135] Affinity maturation: In some aspects, the hIL2 orthologs of the present disclosure may be affinity matured to enhance their activity with respect to orthogonal hCD122. An "affinity matured" polypeptide has one or more mutations in one or more residues that result in an improvement in the affinity (or vice versa) for the cognate orthogonal receptor of the orthogonal polypeptide as compared to the parent polypeptide that does not carry the mutation. Affinity maturation can be performed to increase the binding affinity of the hIL2 ortholog by at least about 10%, or at least about 50%, or at least about 100%, or at least about 150%, or 1 - 5 fold as compared to the "parent" polypeptide. The engineered hIL2 orthologs of the present invention activate their cognate orthogonal receptors as discussed above, but have significantly reduced binding and activation of the native receptor as evaluated by ELISA and / or FACS analysis using a sufficient amount of the molecule under appropriate assay conditions.

[0136] Modification to extend in vivo duration of action In some embodiments, the hIL-2 orthologs of the present disclosure can include modifications to provide an extended in vivo lifespan and / or an extended duration of action in a subject. In some embodiments, the hIL-2 orthologs of the present disclosure have a plasma half-life in a human subject that exceeds 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 hIL2 orthologs with extended lifespans can be achieved by primary sequence modifications and / or conjugation to a carrier molecule.

[0137] Primary sequence modification to extend duration of action The hIL-2 orthologs of the present disclosure can include amino acid substitutions that result in a long-term in vivo lifespan. Examples of positions where amino acid substitutions can be incorporated into the hIL-2 ortholog to provide an extended in vivo duration include one or more of positions V111, R117, and / or T133. In some embodiments, the hIL2 orthologs of the present disclosure include one or more of the V111R, R117K, and / or T133N modifications. Dakshinamurthi, et al. (2009) International Journal of Bioinformatics Research 1 (2):4-13.

[0138] Carrier molecule In some embodiments, the hIL2 ortholog is modified to provide an extended duration of action in a subject, and this modification can be achieved through conjugation to a carrier molecule, providing desired pharmacological properties such as an extended half-life. In one embodiment, the hIL2 ortholog can include a functional domain of a chimeric polypeptide. In some embodiments, the hIL2 ortholog can be covalently linked to the Fc domain of IgG, albumin, or other molecules, such as by pegylation, glycosylation, etc., as known in the art, to extend its half-life.

[0139] Fc fusion In some embodiments, the hIL2 ortholog of the present disclosure is operably linked to a functional domain of an Fc fusion chimeric polypeptide molecule. Fc fusion conjugates have been shown to increase the systemic half-life of biologics, and thus, biopharmaceuticals can be administered less frequently. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels, and upon binding, the Fc fusion molecule is protected from degradation and re-released into circulation, where the molecule is maintained longer. This Fc binding is thought to be the mechanism by which endogenous IgG retains its long plasma half-life. More recently, Fc fusion technology has optimized the pharmacokinetic and pharmacodynamic properties of biologics by linking a single copy of the biologic to the Fc region of an antibody. A "Fc region" useful in the preparation of Fc fusions can be a naturally occurring or synthetic polypeptide that is homologous to the IgG C-terminal domain generated by papain digestion of IgG. The IgG Fc has a molecular weight of approximately 50 kDa. The hIL2 ortholog can provide a smaller portion that retains the ability to extend the circulating half-life of a chimeric polypeptide that is the entire Fc region or a portion thereof. Additionally, the full-length or fragmented Fc region can be a variant of the wild-type molecule. In a typical illustration, each monomer of the dimeric Fc carries a non-identical polypeptide, and the non-identical polypeptides are the same or different.

[0140] In some embodiments, when attempting to administer an hIL2 ortholog in the format of an Fc fusion, particularly in situations where the polypeptide chains conjugated to each subunit of the Fc dimer are different, the Fc fusion may be engineered to possess a "knob-into-hole modification." The knob-into-hole modification is described in more detail by Ridgway, et al. (1996) Protein Engineering 9(7):617-621 and U.S. Patent No. 5,731,168, issued March 24, 1998. The knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domain, where i) in the CH3 domain of the first heavy chain, an amino acid residue is replaced with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan) to create a protrusion from the surface ("knob"), and ii) in the CH3 domain of the second heavy chain, an amino acid residue is replaced with an amino acid residue having a smaller side chain (e.g., alanine or threonine), thereby creating a cavity ("hole") within the interface in the second CH3 domain, into which the protruding side chain ("knob") of the first CH3 domain is received. In one embodiment, the "knob-into-hole modification" includes the amino acid substitutions T366W and optionally S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V, and optionally Y349C in the other antibody heavy chain. Further, the Fc domain may be modified by introduction of cysteine residues at positions S354 and Y349 that result in stable disulfide bridging between the two antibody heavy chains in the Fc region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole format is used to facilitate the expression of a first polypeptide (e.g., an hIL2 ortholog) on a first Fc monomer having a "knob" modification and a second polypeptide on a second Fc monomer bearing a "hole" modification, enabling the expression of a heterodimeric polypeptide conjugate.

[0141] The Fc region can be "soluble" or "insoluble", but is typically insoluble. The insoluble Fc region typically lacks high affinity Fc receptor binding sites and Clq binding sites. The high affinity Fc receptor binding site of murine IgG Fc contains a Leu residue at position 235 of the IgG Fc. Thus, the Fc receptor binding site can be inhibited by mutating or deleting Leu 235. For example, substituting Leu 235 with Glu inhibits the ability of the Fc region to bind to high affinity Fc receptors. The murine Clq binding site can be functionally disrupted by mutating or deleting the Glu 318, Lys 320, and Lys 322 residues of IgG. For example, substituting Glu 318, Lys 320, and Lys 322 with Ala residues renders IgG1 Fc unable to direct antibody-dependent complement lysis. In contrast, the soluble IgG Fc region has high affinity Fc receptor binding sites and Clq binding sites. The high affinity Fc receptor binding site contains a Leu residue at position 235 of the IgG Fc, and the Clq binding site contains the Glu 318, Lys 320, and Lys 322 residues of IgG 1. Soluble IgG Fc has wild-type residues or conservative amino acid substitutions at these sites. Soluble IgG Fc can target cells for antibody-dependent cell cytotoxicity or complement-directed cell lysis (CDC). Appropriate mutations for human IgG are also known (see, e.g., Morrison et al., The Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2: 125, 1994). In some embodiments, the Fc domain monomer contains at least one mutation compared to the wild-type human IgG1, IgG2, or IgG4 Fc region as described in U.S. Patent No. US10,259,859 B2, the entire teachings of which are incorporated herein by reference. In some embodiments, the polypeptide exhibits a reduction in phagocytosis compared to a polypeptide having a wild-type human IgG Fc region in a phagocytosis assay.In some embodiments, the Fc domain monomer is linked to a second polypeptide comprising a second Fc domain monomer to form an Fc domain dimer.

[0142] PEGylation: In some embodiments, the hIL2 ortholog of the present disclosure may be conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of the present invention include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyolefin alcohol), polysaccharides), poly-alpha-hydroxy acids), polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0143] In some embodiments, the hIL2 ortholog is operably linked or "PEGylated" to one or more polyethylene glycol molecules. There can be various methods or sites of attachment of PEG to the hIL2 ortholog, but in certain embodiments, PEGylation does not change or minimally changes the activity of the hIL2 ortholog.

[0144] In some embodiments, a hIL2 ortholog with reduced affinity for one or more subunits of the hIL2 receptor complex (e.g., CD25, CD132) can be generated using selective PEGylation of the hIL2 ortholog (e.g., by incorporation of unnatural amino acids having side chains that facilitate selective PEG conjugation chemistry) as described by Ptacin et al. (PCT International Application No. PCT / US2018 / 045257, filed August 3, 2018 and published as International Publication No. WO 2019 / 028419A1 on February 7, 2019). For example, a hIL2 ortholog incorporating an unnatural amino acid having a specific moiety capable of being PEGylated at such sequences or residues of hIL2 identified as interacting with CD25, including amino acids 34-45, 61-72, and 105-109, provides a hIL2 ortholog with modulated binding to CD25. Similarly, a hIL2 ortholog incorporating an unnatural amino acid having a specific moiety capable of being PEGylated at such sequences or residues of hIL2 identified as interacting with hCD132, including, without limitation, amino acids 18, 22, 109, 126, and / or 133, provides a hIL2 ortholog with modulated binding affinity to hCD132.

[0145] In certain embodiments, the increase in half-life is greater than any decrease in biological activity. PEGs suitable for conjugation to polypeptide sequences are generally water-soluble at room temperature and have the general formula R(O-CH2-CH2) n O-R, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, the protecting group generally has 1 to 8 carbons. The PEG conjugated to the polypeptide sequence can be linear or branched. "Star PEG" and multi-arm PEGs of branched PEG derivatives are contemplated by the present disclosure.

[0146] In the context of the hIL2 orthologs of the present disclosure, the molecular weight of the PEG useful is not necessarily limited to any particular range. The PEG component of the PEG-hIL2 ortholog can have a molecular mass of greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular mass is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa or about 10 kDa to about 30 kDa. The linear or branched PEG molecule has a molecular weight of about 2,000 to about 80,000 daltons, 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 containing two 20kD arms.

[0147] The present disclosure also contemplates conjugate compositions in which the PEG has different n values and thus various different PEGs are present in a specific ratio. For example, some compositions include a mixture of conjugates where n = 1, 2, 3, and 4. In some compositions, the percentage of the conjugate where n = 1 is 18 - 25%, the percentage of the conjugate where n = 2 is 50 - 66%, the percentage of the conjugate where n = 3 is 12 - 16%, and the percentage of the conjugate where n = 4 is at most 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. The conjugate fractions may be separated using chromatography, and then, for example, the fractions containing the conjugate with the desired number of PEGs attached are identified and purified to be free of the unmodified protein sequence and conjugates with other numbers of PEGs attached.

[0148] PEGs suitable for conjugation to IL2 ortholog polypeptides are generally water soluble at room temperature and have the general formula R(O-CH2-CH2) n O-R, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, the protecting group generally has from 1 to 8 carbons.

[0149] Two widely used first-generation activated monomethoxy PEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., U.S. Patent No. 5,650,234 to Dolence et al.), which react preferentially with lysine residues to form carbamate linkages, but are also known to react with histidine and tyrosine residues. The use of PEG-aldehyde linkers targets a single site on the N-terminus of the polypeptide through reductive amination.

[0150] Pegylation can occur at the α-amino group at the N-terminus of the polypeptide, the epsilon amino group on the side chain of lysine residues, and the imidazole group on the side chain of histidine residues. Most recombinant polypeptides possess a single alpha amino group and numerous epsilon amino and imidazole groups, so multiple positional isomers can be generated depending on the linker chemistry. General pegylation strategies known in the art can be applied herein.

[0151] PEG can bind to the hIL2 ortholog of the present disclosure via a terminal reactive group ("spacer") that mediates the bond between one or more free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. PEG having a spacer that can bind to a free amino group includes N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide.

[0152] In some embodiments, PEGylation of the hIL2 ortholog is facilitated by the incorporation of unnatural amino acids having unique side chains that facilitate site-specific PEGylation. The incorporation of unnatural amino acids into a polypeptide to provide a functional moiety for achieving site-specific PEGylation of such a polypeptide is known in the art. See, for example, PCT International Application No. PCT / US2018 / 045257 by Ptacin et al. (filed August 3, 2018 and published as International Publication No. WO 2019 / 028419A1 on February 7, 2019). In one embodiment, the hIL2 ortholog of the present invention incorporates an unnatural amino acid at position D109 of the hIL2 ortholog. In one embodiment of the present invention, the hIL2 ortholog is PEGylated with a PEG molecule having a molecular weight of about 20 kD, or about 30 kD, or about 40 kD at position 109 of the hIL2 ortholog.

[0153] The PEG conjugated to the polypeptide sequence can be linear or branched. "Star PEG" and multi-arm PEG of branched PEG derivatives are contemplated by the present disclosure.In a specific embodiment, useful PEGs in the practice of the present invention include linear PEG-aldehyde of 10 kDa (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), linear PEG-NHS ester of 10 kDa (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), linear PEG-aldehyde of 20 kDa (e.g., Sunbright® ME-200AL, NOF), linear PEG-NHS ester of 20 kDa (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), 2-arm branched-chain PEG-aldehyde of 20 kDa, i.e., 20 kDa PEG-aldehyde containing two 10 kDa linear PEG molecules (e.g., Sunbright® GL2-200AL3, NOF), 2-arm branched-chain PEG-NHS ester of 20 kDa, i.e., 20 kDa PEG-NHS ester containing two 10 kDa linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), 2-arm branched-chain PEG-aldehyde of 40 kDa, i.e., 40 kDa PEG-aldehyde containing two 20 kDa linear PEG molecules (e.g., Sunbright® GL2-400AL3), 2-arm branched-chain PEG-NHS ester of 40 kDa, i.e., 40 kDa PEG-NHS ester containing two 20 kDa linear PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30 kDa PEG-aldehyde (e.g., Sunbright® ME-300AL), and linear 30 kDa PEG-NHS ester.

[0154] In one aspect, the hIL2 ortholog of the present disclosure includes the following structure: [PEG]-[Linker] n -[hoIL2] where n = 0 or 1.

[0155] In certain aspects, the hIL2 ortholog of the present disclosure includes the following structure: [PEG]-[Linker] n -[desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A] where n = 0 or 1.

[0156] In one aspect, the hIL2 ortholog of the present disclosure includes the following structure: [40kDa-PEG]-[Linker] n -[hoIL2] where n = 0 or 1.

[0157] In certain aspects, the hIL2 ortholog of the present disclosure includes the following structure: [40kDa-PEG]-[Linker] n -[desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A] where n = 0 or 1.

[0158] In one aspect, the hIL2 ortholog of the present disclosure includes the following structure: [40kDa branched-chain PEG]-[Linker] n -[hoIL2] where n = 0 or 1.

[0159] In certain aspects, the hIL2 ortholog of the present disclosure includes the following structure: [40KD branched-chain PEG]-[Linker] n -[desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A] where n = 0 or 1.

[0160] In another aspect, the hIL2 ortholog comprises the following structure: TIFF2025108545000069.tif27135 wherein n = 0 or 1.

[0161] In another aspect, the hIL2 ortholog comprises the following structure: TIFF2025108545000070.tif29128 wherein n = 0 or 1.

[0162] Suitable linkers generally include "flexible linkers" that are long enough to allow some movement between components and molecules linked to a modified polypeptide sequence. Linker molecules are generally about 6 - 50 atoms in length. The linker can also be, for example, arylacetylene, an ethylene glycol oligomer containing 2 - 10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof. Suitable linkers can be readily selected and can be of a suitable length such as one 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.

[0163] Examples of flexible linkers include glycine polymers (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., (GmSo)n, (GSGG)n, (GmSoGm)n, (GmSoGmSoGm)n, (GSGGSm)n, (GSGSmG)n, and (GGGGSm)n and combinations thereof, where m, n, and o are each independently selected from integers of at least 1 to 10, such as 1 to 18, 2 to 16, 3 to 14, 4 to 12, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and can therefore serve as neutral linkers between components. Examples of these include GGGSG (SEQ ID NO:139), GGSGG (SEQ ID NO:140), GSGSG (SEQ ID NO:141), GSGGG (SEQ ID NO:142), GGGGSG (SEQ ID NO:143), but are not limited thereto. Additional examples of flexible linkers include glycine polymers (G)n or glycine-serine polymers (e.g., (GS)n, (GSGGS)n, (GGGS)n, and (GGGGS)n, where n = 1 to 50, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 30, 30 to 50 G linkers). Exemplary flexible linkers include GGGGS (SEQ ID NO:144), GGGGS (SEQ ID NO:145), GGSG (SEQ ID NO:146), GGSGG (SEQ ID NO:147), GSGSG (SEQ ID NO:148), GSGGG (SEQ ID NO:149), GGGGSG (SEQ ID NO:150), and GSSSG (SEQ ID NO:151), but are not limited thereto. Multimers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 30, 30 to 50) can be linked together to provide flexible linkers that can be used to conjugate orthologous amino acid sequences to the polypeptides or PEG molecules disclosed herein.Instead of a polypeptide linker, the linker can be a chemical linker, such as a PEG-aldehyde linker.

[0164] Acetylated: In some embodiments, the IL-2 ortholog is acetylated at the N-terminus by an enzyme reaction using N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, the IL-2 ortholog can be acetylated at one or more lysine residues by an enzyme reaction using, for example, lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325 (5942):834-840.

[0165] Flag tag In some embodiments, the IL-2 ortholog is modified to include an additional polypeptide sequence that functions as an antigen tag, such as a FLAG sequence. The FLAG sequence is recognized by biotinylated, highly specific anti-FLAG antibodies as described herein (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 ortholog polypeptide further includes a C-terminal c-myc epitope tag.

[0166] Albumin fusion: In some embodiments, the IL-2 ortholog is conjugated to albumin and is referred to herein as an "IL2 ortholog albumin fusion." The term "albumin," when used in the context of an hIL2 ortholog albumin fusion, includes albumins such as human serum albumin (HSA), cynomolgus monkey serum albumin, and bovine serum albumin (BSA). In some embodiments, the present HSA includes a C34S or K573P amino acid substitution compared to the wild-type HSA sequence. According to the present disclosure, albumin can be conjugated to the hIL2 ortholog at the carboxy terminus, amino terminus, both the carboxyl and amino termini, and internally (see, e.g., USP 5,876,969 and USP 7,056,701). The HSA-hIL2 ortholog polypeptide conjugates contemplated by the present disclosure can use various forms of albumin such as albumin secretion presequences and variants thereof, fragments and variants thereof, and HSA variants. Such forms generally possess one or more desired albumin activities. In additional embodiments, the present disclosure includes fusion proteins comprising hIL2 ortholog polypeptides directly or indirectly fused to albumin, albumin fragments, and albumin variants, wherein the fusion protein has a higher plasma stability than the unfused drug molecule and / or the fusion protein retains the therapeutic activity of the unfused drug molecule. In some embodiments, the indirect fusion is effected by a linker such as a peptide linker or a modified version thereof, as discussed in more detail below.

[0167] Alternatively, the hIL2 ortholog albumin fusion is a fusion protein comprising an albumin binding domain (ABD) polypeptide sequence and an hIL2 ortholog polypeptide, which includes an hIL2 ortholog. As suggested above, a fusion protein comprising an albumin binding domain (ABD) polypeptide sequence and an hIL2 ortholog polypeptide can be achieved, for example, by genetic manipulation such that a nucleic acid encoding HSA or a fragment thereof is joined to a nucleic acid encoding one or more hIL2 ortholog sequences. In some embodiments, the albumin binding peptide comprises the amino acid sequence: DICLPRWGCLW (SEQ ID NO:152).

[0168] His tag In some embodiments, the hIL2 orthologs of the invention (including fusion proteins of such IL-2 orthologs) are expressed as fusion proteins having one or more transition metal chelate polypeptide sequences. Incorporation of such transition metal chelate domains facilitates purification by immobilized metal affinity chromatography (IMAC) as described in U.S. Patent No. 4,569,794 to Smith et al., issued February 11, 1986. Examples of transition metal chelate polypeptides useful in the practice of the present invention are described in U.S. Patent No. 4,569,794 to Smith et al. and U.S. Patent No. 5,320,663 to Dobeli et al., issued May 10, 1995, the entire teachings of which are incorporated herein by reference. A particular transition metal chelate polypeptide useful in the practice of the present invention is a peptide containing 3 to 6 contiguous histidine residues, such as a 6-histidine peptide (His)6, which is often referred to in the art as a "His tag".

[0169] The aforementioned fusion protein may be readily produced by constructing a recombinant vector that contains, by recombinant DNA methodology known in the art, a nucleic acid sequence encoding an hIL2 ortholog in-frame with a nucleic acid sequence encoding a fusion partner at either the N-terminus or C-terminus of the hIL2 ortholog, which sequence optionally further contains a nucleic acid sequence encoding a linker or spacer polypeptide in-frame.

[0170] Targeted hIL2 ortholog molecule In some embodiments, the hIL2 ortholog is provided as a fusion protein having a polypeptide sequence (a "targeting domain") that facilitates selective binding to a particular cell type or tissue that expresses a cell surface molecule that specifically binds to the targeting domain, and optionally, a linker molecule is incorporated between the hIL2 ortholog sequence and the sequence of the targeting domain of the fusion protein. In one embodiment, the targeting domain of the hIL2 ortholog fusion protein specifically binds to a cell surface molecule of the cell type targeted by CAR-T cells that express orthogonal hCD122. For example, if the orthogonal hCD122 CAR-T cells comprise a CAR having an ECD that specifically binds to CD-19, the targeting domain of the hIL2 ortholog fusion protein may also bind to CD-19. Examples of targeting domains include ligands or specific binding molecules to cell surface receptors, antibodies. In one embodiment, the hIL2 ortholog fusion protein comprises a molecule that specifically binds to the same cell type targeted by engineered cells (e.g., hoRb CAR-T cells) that express an orthogonal ligand. In one embodiment where the ECD of the CAR of the hoRb CAR-T cells specifically binds to CD-19, the IL-2 ortholog may be provided as a fusion protein having a CD-19 targeting moiety. For example, in one embodiment where the ECD of the CAR of the hoRb CAR-T cells is an scFv molecule that provides specific binding to CD-19, the IL-2 ortholog is provided as a fusion protein having a CD-19 targeting moiety such as 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 ortholog and an anti-CD19 scFv FMC63 (Nicholson, et al. (1997) Mol Immunol 34: 1157-1165).Similarly, in some embodiments where the ECD of the CAR of the hoRb CAR-T cell specifically binds to BCMA, the IL-2 ortholog is an antibody comprising the CDRs of an anti-BMCA antibody as described in Kalled et al. (U.S. Patent 9,034,324, issued May 9, 2015) or an antibody comprising CDRs as described in Brogdon et al. (U.S. Patent No. 10,174,095, issued January 8, 2019), and is provided as a fusion protein having a BCMA targeting moiety. In some embodiments where the ECD of the CAR of the hoRb CAR-T cell specifically binds to GD2, the IL-2 ortholog is a fusion protein having a GD2 targeting moiety, such as an antibody comprising CDRs as described in Cheung et al. (U.S. Patent No. 9,315,585, issued April 19, 2016), or an antibody comprising CDRs derived from ME36.1 (Thurin et al (1987) Cancer Research 47:1229-1233), 14G2a, 3F8 (Cheung, et al 1985 Cancer Research 45:2642-2649), hu14.18, 8B6, 2E12 or ic9.

[0171] In some embodiments, the targeting moiety of the hIL2 ortholog fusion protein may be the same as or different from that provided by CAR-T cells expressing orthogonal hCD122. In particular, the targeting moiety may be directed to an alternative antigen expressed on the tumor cell type targeted by the CAR. For example, in the context of orthogonal hCD122 scfv 14G2a GD2-targeted CAR-T cells, the hIL2 ortholog may be provided as a targeted fusion construct comprising a specific binding domain for another GD2 tumor antigen.

[0172] In an alternative embodiment, administration of the hIL2 ortholog of the present disclosure in combination with CAR-T cell therapy can provide for targeted delivery of the hIL2 ortholog to CAR-T cells based on the extracellular receptor of the CAR-T cells, for example by an anti-FMC63 antibody, target the hIL2 activity to the CAR-T cells, and can restore depleted CAR-T cells in vivo. Accordingly, embodiments of the present disclosure include targeted delivery of such hIL2 orthologs by conjugation of such hIL2 orthologs to antibodies or ligands designed to interact with specific cell surface molecules of CAR-T cells. Examples of such molecules may include anti-FMC63-hIL2 orthologs.

[0173] In other embodiments, the chimeric polypeptide comprises a mutant IL-2 polypeptide and a heterologous polypeptide that functions to enhance the expression of the mutant IL-2 polypeptide or direct cellular localization, such as an Aga2p lectin subunit (see, e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0174] Protein transduction domain fusion protein: In some embodiments, the hIL2 ortholog may optionally comprise a "protein transduction domain" or "PTD". A PTD is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic molecule that facilitates crossing of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. Incorporation of a PTD into the hIL2 ortholog facilitates the molecule crossing the membrane. In some embodiments, the PTD is covalently linked to the amino or carboxy terminus of the hIL2 ortholog. In some embodiments, the PTD is incorporated into either the N-terminus or C-terminus of the molecule as part of a PTD-IL2 ortholog fusion protein.

[0175] Exemplary protein transduction domains include the minimal decapeptide protein transduction domain (corresponding to residues 47 - 57 of HIV-1 TAT); polyarginine sequences containing a sufficient number of arginine residues to direct entry into cells (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10 - 50 arginines); the VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9 (6):489 - 96); the Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52 (7):1732 - 1737); the truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248 - 1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003 - 13008), transporter (described in Wierzbicki, et al., (2014) Folio Histomchemica et Cytobiologica 52 (4): 270 - 280 and Pooga, et a (1998) FASEB J 12 (1) 67 - 77 and commercially available from AnaSpec under catalog number AS - 61256); KALA (described in Wyman et al., (1997) Biochemistry 36 (10) 3008 - 3017 and commercially available from AnaSpec under catalog number AS - 65459); the antennapedia peptide (described in Pietersz et al., (2001) Vaccine 19:1397 and commercially available from AnaSpec under catalog number AS - 61032); TAT 47 - 57 (commercially available from AnaSpec under catalog number AS - 60023), but are not limited thereto.

[0176] Preparation of hIL2 ortholog The hIL2 ortholog can be produced by any conventional method including recombinant or solid - phase synthesis.

[0177] Solid-phase chemical synthesis: In addition to generating mutant polypeptides via the expression of nucleic acid molecules altered by recombinant molecular biology techniques, the subject hIL2 orthologs can be chemically synthesized. Polypeptides that are chemically synthesized are routinely generated by those of ordinary skill in the art. Chemical synthesis includes the direct synthesis of peptides by chemical means of the protein sequences encoding hIL2 orthologs that exhibit the described properties.

[0178] In some embodiments, the hIL2 orthologs of the present disclosure can be prepared by chemical synthesis. The chemical synthesis of hIL2 orthologs can proceed via a liquid phase or a solid phase. Solid-phase peptide synthesis (SPPS) allows for the incorporation of unnatural amino acids and / or peptide / protein backbone modifications. Various forms of SPPS are available for synthesizing the hIL2 orthologs of the present disclosure and are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero J.A. et al., (2005) Protein Pept Lett. 12:723-8). During the process of chemical synthesis, alpha functions and any reactive side chains can be protected with acid-labile or base-labile groups that are stable under the conditions for linking amide bonds but can be readily cleaved without harming the formed peptide chain.

[0179] In solid-phase synthesis, either the N-terminal or C-terminal amino acid can be attached to a suitable support material. Suitable support materials are inert to the reagents and reaction conditions of the stepwise condensation and cleavage reactions of the synthesis process and are insoluble in the reaction media used. Examples of commercially available support materials include styrene / divinylbenzene copolymers modified with reactive groups and / or polyethylene glycol; chloromethylated styrene / divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene / divinylbenzene copolymers; and the like. The sequential coupling of protected amino acids can be carried out according to conventional methods in peptide synthesis, typically using an automated peptide synthesizer.

[0180] At the end of solid-phase synthesis, the peptide is cleaved from the support material simultaneously with the cleavage of the side-chain protecting groups. The resulting 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 (HPLC), and reverse-phase HPLC.

[0181] Recombinant production: In some embodiments, the hIL2 ortholog (or a fusion protein comprising the hIL2 ortholog) is produced by recombinant methods. A nucleic acid sequence encoding the desired hIL2 ortholog polypeptide (optionally including a secretion leader sequence or signal peptide) is introduced into the cell to be engineered through an expression vector, and the nucleic acid sequence is operably linked to one or more expression control sequences encoded by the vector and functional in the target host cell. If a secretion leader sequence (signal peptide) is incorporated into the polypeptide, the recombinant hIL2 ortholog can be recovered through disruption of the host cell or from the cell culture medium. The recombinant hIL2 ortholog can be purified and concentrated for further use, including incorporation. Processes for the recombinant production of hIL2 polypeptides are known in the art and are described in U.S. Patent No. 4,604,377 to Fernandes and Taforo, issued August 5, 1986, and hIL2 orthologs are described in U.S. Patent No. 4,512,584 to Mark et al., issued May 21, 1985, and U.S. Patent No. 4,401,756 to Gillis, issued August 30, 1983, the entire teachings of which are incorporated herein by reference.

[0182] DNA encoding the hIL2 ortholog can be obtained from various sources and can be designed during the engineering process. Amino acid sequence variants of the hIL2 polypeptide for producing the hIL2 ortholog of the present disclosure are prepared by introducing appropriate nucleotide changes into the coding sequence, as described herein. Such variants represent insertions, substitutions and / or specified deletions of residues as described. Insertions, substitutions and / or specified deletions are arbitrarily combined, subject to the condition that the final construct possesses the desired biological activity as defined herein, to arrive at the final construct.

[0183] Construction of nucleic acid sequences encoding hIL2 ortholog In some embodiments, the hIL2 ortholog is produced by recombinant methods using a nucleic acid sequence encoding the hIL2 ortholog (or a fusion protein comprising the hIL2 ortholog). The nucleic acid sequence encoding the desired hIL2 ortholog can be synthesized by chemical means using an oligonucleotide synthesizer.

[0184] Nucleic acid molecules are not limited to sequences encoding polypeptides; they can also include some or all of the non-coding sequences upstream or downstream of the coding sequence (e.g., the coding sequence of hIL2). Those with ordinary skill in the field of molecular biology are familiar with the routine procedures for isolating nucleic acid molecules. These can be generated, for example, by treating genomic DNA with restriction endonucleases or by performing polymerase chain reaction (PCR). When the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be generated, for example, by in vitro transcription.

[0185] Nucleic acid molecules encoding the hIL2 ortholog (and its fusions) can contain naturally occurring sequences or sequences that are different from naturally occurring ones but encode the same polypeptide due to the degeneracy of the genetic code. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as those produced by phosphoramidite base synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Additionally, the nucleic acid molecule can be double-stranded or single-stranded (i.e., either the sense strand or the antisense strand).

[0186] Nucleic acid sequences encoding hIL2 orthologs can be obtained from various commercial sources that provide custom nucleic acid sequences. Amino acid sequence variants of the hIL2 polypeptide for producing the hIL2 orthologs of the present disclosure are prepared by introducing appropriate nucleotide changes into the coding sequences based on the genetic code well known in the art. Such variants represent insertions, substitutions and / or specified deletions of residues, as described. Insertions, substitutions and / or specified deletions are arbitrarily combined to reach the final construct, provided that the final construct possesses the desired biological activity as defined herein.

[0187] Examples of methods for constructing DNA sequences encoding hIL2 orthologs and expressing those sequences in appropriately transformed host cells include, but are not limited to, using PCR-assisted mutagenesis techniques. Mutations consisting of deletions or additions of amino acid residues to the hIL2 polypeptide can also be generated by standard recombinant techniques. In the case of deletions or additions, the nucleic acid molecule encoding hIL2 is optionally digested with an appropriate restriction endonuclease. The resulting fragments can be expressed directly or further manipulated, for example, by ligation to a second fragment. Ligation can be facilitated if the two ends of the nucleic acid molecule contain complementary nucleotides that overlap with each other, but blunt-ended fragments can also be ligated. Nucleic acids generated by PCR can also be used to generate various variant sequences.

[0188] The hIL2 orthologs of the present disclosure can be recombinantly produced not only as direct recombinant products but also as fusion polypeptides having non-homologous polypeptides, such as signal sequences or other polypeptides having cleavage sites specific to the N-terminus or C-terminus of the mature hIL2 ortholog. Generally, the signal sequence can be a component of the vector or part of the coding sequence inserted into the vector. The non-homologous signal sequence selected is preferably one that is recognized and processed by the host cell (i.e., cleaved by signal peptidase). In some embodiments, the signal sequence is the signal sequence naturally associated with the hIL2 ortholog (i.e., the human IL2 signal sequence). Incorporation of the signal sequence depends on whether it is desirable for the hIL2 ortholog to be secreted from the recombinant cell in which it is made. If the selected cell is prokaryotic, it is generally preferred that the DNA sequence does not encode a signal sequence. If the selected cell is eukaryotic, it is generally preferred that a signal sequence be encoded, and most preferably the wild-type IL2 signal sequence is used. Alternatively, heterologous mammalian signal sequences, such as those derived from secreted polypeptides of the same or related species, as well as viral secretion leaders, such as the herpes simplex gD signal, may be suitable. When the recombinant host cell is a yeast cell such as Saccharomyces cerevisiae, the alpha mating factor secretion signal sequence can be used to achieve extracellular secretion of the IL2 ortholog into the culture medium, as described in Singh U.S. Patent No. 7,198,919 B1, issued April 3, 2007.

[0189] Codon optimization: In some embodiments, the nucleic acid sequences encoding the recombinant proteins (IL2 ortholog, orthogonal hCD122, or CAR) can be "codon optimized" to facilitate expression in a particular host cell type. Techniques for codon optimization in a wide range of expression systems, including mammalian, yeast, and bacterial host cells, are well known in the art, and there are online tools that provide codon-optimized sequences for expression in a variety of host cell types. For example, see Hawash, et al., (2017) 9:46-53 and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols (edited by David Hacker)(Human Press New York). In addition, there are a variety of freely available web-based online software packages to assist in the preparation of codon-optimized nucleic acid sequences.

[0190] Expression vector: Once assembled (by synthesis, site-directed mutagenesis, or another method), the nucleic acid sequence encoding the hIL2 ortholog is inserted into an expression vector. A variety of expression vectors are available for use in a variety of host cells and are typically selected based on the host cell for expression. Vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrative vectors, etc. A plasmid is an example of a non-viral vector. To facilitate transfection of target cells, the target cells can be directly exposed to the non-viral vector under conditions that facilitate uptake of the non-viral vector. Examples of conditions that facilitate uptake of foreign nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g., Lipofectamine®, Thermo-Fisher Scientific), high salt concentration, and magnetic fields (electroporation).

[0191] The hIL2 ortholog is recombinantly produced not only by direct recombinant production but also as a fusion polypeptide having a non-homologous polypeptide, for example, a signal sequence or another polypeptide having a cleavage site specific to the mature protein or polypeptide at the N-terminus. Generally, the signal sequence can be a component of the vector or part of the coding sequence inserted into the vector. The non-homologous signal sequence selected is preferably one that is recognized and processed by the host cell (i.e., cleaved by signal peptidase). In expression in mammalian cells, the native signal sequence can be used or other mammalian signal sequences, such as signal sequences derived from secreted polypeptides of the same or related species, as well as viral secretion leaders, such as the herpes simplex gD signal, can be suitable.

[0192] Selectable marker Expression vectors usually contain a selectable gene, also called a selectable marker. This gene encodes a protein that is necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing the selectable gene will not survive in the culture medium. Typical selectable genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply essential nutrients not obtainable from complex media.

[0193] Regulatory control sequence: The expression vector for the hIL2 ortholog of the present disclosure contains regulatory sequences that are recognized by a host organism and are operably linked to a nucleic acid sequence encoding the hIL2 ortholog. The terms “regulatory control sequence,” “regulatory sequence,” or “expression control sequence” are used interchangeably herein and refer to promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego CA USA). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will recognize that the design of an expression vector can depend on factors such as the choice of host cell to be transformed and the desired level of expression of the protein. A variety of factors understood by those skilled in the art should be considered when selecting expression control sequences. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject hIL2 ortholog, particularly with respect to potential secondary structure.

[0194] Promoter In some embodiments, the regulatory sequence is a promoter, which is selected, for example, based on the cell type in which expression is attempted. The expression vector will contain a promoter recognized by the host organism and operably linked to the orthogonal protein coding sequence. A promoter is an untranslated sequence (generally within about 100 - 1000 bp) located upstream (5') of the start codon of a structural gene that controls the transcription and translation of the particular nucleic acid sequence to which it is operably linked. Such promoters are typically classified into two classes, namely inducible promoters and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription from the DNA under their control in response to some change in the culture conditions, such as the presence or absence of nutrients or a change in temperature. A number of promoters recognized by a wide variety of promising host cells are well known. For example, the T7 promoter can be used in bacteria, the polyhedrin promoter can be used in insect cells, and the cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissue-specific and cell-type-specific promoters are widely available. These promoters are so called because of their ability to direct the expression of nucleic acid molecules in a given tissue or cell type in the body. Those skilled in the art are well aware of a number of promoters and other regulatory elements that can be used to direct the expression of nucleic acids.

[0195] Transcription from a vector in a mammalian host cell can be controlled by a promoter obtained from the genome of a virus such as, for example, polyoma virus, fowlpox virus, adenovirus (such as human adenovirus serotype 5), bovine papilloma virus, Rous sarcoma virus, cytomegalovirus, retrovirus (such as murine stem cell virus), hepatitis B virus, most preferably a promoter obtained from the genome of simian virus 40 (SV40), a heterologous mammalian promoter, for example, an actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, a promoter obtained from a heat shock promoter, provided that such a promoter is compatible with the host cell line. The early and late promoters of the SV40 virus are readily obtained as an SV40 restriction fragment that also contains the SV40 origin of replication.

[0196] Enhancer Transcription by higher eukaryotes is often increased by inserting an enhancer sequence into a vector operably linked to a nucleic acid sequence encoding an hIL2 ortholog. An enhancer is a cis-acting element of DNA, usually about 10 to 300 bp, that acts to increase transcription relative to a promoter. Enhancers are relatively orientation- and position-independent and are found within introns, as well as within the coding sequences themselves, 5' and 3' of the transcription unit. Many enhancer sequences from mammalian genes are currently known (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically, enhancers from eukaryotic viruses are used. Examples include the SV40 enhancer on the late side of the origin of replication, the cytomegalovirus immediate early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer. Enhancers can be inserted into the expression vector at the 5' or 3' position of the coding sequence, but are preferably located 5' of the promoter. Expression vectors used in eukaryotic host cells will also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are generally available from the 5', and sometimes 3', untranslated regions of eukaryotic or viral DNA or cDNA. Construction of suitable vectors containing one or more of the above components uses standard techniques.

[0197] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, the vector can contain an origin of replication and other genes encoding selectable markers. For example, the neomycin resistance (neoR) gene confers G418 resistance to cells in which it is expressed, thus enabling phenotypic selection of transfected cells. Additional examples of marker or reporter genes include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthine-guanine phosphoribosyltransferase (XGPRT). One of ordinary skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

[0198] Proper assembly of the expression vector can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host.

[0199] Host cell for the production of hIL2 ortholog In one aspect, the present disclosure further provides a recombinant cell comprising a nucleic acid sequence encoding an hIL2 ortholog. The cell can be prokaryotic or eukaryotic. The cells of the present disclosure are transfected cells, i.e., cells into which a nucleic acid molecule, such as a nucleic acid molecule encoding a mutant hIL2 polypeptide, has been introduced by recombinant DNA technology. Progeny of such cells are also considered to be within the scope of the present disclosure.

[0200] Host cells for the expression of hIL2 orthologs are typically selected according to their compatibility with the selected expression vector, the toxicity of the product encoded by the DNA sequences of the present invention, their secretion characteristics, their ability to accurately fold polypeptides, their fermentation or culture requirements, and the ease of purification of the product encoded by the DNA sequences. Suitable host cells for cloning DNA into or expressing therein the vectors herein are cells of prokaryotes, yeast, or higher eukaryotes.

[0201] In some embodiments, host cells for the recombinant production of hIL2 orthologs are eukaryotic cells such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors available for protein expression in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in S. cerevisiae of yeast include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.) and pPicZ (Invitrogen Corporation, San Diego, Calif.)); or mammalian cells (examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)).

[0202] Examples of useful mammalian host cell lines include mouse L cells (L-M [TK-], ATCC #CRL-2648), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human fetal kidney line (293 cells, or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2).

[0203] In some embodiments, the resulting hIL2 ortholog is glycosylated or not glycosylated depending on the host organism used to produce the mutein. If bacteria are selected as the host, the resulting hIL2 ortholog is not glycosylated. On the other hand, eukaryotic cells can glycosylate the hIL2 ortholog, but perhaps not in the same way that native IL2 is glycosylated.

[0204] For other additional expression systems for both 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, N.Y.). See Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0205] Transfection: A nucleic acid expression construct encoding an hIL2 ortholog is introduced into a host cell, thereby producing the hIL2 ortholog disclosed herein or a biologically active mutein thereof. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0206] To facilitate transfection of target cells, the target cells can be directly exposed to a non-viral vector under conditions that facilitate uptake of the non-viral vector. Examples of conditions that facilitate uptake of foreign nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g., Lipofectamine®, Thermo-Fisher Scientific), high salt concentrations, and magnetic fields (electroporation).

[0207] Cell culture: Cells can be cultured in conventional nutrient media that have been appropriately modified for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence. Mammalian host cells can be cultured in a wide variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. Any of these media can be supplemented, as necessary, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those that have been used in the past with the host cells selected for expression and will be apparent to those skilled in the art.

[0208] Recovery of recombinant protein: The recombinantly produced hIL2 ortholog polypeptide can be recovered from the culture medium as a secreted polypeptide if a secretion leader sequence is used. Alternatively, the hIL2 ortholog polypeptide can also be recovered from the host cell lysate. To inhibit proteolysis during purification, protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) can be used in the recovery step from the cell lysate, and antibiotics can be included to suppress the growth of accompanying contaminants. In some embodiments, the hIL2 ortholog is produced in Escherichia coli (E. coli), and overexpression of the hIL2 ortholog sequestered in inclusion bodies occurs. Techniques for the isolation and solubilization of inclusion bodies and the recovery of the active protein are well known in the art.

[0209] Various purification processes are known in the art, and for example, affinity chromatography can be utilized. Affinity chromatography separates molecules based on their ability to bind to a specific ligand, typically by exploiting highly specific binding sites that exist in biopolymers. The ligand is covalently attached to an insoluble porous support in such a manner that it is clearly presented to the protein sample, thereby using the natural specific binding of one molecular species to separate and purify a second species from a mixture. Generally, antibodies are used in affinity chromatography. A size selection step may be used, for example, gel filtration chromatography (also known as size exclusion chromatography or molecular sieve chromatography) is used to separate proteins according to their sizes. In gel filtration, a protein solution is passed through a column packed with a semi-permeable porous resin. The semi-permeable resin has a pore size range that determines the sizes of proteins that can be separated in the column.

[0210] Orthogonal hIL2 orthologs may be concentrated, filtered, dialyzed, etc. using methods known in the art. For therapeutic applications, the hIL2 ortholog can be administered to a mammal that contains an appropriately engineered orthogonal receptor. Administration can be intravenous, either as a bolus or by continuous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intrathecal, subcutaneous, intra-articular, intra-synovial, intraspinal, oral, topical, or inhalation routes. Orthogonal hIL2 orthologs can also be appropriately administered by intratumoral, peritumoral, intralesional or perilesional routes or into the lymph to provide local as well as systemic therapeutic effects.

[0211] Route of administration of hIL2 ortholog: In certain aspects of the disclosed methods of treatment, administration of a pharmaceutical formulation comprising an hIL2 ortholog (and / or a nucleic acid encoding an hIL2 ortholog) to a subject in need of treatment is involved. Administration to the subject can be achieved intravenously, either as a bolus or by continuous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intrathecal, subcutaneous, intra-articular, intra-synovial, intraspinal, oral, topical, or inhalation routes. The hIL2 ortholog can also be appropriately administered by intratumoral, peritumoral, intralesional, intranodular or perilesional routes or into the lymph to provide both local and systemic therapeutic effects.

[0212] In some aspects, the subject hIL2 ortholog (and / or a nucleic acid encoding an hIL2 ortholog) can be incorporated into a composition comprising a pharmaceutical composition. Such compositions typically comprise a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. The pharmaceutical composition is formulated to be compatible with its intended route of administration and is suitable for therapeutic use in which the hIL2 ortholog is administered to a subject in need of treatment or prevention.

[0213] Formulation of hIL2 ortholog In some embodiments, the disclosure provides a pharmaceutically acceptable formulation of an hIL2 ortholog. Preferred formulations depend on the intended mode of administration and therapeutic use. Pharmaceutically acceptable formulations of the hIL2 ortholog include physiologically acceptable carriers that are essentially non-toxic and non-therapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride 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. Carriers for topical or gel-based forms of the polypeptide include polysaccharides such as sodium carboxymethyl cellulose or methyl cellulose, polyvinylpyrrolidone, polyacrylate, polyoxyethylene-polyoxypropylene-block polymers, PEG, high molecular weight amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0214] The formulation may also contain a pharmaceutically acceptable non-toxic carrier, excipient, stabilizer or diluent, as defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Formulations to be used for in vivo administration are typically sterile. Sterilization of the compositions of the present invention can be readily achieved by filtration through sterile filtration membranes.

[0215] Typically, the formulation is prepared as an injectable, either as a liquid solution or suspension; solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection can be prepared. The preparation can also be emulsified or encapsulated in liposomes or microparticles, such as polylactic acid, polyglycolide, or copolymers, to enhance the adjuvant effect, as discussed above. Langer, Science (1990) 249: 1527 and Hanes, Advanced Drug Delivery Reviews (1997) 28: 97-119. The agents of the invention can be administered in the form of depot injections or implant preparations, which can be formulated in such a manner as to permit sustained or pulsed release of the active ingredient. Pharmaceutical compositions are generally formulated in accordance with all good manufacturing practice (GMP) regulations of the United States Food and Drug Administration as sterile, substantially isotonic preparations.

[0216] In some embodiments, the methods of the disclosure involve parenteral administration of an hIL2 ortholog. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Parenteral formulations include solutions or suspensions for use in parenteral applications, which can include a vehicle, carrier, and buffer. Pharmaceutical formulations for parenteral administration include sterile aqueous solutions (where water soluble), dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. In one embodiment, the formulation is provided in a pre-filled syringe for parenteral administration.

[0217] Oral formulations, when used, may contain one or more inert diluents and / or edible carriers. For oral therapeutic administration, the hIL2 orthologs can be incorporated with excipients and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the formulation. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel™, or corn starch; lubricants such as magnesium stearate or Sterotes™; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavor.

[0218] For administration by inhalation, the subject hIL2 orthologs, or nucleic acids encoding them, are delivered in the form of an aerosol spray from a suitable nebulizer, e.g., a compressed container or dispenser containing a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0219] Systemic administration of the subject hIL2 ortholog or nucleic acid can also be by transmucosal or transdermal formulations. For transmucosal or transdermal administration, permeants appropriate to permeate the barrier are used in the formulation. Such permeants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved through the use of nasal sprays or suppositories for rectal delivery (using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas. For transdermal administration, the active compound is formulated in an ointment, plaster, gel, or cream, and may incorporate a penetration enhancer such as ethanol or lanolin, as is generally known in the art.

[0220] In some embodiments, the hIL2 ortholog formulation is a sustained release formulation that provides for the sustained delivery of the hIL2 ortholog agent over a period of time or days. Examples of sustained release formulations of injectable compositions can be brought about by including in the composition agents that retard absorption, such as aluminum monostearate and gelatin. In one embodiment, the subject hIL2 ortholog or nucleic acid is prepared with a carrier that prevents the rapid elimination of the variant IL-2 polypeptide from the body, such as a controlled release formulation including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. Liposome suspensions can also be used as a pharmaceutically acceptable carrier.

[0221] Engineered hoCD122 cells: The preparation of recombinant cells useful in the practice of the present invention is accomplished by transforming isolated cells with an expression vector containing a nucleic acid sequence encoding an hCD122 orthogonal receptor. The hIL2 ortholog of the present invention may be used in a method for selectively expanding and growing such engineered hoRB cells (e.g., human T-cells) engineered to express the corresponding orthogonal hCD122 receptor. T-cells useful for engineering with the constructs described herein include naive T-cells, central memory T-cells, effector memory T-cells, or combinations thereof. T-cells for such engineering may be collected from a subject or donor and separated from the cell mixture by techniques for concentrating the desired cells, or may be engineered and cultured without separation. Alternatively, T-cells for engineering may be separated from other cells. Techniques providing accurate separation include fluorescence-activated cell sorting devices. Cells may be selected against dead cells by using a dye associated with dead cells (e.g., propidium iodide). The separated cells may be collected in any suitable medium that maintains cell viability and has a serum cushion at the bottom of the collection tube, usually. A variety of media are commercially available, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., often supplemented with fetal calf serum (FCS), and may be used depending on the nature of the cells. The collected and optionally concentrated cell population may be used immediately for genetic modification or may be frozen and stored at liquid nitrogen temperature, which is thawable and reusable. Cells are usually stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0222] In some embodiments, the engineered cells comprise a complex mixture of immune cells isolated from an individual in need of treatment, such as tumor infiltrating lymphocytes (TIL). 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".

[0223] CAR-T cells In one aspect of the invention, the hoRb cells are T-cells (e.g., human T-cells) ( "hoCAR-T cells") that have been modified to surface-express a chimeric antigen receptor. 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 specifically binds to one or more antigens expressed on the surface of the target cell. The CAR further includes a transmembrane domain that connects the ABD (or linker, if used) to the intracellular cytoplasmic domain of the CAR. The transmembrane domain is composed of any polypeptide sequence that is thermodynamically stable in the eukaryotic cell membrane. The transmembrane domain may be derived from the transmembrane domain of a naturally occurring membrane protein or may be synthetic. When designing a synthetic transmembrane domain, amino acids that prefer an alpha-helix structure are preferred. Transmembrane domains useful in the construction of the CAR are composed of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, or 24 amino acids that prefer a form having an alpha-helix secondary structure. Amino acids that prefer an alpha-helix conformation are well known in the art. See, for example, Pace, et al. (1998) Biophysical Journal 75: 422-427. Amino acids for which an alpha-helix conformation is particularly advantageous 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 membrane protein, such as CD3ζ, CD4, CD8, CD28, etc.

[0224] The cytoplasmic domain (ICD) of the CAR polypeptide contains one or more intracellular signaling domains. In one aspect, the intracellular signaling domain includes the cytoplasmic sequences of the T-cell receptor (TCR) and co-receptors that initiate signaling after antigen receptor engagement, as well as functional derivatives and sub-fragments thereof. Cytoplasmic signaling domains, such as those derived from the T cell receptor zeta chain, are used as part of the CAR to generate stimulatory signals for T lymphocyte proliferation and effector function after the chimeric receptor associates with the target antigen. Examples of cytoplasmic signaling domains include the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also referred to as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also referred to as ICOS), the p110α, β or δ catalytic subunits of PI3 kinase, the human CD3 ζ chain, the cytoplasmic domain of CD134 (also referred to 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, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction such as CD2, CD5 and CD28, but are not limited thereto.

[0225] In some embodiments, the CAR may also provide a co-stimulatory domain. The term "co-stimulatory domain" refers to the stimulatory domain of the CAR, typically the endodomain, that provides a secondary non-specific activation mechanism through which a primary specific stimulus is propagated. The co-stimulatory domain refers to the part of the CAR that enhances the proliferation, survival, or development of memory cells. Examples of co-stimulation include antigen-nonspecific T-cell co-stimulation after antigen-specific signal transduction through the T-cell receptor, and antigen-nonspecific B-cell co-stimulation after signal transduction through the B-cell receptor. Co-stimulation, e.g., T-cell co-stimulation, and the factors involved are described in Chen & Flies. (2013) Nat Rev Immunol 13 (4):227-42. In some embodiments of the present disclosure, the CSD includes one or more members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or combinations thereof. CARs are often referred to as first-generation, second-generation, third-generation, or fourth-generation. The term "first-generation CAR" refers to a CAR whose cytoplasmic domain transmits signals from antigen binding through only one signaling domain, e.g., a signaling domain derived from the high-affinity receptor for IgE FcεR1γ or the CD3ζ chain. The domain contains one or three immunoreceptor tyrosine-based activation motifs [ITAM] for antigen-dependent T-cell activation. The ITAM-based activation signal confers on T-cells the ability to lyse target tumor cells and secrete cytokines in response to antigen binding. Second-generation CARs include a co-stimulatory signal in addition to the CD3ζ signal. The co-delivery of the delivered co-stimulatory signal enhances cytokine secretion and anti-tumor activity induced by CAR-transduced T-cells. The co-stimulatory domain is usually the membrane proximal to the CD3ζ domain. Third-generation CARs include a three-part signaling domain, e.g., including the CD28, CD3ζ, OX40, or 4-1BB signaling regions.In the fourth generation or "armored car", the CAR T-cells are further modified to express or block molecules and / or receptors that enhance immune activity, such as the expression of IL-12, IL-18, IL-7, and / or IL-10; 4-1BB ligand, CD-40 ligand.

[0226] Examples of intracellular signaling domains that can be incorporated into hoCAR-T cells useful in the practice of the present invention include, from amino to carboxy: CD3ζ; CD28-41BB-CD3ζ; CD28-OX40-CD3ζ; CD28-41BB-CD3ζ; 41BB-CD28-CD3ζ, and 41BB-CD3ζ.

[0227] The term CAR includes CAR variants, including but not limited to split CAR, ON switch CAR, bispecific or tandem CAR, inhibitory CAR (iCAR), and induced pluripotent stem (iPS) CAR-T cells.

[0228] The term "split CAR" refers to a CAR in which the extracellular portion of the CAR, the ABD, and the cytoplasmic signaling domain are present on two separate molecules. CAR variants also include, for example, ON switch CARs, which are conditionally activatable CARs that include split CARs in which the conditional heterodimerization of the two parts of the split CAR is pharmacologically controlled. CAR molecules and their derivatives (i.e., CAR variants) are described, for example, in PCT application numbers US2014 / 016527, US1996 / 017060, US2013 / 063083; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20 (2):141-4; Pegram et al. Cancer J (2014) 20 (2):127-33; Cheadle et al. Immunol Rev(2014) 257 (1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304 (the disclosures of which are incorporated herein by reference in their entirety).

[0229] The term "bispecific or tandem CAR" refers to a CAR that includes a secondary CAR binding domain that can amplify or inhibit the activity of a primary CAR.

[0230] The terms "inhibitory chimeric antigen receptor" or "iCAR" are used interchangeably herein and refer to a CAR that, upon binding, uses dual antigen targeting and stops the activation of an active CAR by causing the association of a second inhibitory receptor with an inhibitory signaling domain of a secondary CAR binding domain to result in inhibition of primary CAR activation. Inhibitory CAR (iCAR) is designed to regulate CAR-T cell activity through inhibitory receptor signaling module activation. This approach combines the activities of two CARs, one of which generates a dominant negative signal that restricts the response of CAR-T cells activated by an activating receptor. iCAR can switch off the response of a counteracting activating factor CAR when bound to a specific antigen expressed only by normal tissue. In this way, iCAR-T cells can discriminate between cancer cells and healthy cells and antigen-selectively and reversibly block the function of transduced T cells. The CTLA-4 or PD-1 intracellular domain in iCAR induces an inhibitory signal to T lymphocytes, leading to lower cytokine production, less efficient target cell lysis, and altered lymphocyte motility. The term "tandem CAR" or "TanCAR" refers to a CAR that mediates dual-specific activation of T cells through the association of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to the independent association of two different tumor-associated antigens.

[0231] Typically, chimeric antigen receptor T-cells (CAR-T cells) are T-cells that have been recombinantly modified by transduction with an expression vector encoding a CAR substantially according to the teachings above.

[0232] In some embodiments, hoCAR-T cells are allogeneic with respect to the individual being treated. Graham et al. (2018) Cell 7(10) E155. In some embodiments, the allogeneic engineered T cells are fully HLA-matched. However, not all patients have a donor with a perfect match, and cell products suitable for all patients regardless of HLA type are an option.

[0233] Since hoCAR-T cells are derived from the subject's own T-cells, the cell population to be administered to the subject is necessarily variable, and the response to such an agent can vary. Therefore, prior to administration of the hoCAR-T cell product, intermittent monitoring and management of treatment-related toxicities, which are managed by a series of pharmacological immunosuppression or B-cell depletion, are required. Usually, at least 1×10 6 cells of hoCAR-T cells / kg, at least 1×10 7 cells of hoCAR-T cells / kg, at least 1×10 8 cells of hoCAR-T cells / kg, at least 1×10 9 cells of hoCAR-T cells / kg, at least 1×10 10 cells of hoCAR-T cells / kg, or more, are administered, but usually this is limited by the number of T-cells obtained during the collection process. The engineered hoCAR-T cells may be injected into the subject by any convenient route of administration, usually intravascular administration, in any physiologically acceptable medium, but may also be introduced by other routes by which the cells can find an appropriate site for growth.

[0234] When the hoCAR-T cells are allogeneic T cells, such cells may be modified to reduce graft-versus-host disease. For example, the engineered cells of the present invention can be TCRαβ receptor knockouts achieved by gene editing techniques. TCRαβ is a heterodimer and both alpha and beta chains are required for it to be expressed. Although a single gene encodes the alpha chain (TRAC), there are two genes encoding the beta chain, so for this purpose, the KO of the TRAC locus is deleted. To achieve this deletion, a number of different approaches have been used, such as CRISPR / Cas9; meganucleases; engineered I-CreI homing endonucleases, 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 (related to CAR expression in which TRAC is disrupted by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 without directly integrating CAR into the TRAC locus). An alternative strategy to prevent GVHD is to use, for example, truncated CD3ζ as a TCR inhibitor molecule to modify T cells to express an inhibitor of TCRαβ signaling.

[0235] In one aspect, the present disclosure provides a method for selectively expanding and growing an engineered cell population that expresses an orthogonal hCD122 receptor from a mixed cell population, the method comprising contacting the mixed cell population with the hIL2 ortholog of the present disclosure under conditions that facilitate the growth of the engineered cells. In one aspect, in the case of CAR-T cells that express an orthogonal hCD122 receptor, the CAR-T cells that express the orthogonal receptor can be selectively expanded and grown from the background or mixed population of transduced cells and non-transduced cells through the use of the hIL2 orthologs described herein. The expansion and growth of T cells for therapeutic applications typically involves culturing the cells by contact with a surface to which an agent that stimulates CD3 TCR complex-related signals and an agent that stimulates co-stimulatory molecules on the surface of the T-cells are attached. In conventional practice, engineered T-cells are stimulated by contact with CD3 / D28 prior to administration of the cell therapy product, particularly in the preparation of CAR-T cells for clinical use. To facilitate bead-based activation of T-cells, a wide range of or commercially available products are available, including, without limitation, Invitrogen® CTS Dynabeads® CD3 / 28 (Life Technologies, Inc. Carlsbad CA) or Miltenyi MACS® GMP ExpAct Treg beads or Miltenyi MACS GMP TransAct™ CD3 / 28 beads (Miltenyi Biotec, Inc.). Conditions suitable 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 on January 16, 2015. Target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).In this case, a mixed cell population containing engineered T cells that express the hCD122 orthogonal receptor is cultured in the presence of a certain concentration of the hIL2 ortholog 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 longer in some embodiments. The concentration of the hIL2 ortholog in such an ex vivo situation is sufficient to induce cell proliferation in the cell population. T cell proliferation can be readily evaluated by microscopy, and the determination of the optimal concentration of the hIL2 ortholog will depend on the relative activity of the hIL2 ortholog against the orthogonal hCD122 receptor.

[0236] In one aspect, the present disclosure provides a method of making an engineered cell product in which engineered hoCD122-expressing cells are substantially enriched, comprising: (a) obtaining a biological sample comprising T-cells; (b) contacting the biological sample with a recombinant vector encoding the hoCD122 receptor; (c) contacting the biological sample with an hIL2 ortholog for a time sufficient for the engineered hoCD122-expressing cells to expand and proliferate. The periods of contact and culture can be varied depending on the degree to which the population is enriched.

[0237] When contacting cells with an hIL2 ortholog in vitro, the hIL2 ortholog is added to the engineered cells in a dosage and for a time sufficient to activate signal transduction from the hoCD122 receptor, and natural cellular mechanisms such as accessory proteins, co-receptors, etc. may be utilized. Any suitable culture medium can be used. Such activated cells can be used for any desired purpose, including experimental purposes related to determination of antigen specificity, cytokine profiling, etc., and for in vivo delivery.

[0238] When the contacting step is carried out in vivo, an effective amount of engineered cells, including but not limited to CAR-T cells modified to express an orthogonal hoCD122 receptor, is injected into a recipient in combination with or prior to the administration of an orthogonal cytokine, such as IL-2, to bring the T cells into contact in their natural environment, such as in lymph nodes. The dosage and frequency can vary depending on the agent; the mode of administration; the nature of the hIL2 ortholog, etc. Such guidelines will be understood by those skilled in the art to be adjusted according to the individual circumstances. The dosage may also be varied according to the route of administration, such as intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous injection, etc. Generally, at least about 10 4 engineered cells / kg, at least about 10 5 engineered cells / kg; at least about 10 6 engineered cells / kg, at least about 10 7 engineered cells / kg, or more, are administered.

[0239] When the engineered cells modified to express hoCD122 are T cells, the enhanced immune response can appear as an increase in the cytolytic response of T cells against target cells present in the recipient, such as tumor cells, infected cells; a decrease in the symptoms of autoimmune diseases; etc. In some embodiments where an engineered T cell population is to be administered to a subject, the subject is provided with a course of immunosuppressive treatment, either 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 serum immunoglobulin levels to normal levels. In some embodiments, prior to administration of the CAR-T cell therapy of the present invention, the subject may optionally be subjected to a lymphodepletion regimen. An example of such a lymphodepletion regimen is fludarabine (30 mg / m 2intravenously daily for 4 days) and cyclophosphamide (starting from the first dose of fludarabine at 500 mg / m 2 consisting of daily IV administration for 2 days).

[0240] The engineered hoCD122 cells can be provided in a pharmaceutical composition suitable for therapeutic use, e.g., suitable for human treatment. Therapeutic formulations containing such cells can be frozen together with a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), or prepared in the form of an aqueous solution for administration. The hoCD122 cells are formulated, compounded and administered in a manner consistent with good medical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the dosing schedule and other factors known to the physician.

[0241] The hoCD122 cells can be administered by any suitable means, usually parenterally. Parenteral infusion includes intramuscular, intravenous (bolus or slow infusion), intraarterial, intraperitoneal, intraspinal or subcutaneous administration. In a typical implementation, the engineered T cells are injected into the subject, usually intravascularly, in a physiologically acceptable medium, but may be introduced into any other convenient site where the cells can find a suitable growth site. Usually, at least 1×10 5 cells / kg, at least 1×10 6 cells / kg, at least 1×10 7 cells / kg, at least 1×10 8 cells / kg, at least 1×10 9 cells / kg, or more are administered, but usually this is limited by the number of T cells obtained during the collection process.

[0242] For example, a typical dosage range of cells modified to express an orthogonal hCD122 receptor for use in the practice of the present invention is from about 1×10 5 cells to 5×10 8 cells per kg of subject body weight per treatment unit. Thus, a typical dosage range of viable cells in a human subject adjusted according to body weight is from approximately 1×10 6 cells to approximately 1×10 13 cells, or from approximately 5×10 6 cells to approximately 5×10 12 cells, or from approximately 1×10 7 cells to approximately 1×10 12 cells, or from approximately 5×10 7 cells to approximately 1×10 12 cells, or from approximately 1×10 8 cells to approximately 1×10 12 cells, or from approximately 5×10 8 cells to approximately 1×10 12 cells, or from approximately 1×10 9 cells to approximately 1×10 12 cells per treatment unit. In one aspect, the dosage of cells is in the range of 2.5 to 5×10 9 viable cells per treatment unit.

[0243] Treatment units using hIL2 orthologs and / or hoCD122 cells can include a single dose or multiple doses over a period of time. In some embodiments, hoCD122 cells are administered as a single dose. In some embodiments, hoCD122 cells are administered in two or more divided doses over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. The amount of engineered hoCD122 cells administered in such a divided dosing protocol may be the same for each administration or may be provided at different levels. A dosing protocol over multiple days for a specific period can be provided by one of ordinary skill in the art (e.g., a physician) monitoring the administration of the cells, taking into account the subject's response to the treatment, including the adverse effects of the treatment and their modulation as discussed above.

[0244] The compositions and methods of the present disclosure also provide a method for treating a subject by hoCD122 cell therapy (especially CAR T cell therapy) in the absence of prior lymphocyte depletion. Lymphocyte depletion is typically carried out in a subject in combination with CAR T cell therapy, but the subsequent administration of a mixed cell population and the administration of a non-specific agent (e.g., hIL2) to expand and proliferate the engineered cell population in the subject, when combined with the administration of the cell therapy product, results in significant systemic toxicity (including cytokine release syndrome or "cytokine storm") due to the proliferation and activation of a wide range of immune cells and the presence of a significant proportion of unengineered cells in the cell therapy product itself caused by the administration of an agent that causes widespread activation. The methods and compositions of the present disclosure remove this significant obstacle by providing both (or either) a substantially purified population of engineered cells with little contamination of unengineered cells when the aforementioned ex vivo method is used, and / or the selective activation and expansion of engineered T cells by the hIL2 ortholog of the present invention with a substantially reduced off-target effect of non-specific growth substances such as IL2.

[0245] For example, in current clinical trials of CAR-T cell therapy, CAR-T cells are generally administered in combination with lymphodepletion (e.g., by administration of alemtuzumab (monoclonal anti-CD52), purine analogs, etc.) to facilitate the expansion of CAR-T cells prior to host immune recovery. In some embodiments, the CAR-T cells may be modified for resistance to alemtuzumab. In one aspect of the invention, the lymphodepletion currently used in connection with CAR-T therapy may be obviated or reduced by CAR-T cells expressing an orthogonal ligand of the invention. As described above, lymphodepletion is generally used to enable the expansion of CAR-T cells. However, lymphodepletion is also associated with the major side effects of CAR-T cell therapy. Since the orthogonal ligand provides a means to selectively expand a particular T-cell population, the need for lymphodepletion prior to administration of CAR-T cells expressing the orthogonal ligand can be reduced. The present invention enables the implementation of CAR-T cell therapy that does not require or has reduced lymphodepletion prior to administration of CAR-T cells expressing an orthogonal ligand. In one embodiment, the present disclosure provides a method of treating a subject suffering from a disease, disorder or condition (e.g., cancer) treatable by CAR-T cell therapy, which is not in need of lymphodepletion prior to administration of orthogonal ligand-expressing CAR-T, by administration of orthogonal ligand-expressing CAR-T.

[0246] In one aspect, the present disclosure provides a method of treating a mammalian subject suffering from a neoplastic disease, the method comprising: (a) obtaining a biological sample comprising T-cells from the individual; (b) enriching the biological sample for the presence of T-cells; (c) transfecting the T-cells with one or more expression vectors comprising a nucleic acid sequence encoding a chimeric antigen receptor (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 neoplastic cells; (d) contacting, ex vivo, a cell population comprising CAR-T cells expressing the orthogonal receptor with an hIL2 ortholog such that the cell population is enriched for CAR T-cells expressing hoCD122; (e) administering to the mammal a pharmaceutically effective amount of CAR-T cells expressing the orthogonal receptor; and (f) modulating the growth of CAR-T cells expressing the orthogonal hCD122 receptor by administering a therapeutically effective amount of an hIL2 ortholog that selectively binds to the orthogonal hCD122 receptor expressed on the CAR-T cells. In the foregoing method, when the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the orthogonal hCD122 receptor are provided on the same vector, these sequences may optionally be provided in a polycistronic format in which the nucleic acid sequences are separated by an intervening sequence such as an IRES or a T2A sequence. In one aspect, the foregoing method is accompanied by lymphodepletion or immunosuppression of the mammal prior to the initiation of a course of CAR-T cell therapy. In another aspect, the foregoing method is performed in the absence of lymphodepletion and / or immunosuppression of the mammal.

[0247] Administration of a viral or non-viral vector encoding hIL2 ortholog: Instead of administering the hIL2 ortholog protein, the hIL2 ortholog may be provided to the subject by administering a nucleic acid construct encoding the hIL2 ortholog to the subject to achieve continuous exposure of the subject to the selective hIL2 ortholog. Administration of a recombinant vector encoding the hIL2 ortholog provides extended delivery of the hIL2 ortholog to the subject and long-term activation of the corresponding cells engineered to express the cognate orthogonal receptor associated with such hIL2 ortholog.

[0248] Non-viral vector: In one aspect, the hIL2 ortholog can be administered to a subject in the form of a nucleic acid expression construct containing the hIL2 ortholog in a non-viral delivery system that can be provided by a non-viral delivery system. The non-viral delivery system is typically a complex for facilitating the transduction of target cells with a nucleic acid cargo, and the nucleic acid is complexed with agents such as cationic lipids (DOTAP, DOTMA), surfactants, biologic agents (gelatin, chitosan), metals (gold, magnet), and synthetic polymers (PLG, PEI, PAMAM). Lipid vector systems (Lee et al. (1997) Critical Reviews of Therapeutic Drug Carrier Systems 14:173-206); polymer-coated liposomes (U.S. Patent No. 5,213,804 to Marin et al., issued May 25, 1993; U.S. Patent No. 5,013,556 to Woodle et al., issued May 7, 1991); cationic liposomes (U.S. Patent No. 5,283,185 to Epand et al., issued February 1, 1994; U.S. Patent No. 5,578,475 to Jessee, J. A., issued November 26, 1996; U.S. Patent No. 5,279,833 to Rose et al., issued January 18, 1994; U.S. Patent No. 5,334,761 to Gebeyehu et al., issued August 2, 1994), many aspects of non-viral delivery systems are well known in the art. In one aspect, the nucleic acid sequence in the non-viral vector system encoding the hIL2 receptor is under the control of a regulatable promoter, an inducible promoter, a tissue-specific or tumor-specific promoter, or a temporally regulated promoter.

[0249] Viral vector: In another aspect, the hIL2 ortholog can be administered to a subject in the form of a nucleic acid expression construct in a viral vector encoding the hIL2 ortholog. The terms "viral vector" and "virus" are used interchangeably herein and refer to any obligate intracellular parasite that does not have a protein synthesis or energy generation mechanism. The viral genome can be RNA or DNA and is contained within a protein coat structure of a lipid membrane. The terms virus and viral vector are used interchangeably herein. Viruses useful in the practice of the present invention preferably include DNA and RNA viruses selected from the families Baculoviridae, Parvoviridae, Picornaviridae, Herpesviridae, Poxviridae or Adenoviridae, with or without a recombinant modified envelope. The virus is modified by recombinant DNA techniques to include expression of an exogenous introduced gene (e.g., a nucleic acid sequence encoding the hIL2 ortholog) and may be engineered to be replication-deficient, conditionally replicating or replicable. A minimal vector system may be used in which the viral backbone contains only the sequences necessary for packaging of the viral vector and optionally includes an introduced gene expression cassette. The term "replication-deficient" refers to a vector that is highly attenuated for replication in wild-type mammalian cells. To produce such vectors in large quantities, producer cell lines are generally created by co-transfection with a helper virus or are genomically modified to complement the lost function. The term "replicable viral vector" refers to a viral vector that is capable of infection, DNA replication, packaging and lysis of infected cells. The term "conditionally replicating viral vector" is used herein to refer to a replicable vector designed to achieve selective expression in a particular cell type. Such conditional replication can be achieved by functionally linking tissue-specific, tumor-specific or cell type-specific or other selectively induced regulatory control sequences to an early gene (e.g., the E1 gene of an adenoviral vector).Infection of a subject with a recombinant virus or non-viral vector can provide for long-term expression of an hIL2 ortholog in the subject and for the continuous and selective maintenance of engineered T cells that express the hCD122 orthogonal receptor. In one aspect, the nucleic acid sequence in a viral vector system encoding the hIL2 ortholog is under the control of a regulatable promoter, an inducible promoter, a tissue-specific or tumor-specific promoter, or a temporally regulated promoter.

[0250] Therapeutic combination: The compositions and methods of the present disclosure may be combined with additional therapeutic agents. For example, if the disease, disorder, or condition to be treated is a neoplastic disease (e.g., cancer), the methods of the present disclosure may be combined with conventional chemotherapeutic agents or other biological anti-cancer drugs such as checkpoint inhibitors (e.g., PD1 or PDL1 inhibitors) or therapeutic monoclonal antibodies (e.g., Avastin, Herceptin).

[0251] Examples of chemical agents identified in the art as being useful in the treatment of neoplastic diseases include, but are not limited to, abtrexate, adriamycin, adrucil, amsacrine, asparaginase, anthracycline, azacitidine, azathioprine, bicnu, brexan, busulfan, bleomycin, camptosar, camptothecin, carboplatin, carmustine, cerubidine, chlorambucil, cisplatin, cladribine, cosmege, cytarabine, cytosar, cyclophosphamide, cytoxan, dactinomycin, docetaxel, doxorubicin, daunorubicin, ellence, elsapar, epirubicin, etoposide, fludarabine, fluorouracil, fludara, gemcitabine, gemzar, hycamtin, hydroxyurea, hydrea, idamycin, idarubicin, ifosfamide, ifex, irinotecan, lamvis, leukeran, leustatin, matulane, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, mitramycin, mutamycin, myleran, mylosar, navelbine, 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 bumed.

[0252] The compositions of the present disclosure can be administered in combination with one or more additional therapeutic agents selected from the group consisting of tyrosine kinase inhibitors such as imatinib mesylate (also known as STI-571, sold under the trade name Gleevec®), gefitinib (Iressa®, also known as ZD1839), erlotinib (sold under the trade name Tarceva®), sorafenib (Nexavar®), sunitinib (Sutent®), dasatinib (Sprycel®), lapatinib (Tykerb®), nilotinib (Tasigna®), and bortezomib (Velcade®), Jakafi® (ruxolitinib); Janus kinase inhibitors such as tofacitinib; ALK inhibitors such as crizotinib; Bcl-2 inhibitors such as obatoclax, venetoclax, and gossypol; FLT3 inhibitors such as midostaurin (Rydapt®), 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 conjugated 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 temsirolimus (Torisel®), everolimus (Afinitor®), vemurafenib (Zelboraf®), trametinib (Mekinist), and dabrafenib (Tafinlar®).

[0253] In some embodiments, particularly when the tumor antigen-binding portion of the CAR is directed to BCMA, the engineered CAR-T cells are administered in combination with a γ-secretase inhibitor (GSI), as described in Pont, et al. (2019) "γ-secretase inhibition increases efficacy of BCMA-specific chimeric antigen receptor T cells in multiple myeloma" Blood https: / / doi.org / 10.1182 / blood.2019000050.

[0254] Examples of biopharmaceutical agents identified in the art as being useful in the treatment of neoplastic diseases include, without limitation, cytokines or cytokine antagonists, such as IL-12, INFα, or anti-epidermal growth factor receptor, radiation therapy, irinotecan; tetrahydrofolate antagonists such as pemetrexed; antibodies against tumor antigens, conjugates of monoclonal antibodies and toxins, T-cell adjuvants, bone marrow transplantation, or antigen-presenting cells (e.g., dendritic cell therapy), anti-tumor vaccines, replicable viruses, signal transduction inhibitors (e.g., Gleevec® or Herceptin®) or immunomodulatory agents that achieve additional or synergistic inhibition of tumor growth, cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade® and Enbrel®), interferon-β1a (Avonex®), and interferon-β1b (Betaseron®), and one or more combinations as practiced in known chemotherapy treatment regimens readily recognized by skilled clinicians in the art.

[0255] Tumor-specific monoclonal antibodies that can be administered in combination with the engineered cells can include, without limitation, rituximab (sold under the trade names MabThera or Rituxan), alemtuzumab, panitumumab, ipilimumab (Yervoy), and the like.

[0256] In some embodiments, the compositions and methods of the present disclosure may be combined with immune checkpoint therapy. Examples of immune checkpoint therapies include inhibitors of the binding of PD1 to PDL1 and / or PDL2. Inhibitors of the binding of PD1 to PDL1 and / or PDL2 are well known in the art. Examples of commercially available monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2 include nivolumab (Opdivo®, BMS-936558, MDX1106, commercially available from Bristol Myers Squibb (Princeton NJ)), pembrolizumab (Keytruda®, MK-3475, lambrolizumab, commercially available from Merck and Company (Kenilworth NJ)), and atezolizumab (Tecentriq®, Genentech / Roche, South San Francisco CA). Additional examples of PD1 inhibitory antibodies include durvalumab (MEDI4736, Medimmune / AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, Bristol Myers Squibb), and avelumab (MSB0010718C, Merck Serono / Pfizer) and SHR-1210 (Incyte), but are not limited thereto. Additional PD1 pathway antibody inhibitors are described in U.S. Patent No. 8,217,149 (Genentech, Inc) 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, Inc) issued May 17, 2011. In addition, small molecule inhibitors of the binding of PD1 to PDL1 and / or PDL2 are also 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).

[0257] Although the present invention will be described in more detail hereinafter, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

Examples

[0258] The following examples are presented to more particularly illustrate preferred embodiments of the invention. However, these should in no way be construed as limiting the broad scope of the invention.

[0259] Example 1. Preparation of human IL2 expression vector pcDNA3.1 / hygro(+)-huIL2 Human IL2 DNA ORF (Genbank NM_000586.3) was synthesized (Life Technologies GeneArt Service, Carlsbad, CA), and Platinum SuperFi II DNA Polymerase Kit (item #12361050, ThermoFisher) was used according to the manufacturer's protocol with primers: TIFF2025108545000071.tif12136 (incorporating an NheI restriction site), and TIFF2025108545000072.tif5128 (incorporating an ApaI restriction site) It was amplified via PCR using [relevant reagents]. The PCR fragments were visualized on a 1% agarose gel (item #54803, Lonza, Rockland, ME), excised from the gel, and purified using the QIAquick PCR Purification kit (item #28106, Qiagen, Germany) according to the manufacturer's protocol. The purified PCR fragments and the mammalian expression vector pcDNA 3.1 / Hygro(+) (#V87020, ThermoFisher) were digested with NheI and ApaI (#R0111S and #R0114L, New England Biolabs, Ipswich, MA) restriction enzymes. The expression vector was further treated using the Quick Dephosphorylation kit (#M0508L, New England Biolabs) according to the manufacturer's protocol. The PCR fragments were ligated into pcDNA 3.1 / Hygro(+) using the Rapid DNA Ligation Kit (#11635379001, Sigma Aldrich, St. Louis, MO) according to the manufacturer's protocol, transformed into One Shot TOP10 Chemically Competent E. coli (#C404006, Life Technologies, Carlsbad, CA), plated on LB agar plates containing 100 μg / ml carbenicillin (#L1010, Teknova, Hollister, CA), and grown overnight at 37°C.

[0260] The next day, individual bacterial colonies were picked and used to initiate 3 ml of bacterial cultures in LB Broth (#10855-001, Life Technologies) containing 100 ug / ml of ampicillin (#A9626, Teknova). The cultures were grown overnight at 37°C. The next day, the E. coli was pelleted (10 minutes at 6,000 rpm using a benchtop centrifuge #5424, Eppendorf, Hauppauge, NY), and the DNA expression vector was isolated using the QIAprep Spin Miniprep Kit (#27106, Qiagen). The plasmid DNA was sequence-verified (MCLab, South San Francisco, CA).

[0261] Example 2. Preparation of human IL2 ortholog expression vector pcDNA3.1 / hygro(+)-huIL2-ORTHO An expression vector with six mutations (E35S, H36Q, L39V, D40L, Q42K, and M43A; all numbering is based on the full-length human IL2 ORF NM_000586.3 numbering) introduced into the human IL2 ORF was assembled substantially according to the teachings of Example 1 regarding the pcDNA3.1 / Hygro(+)-human IL2 expression vector, with the following exceptions. The initial template DNA used for PCR was synthesized with the E35S, H36Q, L39V, D40L, Q42K, and M43A mutations added.

[0262] Example 3. Introduction of mutations into the pcDNA3.1 / hygro(+)-huIL2 expression vector or reverse mutations into the pcDNA3.1 / hygro(+)-huIL2 IRTHO expression vector All mutations or reverse mutations (reverting the mutations in pcDNA3.1 / hygro(+)-huIL2-ORTHO to match the wild-type human 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 (#200524, Agilent Technologies, Santa Clara, CA) substantially according to the manufacturer's protocol.

[0263] Table 5 lists the mutations produced, the templates into which the mutations are introduced, and the primer sets used to introduce the mutations. Transformation of the Quik Change PCR reaction into E. coli, as well as isolation and sequence analysis of the plasmid DNA, were carried out using substantially the same protocol as in the preparation of the pcDNA3.1 / Hygro-huIL2 expression vector. The abbreviations of the templates are Template 1 = pcDNA3.1 / hygro(+)-huIL2; Template 2 = pcDNA3.1 / Hygro(+)-huIL2 ortholog.

[0264] (Table 5) QuikChange Mutagenesis and Sequence Information on Mutations TIFF2025108545000073.tif208168TIFF2025108545000074.tif225168TIFF2025108545000075.tif218168TIFF2025108545000076.tif219168TIFF2025108545000077.tif218168TIFF2025108545000078.tif216168TIFF2025108545000079.tif219168TIFF2025108545000080.tif219168TIFF2025108545000081.tif218168TIFF2025108545000082.tif219168TIFF2025108545000083.tif219168TIFF2025108545000084.tif219168TIFF2025108545000085.tif219168TIFF2025108545000086.tif94168

[0265] Example 4. Transient transfection in HEK293 cells All expression vectors were transiently transfected into HEK293 cells (#CRL-1573, ATCC, Manassas, VA). Approximately 1E6 HEK293 cells were plated in 2 ml of DMEM (#10569044, Life Technologies) supplemented with 10% fetal bovine serum (#SH30071.03, Fisher Scientific, Chicago, IL) in each well of a 6-well tissue culture plate and grown overnight at 37 °C and 5% CO2.

[0266] The next day, Lipofectamine 3000 Reagent (#L3000150, Life Technologies) was used according to the manufacturer's protocol, and the cells were transfected using 2.5 μg of DNA, 5 μl of P3000 reagent, and 7.5 μl of Lipofectamine 3000 per transfection. The transfected cells were grown at 37 °C, 5% CO2 for 48 - 72 hours, and then the conditioned medium was harvested.

[0267] Example 5. Analysis of protein expression Protein expression for some mutant proteins was measured by ELISA using a Human IL2 V-PLEX ELISA kit (#K151QQD-4, Mesoscale Diagnostics, Baltimore, MD) according to the manufacturer's protocol (the transfection medium was serially diluted first 1:4 and then 1:2). The plates were read on a Meso Quickplex SQ120 (Mesoscale Diagnostics) using the manufacturer's pre-programmed settings for this ELISA kit. The approximate expression levels in the conditioned medium samples were calculated using the human IL2 standard in the kit. Table 6 below details the approximate expression levels for the expressed proteins.

[0268] (Table 6) Expression levels of human IL2 orthologs TIFF2025108545000087.tif141128

[0269] Example 7. Evaluation of the activity of orthologs in cell lines expressing hoCD122 The IL2 ortholog was evaluated for activity in NKL cells (Robertson, et al (1996) Experimental Hematology 24 (3):406-15). To generate cell lines expressing human orthogonal hCD122 (hoNKL hoRB), NKL cells were infected with a retrovirus encoding hoRB hCD122 and co-expressing YFP (MSCV-hoRb-IRES-YFP) according to procedures known in the art.

[0270] NKL cells and NKL hoRB cells were contacted with supernatants from hIL2 ortholog-transfected 293 cells as follows: Cells were seeded at 500,000 cells / ml in growth medium consisting of RPMI 1640 (ThermoFisher), 10 percent fetal bovine serum (ThermoFisher), 1 percent penicillin / streptomycin (ThermoFisher), and 1 percent glutamax (ThermoFisher). Two days after culturing, the cells were seeded at 25,000 cells / well in 100 μl of growth medium in 96-well plates (Falcon). Two-fold or five-fold serial dilutions of the transfected 293 cell supernatant were made in growth medium, and 100 μl of each dilution was added in duplicate to plates of NKL cells and NKL hoRB cells at final titrations in the range of 1:2 to 1:31,250. The plates were transferred to a humidified incubator (ThermoFisher) and incubated at 37 °C and 5 percent carbon dioxide for 3 days.

[0271] The plate was removed from the incubator and maintained at room temperature for 30 minutes. The plate was centrifuged at 400×g for 5 minutes and the supernatant was discarded. Cells were lysed by adding 50 μl of a 1:1 dilution of Celltiterglo (Promega) in PBS per well. The cell lysates were mixed on an orbital shaker (VWR Scientific) at 600 rpm for 2 minutes and then held at room temperature for 10 minutes. The lysates were transferred to a black clear-bottom 96-well plate (Costar) and the luminescence of the NKL cell lysates (Figure 1) and NKL hoRB cell lysates (Figure 2) was read as counts per second in an Envision 2103 Multilabel Plate Reader (Perkin Elmer).

[0272] To compare the effects of each IL-2 variant on the growth of NKL cells and NKL hoRB cells, the celltiterglo values of cells treated with the supernatant were compared to the values obtained from control cells treated with growth medium alone, empty vector transfection, 293 supernatant from wild-type IL-2 transfection, or supernatant from human orthogonal IL-2 transfection. The data from these experiments are presented in Figures 1 and 2 of the accompanying drawings.

[0273] Example 8. Evaluation of the activity of orthologs in cell lines expressing hoCD122 The IL2 ortholog was evaluated for its activity in CD4-positive human T cell clone 3F8 cells. As described (Yssel and Spits (2002) Current Protocols in Immunology 7.19.1-7.19.12), CD4-positive T cell clone 3F8 was generated by activation of PBMCs from healthy donors by two consecutive mixed leukocyte reactions using the EBV-transformed B cell line JY, followed by single cell cloning by limiting dilution. The 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 (ho3F8 hoRB), 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.

[0274] The 3F8 cells and 3F8 hoRb cells were contacted with the supernatant from 293 cells transfected with the hIL2 ortholog as follows: The cells were grown at 200,000 cells / ml in a growth medium consisting of Yssel medium (Iscove's Modified Dulbecco's Medium (ThermoFisher), 0.25% w / v human albumin (Sigma), 1% penicillin / streptomycin (ThermoFisher), 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), 1% human serum (Gemini) (Yssel et al (1984) J Immunol Methods 72: 219-227)) with 50 Gy irradiated JY cells at 100,000 cells / well and allogeneic PBMC irradiated at 40 Gy at 1,000,000 cells / ml. After 10 days of culture and expansion with 100 pM human IL-2, the cells were washed and seeded at 50,000 cells / well in 50 μl of growth medium in a black clear-bottom 96-well plate (Costar). Two-fold serial dilutions of the transfected 293 cell supernatant were made in growth medium, and 50 μl of each dilution was added to the plates of 3F8 cells and 3F8 hoRB cells at a final titration in the range of 1:2 to 1:31250. The plates were transferred to a humidified incubator (ThermoFisher) and incubated at 37 °C and 5% carbon dioxide for 3 days.

[0275] The plates were removed from the incubator and maintained at room temperature for 30 minutes. The cells were lysed by adding 100 μl / well of Celltiterglo (Promega). The cell lysates were mixed on an orbital shaker (VWR Scientific) at 600 rpm for 2 minutes and then held at room temperature for 10 minutes. Luminescence of the 3F8 cell lysates (Figure 1) and 3F8 hoRB cell lysates (Figure 2) was read as counts per second in an Envision 2103 Multilabel Plate Reader (Perkin Elmer).

[0276] To compare the effects of each IL-2 variant on the growth of 3F8 cells and 3F8 hoRB cells, the celltiterglo values of the cells treated with the supernatant were compared with the values obtained from control cells treated with growth medium alone, empty vector transfection, 293 supernatant from wild-type IL-2 transfection, or supernatant from human orthogonal IL-2 transfection. Data from these experiments are presented in Table 4.

[0277] Sequence Information SEQUENCE LISTING <110> SYNTHEKINE, INC. <120> IL-2 ORTHOLOGS AND METHODS OF USE <150> US 62 / 948,066 <151> 2019-12-13 <160> 438 <170> PatentIn version 3.5 <210> 1 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 1 Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30 Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45 Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60 Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu 65 70 75 80 Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95 Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110 Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125 Ser Gln Ala Ser Asp Phe Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140 Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu 145 150 155 160 Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175 Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190 Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205 Ala Leu Gly Lys Asp Thr 210 <210> 2 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30 Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45 Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60 Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu 65 70 75 80 Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95 Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110 Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125 Ser Gln Ala Ser Asp Phe Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140 Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu 145 150 155 160 Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175 Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190 Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205 Ala Leu Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly 210 215 220 Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn 225 230 235 240 Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr 245 250 255 Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly 260 265 270 Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser 275 280 285 Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg 290 295 300 Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro 305 310 315 320 Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln 325 330 335 Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys 340 345 350 Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu 355 360 365 Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro 370 375 380 Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp 385 390 395 400 Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser 405 410 415 Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser 420 425 430 Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro 435 440 445 Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu 450 455 460 Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg 465 470 475 480 Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe 485 490 495 Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser 500 505 510 Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu 515 520 <210> 3 <211> 524 <212> PRT <213> Homo sapiens <400> 3 Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30 Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45 Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60 Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu 65 70 75 80 Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95 Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110 Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125 Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140 Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu 145 150 155 160 Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175 Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190 Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205 Ala Leu Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly 210 215 220 Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn 225 230 235 240 Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr 245 250 255 Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly 260 265 270 Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser 275 280 285 Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg 290 295 300 Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro 305 310 315 320 Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln 325 330 335 Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys 340 345 350 Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu 355 360 365 Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro 370 375 380 Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp 385 390 395 400 Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser 405 410 415 Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser 420 425 430 Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro 435 440 445 Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu 450 455 460 Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg 465 470 475 480 Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe 485 490 495 Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser 500 505 510 Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu 515 520 <210> 4 <211> 133 <212> PRT <213> Homo sapiens <400> 4 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 5 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 6 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser His 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 7 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Leu Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 8 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 8 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Asp Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 9 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Gln Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 10 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 11 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 12 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Gln 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 13 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 13 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Val Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 14 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 14 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Leu Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 15 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 15 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Lys Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 16 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 16 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 17 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 17 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 18 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 18 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 19 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 19 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 20 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 21 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 21 Ala Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 22 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 22 Ala Pro Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 23 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 23 Ala Pro Cys Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 24 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 24 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 25 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 25 Ala Pro Gly Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 26 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 26 Ala Pro Gln Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 27 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 27 Ala Pro Glu Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 28 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 28 Ala Pro Asn Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 29 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 29 Ala Pro Asp Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 30 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 30 Ala Pro Arg Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 31 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 31 Ala Pro Lys Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 32 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 32 Ala Pro Pro Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 33 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 33 Ala Pro Thr Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 34 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 34 Ala Pro Thr Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 35 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 35 Ala Pro Thr Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 36 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 36 Ala Pro Thr Ser Ser Ser Lys Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 37 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 37 Ala Pro Thr Ser Ser Ser Thr Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 38 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 38 Ala Pro Thr Ser Ser Ser Thr Lys Thr Gln Leu Gln Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 39 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 39 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Leu Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 40 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 40 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Trp Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 41 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 41 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Ser Gln Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 42 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 42 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Met Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 43 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 43 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Trp Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 44 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 44 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Lys Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 45 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 45 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Thr Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 46 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 46 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Leu 1 5 10 15 Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 47 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 47 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Asn 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 48 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 48 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Gly Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 49 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 49 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Met Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 50 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 50 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Phe Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 51 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 51 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Glu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 52 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 52 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu His Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 53 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 53 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Trp Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 54 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 54 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Lys Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 55 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 55 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Gln Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 56 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 56 Ala Pro Thr Ser Ser Ser Thr...

Claims

1. The hIL2 ortholog, wherein the amino acid sequence has at least 90% identity to the polypeptide of formula #1: Wherein, AA1 is A (wild type) or a deletion; AA2 is P (wild type) or a deletion; AA3 is T (wild type), C, A, G, Q, E, N, D, R, K, P, or a deletion; AA4 is S (wild type) or a deletion; AA5 is S (wild type) or a deletion; AA6 is S (wild type) or a deletion; AA7 is T (wild type) or a deletion; AA8 is K (wild type) or a deletion; AA9 is K (wild type) or a deletion; AA13 is Q (wild type), W or a deletion; AA14 is L (wild type), M, W or a deletion; AA15 is E (wild type), K, D, T, A, S, Q, H or a deletion; AA16 is H (wild type), N or Q, or a deletion; AA18 is L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T; AA19 is L (wild type), A, V, I or a deletion; AA20 is D (wild type), T, S, M, L, or a deletion; AA22 is Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, F, or a deletion; AA23 is M (wild type), A, W, H, Y, F, Q, S, V, L, T, or a deletion; AA27 is G (wild type), K, S or a deletion; AA38 is R (wild type), W or G; AA39 is M (wild type), L or V; AA42 is F (wild type) or K; AA51 is T (wild type), I or a deletion AA55 is H (wild type) or Y; AA74 is Q (wild type), N, H, S; AA80 is L (wild type), F or V; AA81 is R (wild type), I, D, Y, T or a deletion AA85 is L (wild type) or V; AA86 is I (wild type) or V; AA88 is N (wild type), E or Q, or a deletion; AA89 is I (wild type) or V; AA91 is V (wild type), R or K; AA92 is I (wild type) or F; AA97 is K (wild type) or Q; AA104 is M (wild type) or A; AA109 is D (wild type), C or a non-natural amino acid having an activated side chain; AA113 is T (wild type) or N; AA125 is C (wild type), A or S; AA126 is Q (wild type) or H, M, K, C, D, E, G, I, R, S, or T; and / or AA130 is S (wild type), T or R. **Claim 2** The hIL2 ortholog according to claim 1, comprising modifications at positions AA15, AA16, AA19, AA20 and AA24. **Claim 3** The hIL2 ortholog according to claim 2, further comprising an amino acid substitution at one or more positions selected from the group consisting of L12, Q13, H16, L19, D20, M23, R81, D84, S87, N88, V91, I92 and E95. **Claim 4** The IL2 ortholog according to claim 3, wherein one or more substitutions are selected from the group consisting of Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I92F. **Claim 5** The IL2 ortholog according to claim 3, wherein one or more amino acid substitutions are a set of the following amino acid substitutions: selected from **Claim 6** The hIL2 ortholog according to claim 2, further comprising an amino acid substitution 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. **Claim 7** The IL2 ortholog of claim 6, wherein one or more substitutions are 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.

8. The IL2 ortholog of claim 6, wherein one or more amino acid substitutions are selected from the following set of amino acid substitutions:

9. The IL2 ortholog of claim 2, further comprising an amino acid substitution at one or more positions selected from the group consisting of Q11, L18, Q22, E110, N119, T123, Q126, S127, Q126, S127, I129, S130, and T133.

10. The hIL2 ortholog of claim 9, further comprising an amino acid substitution at one or more positions selected from the group consisting of L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18T, Q22E, Q22G, 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.

11. The hIL2 ortholog of claim 1, comprising one of the following sets of amino acid modifications:

12. The hIL2 ortholog of claim 1, selected from the group consisting of SEQ ID NOs: 5 - 138.

13. The hIL2 ortholog of claim 1, functionally linked to at least one carrier molecule.

14. The hIL2 ortholog according to claim 13, wherein the carrier molecule is independently selected from the group consisting of a water-soluble polymer, the Fc domain of IgG, a sugar, and / or albumin.

15. The hIL2 ortholog according to claim 14, wherein at least one carrier molecule is polyethylene glycol (PEG).

16. The hIL2 ortholog according to claim 14, wherein at least one carrier molecule is polyethylene glycol (PEG).

17. Structure: [PEG]-[Linker] n -[hoIL2] comprising wherein n = 0 or 1, and hoIL2 is a human orthogonal IL2 polypeptide variant. The hIL2 ortholog according to claim 14.

18. The hIL2 ortholog according to claim 17, wherein PEG has a molecular weight of 5 kDa to 80 kDa.

19. The hIL2 ortholog according to claim 17, wherein PEG has a molecular weight of approximately 40 kDa.

20. The hIL2 ortholog polypeptide according to claim 19, wherein hoIL2 is an IL2 polypeptide variant comprising the set of amino acid substitutions [desAla1-E15S-H16Q-L19V-D20L-Q22K-M23A].

21. hoIL2 is an amino acid sequence: The hIL2 ortholog polypeptide according to claim 19, which is an IL2 polypeptide variant.

22. A nucleic acid sequence encoding the hIL2 ortholog polypeptide according to any one of claims 1.

23. A recombinant vector comprising the nucleic acid sequence according to claim 22.

24. a. A nucleic acid sequence encoding a transmembrane receptor molecule comprising the extracellular domain (ECD) of orthogonal hCD122, functionally linked to one or more expression control elements capable of achieving the expression and surface presentation of the ECD of the transmembrane receptor molecule administering the engineered mammalian cell comprising to a subject suffering from a disease, disorder, or condition; and b. Administering a therapeutically effective dose of the hIL2 ortholog according to claim 1 to the subject A method of treating the disease, disorder, or condition in the subject thereby.

25. The method according to claim 24, wherein the engineered mammalian cell is an engineered T cell.

26. The method according to claim 25, wherein the engineered T cell is a CAR-T cell.

27. The method according to claim 26, wherein the hIL2 ortholog is the hIL2 ortholog according to claim 12.

28. The method according to claim 24, wherein the hIL2 ortholog is the hIL2 ortholog according to claim 17. **Claim 29** A method for preparing an engineered T cell product comprising at least 20% hoCD122 T cells, comprising the following steps: a. isolating a population of T cells from a mammalian subject; b. contacting the isolated population of T cells ex vivo with a recombinant vector comprising a nucleic acid sequence encoding hoCD122 operably linked to one or more expression control sequences to facilitate expression in mammalian T cells, under conditions that allow uptake of the recombinant vector by the T cells; c. contacting the isolated population of T cells with an effective amount of the hIL2 ortholog according to claim 1. **Claim 30** A cell population product of the method according to claim 29, wherein the cell population comprises at least 20% engineered hoCD122 T cells.

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