Trikine engineered signaling proteins

Abstract

Engineered synthetic signaling molecules, herein termed "trikines", are provided. Trikines are genetically engineered, tri-specific ligands of cell surface receptors, where the trikine specifically binds at high affinity to the extracellular domains of three different cell surface receptor polypeptides. In some embodiments, generation of a receptor multimer by binding to a trikine results in intracellular trans-phosphorylation of receptor subunits. In some embodiments trikines modulate STAT signaling that results from the receptor binding and mutimerization.

Description

TRIKINE ENGINEERED SIGNALING PROTEINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 1 19 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 608660, filed December 1 1 , 2023, the disclosure of which application is herein incorporated by reference.GOVERNMENT SUPPORT RESEARCH

[0002] This invention was made with Government support under contract AI51321 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] The ability to manipulate receptors to provide novel intracellular signal combinations is a significant challenge in protein engineering. Of particular interest is the ability to “tune” cytokine receptor signaling in way that can provide for a desired activation of specific intracellular pathways.

[0004] Cytokines are secreted glycoproteins that act as intercellular messengers to control hematopoietic and immune systems. A number of cytokines signal through binding to cell surface receptors that transduce signaling through the JAK / STAT cascade. The JAK / STAT cascade utilizes a receptor, kinase, and transcription factor to elicit a response. A general rule of cytokine signaling is that each cytokine binds to a specific receptor; this binding induces activation of specific JAK(s) and STAT(s).

[0005] Cytokines, chemokines, growth factor agonists and the like activate JAK / STAT; RTK linked; or death domain (TNF super family) receptors by multimerization, e.g. generating homo or hetero-dimers, or higher order oligomers, to elicit signaling through intracellular transphosphorylation. The identity of the specific receptor chains within a multimer (e.g. dimer or trimer) determines the signaling and functional response. Cytokines act as bi-specific ligands to specify which receptors are included in the dimers, by forming specific contacts with each of the two receptor extracellular domains, thus acting to bridge or cross-link the dimeric signaling complex. Cytokine receptor dimerization leads to the activation of an intracellular JAK / STAT signaling pathway, comprised of four Janus Kinases (JAK1 -3, TYK2) and seven signal transducer and activator of transcription (STAT1 - 6) proteins.

[0006] JAKs are inactive, but specific binding of a cognate cytokine to the extracellular domain (ECD) receptor induces their auto-activation by transphosphorylation. Once activated, JAKs phosphorylate the intracellular tails of the receptors on specific tyrosines which in turn act as docking sites for members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors. Receptor-localized STATs are then phosphorylated by JAK whichleads to their disassociation from the receptor and translocation to the nucleus, where they drive the expression of cytokine-responsive genes.

[0007] The human JAK family contains four JAKs: JAK1 , JAK2, JAK3 and TYK2. These proteins are tyrosine kinases, TWO regions on the cytoplasmic tail of receptors, termed Box 1 and Box 2, are critical for the association of JAKs. Box 1 is proline rich and is located approximately 10 residues from the C-terminus of the transmembrane region of the receptor; Box 2 is about 10-50 residues further downstream and is rich in hydrophobic residues. Sequence differences within the Box 1 and Box 2 motifs of different receptors determine which JAK is bound by the receptor.

[0008] JAK phosphorylation of distal tyrosines on the receptor intracellular domains enables those sequences to act as docking sites for STAT proteins, as well as other non-STAT family proteins that also can be recruited to the receptor and activate important signaling pathways. The human STAT family contains seven STATs: STAT1 , STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. Just as different receptors bind different JAKs, they also bind different STATs. The ability of a certain cytokine to induce activation of a particular set of STATs is driven purely by the STAT-binding sites contained within the receptor ICD. STAT-binding sites from one receptor can be replaced with binding sites for different STATs from other receptors and thereby activate non-physiological STATs (see, for example, Stahl et al. (1995) Science 267:1349-1353). It has been proposed that the affinity for one STAT over another is a function of the sequence immediately surrounding the phosphotyrosine, for example pYxxP, pYxxQ, pYxxL, and pYxxF sequences are associated with recruitment of STAT1 , STAT3, STAT5, and STAT6, respectively.

[0009] The activation of transcription by STAT proteins drives the phenotype of immune cells during the processes of activation, effector function, memory, and the like. Activated STAT proteins translocate to the cell nucleus, bind to specific regulatory elements, and in coordination with other transcription factors, regulate transcription of particular genes to provide a specific transcriptional profile. It is this gene profile, is in the context of a network of many other signaling pathways and combination of transcription factors, that yield specific but diverse transcriptional profiles that result in the different phenotypes and functions of immune cells. Tools to tune these responses are of great interest, for example, for cell mediated therapies. The present disclosure provides such tools.

[0010] While the ligands are specific for the extracellular domains of their receptors, the JAK / TYK / STAT signaling modules are found in many combinations in endogenous receptor signaling complexes, and thus are capable of extensive cross-talk. Ligands for RTK receptors (such as EGF, VEGF, etc.) also compel signaling through receptor dimerization, although the molecular mechanisms can be quite distinct from cytokines. In both cases however, the ligands induce positioning of their specific receptor subunits into dimers, such that the intracellular kinases domains are in an orientation and proximity to enable trans-phosphorylation of both the kinases and the receptor intracellular domains. The sequence requirements (i.e. substratespecificity) of these tyrosine kinases can be rather degenerate, raising the possibility that these enzymes can be redirected by alternative receptor dimerizing ligands to phosphorylate receptor substrates other than those they are normally presented with in nature.

[0011] The ability to selectively modulate signaling pathways of interest is of great interest. The present invention provides compositions and methods for this purpose.SUMMARY

[0012] Engineered synthetic signaling molecules, herein termed “trikines”, are provided. Trikines are genetically engineered, tri-specific ligands of cell surface receptors, where the trikine specifically binds at high affinity to the extracellular domains of three different cell surface receptor polypeptides. Each of the cell surface receptors are characterized by activation of signaling upon multimerization. In some embodiments, generation of a receptor multimer by binding to a trikine results in intracellular trans-phosphorylation of the receptor. In some embodiments trikines modulate STAT signaling that results from the receptor binding and mutimerization.

[0013] Cytokines that signal through receptor heterodimers, see for example Table 1 , initiate a known pattern of STAT activation, by binding to and multimerizing a receptor subunit (i) and a receptor subunit (ii). The receptor subunit (ii) is frequently a common chain, e.g. CD132 (common y chain), CDI ORb (CDw210b), gp130, [3c, etc. The receptor (i) is usually specific for the cytokine. Trikines bind to the receptor (i) and receptor (ii), and further recruit an additional receptor (iii), where binding to receptor (iii) alters the pattern of STAT signaling relative to a cytokine that binds only to receptors (i) and (ii). In some embodiments the pattern of signaling for one, two or all of STAT5, STAT3 and STAT1 is altered. Trikines induce non-natural signaling by simultaneous binding of (i), (ii) and (iii), and by bringing the three receptors into proximity with each other, e.g. to allow activation by cross-phosphorylation. Trikines not only restrict their activity to cells expressing receptor subunits (i), (ii) and (iii), but also enable tunable signaling of the (iii) STATs relative to the STATs induced by native cytokines, by the inclusion of linkers, orientation, binding epitope of the third receptor chain binder, etc. In some embodiments the receptor subunits (i) and (iii) do not naturally multimerize in response to contacting the cell with a naturally occurring cytokine ligand.

[0014] Trikines are polypeptides comprised of two or more linked binding domains. In some embodiments, a trikine comprises two binding domains, which may be joined by a peptide linker. Where reference is made to binding domain 1 or 2 it should be understood that the order can be reversed with respect to the orientation of the polypeptide sequences. The binding domains usually bind to the desired receptor extracellular domain at an affinity of, e.g. a Kd of not more than about 1 x 107M, not more than about 1 x 108M, not more than about 1 x 109M, or not more than about 1 x 101° M, not more than 1011M, or less. In some embodiments the second binding domain, which is specific for receptor subunit (iii), has a low binding affinity, e.g. a Kdgreater than about 108M, greater than about 107M, greater than 106M, greater than 105M, etc. Use of a low affinity binding domain favors binding only binds to cells that express all 3 receptor subunits. The binding domains may be contiguous within one globular domain, or separated by a linker, e.g. a polypeptide linker, etc. The length of the linker, and therefore the spacing between the binding domains can be used to modulate the proximity between receptors, can be selected depending on the desired use of the trikine. The enforced distance between binding domains can vary, but in certain embodiments may be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, or less than about 50 angstroms.

[0015] Binding domain 1 of a trikine binds to a receptor subunit (i) and receptor (ii), as discussed above. Binding domain 1 activates detectable signaling through receptor subunit (i) and (ii). In some embodiments binding domain 1 is a wild-type cytokine or analog thereof, e.g. a modified version of a cytokine, e,g, with increased activity relative to wild-type, or other modifications that retain detectable activity.

[0016] Binding domain 2 of the trikine binds to receptor subunit (iii). In some embodiments binding domain 2 does not, by itself, result in detectable activity from any of receptor (i), (ii) or (iii), but modifies signaling induced by binding domain 1. In some embodiments, binding domain 2 is a monomeric antibody, also referred to as an immunoglobulin single variable domain, e.g. a VHH sequence, an scFv sequence, etc. In other embodiments binding domain 2 of the trikine is an inactive cytokine, e.g. a dominant-negative cytokine, etc.

[0017] A trikine can be multimerized, e.g. through an Fc domain, by concatenation, coiled coils, polypeptide zippers, biotin / avidin or streptavidin multimerization, and the like. The trikine can also be joined to a moiety such as PEG, Fc, etc. as known in the art to enhance stability in vivo.

[0018] In some embodiments a trikine is a heterodimer. In some such embodiments a first polypeptide of the heterodimer is a trikine polypeptide as disclosed above, and a second polypeptide of the heterodimer is a second polypeptide of a heterodimeric cytokine. In an embodiment, the trikine comprises an IL-12 polypeptide, e.g. p40 or p35; and the second polypeptide of the heterodimer comprises a second IL-12 polypeptide, e.g. p40 or p35. The two polypeptides are co-expressed or combined after expression, and allowed to form a heterodimer.

[0019] In some embodiments receptor subunits (i), (ii) and (iii) are associated with JAK / STAT mediated signaling. In some embodiments the receptor subunits are those set forth in Table 1. In some embodiments binding domain 1 is selected from IL-2; IL-4; IL-7; IL-9; IL-15; IL-21 ; IL-3; IL-5; GM-CSF; IL-6; LIF; CNTF; CT1 ; CLC; OSM; IL-31 ; NP; IL-12; IL-13; IL-23; IL-10; IL-19; IL- 20; IL-22; IL-24; IL-26; or an analog thereof.

[0020] In some embodiments binding domain 1 binds to (i) I L-2R[3, IL-4Roc, IL-7Rot, IL-9Ra, IL- 15Roc, or IL-21 Roc and (ii) yc (CD132). In some embodiments binding domain 1 binds to (i) IL- 2Rp, or IL-21 Roc and (ii) yc (CD132).

[0021] In other embodiments binding domain 1 binds to (i) IL1 ORa, IL28R, IL20Ra, or IL22R; and(ii) IL1 OR , or IL2OR .

[0022] In other embodiments binding domain 1 binds to (i) IL12Rpi or IL23R, and (ii) IL12R[32.

[0023] Binding domain 2 binds to a receptor subunit (iii) that can, in forced proximity, interact with subunits (i) and (ii). Binding domain 2 lacks intrinsic agonist activity, unless it is linked to binding domain 1 . Binding domain 2 uses the proximity of receptor subunits (i) and (ii) to activate signaling by receptor subunit (iii). In some embodiments binding domain 2 binds to (iii) IL-2RP, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, IL-21 Ra, IL10Ra, IL28R, IL20Ra, IL22R, IL1 OR , IL2OR , IL12R 1 , IL23R or IL12R 2, with the proviso that subunits (i) and (iii) are different.

[0024] Certain specific trikines of interest bind to:• (i) IL-2R , (ii) CD132, (iii) IL-21 Ra;• (i) IL-21 Ra, (ii) CD132, (iii) IL-2R0;• (i) IL-10Ra, (ii) IL-1 ORp, (iii) IL-2Rp;• (i) IL-1 ORa, (ii) IL-1 ORp, (iii) IL-21 Ra;• (i) IL-12Rb1 , (ii) IL-12Rb2, (iii) IL-2Ra;• (i) IL-12Rb1 , (ii) IL-12Rb2, (iii) IL-21 Ra.

[0025] Examples of sequences for trikines of interest are set forth in SEQ ID NO:1 -91 . The provided polypeptide sequences are mature forms of the proteins, and as shown include optional terminal sequences, e.g. SEQ ID NO:144, GAPGSGLNDIFEAQKIEWHEHHHHHH, that are not required for binding. Examples of binding domains are provided in SEQ ID NO:92-141. In some embodiments a trikine comprises a binding domain (1 ) selected from SEQ ID NO:128-141 and a binding domain selected from SEQ ID NO:92-127, optionally joined by a polypeptide linker.

[0026] Compositions of interest include, without limitation, an effective dose of a trikine in a pharmaceutically acceptable excipient. Compositions may comprise additional agents, e.g. adjuvants and the like. Trikines may be produced synthetically; by various suitable recombinant methods, and the like, as known in the art. Also of interest are nucleic acids encoding trikines, vectors comprising such coding sequences, and cells comprising the coding sequences and / or vectors.

[0027] In some aspects of the invention, a method is provided for modifying selected JAK / STAT signaling in a cell. In such methods, a cell expressing the cognate receptor polypeptides for a trikine of interest is contacted with a concentration of a trikine that is effective to alter signaling, e.g. to change the relative level of one or more of STAT1 , STAT3 and STAT5 by 25%, 50%, 75%, 90%, 95%, or more, relative to the signaling by a wild-type cytokine binding to receptor subunits (i) and (ii). Such signaling activation may include, without limitation, modulation of immune responses, growth factor responses, and the like. In some methods, the receptor-expressing cell is contacted in vitro. In other embodiments, the receptor-expressing cell is contacted in vivo.

[0028] In some aspects of the invention, a method is provided for treating or preventing a disease or disorder in a subject in need thereof, the method comprising providing to the subject an effective amount of a trikine. In particular embodiments, the subject has an immune disease or dysfunction. In other embodiments the subject has cancer.

[0029] Trikines are shown herein to modulate the balance of STAT signaling compared to the wild-type cytokine by rebalancing signaling. Trikines allow tuning of the signaling response, and extend the STAT signaling range of natural cytokines by recruiting a third receptor that has its own STAT. By linking a natural cytokine to a binding domain 2 through a linker, and then recruiting the subunit (iii) the natural complex of subunits (i) and (ii), new combinations of STAT signals are produced that are not found in nature. Trikines only act on cells expressing all three receptors (i), (ii) and (iii), which reduces off target effects.

[0030] In an example, STAT5 signaling induced by IL-2 mediates proliferation of T cells, but signaling from IL-21 and IL-10 induce STAT3, which promotes T cell sternness and mitigates exhaustion. IL-based trikines that recruited IL-21 R chain showed enhanced sternness, while IL- 21 and IL-10 based trikines that recruited STAT5 on IL2Rb showed enhanced proliferation. These molecules demonstrate therapeutically relevant STAT rebalancing.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The invention may be understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity.

[0032] Fig. 1 A-1 B. A. Conceptual overview of trikines. Trikines are comprised of linked binding domains against multiple extracellular domains (ECDs) of cytokine receptors (wild type, engineered, or dominant-negative cytokine; scFv; or VHH). Trikines induce non-natural signaling via proximity of three cytokine receptors by binding to their ECDs simultaneously. IL-2 based, linking IL-21 Ra binding domain to IL-2 IL-2 signals mainly through pSTAT5, IL-21 R signals through pSTAT3 and pSTATI . The design goal of this trikine is that we enhance the sternness properties of IL-2 on T cells by bringing in pSTAT3. B. IL-21 based, linking IL-2Rb binding domain to IL-21 . IL-21 signals mainly through pSTAT5, IL-21 R signals through pSTAT3 and pSTATI . The design goal of this trikine is that we enhance the proliferative properties of IL-21 on T cells by bringing in pSTAT5.

[0033] Fig. 2A-2G. IL-2 - alL21 R trikines rebalance the pSTAT signaling profile and phenotypic effects of IL-2. (A) Schematic of IL-2 - oclL21 R trikine. (B & C) List of binding domains comprising (B) human- and (C) mouse-reactive IL-2 - oclL21 R trikines. (D) Dose response curves for phospho-STATs 1 , 3 and 5 in pre-activated and rested human CD8+ T cells stimulated for 20 minutes with controls and selected IL-2 - alL21 R trikines, GER-023 and GER-046. (E) pSTATsignaling profiles of IL-2 - alL21 R trikines with identical binding modules but varied number of residues of flexibility between binding modules. (F) Cell counts of CD4+ and CD8+ cells after preactivated and rested human T cells were incubated with 10 nM ligand for 3 days. (G) Cytokine production of human CD8+ T cells treated with 10nM ligand every 48h for 8 days, followed by 5h treatment with PMA, ionomycin, brefeldin A, and monensin.

[0034] FIGS. 3A-3F. IL-21 - oclL2Rb trikines rebalance the pSTAT signaling profile and phenotypic effects of IL-21 . (A) Schematic of IL-21 - oclL2Rb trikine. (B & C) List of binding domains comprising (B) human- and (C) mouse-reactive IL-21 - oclL2Rb trikines. (D) Dose response curves for phospho-STATs 1 , 3 and 5 in pre-activated and rested human CD8+ T cells stimulated for 20 minutes with controls and selected IL-21 - oclL2Rb trikines, GER-060, GER- 061 , and GER-064. (E) Cell counts of CD4+ and CD8+ cells after preactivated and rested human T cells were incubated with 10 nM ligand for 3 days. (F) Cytokine production of human CD8+ T cells treated with 10nM ligand every 48h for 8 days, followed by 5h treatment with PMA, ionomycin, brefeldin A, and monensin.

[0035] FIGS. 4A-4C. Mono-super-IL-10 - alL2Rb trikines rebalance the pSTAT signaling profile of IL-10. (A) Schematic of mono-super-IL-10 - alL2Rb trikine. (B) List of binding domains comprising human-reactive mono-super-IL-10 - alL2Rb trikines. (C) Dose response curves for phospho-STATs 3 and 5 in pre-activated and rested human T cells stimulated for 20 minutes controls and selected IL-21 - oclL2Rb trikines at 100nM concentration.

[0036] FIG. 5A-FIG. 5B. A schematic showing the difference between linked cytokines (A) and trikines (B). Trikines induce non-natural signaling by simultaneous binding of (i), (ii) and (iii), and by bringing the three receptors into proximity with each other, e.g. to allow activation by crossphosphorylation, thereby limiting pleiotrophic effects. Trikines do not signal through receptor subunit (iii) and the absence of subunits (i) and (ii), unlike linked cytokines.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] In order for the present disclosure to be more readily understood, certain terms and phrases are defined below as well as throughout the specification. The definitions provided herein are non-limiting and should be read in view of what one of skill in the art would know at the time of invention.Definitions

[0038] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0039] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0041] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0042] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0043] By “comprising” it is meant that the recited elements are required in the composition / method / kit, but other elements may be included to form the composition / method / kit etc. within the scope of the claim. For example, a composition comprising a trikine is a composition that may comprise other elements in addition to trikine(s), e.g. functional moieties such as polypeptides, small molecules, or nucleic acids bound, e.g. covalently bound, to the trikine; agents that promote the stability of the trikine composition, agents that promote the solubility of the trikine composition, adjuvants, etc. as will be readily understood in the art, with the exception of elements that are encompassed by any negative provisos.

[0044] By “consisting essentially of”, it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention. For example, a trikine “consisting essentially of” a disclosed sequence has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence based upon the sequence from which it was derived, e.g. about 5 residues, 4 residues, 3 residues, 2 residues or about 1 residue less than the recited bounding amino acid residue, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited bounding amino acid residue.

[0045] By “consisting of”, it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, a trikine “consisting of” a disclosed sequence consists only of the disclosed amino acid sequence.

[0046] By “functional moiety” or “FM” it is meant a polypeptide, small molecule or nucleic acid composition that confers a functional activity upon a composition. Examples of functional moieties include, without limitation, therapeutic moieties, binding moieties, and imaging moieties.

[0047] The terms "treatment”, "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0048] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., CSH Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0049] The term "identity," as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit (e.g., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.

[0050] The term "polypeptide," "protein" or "peptide" refer to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation).

[0051] By "protein variant" or "variant protein" or "variant polypeptide" herein is meant a protein that differs from a wild-type protein by virtue of at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified version of a WT polypeptide. Variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence that encodes it. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g. from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.

[0052] By "parent polypeptide", "parent protein", "precursor polypeptide", or "precursor protein" as used herein is meant an unmodified polypeptide that is subsequently modified to generate a variant. A parent polypeptide may be a wild-type (or native) polypeptide, or a variant or engineered version of a wild-type polypeptide. Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it.

[0053] By "wild type" or "WT" or "native" herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.

[0054] The terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. Preferably, the mammal is human.

[0055] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent, e.g. trikine, sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., delay or minimize the spread of cancer, or the amount effect to decrease or increase signaling from a receptor of interest. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0056] As used herein, the terms "prevent", "preventing" and "prevention" refer to the prevention of the recurrence or onset of one or more symptoms of a disorder in a subject as result of the administration of a prophylactic or therapeutic agent.

[0057] As used herein, the term "in combination" refers to the use of more than one prophylactic and / or therapeutic agents. The use of the term "in combination" does not restrict the order in which prophylactic and / or therapeutic agents are administered to a subject with a disorder. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.

[0058] Cytokines that signal through the JAK / STAT pathway. A number of cytokines utilize the JAK / STAT pathway to activate transcription programs and produce changes in phenotype of responding cells. Included are the cytokines and receptors listed below in Table 1 .Table 1

[0059] STAT activation profile. JAK phosphorylation of distal tyrosines on the receptor intracellular domains enables those sequences to act as docking sites for STAT proteins, where a specific set of STAT proteins is associated with signaling from a specific subunit intracellular domain (ICD). The ability of a ligand to induce activation of a particular set of STATs is driven by the STAT-binding sites contained within the receptor ICD.

[0060] The human STAT family contains seven STATs: STAT1 , STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. It has been proposed that the affinity for one STAT over another is a function of the sequence immediately surrounding the phosphotyrosine, for example pYxxP, pYxxQ, pYxxL, and pYxxF sequences are associated with recruitment of STAT1 , STAT3, STAT5, and STAT6, respectively.

[0061] The set of STAT proteins activated from a specific receptor ICD may be referred to as the STAT activation profile. An ICD may activate one or more STAT proteins, and may further be characterized by degree of activation, in addition to the characterization of specific STAT proteins that are activated. The level of activation of STAT1 , STAT3 and STAT5 are of particular interest.

[0062] The level of activation of STAT proteins can be determined by any convenient method, including without limitation assays set forth in the Examples. For example, cells expressing a cytokine receptor can be stimulated with an appropriate ligand in vitro for a period of time sufficient to activate the JAK / STAT pathway. The level of phosphorylated STAT proteins in the cell is then determined, e.g. by binding with specific, detectably labeled antibodies. The level of binding provides quantitation of the level of a phosphorylated STAT protein of interest. The STAT activation profile is used to determine the signaling properties of an ICD of interest.

[0063] As exemplary controls for the level of signaling, IL-2R provides a strong (+++) signal for activation of STAT5, and very low (-) for STAT1 and STAT3. Interferon receptors, e.g. IFNAB1 provide a strong (+++) signal for STAT 1 . IL-22R provides a strong (+++) signal for STAT3.

[0064] Sternness. A clinical correlate of effective T cell therapy relates to the T cell properties of sternness, which may be defined by reduced effector cell differentiation, enhanced expression of co-stimulatory receptors, retention of key sternness-related transcription factors such as TCF1 (TCF7) and enhanced self-renewal capacity, relative to, for example, bulk peripheral blood T cells. The presence of stem-like T cells can play a role in mediating responses to immune checkpoint inhibitor therapy. Cell surface markers for sternness include, for example, CD39-CD69- T cells. These cells also exhibited increased persistence after infusion. Retaining a population of cells with self-renewal characteristics is an important goal to deliver effective T cell mediated therapy.

[0065] Manipulation of signaling pathways with trikines can enhance the sternness of a T cell population. For example, activation of STAT3 is associated with stem cell properties in a number of cell types, including T cells. Tunable receptors that provide for increased STAT3 activation in the STAT activation profile can be useful in increasing sternness in a T cell population. The level of sternness can be determined by, for example, quantitating the retention of TCF1 in the cell population, determining the number of CD39_CD69“ T cells in the population, determining persistence after infusion of the T cells, and the like.

[0066] It will be understood by one of skill in the art that desirable cell populations may be balanced between the level of activation, e.g. STAT5 activation, and the level of sternness, e.g. STAT3 activation.

[0067] Proliferation. Another clinical correlate of effective T cell therapy is the proliferation of T cells, which can be associated with activation. The level of proliferation can readily be measured by means known in the art, e.g. uptake of 3H-thymidine, dilution of dyes, and the like.

[0068] Immunoglobulin sequences, such as antibodies and antigen binding fragments derived there from (e.g., immunoglobulin single variable domains or ISVs) can used to specifically target the receptor subunits disclosed herein. The generation of monomeric antibodies, e.g. immunoglobulin single variable domains such as e.g., HHS, ISV, scFv, may involve selection from phage display or yeast display, for example ISV can be selected by utilizing surface display platforms where the cell or phage surface display a synthetic library of ISV, in the presence of tagged antigen. A fluorescent secondary antibody directed to the tagged antigen is added to the solution thereby labeling cells bound to antigen. Cells are then sorted using any cell sorting platform of interest e.g., magnetic-activated cell sorting (MACS) or fluorescence-activated cellsorting (FACS). Sorted clones are amplified, resulting in an enriched library of clones expressing ISV that bind antigen. The enriched library is then re-screened with antigen to further enrich for surface displayed antigen binding ISV. These clones can then be sequenced to identify the sequences of the ISV of interest and further transferred to other heterologous systems for large scale protein production.

[0069] As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain is comprised of at least four domains (each about 1 10 amino acids long)- an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CH1 , CH2, and the carboxyterminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1 , CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1 , FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure.

[0070] The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments,antibodies produced and / or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation.

[0071] Any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and / or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art.

[0072] Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgG, IgE and IgM, bi- or multi- specific antibodies (e.g., Zybodies®, etc), single chain Fvs, Fabs, Small Modular ImmunoPharmaceuticals (“SMIPs™’), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, a TrimerX®, MicroProteins, Fynomers®, Centyrins®, and a KALBITOR®. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.]

[0073] Alternatively, similar immunoglobulin single variable domains can be generated and selected by the immunization of an experimental animal such as a llama, construction of phage libraries from immune tissue, and

[0074] Unless indicated otherwise, the term "immunoglobulin single variable domain" or "ISV" is used as a general term to include but not limited to antigen-binding domains or fragments such as VHH domains or VHor VLdomains, respectively. VHH domains are of interest for the present disclosure. The terms antigen-binding molecules or antigen-binding protein are used interchangeably and include also the term NANOBODIES®. The immunoglobulin single variable domains can be light chain variable domain sequences [e.g., a Vi_-sequence), or heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, they can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. Accordingly, the immunoglobulin single variable domains can be single domain antibodies, or immunoglobulin sequences that are suitable for use as single domain antibodies, "dAbs", or immunoglobulinsequences that are suitable for use as dAbs, or NANOBODIES™, including but not limited to VHH sequences.

[0075] The invention includes immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The immunoglobulin single variable domain includes fully human, humanized, otherwise sequence optimized or chimeric immunoglobulin sequences. The immunoglobulin single variable domain and structure of an immunoglobulin single variable domain can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region 1" or "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. It is noted that the terms Nanobody or Nanobodies are registered trademarks of Ablynx N.V. and thus may also be referred to as NANOBODY® or NANOBODIES®, respectively.

[0076] An amino acid sequence such as, e.g. an immunoglobulin single variable domain or polypeptide according to the invention, is said to be a "VHH1 type immunoglobulin single variable domain" or "VHH type 1 sequence", if said VHH1 type immunoglobulin single variable domain or VHH type 1 sequence has 85% identity (using the VHH1 consensus sequence as the query sequence and use the blast algorithm with standard setting, i.e., blosom62 scoring matrix) to the VHH1 consensus sequence and mandatorily has a cysteine in position 50, i.e., C50 (using Kabat numbering). See, for example, VHH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1 -2): 185-195. Specific VHH useful in constructing trikines include, for example, any of SEQ ID NO:92-127.

[0077] Such immunoglobulin single variable domains may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring VHH sequences (i.e., from a suitable species of Camelid, e.g., llama) or synthetic or semi-synthetic VHs or VLs (e.g., from human). Such immunoglobulin single variable domains may include "humanized" or otherwise "sequence optimized" VHHs, "camelized" immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences, i.e., camelized VHs), as well as human VHs, human VLs, camelid VH Hs that have been altered by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein.

[0078] Immunoglobulin single variable domains may comprise an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g. indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art on humanization referred to herein. Again, it should be noted that such humanized immunoglobulin single variable domains of the invention can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.

[0079] Another class of immunoglobulin single variable domains of the invention comprises immunoglobulin single variable domains with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHdomain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the description herein. Such "camelizing" substitutions may be inserted at amino acid positions that form and / or are present at the VH-VL interface, and / or at the so-called Camelidae hallmark residues (see for example WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). The VH sequence that is used as a starting material or starting point for generating or designing the camelized immunoglobulin single variable domains is preferably a VH sequence from a mammal, more preferably the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized immunoglobulin single variable domains can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHdomain as a starting material.

[0080] Linker. The binding domains of a trikine may be separated by a linker, e.g. a polypeptide linker. The amino acid linkers that join domains can play an important role in the structure and function of multi-domain proteins. The length of the linker and therefore the spacing between the binding domains, can be used to modulate the signal strength of the surrogate, and can be selected depending on the desired use of the surrogate. The enforced distance between binding domains of a surrogate can vary, but in certain embodiments may be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70angstroms, less than about 60 angstroms, less than about 50 angstroms, less than about 40 angstroms, less than about 30 angstroms, less than about 20 angstroms.

[0081] In some embodiments the linker is a rigid linker, in other embodiments the linker is a flexible linker. In some embodiments, the linker moiety is a peptide linker. In some embodiments, the peptide linker comprises 2 to 100 amino acids. In some embodiments, the peptide linker comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25,26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50,51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75,76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 but no greater than 100 amino acids. In some embodiments, the peptide linker is between 3 to 75, 3 to 50, 3 to 25, 3 to 20, 3 to 15, 3 to 10 or 3 to 6 amino acids in length. Exemplary linkers include linear peptides having at least two amino acid residues such as GSG, GG, GAG, GPA, (SEQ ID NO:145) GGGGS. Suitable linear peptides include poly glycine, polyserine, polyproline, polyalanine and oligopeptides consisting of alanyl and / or serinyl and / or prolinyl and / or glycyl amino acid residues. Many such linkers are known and used in the art and may serve this purpose.

[0082] Variants. In some embodiments the binding domains comprise cytokines or variants thereof, e.g. derivatives, variants, and biologically active fragments of cytokines. A "variant" polypeptide means a biologically active polypeptide as defined below having less than 100% sequence identity with a provided sequence. Such variants include polypeptides comprising one or more amino acid modifications, e.g., insertions, deletions or substitutions, as compared to the provided sequence, e.g., wherein one or more amino acid residues are added at the N- or C- terminus of, or within, the native sequence; from about one to forty amino acid residues are deleted, and optionally substituted by one or more amino acid residues; and derivatives of the above polypeptides, wherein an amino acid residue has been covalently modified so that the resulting product has a non-naturally occurring amino acid. Ordinarily, a biologically active variant will have an amino acid sequence having at least about 90% amino acid sequence identity with a native sequence or a reference sequence polypeptide, preferably at least about 95%, more preferably at least about 99%.

[0083] A "functional derivative" of a sequence is a compound having a qualitative biological property in common with an initial sequence. "Functional derivatives" include, but are not limited to, fragments of a sequence and derivatives of a sequence, provided that they have a biological activity in common. The term "derivative" encompasses both amino acid sequence variants of polypeptide and covalent modifications thereof.

[0084] Binding domains for use in the subject compositions and methods may be modified using ordinary molecular biological techniques and synthetic chemistry so as to improve theirresistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

[0085] Expression construct: A trikine may be produced by recombinant methods. The trikine may be introduced on an expression vector into the cell to be engineered. DNA encoding a trikine may be obtained from various sources as designed during the engineering process. Amino acid sequence variants can be 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 noted. Any combination of insertion, substitution, and / or specified deletion is made to arrive at the final construct, provided that the final construct possesses the desired biological activity as defined herein.

[0086] The nucleic acid encoding a trikine may be inserted into a replicable vector for expression. Many such vectors are available. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, and the like. Alternatively a coding sequence can be inserted into the genome of the cell by genomic editing, e.g. using CRISPR / cas9 technology.

[0087] A trikine may be produced recombinantly as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or other polypeptide having a specific cleavage site at the N- terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the coding sequence that is inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression the native signal sequence may be used, or other mammalian signal sequences may be suitable, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex gD signal.

[0088] Expression vectors usually contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.

[0089] Expression vectors will contain a promoter that is recognized by the host organism and is operably linked to a trikine coding sequence. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known.

[0090] Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), lentivirus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.

[0091] Transcription by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, cx-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5' from the promoter.

[0092] Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. Construction of suitable vectors containing one or more of the above-listed components employs standard techniques.

[0093] Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

[0094] Recombinantly produced trikines can be recovered from the culture medium as a secreted polypeptide, although it can also be recovered from host cell lysates. A protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants. Various purification steps are known in the art and find use, e.g. affinity chromatography. Affinity chromatography makes use of the highly specific binding sites usually present in biological macromolecules, separating molecules on their ability to bind a particular ligand. Covalent bonds attach the ligand to an insoluble, porous support medium in a manner that overtly presents the ligand to the protein sample, thereby using natural biospecific binding of one molecular species to separate and purify a second species from a mixture. Antibodies are commonly used in affinity chromatography. Size selection steps may also be used, e.g. gel filtration chromatography (also known as size-exclusion chromatography or molecular sieve chromatography) is used to separate proteins according to their size. In gel filtration, a protein solution is passed through a column that is packed with semipermeable porous resin. The semipermeable resin has a range of pore sizes that determines the size of proteins that can be separated with the column. Also of interest is cation exchange chromatography.

[0095] The final trikine composition may be concentrated, filtered, dialyzed, etc., using methods known in the art. For therapeutic applications, the trikine can be administered to a mammal comprising the appropriate combination of receptor polypeptides. Administration may be intravenous, as a bolus or by continuous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intra-cerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The trikines also are suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes or to the lymph, to exert local as well as systemic therapeutic effects.

[0096] Such dosage forms encompass physiologically acceptable carriers that are inherently 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, polyvinyl pyrrolidone, cellulose-based substances, and PEG. Carriers fortopical or gel-based forms of polypeptides include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene- polyoxypropylene-block polymers, PEG, and wood wax alcohols. For all administrations, conventional depot forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and sustained-release preparations. The polypeptide will typically be formulated in such vehicles at a concentration of about 0.1 pig / ml to 100 |ig / ml.

[0097] In the event the trikine is "substantially pure," the trikine can be at least about 60% by weight (dry weight) the polypeptide of interest, for example, a polypeptide containing the trikine amino acid sequence. For example, the polypeptide can be at least about 75%, about 80%, about 85%, about 90%, about 95% or about 99%, by weight, the polypeptide of interest. Purity can be measured by any appropriate standard method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0098] In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of the conditions described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is the trikine. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. Further container(s) may be provided with the article of manufacture which may hold, for example, a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution or dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.Compositions

[0099] Engineered synthetic signaling molecules, herein termed “trikines”, are provided. Trikines are genetically engineered, tri-specific ligands of cell surface receptors, where the trikine specifically binds at high affinity to the extracellular domains of three different cell surface receptor polypeptides. The cell surface receptors are characterized by activation of signaling upon multimerization. In some embodiments, generation of a receptor multimer by binding to a trikine results in intracellular trans-phosphorylation of the receptor. In some embodiments trikines modulate STAT signaling that results from the receptor binding and mutimerization.

[0100] Cytokines that signal through receptor heterodimers, see for example Table 1 , initiate a known pattern of STAT activation, by binding to and multimerizing a receptor subunit (i) and a receptor subunit (ii). The receptor subunit (ii) is frequently a common chain, e.g. CD132 (common y chain), CDI ORb (CDw210b), gp130, pc, etc. The receptor (i) is usually specific for the cytokine. Trikines bind to the receptor (i) and receptor (ii), and further recruit an additional receptor (iii), where binding to receptor (iii) alters the pattern of STAT signaling relative to a cytokine that binds only to receptors (i) and (ii). In some embodiments the pattern of signaling for one, two or all of STAT5, STAT3 and STAT1 is altered. Trikines induce non-natural signaling by simultaneous binding of (i), (ii) and (iii), and by bringing the three receptors into proximity with each other, e.g. to allow activation by cross-phosphorylation.

[0101] Trikines are polypeptides comprised of two or more linked binding domains. In some embodiments, a trikine comprises two binding domains, which may be joined by a peptide linker. Where reference is made to binding domain 1 or 2 it should be understood that the order can be reversed with respect to the orientation of the polypeptide sequences. The binding domains usually bind to the desired receptor extracellular domain at high affinity, e.g. a Kd of not more than about 1 x 107M, not more than about 1 x 108M, not more than about 1 x 109M, or not more than about 1 x 1010M. The binding domains may be contiguous within one globular domain, or separated by a linker, e.g. a polypeptide linker, etc. The length of the linker, and therefore the spacing between the binding domains can be used to modulate the proximity between receptors, can be selected depending on the desired use of the trikine. The enforced distance between binding domains can vary, but in certain embodiments may be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, or less than about 50 angstroms.

[0102] Binding domain 1 of a trikine binds to a receptor subunit (i) and receptor (ii), as discussed above. Binding domain 1 activates detectable signaling through receptor subunit (i) and (ii). In some embodiments binding domain 1 is a wild-type cytokine or analog thereof, e.g. a modified version of a cytokine, e,g, with increased activity relative to wild-type, or other modifications that retain detectable activity.

[0103] Binding domain 2 of the trikine binds to receptor subunit (iii). In some embodiments binding domain 2 does not, by itself, result in detectable activity from any of receptor (i), (ii) or (iii), but modifies signaling induced by binding domain 1. In some embodiments, binding domain 2 is a monomeric antibody, also referred to as an immunoglobulin single variable domain, e.g. a VHH sequence, an scFv sequence, etc. In other embodiments binding domain 2 of the trikine is an inactive cytokine, e.g. a dominant-negative cytokine, etc.

[0104] In some embodiments, binding domain 1 comprises the binding domain of a native ligand, i.e. IL-1 d, IL-1 p, IL-1 RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-4, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-1 1 , G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-a, IFN-p, IFN-y, LT-p, TNF-a, TNF-p, 4-1 BBL, CD70, CD153, CD178, TGF- 1 , TGF- 2, TGF- 3, Epo, Tpo, Flt-3L, SCF, M- CSF, MSP; etc. where the binding domain activates the native receptor for the ligand.

[0105] The term "specific binding" refers to that binding which occurs between such paired species as enzyme / substrate, receptor / ligand, antibody / antigen, and lectin / carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non- covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody / antigen or ligand / receptor interaction. One may determine the biological activity of a trikine in a composition by determining the level of activity in a functional assay after in vivo administration, e.g. accelerating bone regeneration, enhancing stem cell proliferation, etc., nuclear localization of [3-catenin, increased transcription of -responsive genes; etc.

[0106] Each binding domain and can be selected from any domain that binds the desired receptor extracellular domain at high affinity, e.g. a KDof less than about 1 x 107M, less than about 1 x 108M, less than about 1 x 109M, less than about 1 x 1010M. Suitable binding domains include, without limitation, cytokines, antibody derived binding proteins, e.g. scFv, Fab, etc. and other portions of antibodies that specifically bind to one or more proteins; nanobody derived binding domains; knottin-based engineered scaffolds; and the like.

[0107] A binding domain may be affinity selected to enhance binding to a desired extracellular domain. Methods of affinity selection for this purpose may optionally utilize one or more rounds of selection by introducing targeted amino acid changes and generating a library of candidate coding sequences, transforming a population of cells with the candidate coding sequence, e.g. into yeast cells, selecting (for example using paramagnetic microbeads) for the desired specificity. Typically multiple rounds of selection will be performed, and the resulting vectors sequenced and used as the basis for protein engineering. For example, the binding domain, including without limitation a modified cytokine, an antibody or nanobody derived domain, an engineered protein, etc. can be selected to bind selectively to an extracellular domain of interest.

[0108] Variants. Binding domains may also include derivatives, variants, and biologically active fragments of polypeptides described above, e.g. variants of native ligands. A "variant" polypeptide means a biologically active polypeptide as defined below having less than 100% sequence identity with a provided sequence. Such variants include polypeptides comprising one or more amino acid modifications, e.g., insertions, deletions or substitutions, as compared to the provided sequence, e.g., wherein one or more amino acid residues are added at the N- or C- terminus of, or within, the native sequence; from about one to forty amino acid residues are deleted, and optionally substituted by one or more amino acid residues; and derivatives of theabove polypeptides, wherein an amino acid residue has been covalently modified so that the resulting product has a non-naturally occurring amino acid. Ordinarily, a biologically active variant will have an amino acid sequence having at least about 90% amino acid sequence identity with a native sequence polypeptide, preferably at least about 95%, more preferably at least about 99%.

[0109] A "functional derivative" of a sequence is a compound having a qualitative biological property in common with an initial sequence. "Functional derivatives" include, but are not limited to, fragments of a sequence and derivatives of a sequence, provided that they have a biological activity in common. The term "derivative" encompasses both amino acid sequence variants of polypeptide and covalent modifications thereof.

[0110] Binding domains for use in the subject compositions and methods may be modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

[0111] A trikine may be fused or bonded to an additional polypeptide sequence. Examples include immunoadhesins, which combine a trikine with an immunoglobulin sequence particularly an Fc sequence, and epitope tagged polypeptides, which comprise a native inhibitors polypeptide or portion thereof fused to a "tag polypeptide". A trikine may be pegylated.

[0112] The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with biological activity of the native inhibitors polypeptide. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 6-60 amino acid residues. The trikine may also be fused or combined in a formulation, or co-administered with an agent that enhances activity, e.g. cytokines, growth factors, chemotherapeutic agents, immunosuppressants, etc.

[0113] In some embodiments, the trikine is conjugated to additional molecules to provide desired pharmacological properties such as extended half-life. In one embodiment, a trikine is fused to the Fc domain of IgG, albumin, or other molecules to extend its half-life, e.g. by pegylation, glycosylation, and the like as known in the art. In some embodiments the trikine is conjugated to a polyethylene glycol molecules or “PEGylated.” The molecular weight of the PEG conjugated to the trikine include but are not limited to PEGs having molecular weights between 5kDa and 80kDa, in some embodiments the PEG has a molecular weight of approximately 5kDa, in some embodiments the PEG has a molecular weight of approximately 10kDa, in some embodiments the PEG has a molecular weight of approximately 20kDa, in some embodiments the PEG has amolecular weight of approximately 30kDa, in some embodiments the PEG has a molecular weight of approximately 40kDa, in some embodiments the PEG has a molecular weight of approximately 50kDa, in some embodiments the PEG has a molecular weight of approximately 60kDa in some embodiments the PEG has a molecular weight of approximately 80kDa. In some embodiments, the molecular mass is from about 5kDa to about 80kDa, from about 5kDa to about 60kDa, from about 5kDa to about 40kDa, from about 5kDa to about 20kDa. The PEG conjugated to the polypeptide sequence may be linear or branched. The PEG may be attached directly to the polypeptide or via a linker molecule. The processes and chemical reactions necessary to achieve PEGylation of biological compounds is well known in the art.

[0114] Trikines can be provided in single-chain form, which means that the binding domains are linked by peptide bonds through a linker peptide. In other embodiments, the binding domains are individual peptides and can be joined through a non-peptidic linker.

[0115] Chemical groups that find use in linking binding domains include carbamate; amide (amine plus carboxylic acid); ester (alcohol plus carboxylic acid), thioether (haloalkane plus sulfhydryl; maleimide plus sulfhydryl), Schiff's base (amine plus aldehyde), urea (amine plus isocyanate), thiourea (amine plus isothiocyanate), sulfonamide (amine plus sulfonyl chloride), disulfide; hyrodrazone, lipids, and the like, as known in the art.Pharmaceutical Compositions

[0116] For therapeutic applications, the trikine is administered to a mammal, preferably a human, in a physiologically acceptable dosage form, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time. Alternative routes of administration include topical, intramuscular, intraperitoneal, intra-cerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The trikines also are suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes or to the lymph, to exert local as well as systemic therapeutic effects.

[0117] Pharmaceutical compositions may also comprise combinations of the molecules of the invention with cells, including stem cells, progenitor cells, immune effector cells, and the like. In such combinations, cells can be pre-treated with a molecule of the invention prior to use, e.g. ex vivo treatment of immune effector cells with the trikine; cells can be administered concomitantly with a molecule of the invention in a separate or combined formulation; cells can be provided to an individual prior to treatment with a molecule of the invention, and the like.

[0118] Pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are defined as vehicles 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. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution,dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.

[0119] The composition can also include any of a variety of stabilizing agents, such as an antioxidant for example. When the pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate. The polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0120] Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

[0121] The pharmaceutical compositions can be administered for prophylactic and / or therapeutic treatments. Toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determining the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5o / ED5OCompounds that exhibit large therapeutic indices are preferred.

[0122] The data obtained from cell culture and / or animal studies can be used in formulating a range of dosages for humans. The dosage of the active ingredient typically lines within a range of circulating concentrations that include the ED5o with low toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[0123] For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similardiluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0124] The active ingredient, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen.

[0125] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0126] The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

[0127] The effective amount of a therapeutic composition to be given to a particular patient will depend on a variety of factors, several of which will be different from patient to patient. A formulation may be provided, for example, in a unit dose. A competent clinician will be able to determine an effective amount of a therapeutic agent to administer to a patient. Dosage of the trikine will depend on the treatment, route of administration, the nature of the therapeutics, sensitivity of the disease to the therapeutics, etc. Utilizing LD5o animal data, and other information available, a clinician can determine the maximum safe dose for an individual, depending on the route of administration. Compositions which are rapidly cleared from the body may be administered at higher doses, or in repeated doses, in order to maintain a therapeutic concentration. Utilizing ordinary skill, the competent clinician will be able to optimize the dosage of a particular therapeutic or imaging composition in the course of routine clinical trials. Typically the dosage will be 0.001 to 100 milligrams of agent per kilogram subject body weight.

[0128] The compositions can be administered to the subject in a series of more than one administration. For therapeutic compositions, regular periodic administration (e.g., every 2-3 days) will sometimes be required, or may be desirable to reduce toxicity. For therapeutic compositions which will be utilized in repeated-dose regimens, moieties which do not provoke immune responses are preferred.

[0129] In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of the conditions described herein is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is the trikine. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. Further container(s) may be provided with the article of manufacture which may hold, for example, a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution or dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0130] As used herein, the term “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, stabilizes one or more characteristics of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.

[0131] For example, in some embodiments, term "therapeutically effective amount", refers to an amount which, when administered to an individual in need thereof in the context of inventive therapy, will block, stabilize, attenuate, or reverse a disease process occurring in said individual.

[0132] In certain embodiments, multiple therapeutically effective doses are administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By "intermittent" administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days,every three days, once a week, once every two weeks, once every three weeks, once a month, and so forth. For example, in some embodiments, an antibody is administered once every two to four weeks for an extended period of time, such as for 1 , 2, 3, 4, 5, 6, 7, 8, 10, 15, 24 months, and so forth. By "twice-weekly" or "two times per week" is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By "thrice weekly" or "three times per week" is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as "intermittent" therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy for one or more weekly or monthly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below.

[0133] In certain embodiments, multiple therapeutically effective doses are administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By "intermittent" administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, once every two weeks, once every three weeks, once a month, and so forth. For example, in some embodiments, an antibody is administered once every two to four weeks for an extended period of time, such as for 1 , 2, 3, 4, 5, 6, 7, 8, 10, 15, 24 months, and so forth. By "twice-weekly" or "two times per week" is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By "thrice weekly" or "three times per week" is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as "intermittent" therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy for one or more weekly or monthly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below.

[0134] The therapeutic dose may be at least about 0.01 pig / kg body weight, at least about 0.05 pig / kg body weight; at least about 0.1 itg / kg body weight, at least about 0.5 pig / kg body weight, at least about 1 pig / kg body weight, at least about 2.5 ,ng / kg body weight, at least about 5 ug / kg body weight, and not more than about 100 .g / kg body weight. It will be understood by one of skillin the art that such guidelines will be adjusted for the molecular weight of the active agent. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, efc., or for systemic administration, e.g. i.m., i.p., i.v., and the like.Methods of Use

[0135] The trikines are useful for both prophylactic and therapeutic purposes. Thus, as used herein, the term “treating” is used to refer to both prevention of disease, and treatment of a preexisting condition. In certain instances, prevention indicates inhibiting or delaying the onset of a disease or condition, in a patient identified as being at risk of developing the disease or condition. The treatment of ongoing disease, to stabilize or improve the clinical symptoms of the patient, is a particularly important benefit provided by the present invention. Such treatment is desirably performed prior to loss of function in the affected tissues; consequently, the prophylactic therapeutic benefits provided by the invention are also important. Evidence of therapeutic effect may be any diminution in the severity of disease. The therapeutic effect can be measured in terms of clinical outcome or can be determined by immunological or biochemical tests. Patients for treatment may be mammals, e.g. primates, including humans, may be laboratory animals, e.g. rabbits, rats, mice, etc., particularly for evaluation of therapies, horses, dogs, cats, farm animals, etc.

[0136] The dosage of the therapeutic formulation, e.g., pharmaceutical composition, will vary widely, depending upon the nature of the condition, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. In particular embodiments, the initial dose can be larger, followed by smaller maintenance doses. In certain embodiments, the dose can be administered as infrequently as weekly or biweekly, or more often fractionated into smaller doses and administered daily, semi-weekly, or otherwise as needed to maintain an effective dosage level.

[0137] In some embodiments of the invention, administration of the composition or formulation comprising the trikine is performed by local administration. Local administration, as used herein, may refer to topical administration, but also refers to injection or other introduction into the body at a site of treatment. Examples of such administration include intramuscular injection, subcutaneous injection, intraperitoneal injection, and the like. In other embodiments, the composition or formulation comprising the trikine is administered systemically, e.g., orally or intravenously. In one embodiment, the composition of formulation comprising the trikine is administered by infusion, e.g., continuous infusion over a period of time, e.g., 10 min, 20 min, 3 min, one hour, two hours, three hours, four hours, or greater.

[0138] In some embodiments of the invention, the compositions or formulations are administered on a short term basis, for example a single administration, or a series of administrations performed over, e.g. 1 , 2, 3 or more days, up to 1 or 2 weeks, in order to obtain a rapid, significantincrease in activity. The size of the dose administered must be determined by a physician and will depend on a number of factors, such as the nature and gravity of the disease, the age and state of health of the patient and the patient's tolerance to the drug itself.

[0139] In certain methods of the present invention, an effective amount of a composition comprising a trikine is provided to cells, e.g. by contacting the cell with an effective amount of that composition to achieve a desired effect, e.g. to enhance signaling, proliferation, etc. In particular embodiments, the contacting occurs in vitro, ex vivo or in vivo. In particular embodiments, the cells are derived from or present within a subject in need or increased signaling.

[0140] In some methods of the invention, an effective amount of the subject composition is provided to enhance signaling in a cell. Biochemically speaking, an effective amount or effective dose of a trikine is an amount to alter STAT profiles in a cell by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or by 100% relative to the signaling by the native cytokine. The amount of modulation of a cell’s activity can be determined by a number of ways known to one of ordinary skill in the art of biology.

[0141] In a clinical sense, an effective dose of a trikine composition is the dose that, when administered to a subject for a suitable period of time, e.g., at least about one week, and maybe about two weeks, or more, up to a period of about 4 weeks, 8 weeks, or longer, will evidence an alteration in the symptoms associated with lack of signaling. In some embodiments, an effective dose may not only slow or halt the progression of the disease condition but may also induce the reversal of the condition. It will be understood by those of skill in the art that an initial dose may be administered for such periods of time, followed by maintenance doses, which, in some cases, will be at a reduced dosage.

[0142] The calculation of the effective amount or effective dose of trikine composition to be administered is within the skill of one of ordinary skill in the art, and will be routine to those persons skilled in the art. Needless to say, the final amount to be administered will be dependent upon the route of administration and upon the nature of the disorder or condition that is to be treated.

[0143] Cells suitable for use in the subject methods are cells that comprise one or more receptors. The cells to be contacted may be in vitro, that is, in culture, or they may be in vivo, that is, in a subject. Cells may be from / in any organism, but are preferably from a mammal, including humans, domestic and farm animals, and zoo, laboratory or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebrafish, fruit fly, worm, etc. Preferably, the mammal is human. Cells may be from any tissue. Cells may be frozen, or they may be fresh. They may be primary cells, or they may be cell lines. Often cells are primary cells used in vivo, or treated ex vivo prior to introduction into a recipient.

[0144] Cells in vitro may be contacted with a composition comprising a trikine by any of a number of well-known methods in the art. For example, the composition may be provided to the cells in the media in which the subject cells are being cultured. Nucleic acids encoding the trikine may be provided to the subject cells or to cells co-cultured with the subject cells on vectors under conditions that are well known in the art for promoting their uptake, for example electroporation, calcium chloride transfection, and lipofection. Alternatively, nucleic acids encoding the trikine may be provided to the subject cells or to cells cocultured with the subject cells via a virus, i.e. the cells are contacted with viral particles comprising nucleic acids encoding the peptide trikine polypeptide. Retroviruses, for example, lentiviruses, are particularly suitable to the method of the invention, as they can be used to transfect non-dividing cells (see, for example, Uchida et al. (1998) P.N.A.S. 95(20) :11939-44). Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line.

[0145] Likewise, cells in vivo may be contacted with the subject trikine compositions by any of a number of well-known methods in the art for the administration of peptides, small molecules, or nucleic acids to a subject. The trikine composition can be incorporated into a variety of formulations or pharmaceutical compositions, which in some embodiments will be formulated in the absence of detergents, liposomes, etc., as have been described for the formulation of full- length proteins.

[0146] In some embodiments trikines act on immune effector cells, and modulate immune responsiveness. For example, pathways involved in inflammatory disease may be targeted. Inflammation is a process whereby the immune system responds to infection or tissue damage. Inflammatory disease results from an activation of the immune system that causes illness, in the absence of infection or tissue damage, or at a response level that causes illness. Inflammatory disease includes autoimmune disease, which are any disease caused by immunity that becomes misdirected at healthy cells and / or tissues of the body. Autoimmune diseases are characterized by T and B lymphocytes that aberrantly target self-proteins, -polypeptides, -peptides, and / or other self-molecules causing injury and or malfunction of an organ, tissue, or cell-type within the body (for example, pancreas, brain, thyroid or gastrointestinal tract) to cause the clinical manifestations of the disease. Autoimmune diseases include diseases that affect specific tissues as well as diseases that can affect multiple tissues, which can depend, in part on whether the responses are directed to an antigen confined to a particular tissue or to an antigen that is widely distributed in the body.

[0147] The immune system employs a highly complex mechanism designed to generate responses to protect mammals against a variety of foreign pathogens while at the same time preventing responses against self-antigens. In addition to deciding whether to respond (antigenspecificity), the immune system must also choose appropriate effector functions to deal with each pathogen (effector specificity). Inflammatory diseases of interest include, without limitation Secondary Progressive Multiple Sclerosis (SPMS); Primary Progressive Multiple Sclerosis (PPMS); Neuromyelitis Optica (NMO); Psoriasis; Systemic Lupus Erythematosis (SLE); Ulcerative Colitis; Crohn's Disease; Ankylosing Spondylitis (see, for example, Mei et al. (201 1 ) Clin. Rheumatol. 30:269-273; type 1 (IDDM); Asthma; Chronic Obstructive Pulmonary Disorder (COPD); Chronic Hepatitis; Amyotrophic Lateral Sclerosis (ALS); Alzheimer's Disease (AD); Parkinson’s Disease; Frontotemporal Lobar Degeneration (FTLD), systemic lupus erythematosus, atherosclerosis / cardiovascular disease, and obesity / metabolic syndrome.

[0148] In other embodiments, a trikine activates an immune effector cell for the treatment of cancer, or activates a pathway for inducing death or reducing growth of cancer cells. The term “cancer”, as used herein, refers to a variety of conditions caused by the abnormal, uncontrolled growth of cells. Cells capable of causing cancer, referred to as "cancer cells", possess characteristic properties such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or certain typical morphological features. A cancer can be detected in any of a number of ways, including, but not limited to, detecting the presence of a tumor or tumors (e.g., by clinical or radiological means), examining cells within a tumor or from another biological sample (e.g., from a tissue biopsy), measuring blood markers indicative of cancer, and detecting a genotype indicative of a cancer. However, a negative result in one or more of the above detection methods does not necessarily indicate the absence of cancer, e.g., a patient who has exhibited a complete response to a cancer treatment may still have a cancer, as evidenced by a subsequent relapse.Therapeutic Methods

[0149] In some embodiments the subject compositions, methods and kits are used to enhance a T cell mediated immune response. In some embodiments the immune response is directed towards a condition where it is desirable to deplete or regulate target cells, e.g., cancer cells, infected cells, autoimmune cells, regulation of immune cells, including without limitation immune cells involved in autoimmune disease, immune cells involved in transplantation, undesirable inflammatory responses, enhancing erythropoiesis, enhancing thrombopoiesis, etc. Immune conditions may include, without limitation, autoimmune diseases, graft v host disease, hematopoietic bone marrow transplantation, adoptive cell therapy, tumor infiltrating cell (TIL) therapy, inflammation, graft rejection, and the like.

[0150] Any cell can be used for this purpose. In some embodiments the cell is a T cell, including without limitation naive CD8+T cells, cytotoxic CD8+T cells, naive CD4+T cells, helper T cells, e.g. TH1 , TH2, TH9, TH11 , TH22, TFH; regulatory T cells, e.g. TR1 , natural TReg, inducible TReg; memory T cells, e.g. central memory T cells, effector memory T cells, NKT cells, yS T cells andengineered variants of such T-cells including CAR-T cells; etc. In other embodiments the cell is a stem cell, e.g. a hematopoietic stem cell, an NK cell, a macrophage, or a dendritic cell. In some embodiments the cell is genetically modified in an ex vivo procedure, prior to transfer into a subject. The cell can be provided in a unit dose for therapy, and can be allogeneic, autologous, etc. with respect to an intended recipient.

[0151] In some embodiments the condition is cancer. As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative," and "neoplastic" to refer to cells having the capacity for autonomous or unregulated growth (e.g., an abnormal state or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic disease states may be categorized as pathologic (e.g., characterizing or constituting a disease state), or they may be categorized as non- pathologic (e.g., as a deviation from normal but not associated with a disease state). The terms are meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. The terms "cancer" or "neoplasm" are used to refer to malignancies of the various organ systems, including those affecting the lung, breast, thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, and the genitourinary tract, as well as to adenocarcinomas which are generally considered to include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, nonsmall cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0152] The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0153] Examples of tumor cells include but are not limited to AML, ALL, CML, adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g. Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasalcancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma skin cancer, non-melanoma skin cancers, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer (e.g. uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarinoma, head and neck cancers, teratocarcinoma, or Waldenstrom's macroglobulinemia. Any cancer is a suitable cancer to be treated by the subject methods and compositions.

[0154] The compositions and methods of the present disclosure may be combined with additional therapeutic agents. For example, when the disease, disorder or condition to be treated is a neoplastic disease (e.g. cancer) the methods 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®).

[0155] Examples of chemical agents identified in the art as useful in the treatment of neoplastic disease, include without limitation, abitrexate, adriamycin, adrucil, amsacrine, asparaginase, anthracyclines, azacitidine, azathioprine, bicnu, blenoxane, busulfan, bleomycin, camptosar, camptothecins, carboplatin, carmustine, cerubidine, chlorambucil, cisplatin, cladribine, cosmegen, cytarabine, cytosar, cyclophosphamide, cytoxan, dactinomycin, docetaxel, doxorubicin, daunorubicin, ellence, elspar, epirubicin, etoposide, fludarabine, fluorouracil, fludara, gemcitabine, gemzar, hycamtin, hydroxyurea, hydrea, idamycin, idarubicin, ifosfamide, ifex, irinotecan, lanvis, leukeran, leustatin, matulane, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, mithramycin, 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 vumon.

[0156] Targeted therapeutics that can be administered in combination may include, without limitation, tyrosine-kinase inhibitors, such as Imatinib mesylate (Gleevec, also known as STI- 571 ), Gefitinib (Iressa, also known as ZD1839), Erlotinib (marketed as 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, venclexta, 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 linked to [D-Lys(6)]-LHRH; Braf inhibitors, such as vemurafenib, dabrafenib, and LGX818; MEK inhibitors, such as trametinib; CDK inhibitors, such as PD-0332991 and LEE01 1 ; 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).

[0157] Examples of biological agents identified in the art as useful in the treatment of neoplastic disease, include without limitation, cytokines or cytokine antagonists such as IL-12, INFa, or anti- epidermal growth factor receptor, radiotherapy, irinotecan; tetrahydrofolate antimetabolites such as pemetrexed; antibodies against tumor antigens, a complex of a monoclonal antibody and toxin, a T cell adjuvant, bone marrow transplant, or antigen presenting cells (e.g., dendritic cell therapy), anti-tumor vaccines, replication competent viruses, signal transduction inhibitors (e.g., Gleevec® or Herceptin®) or an immunomodulator to achieve additive or synergistic suppression of tumor growth, cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade® and Enbrel®), interferon-|31 a (Avonex®), and interferon-(31 b (Betaseron®) as well as combinations of one or more of the foregoing as practiced in known chemotherapeutic treatment regimens readily appreciated by the skilled clinician in the art.

[0158] Tumor specific monoclonal antibodies that can be administered in combination with an engineered cell may include, without limitation, Rituximab (marketed as MabThera® or Rituxan®), Alemtuzumab, Panitumumab, Ipilimumab (Yervoy®), etc.

[0159] In some embodiments the compositions and methods of the present invention may be combined with immune checkpoint therapy. Examples of immune checkpoint therapies include inhibitors of the binding of PD1 to PDL1 and / or PDL2. PD1 to PDL1 and / or PDL2 inhibitors 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, MDX1 106, commercially available from BristolMyers 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 but are not limited to 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). Additional antibody PD1 pathway inhibitors are described in United States Patent No. 8,217,149 (Genentech, Inc) issued July 10, 2012; United States Patent No. 8,168,757 (Merck Sharp and Dohme Corp.) issued May 1 , 2012, United States Patent No. 8,008,449 (Medarex) issued August 30, 201 1 , United States Patent No. 7,943,743 (Medarex, Inc) issued May 17, 2011 . Additionally, small molecule PD1 to PDL1 and / or PDL2 inhibitors are known in the art. See, e.g. Sasikumar, et al as WO2016142833A1 and Sasikumar, et al. WO2016142886A2, BMS-1 166 and BMS-1001 (Skalniak, et al (2017) Oncotarget 8(42): 72167- 72181 ).

[0160] In other embodiments the methods of the invention are used in the treatment of infection. As used herein, the term “infection” refers to any state in at least one cell of an organism (i.e. , asubject) is infected by an infectious agent (e.g., a subject has an intracellular pathogen infection, e.g., a chronic intracellular pathogen infection). For example, infectious agents include, but are not limited to bacteria, viruses, protozoans, and fungi. Intracellular pathogens are of particular interest. Infectious diseases are disorders caused by infectious agents. Some infectious agents cause no recognizable symptoms or disease under certain conditions, but have the potential to cause symptoms or disease under changed conditions. The subject methods can be used in the treatment of chronic pathogen infections, for example including but not limited to viral infections, e.g. retrovirus, lentivirus, hepadna virus, herpes viruses, pox viruses, human papilloma viruses, etc.; intracellular bacterial infections, e.g. Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp, Helicobacter pylori etc.; and intracellular protozoan pathogens, e.g. Plasmodium sp, Trypanosoma sp., Giardia sp., Toxoplasma sp., Leishmania sp., etc.

[0161] Treatment may be combined with other active agents. Classes of antibiotics include penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafci Ilin, ampicillin, etc.; penicillins in combination with p-lactamase inhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc. Cytokines may also be included, e.g. interferon y, tumor necrosis factor a, interleukin 12, etc. Antiviral agents, e.g. acyclovir, gancyclovir, etc., may also be used in treatment.

[0162] In yet other embodiments, T cells are treated for the treatment of autoimmune disease. The spectrum of inflammatory diseases and diseases associated with inflammation is broad and includes autoimmune diseases such rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and autoimmune hepatitis; insulin dependent diabetes mellitus, degenerative diseases such as osteoarthritis (OA), Alzheimer’s disease (AD), and macular degeneration.

[0163] Many, if not most, autoimmune and inflammatory diseases involve multiple types of T cells, e.g. TH1 , TH2, TH17, and the like. Autoimmune diseases are characterized by T and B lymphocytes that aberrantly target self-proteins, -polypeptides, -peptides, and / or other selfmolecules causing injury and or malfunction of an organ, tissue, or cell-type within the body (for example, pancreas, brain, thyroid or gastrointestinal tract) to cause the clinical manifestations of the disease. Autoimmune diseases include diseases that affect specific tissues as well as diseases that can affect multiple tissues, which can depend, in part on whether the responses are directed to an antigen confined to a particular tissue or to an antigen that is widely distributed in the body.

[0164] In another embodiment of the invention a T-cell that has been modified to surface express a chimeric antigen receptor (a ‘CAR-T’ cell) is stimulated with a trikine, e.g. to enhance sternness and reduce exhaustion by IL-2 stimulation. As used herein, the terms “chimeric antigen receptor T-cell” and “CAR-T cell” are used interchangeably to refer to a T-cell that has been recombinantly modified to express a chimeric antigen receptor. As used herein, the terms “chimeric antigen receptor” and “CAR” are used interchangeably to refer to a polypeptide comprising multiple functional domains arranged from amino to carboxy terminus in the sequence: (a) an antigen binding domain (ABD), (b) a transmembrane domain (TD); and (c) one or more cytoplasmic signaling domains (CSDs) wherein the foregoing domains may optionally be linked by one or more spacer domains. The CAR may also further comprise a signal peptide sequence which is conventionally removed during post-translational processing and presentation of the CAR on the cell surface. CARs useful in the practice of the present invention are prepared in accordance with principles well known in the art. See e.g., Eshhaar etal. United States Patent No. 7,741 ,465 B1 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 a receptor include axicabtagene ciloleucel (marketed as Yescarta® commercially available from Gilead Pharmaceuticals) and tisagenlecleucel (marketed as Kymriah® commercially available from Novartis).

[0165] 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 may be any polypeptide that specifically binds to one or more antigens expressed on the surface of a target cell. In certain embodiments, the target cell antigen is a tumor antigen. Examples of tumor antigens that may be targeted by the ABD of the CAR include one or more antigens selected from the group including, but not limited to, the CD19, CD20, HER2, NY-ESO-1 , MUC1 , CD123, FLT3, B7-H3, CD33, IL1 RAP, CLL1 (CLEC12A)PSA, CEA, VEGF, VEGF-R2, CD22, ROR1 , mesothelin, c-Met, Glycolipid F77, FAP, EGFRvlll, MAGE A3, 5T4, WT1 , KG2D ligand, a folate receptor (FRa), and Wnt1 antigens.

[0166] In one embodiment, the ABD is a single chain Fv (ScFv). An ScFv is a polypeptide comprised of the variable regions of the immunoglobulin heavy and light chain of an antibody covalently connected by a peptide linker (Bird, et al. (1988) Science 242:423-426; Huston, et al. (1988) PNAS(USA) 85:5879-5883; S-z Hu, et al. (1996) Cancer Research, 56, 3055-3061 . The generation of ScFvs based on monoclonal antibody sequences is well known in the art. See, e.g. The Protein Protocols Handbook, John M. Walker, Ed. (2002) Humana Press Section 150 “Bacterial Expression, Purification and Characterization of Single-Chain Antibodies” Kipriyanov, S. Antibodies used in the preparation of scFvs may be optimized to select for those moleculeswhich possess particular desirable characteristics (e.g. enhanced affinity) through techniques well known in the art such as phage display and directed evolution. In some embodiments, the ABD comprises an anti-CD19 scFv, an anti-PSA scFv, an anti-HER2 scFv, an anti-CEA scFv, an anti-EGFR scFv, an anti-EGFRvlll scFv, an anti-NY-ESO-1 scFv, an anti-MAGE scFv, an anti- 574 scFv, or an anti-Wnt1 scFv. In another embodiment, the ABD is a single domain antibody obtained through immunization of a camel or llama with a target cell derived antigen, in particular a tumor antigen. See, e.g. Muyldermans, S. (2001 ) Reviews in Molecular Biotechnology 74: ~l- 302. Alternatively, the ABD may be generated wholly synthetically through the generation of peptide libraries and isolating compounds having the desired target cell antigen binding properties in substantial accordance with the teachings or Wigler, et al. United States Patent No. 6303313 B1 issued November 12, 1999; Knappik, et al., United States Patent No 6,696,248 B1 issued February 24, 2004, Binz, etal. (2005) Nature Biotechnology 23:1257-1268, and Bradbury, et al. (2011) Nature Biotechnology 29:245-254.

[0167] The ABD may have affinity for more than one target antigen. For example, an ABD of the present invention may comprise chimeric bispecific binding members, i.e. have capable of providing for specific binding to a first target cell expressed antigen and a second target cell expressed antigen. Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann (2012) MAbs. 4(2): 182-197; Stamova et al. (2012) Antibodies, 1 (2), 172-198; Farhadfar et al. (2016) Leuk Res. 49:13-21 ; Benjamin et al. TherAdv Hematol. (2016) 7(3) :142-56; Kiefer et al. Immunol Rev. (2016) 270(1 ):178-92; Fan et al. (2015) J Hematol Oncol. 8:130; May et al. (2016) Am J Health Syst Pharm. 73(1):e6-e13. In some embodiments, the chimeric bispecific binding member is a bivalent single chain polypeptides. See, e.g. Thirion, et al. (1996) European J. of Cancer Prevention 5(6):507-511 ; DeKruif and Logenberg (1996) J. Biol. Chem 271 (13)7630-7634; and Kay, et al. United States Patent Application Publication Number 2015 / 0315566 published November 5, 2015. In some instances, a chimeric bispecific binding member may be a bispecific T cell engager (BiTE). A BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3. In some instances, a chimeric bispecific binding member may be a CAR T cell adapter. As used herein, by “CAR T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR T cell adapter will have to binding regions, one specific for an epitope on the CAR to which it is directed and a second epitopedirected to a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR T cell adapters include but are not limited to e.g., those described in Kim et al. (2015) J Am Chem Soc. 137(8):2832-5; Ma et al. (2016) Proc Natl Acad Sci U S A. 1 13(4):E450- 8 and Cao et al. (2016) Angew Chem Int Ed Engl. 55(26):7520-4.

[0168] In some embodiments, a linker polypeptide molecule is optionally incorporated into the CAR between the antigen binding domain and the transmembrane domain to facilitate antigen binding. Moritz and Groner (1995) Gene Therapy 2(8) 539-546. In one embodiment, the linker is the hinge region from an immunoglobulin, e.g. the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. In those instances where the ABD is an scFv, an IgG hinge may be employed. In some embodiments the linker comprises the amino acid sequence (G4S)nwhere n is 1 , 2, 3, 4, 5, etc., and in some embodiments n is 3.

[0169] CARs useful in the practice of the present invention further comprise a transmembrane domain joining the ABD (or linker, if employed) to the intracellular cytoplasmic domain of the CAR. The transmembrane domain is comprised of any polypeptide sequence which is thermodynamically stable in a eukaryotic cell membrane. The transmembrane spanning domain may be derived from the transmembrane domain of a naturally occurring membrane spanning protein or may be synthetic. In designing synthetic transmembrane domains, amino acids favoring alpha-helical structures are preferred. Transmembrane domains useful in construction of CARs are comprised of approximately 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 22, 23, or 24 amino acids favoring the formation having an alpha-helical secondary structure. Amino acids having a to favor alpha-helical conformations are well known in the art. See, e.g Pace, et al. (1998) Biophysical Journal 75: 422-427. Amino acids that are particularly favored in alpha helical conformations include methionine, alanine, leucine, glutamate, and lysine. In some embodiments, the CAR transmembrane domain may be derived from the transmembrane domain from type I membrane spanning proteins, such as CD3^, CD4, CD8, CD28, etc.

[0170] The cytoplasmic domain of the CAR polypeptide comprises one or more intracellular signal domains. In one embodiment, the intracellular signal domains comprise the cytoplasmic sequences of the T-cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement and functional derivatives and sub-fragments thereof. A cytoplasmic signaling domain, such as those derived from the T cell receptor -chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Examples of cytoplasmic signaling domains include but are not limited to the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also referred to as 4-1 BB and TNFRSF9), the cytoplasmic domain of CD278 (also referred to as ICOS), p110a, p, or 6 catalytic subunit of PI3 kinase, the human CD3 chain, cytoplasmic domain of CD134 (alsoreferred to as 0X40 and TNFRSF4), FceRl y and p chains, MB1 (Iga) chain, B29 (lg|3) chain, etc.), CD3 polypeptides (0, A and E), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28.

[0171] In some embodiments, the CAR may also provide a co-stimulatory domain. The term “costimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. The co-stimulatory domain refers to the portion of the CAR which enhances the proliferation, survival or development of memory cells. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. (2013) Nat Rev Immunol 13(4):227-42. In some embodiments of the present disclosure, the CSD comprises one or more of members of the TNFR superfamily, CD28, CD137 (4-1 BB), CD134 (0X40), Dap10, CD27, CD2, CD5, ICAM-1 , LFA-1 (CD1 1 a / CD18), Lek, TNFR- I, TNFR-II, Fas, CD30, CD40 or combinations thereof.

[0172] Typically, the chimeric antigen receptor T-cells (CAR-T cells) are T-cells which have been recombinantly modified by transduction with an expression vector encoding a CAR in substantial accordance with the teaching above.

[0173] Cells may be prepared using the patient’s own T-cells for engineering. Consequently, the population of the cells to be administered is to the subject is necessarily variable. Additionally, since the CAR-T cell agent is variable, the response to such agents can vary and thus involves the ongoing monitoring and management of therapy related toxicities which are managed with a course of pharmacologic immunosuppression or B cell depletion prior to the administration of the CAR-T cell treatment. Examples of such immunosuppressive regimens including systemic corticosteroids (e.g., methylprednisolone). Therapies for B cell depletion include intravenous immunoglobulin (IVIG) by established clinical dosing guidelines to restore normal levels of serum immunoglobulin 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 lymphodepleting regimen. One example of a such lymphodepleting regimen consists of the administration to the subject of fludarabine (30 mg / m2intravenous [IV] daily for 4 days) and cyclophosphamide (500 mg / m2IV daily for 2 days starting with the first dose of fludarabine).

[0174] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.EXAMPLE 1Methods

[0175] Cloning and protein expression. Cytokines and trikines were cloned into the expression vector pD649 with a C-terminal Avitag and 6xHis tag. Proteins were expressed in Expi293 cells maintained in Expi293 Media at 37°C in a humidified atmosphere with 5% CO2 with gentle agitation.

[0176] Protein purification. IL-2, IL-21 , and trikines were purified by Ni-NTA chromatography followed by size exclusion chromatography. Fractions containing the desired molecule were pooled and concentrated using Amicon Ultra Centrifugal Filters. The concentrated proteins were each aliquoted, flash frozen in liquid nitrogen, and then stored at -80°C until use.

[0177] Tissue culture. Peripheral blood mononuclear cells (PBMCs) isolated from human blood were cultured in complete RPMI medium (RPMI medium supplemented with 10% FBS, penicillinstreptomycin, Glutamax, sodium pyruvate, Non-Essential Amino Acids, and HEPES) and maintained at 37°C in a humidified atmosphere with 5% CO2. PBMCs were thawed and activated with anti-CD3 and anti-CD28 for three days to prepare activated T cells. The T cells were then washed with PBS and complete medium and rested overnight before use in assays.

[0178] Phospho-STAT signaling assays. Rested human T cells were counted and washed with FACS buffer (PBS, 2% FBS) and incubated with Human TruStain FcX prior to staining with fluorophore-conjugated anti-CD4 and anti-CD8 antibodies for 15 min, shaking at room temperature. The cells were then washed twice with FACS buffer and plated in a 96 well plate, approximately 105cells per well. The cells were then stimulated with up to 10 nM cytokine or trikine for 20 min at 37°C. The cells were immediately fixed with 16% paraformaldehyde followed by incubation, shaking for 10 min at room temperature. The cells were washed twice with FACS buffer and then permeabilized with 100% ice-cold methanol overnight at -80°C. The fixed and permeabilized cells were washed twice with FACS buffer and incubated with fluorescently- labeled pSTAT5, pSTAT3, and / or pSTATI detection antibodies diluted in FACS buffer for 1 hr. Cells were then washed twice and mean fluorescence intensity (MFI) was quantified on a CytoFLEX S Flow Cytometer (Beckman Coulter), and single-dose bar graphs or dose-response curves were plotted and fit in Prism 9.5.1 (GraphPad).

[0179] Proliferation assays. Approximately 104rested T cells were plated per well in a 96 well plate and stimulated with 10 nM ligand. After incubation for three days, the cells were washed with FACS buffer twice and incubated with Human TruStain FcX for 5 min prior to staining with fluorophore-conjugated anti-CD4 and anti-CD8 antibodies for 15-20 min, shaking at room temperature. Cells were then washed twice and cell counts of CD4+ and CD8+ cells were quantified on a CytoFLEX S Flow Cytometer (Beckman Coulter). Single-dose bar graphs were plotted in Prism 9.5.1 (GraphPad).

[0180] Cytokine production assays. Approximately 104rested T cells were plated per well in a 96 well plate and stimulated with 10 nM ligand every 48 hours. Cells were split 1 :3 as necessary to prevent overgrowth. On day 8, cells were washed with FACS buffer (PBS, 2% FBS) twice and incubated with Human T ruStain FcX for 5 min prior to staining with a f luorophore-conjugated anti- CD8 antibody for 15-20 min. The cells were then incubated with either eBioscience Protein Transport Inhibitor Cocktail or eBioscience Cell Stimulation Cocktail (plus protein transport inhibitors) for 5 hours, followed by fixation and permeabilization via BD Cytofix / Cytoperm Fixation / Permeabilization Kit. Cells were then intracellularly stained for IFNy, IL-2, and TNF. Cells were then washed twice prior to quantification of intracellular cytokine staining of CD8+ cells on a CytoFLEX S Flow Cytometer (Beckman Coulter). Single-dose bar graphs were plotted in Prism 9.5.1 (GraphPad).AntibodiesAlexa Fluor® 488 Anti-Stat5 (pY694)Alexa Fluor® 647 Mouse Anti-Human IFN-yAlexa Fluor® 647 Mouse Anti-Stat3 (pY705) Alexa Fluor® 647 Mouse Anti-Stat5 (pY694) Brilliant Violet 785™ anti-human CD8 Antibody FITC anti-human CD4 AntibodyHuman TruStain FcX™ (Fc Receptor Blocking Solution)Pacific Blue™ anti-human CD8a AntibodyPE anti-human IL-2 AntibodyPE-Cy7 Mouse Anti-Human TNF Clone MAb11 Phospho-Statl (Tyr701 ) (58D6) Rabbit mAb (Alexa Fluor® 647 Conjugate) #8009 Ultra-LEAF™ Purified anti-human CD28 Antibody (1 mg) Ultra-LEAF™ Purified anti-human CD3 AntibodyResults

[0181] Trikines are comprised of linked binding domains against multiple extracellular domains (ECDs) of cytokine receptors (wild type, engineered, or dominant-negative cytokine; scFv; or VHH). Trikines induce non-natural signaling via proximity of three cytokine receptors by binding to their ECDs simultaneously. Shown in FIG. 1 is a schematic of an IL-2 based, linking IL-21 Ra binding domain to IL-2. IL-2 signals mainly through pSTAT5, IL-21 R signals through pSTAT3 and pSTATI . The design goal of this trikine is to enhance the sternness properties of IL-2 on T cells by bringing in pSTAT3. An alternative configuration is shown in FIG. 2B, which is IL-21 based, linking IL-2Rb binding domain to IL-21. IL-21 signals mainly through pSTAT5, IL-21 R signals through pSTAT3 and pSTATI . The design goal of this trikine is to enhance the proliferative properties of IL-21 on T cells by bringing in pSTAT5.

[0182] As shown in FIG. 2, IL-2 - alL21 R trikines rebalance the pSTAT signaling profile and phenotypic effects of IL-2. FIG. 2B,C provide a list of of binding domains comprising (B) human- and (C) mouse-reactive IL-2 - alL21 R trikines. A dose response curve for phospho-STATs 1 , 3 and 5 in pre-activated and rested human CD8+ T cells stimulated for 20 minutes with controls and selected IL-2 - alL21 R trikines, GER-023 and GER-046 Is shown in FIG. 2D. pSTAT signaling profiles of IL-2 - oclL21 R trikines with identical binding modules but varied number of residues of flexibility between binding modules is shown in (E). (F) Cell counts of CD4+ and CD8+ cells after preactivated and rested human T cells were incubated with 10 nM ligand for 3 days. (G) Cytokine production of human CD8+ T cells treated with 10nM ligand every 48h for 8 days, followed by 5h treatment with PMA, ionomycin, brefeldin A, and monensin.

[0183] FIGS. 3A-3F. IL-21 - cxlL2Rb trikines rebalance the pSTAT signaling profile and phenotypic effects of IL-21 . (A) Schematic of IL-21 - odL2Rb trikine. (B & C) List of binding domains comprising (B) human- and (C) mouse-reactive IL-21 - alL2Rb trikines. (D) Dose response curves for phospho-STATs 1 , 3 and 5 in pre-activated and rested human CD8+ T cells stimulated for 20 minutes with controls and selected IL-21 - oclL2Rb trikines, GER-060, GER- 061 , and GER-064. (E) Cell counts of CD4+ and CD8+ cells after preactivated and rested human T cells were incubated with 10 nM ligand for 3 days. (F) Cytokine production of human CD8+ T cells treated with 10nM ligand every 48h for 8 days, followed by 5h treatment with PMA, ionomycin, brefeldin A, and monensin.

[0184] FIGS. 4A-4C. Mono-super-IL-10 - oclL2Rb trikines rebalance the pSTAT signaling profile of IL-10. (A) Schematic of mono-super-IL-10 - alL2Rb trikine. (B) List of binding domains comprising human-reactive mono-super-IL-10 - cdL2Rb trikines. (C) Dose response curves for phospho-STATs 3 and 5 in pre-activated and rested human T cells stimulated for 20 minutes controls and selected IL-21 - alL2Rb trikines at 100nM concentration.SequencesTable 2Sequence identifier Amino acid sequenceTable 3 Binding DomainsTable 4 Components of GER constructsco-expressed with IL-12 p35, SEQ ID NO:142

Claims

WHAT IS CLAIMED IS:1 . A trikine polypeptide, comprising: a first binding domain that binds to a receptor subunit (i) and receptor (ii), wherein (i) and (ii) multimerize in response association with the binding domain and activate signaling as a result; and a second binding domain that binds to a receptor subunit and does not activate signaling; wherein upon binding a trikine to receptor subunits (i), (ii), and (iii) on a cell, signaling from (i) and (ii) produces an altered STAT profile.

2. The trikine of claim 1 , wherein the first binding domain is a cytokine or biologically active variant thereof.

3. The trikine of claim 1 or claim 2, wherein the first binding domain binds to a cytokine set forth in Table 1 .

4. The trikine of any of the previous claims, wherein binding domain 1 is selected from IL- 2; IL-4; IL-7; IL-9; IL-15; IL-21 ; IL-3; IL-5; GM-CSF; IL-6; LIF; CNTF; CT1 ; CLC; OSM; IL-31 ; NP; IL-12; IL-13; IL-23; IL-10; IL-19; IL-20; IL-22; IL-24; IL-26; or an analog thereof.

5. The trikine of any of the previous claims, wherein binding domain 1 binds to receptor subunit (i) IL-2R , IL-4Ra, IL-7Rcc, IL-9Roc, IL-15Ra, or IL-21 Ret and receptor subunit 2 (ii) 7c (CD132).

6. The trikine of any of the previous claims, wherein binding domain 1 binds to receptor subunit (i) IL-2R , or IL-21 Ra and receptor subunit (ii) yc (CD132).

7. The trikine of any of the previous claims, wherein binding domain 1 binds to receptor subunit (i) ILI ORa, IL-1 Oct and IL-1 Op, IL28R, IL20Ra, or IL22R; and receptor subunit (ii) IL1 OR , IL2OR , or IL-21 R.

8. The trikine of any of the previous claims, wherein binding domain 1 binds to receptor subunit (i) IL12Rpi , or IL23R and receptor subunit (ii) IL12Rp2.

9. The trikine of any of the previous claims, wherein binding domain 2 is a monovalent antibody or a dominant negative cytokine.

10. The trikine of any of the previous claims, wherein binding domain 2 is a VHH antibody.

11. The trikine of any of the previous claims, wherein binding domain binds to receptor subunit (iii) that will, in forced proximity, interact with subunits (i) and (ii).

12. The trikine of any of the previous claims, wherein binding domain 2 binds to receptor subunit (iii) IL-2RP, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, IL-21 Ra, ILIORa, IL28R, IL20Ra, IL22R, IL10RP, IL20RP, IL12Rp1 , IL23R or IL12RP2, with the proviso that subunits (i) and (iii) are different.

13. The trikine of any of the previous claims, which binds to: a. (i) IL-2Rp, (ii) CD132, (iii) IL-21 Ra; b. (i) IL-21 Ra, (ii) CD132, (iii) IL-2Rp; c. (i) IL-10Ra, (ii) IL-1 ORp. (iii) IL-2Rp; d. (i) IL-10Ra, (ii) IL-1 ORp. (iii) IL-21 Ra; e. (i) IL-12Rb1 , (ii) IL-12Rb2, (iii) IL-2Ra; or f. (i) IL-12Rb1 , (ii) IL-12Rb2, (iii) IL-21 Ra.

14. A trikine comprising a mature protein sequence of any of SEQ ID NO:1-91 , or SEQ ID NO:146-156.

15. A trikine comprising a binding domain 1 selected from SEQ ID NO:128-141 and 157- 160 and a binding domain 2 selected from SEQ ID NO:92-127, optionally joined by a polypeptide linker.

16. A trikine of any of the previous claims, conjugated to an Fc region sequence.

17. A trikine of any of the previous claims, conjugated to a PEG moiety.

18. A trikine of any of the previous claims, wherein STAT profile for one, two or all of STAT5, STAT3 and STAT1 is altered relative to the STAT profile from wild-type cytokine activation of receptor subunits (i) and (ii).

19. A trikine of any of claims 1 -8, wherein binding domain 1 is complexed with an IL12 subunit to form a heterodimer, and binds to receptor subunits IL12Rp and IL-12Ra.

20. A pharmaceutical formulation comprising a trikine according to any of the previous claims, and a pharmaceutically acceptable excipient.21 . A nucleic acid encoding comprising a trikine according to any of the previous claims.

22. A vector comprising a nucleic acid sequence of claim 21 .

23. A cell comprising a nucleic acid of claim 21 or a vector of claim 22.

24. A method for modulating STAT signaling from receptor subunits (i) and (ii), the method comprising contacting a cell expressing receptor subunits (i), (ii) and (iii) with an effective dose of a trikine according to any of the previous claims.

25. The method of 24, wherein the cell is an immune cell or a stem cell.

26. The method of claim 25, wherein the immune cell is a T cell.

27. The method of claim 25, wherein the cell is a naive CD8+T cells, cytotoxic CD8+T cells, naive CD4+T cells, helper T cells, e.g. TH1 , TH2, TH9, TH1 1 , TH22, TFH; regulatory T cells, e.g. TR1 , natural TReg, inducible TReg; memory T cells, e.g. central memory T cells, effector memory T cells, NKT cells, y§ T cells or engineered variants of such T-cells.

28. The method of claim 26 or 27, wherein the T cell is present in a tumor infiltrating lymphocyte population.

29. The method of any of claims 26-28, wherein the T cell is a CAR T cell.

30. A method of treating an individual, the method comprising administering an effective dose of a trikine or pharmaceutical formulation according to any of claims 1 -18.31 . The method of claim 30, wherein the individual is treated for cancer.

32. The method of claim 30, wherein the individual is treated for autoimmune disease.

33. The method of claim 30, wherein the individual is treated for infection.

34. A trikine, comprising a cytokine linked to a second binding domain that binds to a receptor subunit expressed on an immune cell, that does not activate signaling.

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