COMPOSITIONS AND METHODS FOR REGULATING IMMUNE SYSTEM ACTIVITY
20230159619 · 2023-05-25
Inventors
- Peter J. Kushner (San Francisco, CA)
- Leslie Hodges Gallagher (Alameda, CA, US)
- Cyrus L. Harmon (Bolinas, CA)
- David C. Myles (Berkeley, CA)
- Richard Sun (San Francisco, CA, US)
Cpc classification
C07K2319/90
CHEMISTRY; METALLURGY
C07K14/70567
CHEMISTRY; METALLURGY
C12Y207/10002
CHEMISTRY; METALLURGY
C07K14/721
CHEMISTRY; METALLURGY
C12N9/12
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
A trigger-responsive immune-inactivating signaling polypeptide disclosed herein can include a modulating domain and an immune-inactivating moiety, such as a dominant negative signaling moiety or constitutively active signaling moiety. A modulating domain can be characterized by an ability to adopt a first state and a second state, and to transition between the first state and the second state when exposed to a trigger. When the modulating domain is in its first state, the immune-inactivating signaling moiety can be inhibited, and when the modulating domain is in its second state, the inhibition can be relieved. Further disclosed herein are compositions for the delivery of a trigger-responsive immune-inactivating signaling polypeptide. Also, methods for using a trigger-responsive immune-inactivating signaling polypeptide, including to regulate an activity of immune system cells, are disclosed.
Claims
1. A trigger-responsive immune-inactivating signaling polypeptide comprising: a modulating domain characterized by an ability to adopt a first state and a second state, and to transition between the first state and the second state when exposed to a trigger; and an immune-inactivating moiety; wherein, when the modulating domain is in its first state, the immune-inactivating moiety is inhibited, and when the modulating domain is in its second state, the inhibition is relieved.
2. The trigger-responsive immune-inactivating signaling polypeptide of claim 1, wherein the modulating domain comprises a nuclear receptor or a fragment thereof.
3. (canceled)
4. The trigger-responsive immune-inactivating signaling polypeptide of claim 1, wherein the modulating domain comprises a hormone receptor, retinoic acid receptor, vitamin D receptor, peroxisome proliferator-activated receptor, farnesoid X receptor, or liver X receptor.
5.-7. (canceled)
8. The trigger-responsive immune-inactivating signaling polypeptide of claim 4, wherein the hormone receptor is an estrogen receptor.
9. The trigger-responsive immune-inactivating signaling polypeptide of claim 8, wherein the estrogen receptor is estrogen receptor-α.
10. The trigger-responsive immune-inactivating signaling polypeptide of claim 8, wherein the modulating domain includes an amino acid sequence that has at least 90% sequence identity with an amino acid sequence that starts at residue 251, 282, or 305 of SEQ ID NO: 12 and ends at residue 545 or 595 of SEQ ID NO: 12.
11. The trigger-responsive immune-inactivating signaling polypeptide of claim 8, wherein the modulating domain includes an amino acid sequence that has 90% sequence identity with SEQ ID NO: 4 or wherein the modulating domain includes an amino acid sequence that has 90% sequence identity with SEQ ID NO: 13.
12.-13. (canceled)
14. The trigger-responsive immune-inactivating signaling polypeptide of claim 8, wherein the modulating domain includes one or more mutations that (i) confer on the modulating domain a reduced affinity to at least one naturally occurring estrogen; (ii) confer on the modulating domain a preferential binding to at least one synthetic estrogen receptor ligand; or (iii) confer increased affinity for at least one chaperone protein.
15. The trigger-responsive immune-inactivating signaling polypeptide of claim 14, wherein the at least one naturally occurring estrogen includes an estradiol.
16.-17. (canceled)
18. The trigger-responsive immune-inactivating signaling polypeptide of claim 14, wherein the at least one synthetic estrogen receptor ligand includes tamoxifen, endoxifen, 4-hydroxytamoxifen, fulvestrant, OP-1250, OP-1074, or OP-1124.
19. (canceled)
20. The trigger-responsive immune-inactivating signaling polypeptide of claim 14, wherein the at least one chaperone protein includes HSP90.
21. The trigger-responsive immune-inactivating signaling polypeptide of claim 14, wherein the modulating domain includes at least one mutation selected from the group consisting of G400V, G400M, G400A, G400L, G521R, G521T, L539A, L540A, M543A and L544A, wherein the residue numbering is based on SEQ ID NO: 12.
22. The trigger-responsive immune-inactivating signaling polypeptide of claim 14, wherein the modulating domain includes at least one mutation selected from the group consisting of G400V, G400M, G400A, G400L, G521R, and G521T, and at least one mutation selected from the group consisting of L539A, L540A, M543A, and L544A, wherein the residue numbering is based on SEQ ID NO: 12.
23. The trigger-responsive immune-inactivating signaling polypeptide of claim 14, wherein the modulating domain includes at least one mutation selected from the group consisting of G400V, G400M, G400A, G400L, G521R, and G521T, and at least one mutation selected from (i) the group consisting of L539A and L540A, or (ii) the group consisting of M543A and L544A, wherein the residue numbering is based on SEQ ID NO: 12.
24. The trigger-responsive immune-inactivating signaling polypeptide of claim 1, wherein the trigger-responsive immune-inactivating signaling polypeptide is a trigger-responsive dominant negative signaling polypeptide and the immune-inactivating moiety is a dominant negative signaling moiety.
25.-33. (canceled)
34. The trigger-responsive immune-inactivating signaling polypeptide of claim 24, wherein the dominant negative signaling moiety includes a dominant negative kinase moiety that is a dominant negative variant of a Zap70 kinase.
35. The trigger-responsive immune-inactivating signaling polypeptide of claim 34, wherein the dominant negative Zap70 kinase moiety has a sequence that has at least 90% sequence identity to SEQ ID NO: 2.
36. (canceled)
37. The trigger-responsive immune-inactivating signaling polypeptide of claim 24, wherein the dominant negative signaling moiety includes a dominant negative kinase moiety that is a dominant negative variant of a LCK kinase.
38. The trigger-responsive immune-inactivating signaling polypeptide of claim 37, wherein the dominant negative LCK kinase moiety has a sequence that has at least 90% sequence identity to SEQ ID NO: 17.
39. The trigger-responsive immune-inactivating signaling polypeptide of claim 1, wherein the trigger-responsive immune-inactivating signaling polypeptide is a trigger-responsive constitutively active signaling polypeptide and the immune-inactivating moiety is a constitutively active signaling moiety.
40.-48. (canceled)
49. The trigger-responsive immune-inactivating signaling polypeptide of claim 39, wherein the constitutively active signaling moiety includes a constitutively active phosphatase moiety that is a constitutively active variant of a SHP1 phosphatase.
50. The trigger-responsive immune-inactivating signaling polypeptide of claim 49, wherein the constitutively active SHP1 phosphatase moiety has a sequence that has at least 90% sequence identity to SEQ ID NO: 23.
51.-53. (canceled)
54. A nucleic acid encoding the trigger-responsive immune-inactivating signaling polypeptide of claim 1.
55. (canceled)
56. A cell including the nucleic acid of claim 54.
57.-65. (canceled)
66. A pharmaceutical composition that delivers the trigger-responsive dominant negative signaling polypeptide of claim 1.
67. A method of regulating activity of T cells in vivo comprising the step of administering a composition that delivers the trigger-responsive dominant negative signaling polypeptide of claim 1 to a subject.
68.-70. (canceled)
71. A method of treating cancer, the method comprising the step of administering a composition that delivers the trigger-responsive dominant negative signaling polypeptide of claim 1 to a subject.
72.-76. (canceled)
77. A method of manufacturing a trigger-responsive dominant negative signaling polypeptide of claim 1, comprising expressing the trigger-responsive dominant negative signaling polypeptide in a host cell.
78. (canceled)
79. A method of manufacturing a genetically modified T cell, comprising introducing the nucleic acid of claim 54 into a T cell.
80. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWING
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DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0132] The present disclosure provides technologies for regulating the activity of immune system cells (e.g., monocytes, eosinophils, neutrophils, basophils, macrophages, dendritic cells, natural killer cells, T cells (including helper T cells and cytotoxic T cells), T regulatory cells, and/or B cells). Particularly, the present disclosure provides technologies for regulating T cell activity and encompasses the insight that systems for controlling T cell activation and/or activity can greatly enhance therapies that utilize and/or rely on T cells.
Adoptive T Cell Therapy
[0133] Adoptive T Cell Therapy (ATCT) is one current approach that shows promise in treating various conditions and/or diseases (e.g., cancer). ATCT entails collection and isolation of T cells from a subject (e.g., a patient). Isolated T cells are then clonally enriched, modified, and/or engineered to achieve a T cell population having desired properties and/or characteristics. The T cell population can then be expanded through ex-vivo growth and reintroduced into the subject to allow the enriched, modified, and/or engineered T cells to specifically attack cells of interest.
[0134] One type of ATCT that has been particularly effective in treating cancers (such as leukemias and lymphomas) utilizes T cells that have been engineered to express a chimeric antigen receptor (a “CAR”); such T cells are often referred to as CAR-T cells.
[0135] While initial CAR T cell approaches did not produce the desired clinical results, second-generation CAR T cells that were engineered to express, e.g., a chimeric fusion protein that contains an extracellular domain that recognizes antigens present on tumor cells, a hinge/transmembrane domain, a costimulatory domain, and a CD3 zeta chain, showed promise. For instance, a group at the University of Pennsylvania and the pharmaceutical company Novartis reported positive clinical results in patients with Chronic Leukocytic Lymphoma (CLL) (see Porter, et al., Chimeric Antigen Receptor—Modified T Cells in Chronic Lymphoid Leukemia, NEJM (2011)), Acute Lymphocytic Leukemia (ALL) (see Maude, et al., Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia, NEJM (2014)), and Non-Hodgkins Lymphoma (NHL)(see Schuster, S. J., “183 Sustained Remissions Following Chimeric Antigen Receptor Modified T Cells Directed Against CD19 (CTL019) in Patients with Relapsed or Refractory CD19+ Lymphomas,” ASH 57.sup.th Annual Meeting & Exposition, Session 624, Poster, (2015)). Subsequently, many groups have rushed into the field to develop optimized CAR T cells, and other related T cell based therapies.
[0136] Another type of ATCT that has been effective involves use of specific high-affinity T Cell Receptors (TCRs) to bind target antigens. Such high-affinity TCRs can be used, e.g., in place of a CAR, and can bind to cell surface proteins, such as CD19.
[0137] Despite the extremely promising effectiveness of ATCTs, the usefulness of the method has been hampered by potential adverse effects of the treatment. A prominent adverse effect has been the occurrence of cytokine storm in many patients, which can involve damage to multiple organs, fever, neurotoxicity, and/or death. Another adverse event has been tumor lysis syndrome. Still another adverse effect has been allogenic therapy graft versus host disease.
[0138] Efforts to minimize the chances of such side effects have included introduction of control elements into engineered T cells. One example of such an approach is described by Wendell Lim, et al. (Wu, et al., 2015, “Remote control of therapeutic T cells through a small molecule-gated chimeric receptor,” Science, (350) 6258), in which a CAR is divided into two pieces that are inactive unless brought together by a small molecule compound that can mediate association of the two pieces. Unfortunately, the small molecule compound used to demonstrate the method is not practical to use on human patients for a number of reasons, including a short half-life.
[0139] As another example, Bellicum, Inc. has employed a so-called “suicide switch” that, when activated by a small molecule drug, leads to programmed cell death of engineered T cells (Di Stasi, A., et al., “Inducible apoptosis as a safety switch for adoptive cell therapy,” N Engl J Med., 365(18):1673-83; doi: 10.1056/NEJMoa1106152 (2011)). This suicide switch may be activated, for example, if the engineered T cells are mediating graft versus host disease. This approach can be effective to “turn off” the engineered T cells. However, the present disclosure appreciates that there is a problem with the strategy, as it inactivates engineered T cells by destroying them, which wastes time and resources and also may result in the subject (e.g., patient) having to undergo additional procedures to replace the destroyed T cells, which can be painful, expensive and time consuming.
Trigger-Responsive Modulation of T Cell Activity
[0140] The present disclosure provides a system that can allow for fine-tuned regulation of T cell activity, including specifically of CAR-T and TCR T cell activity using a trigger, for example, an innocuous, practical, and approved small molecule. In some embodiments, such regulation is reversible (e.g., by alternating initiation and termination of exposure to the trigger). Alternatively or additionally, in some embodiments, such regulation may be sensitive to degree of exposure to the trigger (e.g., to trigger concentration and/or frequency, etc). In some embodiments, exposure to a trigger can “dial down” cytokine release and/or one or more other activities of T cells, including of reintroduced and/or engineered T cells.
[0141] In some embodiments, exposure to a trigger involves administration of a trigger agent (e.g., a small molecule agent). In some embodiments, exposure to a trigger may be for a finite (and/or predetermined) period of time, for example due to clearance (e.g., by degradation, removal, sequestering, or other means etc) of the trigger agent. In some embodiments, cessation of exposure to the agent relieves the modification of T cell activity that occurred during exposure to the agent.
[0142] In some particular embodiments, administration of a trigger agent results in a decrease in one or more hallmarks of T cell activity (e.g., cytokine release). In some embodiments, such decrease may be commensurate with concentration (e.g., local concentration and/or plasma concentration) of administered agent, and/or with frequency and/or magnitude of dose administration). Alternatively or additionally, in some embodiments, clearance of the agent (e.g., via natural mechanisms or by induced removal or degradation, for example as may be achieved by administration of a follow-on agent that stimulates clearance of the trigger agent) relieves the decrease. In some embodiments, subsequent administration of the trigger agent re-establishes the decrease. In some embodiments, the system remains sensitive to multiple cycles of administration and clearance of the trigger agent.
[0143] In some embodiments, the present disclosure achieves regulation of T cell activity through use of a immune-inactivating moiety of a T cell activation pathway component. Moreover, in some embodiments, the present disclosure provides an insight that association of such an immune-inactivating moiety with a modulating domain whose inhibitory or masking action can be relieved by a trigger creates an agent that can regulate T cell activity in a trigger-responsive, and, in many embodiments, reversible (even serially reversible) fashion.
[0144] In some embodiments, the present disclosure achieves regulation of T cell activity through use of a dominant negative signaling moiety of a T cell activation pathway component. Moreover, in some embodiments, the present disclosure provides an insight that association of such a dominant negative signaling moiety with a modulating domain whose inhibitory or masking action can be relieved by a trigger creates an agent that can regulate T cell activity in a trigger-responsive, and, in many embodiments, reversible (even serially reversible) fashion.
[0145] In some embodiments, the present disclosure achieves regulation of T cell activity through use of a constitutively active signaling moiety of a T cell activation pathway component. Moreover, in some embodiments, the present disclosure provides an insight that association of such a constitutively active signaling moiety with a modulating domain whose inhibitory or masking action can be relieved by a trigger creates an agent that can regulate T cell activity in a trigger-responsive, and, in many embodiments, reversible (even serially reversible) fashion.
[0146] In particular embodiments, the present disclosure provides insights that connect technologies from disparate fields to provide new strategies for regulating T cell activity that achieve surprising advantages relative to existing approaches. For example, among other things, the present disclosure appreciates that developments providing immune-inactivating moieties of T cell activation pathway components (such as a dominant negative kinase moiety or a constitutively active phosphatase moiety) can be combined with features of ligand-responsive nuclear receptors to provide a system for trigger-responsive regulation of T cell activity. Furthermore, the present disclosure appreciates that application of such a system to ATCT technologies, including CAR-T and/or TCR T cells, provides new and remarkably useful therapeutic T cell modalities.
[0147] Approaches for regulation of T cell activity described herein provide a variety of advantages relative to available systems including, for example, that T cell activity can be inhibited without destroying T cells. Furthermore, in many embodiments, provided systems provide for reversible inhibition of T cell activity. Thus, the present disclosure provides systems in which activity of a T cell population (which may be a maintained T cell population) can be reversibly decreased and increased through application and removal of a trigger. Combining trigger-responsiveness with maintenance of T cell levels (and, in at least some embodiments, reversibility and/or tunability through adjustment of trigger “intensity”—e.g., concentration, level and/or frequency of application, etc) provides a remarkably sophisticated and effective system that, moreover, is applicable to any of a variety of T cell populations including, for example, existing ATCT (e.g., CAR-T and/or TCR) T cell populations. Yet another advantage of provided systems is that they utilize and/or impact existing T cell biological cascades, rather than requiring that a new signaling cascade be introduced as is required, for example, for the recently-reported Notch-signaling-based system developed by Lim, et al. (see, for example, Roybal, et al., Cell 167:419, “Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors,” Oct. 6, 2016).
[0148] Those of skill in the art will appreciate from the present disclosure that a further advantage of regulating T cell activity is that methods and compositions of the present disclosure can be used to treat T cell exhaustion of T cells (e.g., T cells introduced into a subject, e.g., CAR-T cells) that include, express, or encode a trigger-responsive immune-inactivating polypeptide. For instance, in certain embodiments, exposure of subject including one or more exhausted T cells that include, express, or encode a trigger-responsive immune-inactivating polypeptide to a trigger can treat exhaustion of T cells in the subject. In such instances, treatment of T cell exhaustion can include a change in the state of one or more exhausted T cells such that the T cells are capable of functioning as non-exhausted T cells, e.g., during or after cessation of exposure of the T cell to trigger.
[0149] Thus, among other things, the present disclosure provides trigger-responsive T cell activity modulating agents that comprise a immune-inactivating moiety (i.e., a moiety that, when present in a T cell that includes a functional T cell activation signaling cascade, interferes with the cascade such that T cell activation signaling is disrupted) and a modulating domain that, in many embodiments, is characterized by an ability to adopt a first state and a second state, and to transition between the first state and the second state when exposed to a trigger. When such a modulating domain is in its first state, the immune-inactivating moiety with which it is associated is inhibited, and when the modulating domain is in its second state, the inhibition is relieved. In accordance with the present disclosure, introduction of such a trigger-responsive T cell activity modulator into a T cell (e.g., by introduction and/or expression of a nucleic acid that encodes it) renders activity of the T cell responsive to presence of the trigger: when the trigger is absent, the modulating domain adopts its first state and the immune-inactivating moiety is inhibited so that the T cell activation cascade is functional; when the trigger is present, the modulating domain adopts its second state and the immune-inactivating moiety is active so that the T cell activation cascade is inhibited. Those of skill in the art will appreciate that, in some embodiments, degree of inhibition (or functionality) of the T cell activation cascade may be tuned through adjustment of level and/or frequency of trigger exposure (e.g., by concentration of the trigger) and/moreover, that such inhibition (or functionality) may, in many embodiments, be reversible, optionally through several cycles.
[0150] Thus, among other things, the present disclosure provides trigger-responsive T cell activity modulating agents that comprise a dominant negative signaling moiety (i.e., a moiety that, when present in a T cell that includes a functional T cell activation signaling cascade, interferes with the cascade such that T cell activation signaling is disrupted) and a modulating domain that, in many embodiments, is characterized by an ability to adopt a first state and a second state, and to transition between the first state and the second state when exposed to a trigger. When such a modulating domain is in its first state, the dominant negative signaling moiety with which it is associated is inhibited, and when the modulating domain is in its second state, the inhibition is relieved. In accordance with the present disclosure, introduction of such a trigger-responsive T cell activity modulator into a T cell (e.g., by introduction and/or expression of a nucleic acid that encodes it) renders activity of the T cell responsive to presence of the trigger: when the trigger is absent, the modulating domain adopts its first state and the dominant negative signaling moiety is inhibited so that the T cell activation cascade is functional; when the trigger is present, the modulating domain adopts its second state and the dominant negative signaling moiety is active so that the T cell activation cascade is inhibited. Those of skill in the art will appreciate that, in some embodiments, degree of inhibition (or functionality) of the T cell activation cascade may be tuned through adjustment of level and/or frequency of trigger exposure (e.g., by concentration of the trigger) and/moreover, that such inhibition (or functionality) may, in many embodiments, be reversible, optionally through several cycles.
[0151] The present disclosure provides trigger-responsive T cell activity modulating agents that comprise a constitutively active signaling moiety (i.e., a moiety that, when present in a T cell that includes a functional T cell activation signaling cascade, interferes with the cascade such that T cell activation signaling is disrupted) and a modulating domain that, in many embodiments, is characterized by an ability to adopt a first state and a second state, and to transition between the first state and the second state when exposed to a trigger. When such a modulating domain is in its first state, the constitutively active signaling moiety with which it is associated is inhibited, and when the modulating domain is in its second state, the inhibition is relieved. In accordance with the present disclosure, introduction of such a trigger-responsive T cell activity modulator into a T cell (e.g., by introduction and/or expression of a nucleic acid that encodes it) renders activity of the T cell responsive to presence of the trigger: when the trigger is absent, the modulating domain adopts its first state and the constitutively active signaling moiety is inhibited so that the T cell activation cascade is functional; when the trigger is present, the modulating domain adopts its second state and the constitutively active signaling moiety is active so that the T cell activation cascade is inhibited. Those of skill in the art will appreciate that, in some embodiments, degree of inhibition (or functionality) of the T cell activation cascade may be tuned through adjustment of level and/or frequency of trigger exposure (e.g., by concentration of the trigger) and/moreover, that such inhibition (or functionality) may, in many embodiments, be reversible, optionally through several cycles.
[0152] In some embodiments, a dominant negative signaling moiety may be or comprise a dominant negative signaling moiety of a T cell activation pathway component. In some embodiments, a dominant negative signaling moiety may be or comprise a dominant negative kinase moiety (e.g., of a kinase that operates in a T cell activation pathway).
[0153] In some embodiments, a constitutively active signaling moiety may be or comprise a constitutively active signaling moiety of a T cell activation pathway component. In some embodiments, a constitutively active signaling moiety may be or comprise a constitutively active phosphatase moiety (e.g., of a phosphatase that operates in a T cell activation pathway).
[0154] In some embodiments, a modulating domain can be or comprise a nuclear receptor (e.g., a hormone receptor) or portion thereof (e.g., a ligand binding domain thereof). For example, in some embodiments, a modulating domain can be or comprise a ligand binding domain of an estrogen receptor, e.g., an estrogen receptor in which mutations have been introduced. In some embodiments, mutations are introduced in an estrogen receptor to increase its ability to form inactivating complexes with heat shock proteins, to lose affinity to estrogen, and/or to retain affinity for synthetic ligands such as raloxifene, tamoxifen, 4-hydroxy tamoxifen and endoxifen (e.g., in ER(T2) or ER(T12)).
Signaling Moieties
T Cell Activation Pathway and Associated Signaling Entities
[0155] A T cell-receptor (TCR) is a polypeptide complex found on the surface of T cells. A TCR comprises a heterodimer of α and β polypeptide chains that is non-covalently associated with a CD3 dimer of γε, δε, or ζζ polypeptide chains. Each of the γ, δ, ε, and ζ polypeptides includes at least one polypeptides include three) so-called immunoreceptor tyrosine-based activation motifs (ITAMs) characterized by two tyrosine residues flanking a series of amino acids that include key leucine/isoleucine residues with stereotypic spacing.
[0156] TCR signaling in response to antigen recognition initiates T cell activation, which plays a central role in the adaptive immune response. As explained by, e.g., Huse, M., “The T-cell-receptor signaling network,” Journal of Cell Science, 122, p. 1269-1273 (2009) and shown in
[0157] Upon localization to a TCR complex, Zap70 phosphorylates multiple tyrosine residues within Linker for the Activation of T cells (LAT), a membrane-associated scaffolding protein. Phosphorylated LAT recruits a second molecular scaffold, SH2-domain-containing leukocyte protein of 76 kDa (Slp76), which binds to LAT via an intervening protein Gads (Grb2-related adapter protein 2 or GRAP2). Slp76 is then phosphorylated by Zap70, and the resulting LAT-Slp76 complex acts as a platform for recruitment of signaling effectors, many of which bind directly to phosphotyrosine-based motifs. One of the signaling effectors is phospholipase C-γ (PLCγ), which can interact directly with both LAT and Slp76. PLCγ transduces TCR signals by hydrolyzing phosphatidylinositol bisphosphate (PIP2) to yield diacylglycerol (DAG), a membrane-associated lipid, and inositol trisphosphate (IP3), a diffusible second messenger. DAG recruits a number of downstream proteins to the membrane, among them protein kinase C-θ (PKCθ) and RasGRP (RAS guanyl nucleotide-releasing protein), which is a guanine nucleotide exchange factor (GEF). RasGRP activates the small GTPase, Ras, an activator of mitogen-activated protein kinase (MAPK) signaling pathways in many cell types. Ras can also be activated by the exchange factor son of sevenless (SOS), which is recruited to LAT via the adaptor molecule Grb2 (growth-factor-receptor-bound protein 2).
[0158] Phosphorylated Slp76 binds directly to the Tec family kinase interleukin inducible T cell kinase (ITK). Together with Zap70 and Lck, ITK has an essential role in the phosphorylation and activation of PLCγ. In addition, Slp76 recruits the GEF, Vav, which activates the small GTPases, Rac and Cdc42. The adaptor proteins Nck and adhesion- and degranulation-promoting adaptor protein (ADAP) are also recruited into the complex. The LAT-Slp76 complex may be a highly cooperative signalosome. Many of its constituent proteins interact with several partners, and the loss of any one protein disrupts signaling through other effectors. This cooperative behavior may be important for coordinating and coupling different branches of the TCR signaling network.
[0159] These early membrane-proximal signaling steps are subject to inhibition on a number of levels. The tyrosine phosphatase SH2-domain containing phosphatase 1 (SHP1) dephosphorylates and deactivates both Zap70 and Lck (see, e.g.,
[0160] Lck tail phosphorylation is removed by CD45, a tyrosine phosphatase, which restores TCR signaling. Under certain conditions, however, CD45 can inhibit Lck and other effectors by dephosphorylating phosphotyrosine residues that are required for their optimal activity.
[0161] In addition to early TCR signals, TCR stimulation results in signal transduction to the nucleus, which leads to profound changes in gene expression. Many of these changes are mediated by the transcription factors activator protein 1 (AP1, a heterodimer of Fos and Jun), nuclear factor of activated T cells (NFAT) and nuclear factor-κB (NF-κB). These three factors act together to activate transcription of the interleukin-2 gene.
[0162] Activation of Fos and Jun occurs as a downstream event of three MAPK signaling pathways. Each pathway consists of an effector MAPK [extracellular signal-regulated kinase (Erk), Jun kinase (JNK) and protein of 38 kDa (p38)], an upstream MAPK kinase [MAPK or ERK kinase (MEK), JNK kinase (JNKK) and MAPK kinase 3/6 (MKK3/6)], and a MAPK kinase kinase [MEK kinase 1 (MEKK1) and Raf]. The Erk pathway is stimulated by the association of active Ras with Raf, whereas the JNK and p38 pathways respond to activated Rac in addition to Ras. MAPK signaling cascades stimulate AP1 activity via the upregulation of Fos and Jun transcription, and also by direct phosphorylation of the Fos and Jun proteins. In addition, Erk engages in positive feedback by phosphorylating Lck. This phosphorylation event blocks inhibitory interactions between Lck and SHP1.
[0163] NFAT activity is regulated by intracellular Ca.sup.2+ concentration. When Ca.sup.2+ levels are low, phosphorylation by a kinase known as glycogen synthase kinase 3 (GSK3) induces nuclear export of NFAT. Increases in intracellular Ca.sup.2+ lead to dephosphorylation and nuclear import of NFAT. NFAT dephosphorylation is mediated by the phosphatase calcineurin (CN), which is activated by its association with the Ca.sup.2+-binding protein calmodulin (CaM). Cytoplasmic Ca.sup.2+ levels are coupled to TCR activation through PLCγ. Production of IP3 by PLCγ stimulates the opening of Ca.sup.2+-permeable ion channels known as IP3 receptors (IP3Rs) in the endoplasmic reticulum (ER). This leads to the depletion of Ca.sup.2+ from the ER, which induces the aggregation of the Ca.sup.2+ sensors stromal interaction molecule 1 (STIM1) and STIM2 in regions of close ER-plasma-membrane apposition. These STIM clusters are thought to trigger the opening of Orail channels in the cell membrane, leading to a large and sustained influx of Ca.sup.2+ into the cytoplasm. This second, Orail-dependent, rise in Ca.sup.2+ drives NFAT into the nucleus.
[0164] NFAT translocation is also regulated by phosphatidylinositol 3-kinase (PI3K), which is activated downstream of several TCR signaling effectors, including Ras. PI3K phosphorylates PIP2 to yield PIP3, a phospholipid that recruits a variety of cytoplasmic proteins to the cell membrane. One of the most important of these is the kinase AKT, which promotes cell survival via several distinct pathways. AKT phosphorylates GSK3, thereby inhibiting the phosphorylation of NFAT and promoting its nuclear translocation. PI3K signaling is regulated by the opposing activity of the phosphatase and tensin homolog (PTEN).
[0165] Under resting conditions, NF-κB is sequestered in the cytoplasm by inhibitor of κB (IκB). Phosphorylation of IκB by the IκB kinase (IKK) complex leads to the ubiquitylation and degradation of IκB, allowing NF-κB to translocate to the nucleus. IKK is activated by MEKK1 and also by a protein complex comprising the adaptors caspase recruitment domain containing membrane-associated guanylate kinase protein 1 (CARMA1), B-cell lymphoma 10 (Bcl10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1). This complex functions downstream of PKCθ, which is recruited to the cell membrane by DAG. Thus, both NFAT and NF-κB rely on different branches of the PLCγ signaling pathway for their activation.
[0166] Optimal T cell stimulation that leads to proliferation and other effector functions requires that a second, ‘costimulatory’ signal be delivered through a distinct cell-surface receptor. Although several transmembrane proteins, including LFA1 and CD2, can provide costimulation in certain contexts, the archetypal costimulatory receptor is CD28. CD28 binds to B7-1 (also known as CD80) and B7-2 (also known as CD86), which are highly expressed by antigen presenting cells (APCs), such as dendritic cells. Ligand binding of CD28 induces the phosphorylation of tyrosine-containing sequences in its cytoplasmic tail by Src-family kinases. This event leads to the recruitment of several downstream proteins, including PI3K, Grb2, Vav and ITK.
[0167] The inhibitory receptor cytotoxic T-lymphocyte antigen 4 (CTLA4) is closely related to CD28 and also binds to B7-1 and B7-2, but with significantly higher affinity than CD28. In resting T cells, almost all CTLA4 is sequestered in intracellular compartments such as endosomes via a mechanism that depends on the sorting adaptor AP2 (adaptor protein 2). TCR stimulation induces the trafficking of CTLA4 to the cell surface, where it can bind to its ligand and trigger signals that attenuate TCR signaling. Similarly to CD28, CTLA4 is phosphorylated by Src kinases at tyrosine residues in its cytoplasmic tail. The phosphatases protein phosphatase 2A (PP2A) and Src-homology 2 domain-containing phosphatase 2 (SHP2) both bind to phosphorylated CTLA4, as does PI3K. PP2A and SHP2 might inhibit TCR signaling by dephosphorylating membrane-proximal effectors, although it is also possible that CTLA4 mediates its inhibitory effects by competing with CD28 for binding to B7 ligands that are common to both receptors, which would crowd CD28 out of the immunological synapse.
[0168] TCR signaling stimulates the expression of two other CD28 family members known as inducible costimulatory molecule (ICOS) and programmed cell death 1 (PD1). After trafficking to the surface, both of these proteins can regulate the sustained phase of T cell signaling when activated by their respective ligands. ICOS enhances T cell effector functions but, unlike CD28, does not stimulate proliferation. By contrast, PD1 is a potent inhibitor of TCR signaling, similarly to CTLA4. It appears to act in different contexts than CTLA4, however, because PD1 ligand (PD-L) is expressed by different cell types than those that express B7-1 and B7-2.
[0169] TCR signaling also induces dramatic changes in cytoskeletal architecture. Antigen recognition by the T cell stimulates a burst of actin polymerization at the immunological synapse, generating a lamellapodial sheet structure that spreads over the surface of the APC. The actin-related protein 2/3 (Arp2/3) complex, which stimulates the growth of branched actin arrays, has a central role in this process. Arp2/3 is coupled to the LAT-Slp76 signalosome through Vav, which activates Cdc42 and Rac. Cdc42 triggers Arp2/3 activation by recruiting and activating the Wiskott-Aldrich syndrome protein (WASP), whereas Rac activates Arp2/3 through the WAVE (WASP family verprolin-homologous protein) complex. Actin polymerization is also stimulated by the cortactin homolog HS1 (hematopoietic lineage cell-specific protein 1), as well as the GTPase dynamin 2 (Dyn2), both of which interact with Vav.
[0170] TCR-stimulated actin polymerization is temporally correlated with an increase in integrin-mediated adhesion, which occurs via an ‘inside-out’ signaling mechanism. The upregulation of the function of integrins, primarily of the αLβ2 integrin LFA1 (lymphocyte function-associated antigen 1) is directly affected by Vav, PLCγ and other components of the LAT-Slp76 complex. Vav-dependent actin polymerization can induce integrin activation via recruitment of the cytoskeletal linker talin, which binds directly to integrin tails. PLCγ, for its part, activates integrins via the small GTPase, Rap. This occurs through the generation of DAG by PLCγ, which stimulates Rap by recruiting a protein complex containing PKCθ and the Rap exchange factor RapGEF2. Rap can also be activated by the exchange factor C3G (RapGEF1), which is recruited together with the tyrosine kinase Abl to the WAVE complex. Once Rap is loaded with GTP, it associates with LAT-Slp76 through a protein complex that contains ADAP, Src kinase-associated phosphoprotein of 55 kDa (SKAP55) and Rap-GTPinteracting adapter molecule (RIAM) and mediates integrin activation.
[0171] Integrin activation promotes enhanced adhesion of the T cell to the APC, facilitating the establishment of a long-lived T cell-APC contact. Activated integrins also induce intracellular signals that promote further cytoskeletal remodeling. For example, the exchange factor p21-activated kinase (PAK)-interacting exchange factor (PIX), which is associated with the adaptor G-protein-coupled receptor kinase interactor (GIT), is activated downstream of integrin adhesion. PIX-mediated activation of Rac in this context stimulates the kinase activity of PAK, which phosphorylates LIM kinase (LIMK) and myosin light chain kinase (MLCK). PAK phosphorylation activates LIMK, which promotes actin polymerization by phosphorylating and inhibiting the actin-severing protein cofilin. Phosphorylation of MLCK inhibits its kinase activity, and thereby its ability to promote myosin-based contraction. Taken together, these effects promote the growth and maintenance of actin-based structures in the cell.
[0172] TCR signaling also induces the polarization of the microtubule-organizing center (MTOC) to the immunological synapse. MTOC reorientation appears to depend on the negatively directed microtubule motor dynein. Microtubules radiate from the MTOC with positive ends facing outwards and negative ends facing inwards. Therefore, dynein that is localized at the immunological synapse can bind to microtubule tips and ‘reel’ the MTOC in towards itself.
Moieties Based on T Cell Activation Cascade Signaling Entities
[0173] The present disclosure provides a particular insight that T cell activation involves signaling pathways that may provide particularly attractive opportunities to control T cell activation and/or activity, which can greatly enhance therapies that utilize and/or rely on T cells. For example, a number of kinases and phosphatases play a role in T cell activation. Still further, the present disclosure appreciates that dominant negative moieties based on signaling entities, e.g., kinases and/or phosphatases, within a T cell activation pathway are available and/or can be readily generated.
[0174] In certain embodiments, the present disclosure provides technologies that utilize a dominant negative signaling moiety based on a kinase in a T cell activation pathway to regulate T cell activity. In certain particular embodiments, the present disclosure provides trigger-responsive dominant negative signaling polypeptides—i.e., constructs that can adopt at least first and second conformations, and can switch from one to the other in response to a particular trigger—in which a dominant negative signaling moiety (e.g., kinase moiety) is inhibited in one state relative to the other state. In particular embodiments, such a trigger-responsive dominant negative signaling polypeptide comprises a dominant negative variant based on a signaling entity (e.g., a kinase) that participates in a T cell activation pathway operably linked with a modulating domain as described herein.
[0175] In certain embodiments, the present disclosure provides technologies that utilize a constitutively active signaling moiety based on a phosphatase in a T cell activation pathway to regulate T cell activity. In certain particular embodiments, the present disclosure provides trigger-responsive constitutively active signaling polypeptides—i.e., constructs that can adopt at least first and second conformations, and can switch from one to the other in response to a particular trigger—in which a constitutively active signaling moiety (e.g., phosphatase moiety) is inhibited in one state relative to the other state. In particular embodiments, such a trigger-responsive constitutively active signaling polypeptide comprises a constitutively active variant based on a signaling entity (e.g., a phosphatase) that participates in a T cell activation pathway operably linked with a modulating domain as described herein.
[0176] A list of exemplary signaling entities that play a role in a T cell activation pathway, which are included in Table 4 (below).
TABLE-US-00004 TABLE 4 Exemplary Kinases in a T cell Activation Pathway zeta-chain-associated protein kinase 70 (“Zap-70”) lymphocyte-specific protein tyrosine kinase (“Lck”) phosphatidylinositol-4,5-bisphosphate 3-kinase (“PI3K”) pyruvate dehydrogenase lipoamide kinase isozyme 1 (“PDK1”) protein kinase C theta (“PKCθ”) serine/threonine-protein kinase (“Raf”) mitogen-activated protein kinase kinase 1 (“MEK1” or “MAP2K1”) mitogen-activated protein kinase kinase 2 (“MEK2” or “MAP2K2”) mitogen-activated protein kinase 3 (“ERK1” or “MAPK3”) mitogen-activated protein kinase 1 (“ERK2” or “MAPK1”) mitogen-activated protein kinase kinase kinase 1 (“MEKK1” or “MAP3K1”) mitogen-activated protein kinase kinase 4 (“MKK4” or “MAP2K4” or “JNKK”) mitogen-activated protein kinase kinase 7 (“MKK7” or “MAP2K7”) mitogen-activated protein kinase 3/6 (“MAPK 3/6”) c-Jun N-terminal kinase 1 (“JNK1”) p38 mitogen-activated protein kinase (“p38 MAPK”) c-Jun N-terminal kinase 2 (“JNK2”) inhibitor of nuclear factor kappa-B kinase subunit gamma (“IKKγ”) inhibitor of nuclear factor kappa-B kinase subunit beta (“IKKβ”) inhibitor of nuclear factor kappa-B kinase subunit alpha (“IKKα”) protein kinase B (“Akt” or “PKB”) mechanistic target of rapamycin (“mTOR”) calcium/calmodulin-dependent protein kinase type IV (“CaMKIV”) mitogen-activated protein kinase kinase kinase kinase 1 (“HPK1” or “MAP4K1”) TGF-beta-activated kinase 1 (“TAK1” or “MAP3K7”) inducible T cell kinase (“ITK”) C-terminal Src kinase (“Csk”) glycogen synthase kinase 3 (“GSK3”) Other Exemplary Enzymes in a T cell Activation Pathway calcineurin (“CaN”) Calpain phospholipase Cγ1 (“PLCγ1”) cell division control protein 42 homolog (“Cdc42”) ras-related C3 botulinum toxin substrate (“Rac”) Ras Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (“MALT1”) CD45 receptor tyrosine phosphatase Tyrosine phosphatase SH2-domain containing phosphatase 1 (SHP1) phosphatases protein phosphatase 2A (PP2A)
[0177] In some embodiments, a dominant negative signaling moiety based on a signaling entity in a T cell activation pathway (e.g., as listed in Table 4) can be used in a trigger-responsive dominant negative signaling polypeptide described here. Dominant negative moieties based on kinases within a T cell activation pathway are available and/or can be readily generated. As one specific example, a dominant negative form of Zeta-associated Protein (Zap)-70 has been described by Qian, et al. (Qian, D., et al., “Dominant-negative Zeta-associated Protein 70 Inhibits T Cell Antigen Receptor Signaling,” J. Exp. Med., Vol. 183, p. 611-620 (1996)). As discussed above, Zap-70 is a cytoplasmic protein tyrosine kinase that is essential for T cell activity. In wild type cells, a T cell Receptor (TCR) binds antigens and recruits a CD3-zeta chain protein, leading to phosphorylation of ITAMs on CD3 and recruitment and activation of Zap-70. Activation of Zap-70 triggers an intracellular signaling cascade that drives T Cell activity. Qian, et al. made dominant negative mutants of Zap-70 that inactivated the kinase activity of Zap-70, and therefore, were able to disrupt Zap-70 signaling. Qian, et al. achieved the inactivation of Zap-70 kinase activity using two general approaches: point mutations or a truncation of the kinase domain.
[0178] In some embodiments, a dominant negative Zap70 moiety can be encoded by DNA having a nucleotide sequence according to SEQ ID NO: 1. As disclosed herein, SEQ ID NO: 1 represents an exemplary nucleotide sequence encoding a dominant negative Zap70 moiety. In some embodiments, a dominant negative Zap70 moiety can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 1. In some embodiments, a dominant negative Zap70 moiety can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
TABLE-US-00005 SEQ ID NO: 1 ATGCCAGACCCCGCGGCGCACCTGCCCTTCTTCTACGGCAGCATCTCGCG TGCCGAGGCCGAGGAGCACCTGAAGCTGGCGGGCATGGCGGACGGGCTCT TCCTGCTGCGCCAGTGCCTGCGCTCGCTGGGCGGCTATGTGCTGTCGCTC GTGCACGATGTGCGCTTCCACCACTTTCCCATCGAGCGCCAGCTCAACGG CACCTACGCCATTGCCGGCGGCAAAGCGCACTGTGGACCGGCAGAGCTCT GCGAGTTCTACTCGCGCGACCCCGACGGGCTGCCCTGCAACCTGCGCAAG CCGTGCAACCGGCCGTCGGGCCTCGAGCCGCAGCCGGGGGTCTTCGACTG CCTGCGAGACGCCATGGTGCGTGACTACGTGCGCCAGACGTGGAAGCTGG AGGGCGAGGCCCTGGAGCAGGCCATCATCAGCCAGGCCCCGCAGGTGGAG AAGCTCATTGCTACGACGGCCCACGAGCGGATGCCCTGGTACCACAGCAG CCTGACGCGTGAGGAGGCCGAGCGCAAACTTTACTCTGGGGCGCAGACCG ACGGCAAGTTCCTGCTGAGGCCGCGGAAGGAGCAGGGCACATACGCCCTG TCCCTCATCTATGGGAAGACGGTGTACCACTACCTCATCAGCCAAGACAA GGCGGGCAAGTACTGCATTCCCGAGGGCACCAAGTTTGACACGCTCTGGC AGCTGGTGGAGTATCTGAAGCTGAAGGCGGACGGGCTCATCTACTGCCTG AAGGAGGCCTGCCCCAACAGCAGTGCCAGCAACGCCTCAGGGGCTGCTGC TCCCACACTCCCAGCCCACCCATCCACGTTGACG
[0179] In some embodiments, a dominant negative Zap70 moiety can have an amino acid sequence according to SEQ ID NO: 2. As disclosed herein, SEQ ID NO: 2 represents an exemplary amino acid sequence of a dominant negative Zap70 moiety. In some embodiments, a dominant negative Zap70 moiety can have an amino acid sequence substantially similar to SEQ ID NO: 2. In some embodiments, a dominant negative Zap70 moiety can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.
TABLE-US-00006 SEQ ID NO: 2 M P D P A A H L P F F Y G S I S R A E A E E H L K L A G M A D G L F L L R Q C L R S L G G Y V L S L V H D V R F H H F P I E R Q L N G T Y A I A G G K A H C G P A E L C E F Y S R D P D G L P C N L R K P C N R P S G L E P Q P G V F D C L R D A M V R D Y V R Q T W K L E G E A L E Q A I I S Q A P Q V E K L I A T T A H E R M P W Y H S S L T R E E A E R K L Y S G A Q T D G K F L L R P R K E Q G T Y A L S L I Y G K T V Y H Y L I S Q D K A G K Y C I P E G T K F D T L W Q L V E Y L K L K A D G L I Y C L K E A C P N S S A S N A S G A A A P T L P A H P S T L T
[0180] In some embodiments, a dominant negative LCK moiety can be encoded by DNA having a nucleotide sequence according to SEQ ID NO: 19. As disclosed herein, SEQ ID NO: 19 represents an exemplary nucleotide sequence encoding a dominant negative LCK moiety. In some embodiments, a dominant negative LCK moiety can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 19. In some embodiments, a dominant negative LCK moiety can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 19.
TABLE-US-00007 SEQ ID NO: 19 ATGGGCTGTGGCTGCAGCTCACACCCGGAAGATGACTGGATGGAAAACAT CGATGTGTGTGAGAACTGCCATTATCCCATAGTCCCACTGGATGGCAAGG GCACGCTGCTCATCCGAAATGGCTCTGAGGTGCGGGACCCACTGGTTACC TACGAAGGCTCCAATCCGCCGGCTTCCCCACTGCAAGACAACCTGGTTAT CGCTCTGCACAGCTATGAGCCCTCTCACGACGGAGATCTGGGCTTTGAGA AGGGGGAACAGCTCCGCATCCTGGAGCAGAGCGGCGAGTGGTGGAAGGCG CAGTCCCTGACCACGGGCCAGGAAGGCTTCATCCCCTTCAATTTTGTGGC CAAAGCGAACAGCCTGGAGCCCGAACCCTGGTTCTTCAAGAACCTGAGCC GCAAGGACGCGGAGCGGCAGCTCCTGGCGCCCGGGAACACTCACGGCTCC TTCCTCATCCGGGAGAGCGAGAGCACCGCGGGATCGTTTTCACTGTCGGT CCGGGACTTCGACCAGAACCAGGGAGAGGTGGTGAAACATTACAAGATCC GTAATCTGGACAACGGTGGCTTCTACATCTCCCCTCGAATCACTTTTCCC GGCCTGCATGAACTGGTCCGCCATTACACCAATGCTTCAGATGGGCTGTG CACACGGTTGAGCCGCCCCTGCCAGACCCAGAAGCCCCAGAAGCCGTGGT GGGAGGACGAGTGGGAGGTTCCCAGGGAGACGCTGAAGCTGGTGGAGCGG CTGGGGGCTGGACAGTTCGGGGAGGTGTGGATGGGGTACTACAACGGG
[0181] In some embodiments, a dominant negative LCK moiety can have an amino acid sequence according to SEQ ID NO: 17. As disclosed herein, SEQ ID NO: 17 represents an exemplary amino acid sequence of a dominant negative LCK moiety. In some embodiments, a dominant negative LCK moiety can have an amino acid sequence substantially similar to SEQ ID NO: 17. In some embodiments, a dominant negative LCK moiety can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 17.
TABLE-US-00008 SEQ ID NO: 17 MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGTLLIRNGSEVRDPLVT YEGSNPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKA QSLTTGQEGFIPFNFVAKANSLEPEPWFFKNLSRKDAERQLLAPGNTHGS FLIRESESTAGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYISPRITFP GLHELVRHYTNASDGLCTRLSRPCQTQKPQKPWWEDEWEVPRETLKLVER LGAGQFGEVWMGYYNG
[0182] Additional examples of dominant negative forms of signaling entities in a T cell activation pathway have been described and could be utilized in dominant negative signal moieties according to the present disclosure. See, e.g., Herskowitz, Functional inactivation of genes by dominant negative mutations,” Nature, Vo. 329 (1987). Examples of such dominant negative forms of signaling entities in a T cell activation pathway include: Lck (see, e.g., Levin, et al., A dominant-negative transgene defines a roles for p56lck in thymopoiesis, EMBO, 12(4), 1671-1680 (1993)), Ras (see, e.g., Stoll, et al., Dominant negative inhibitors of signaling through the phophoinositol 3-kinase pathway for gene therapy of pancreatic cancer, Gut, 54, 109-116 (2005)), PI3K (see, e.g., Pugazhenthi, S., et al., “Akt/Protein Kinase B Up-regulates Bcl-2 Expression through cAMP-response Element-binding Protein,” J Biol Chem, 275(15), 10761-10766 (2000)), PDK1 (see, e.g., Nirula, A., et al., “Phosphoinositide-dependent kinase 1 targets protein kinase A in a pathway that regulates interleukin 4,” JEM, 203(7), 1733-1744 (2006)), p38 MAPK (see, e.g., Somwar, R., et al., “A Dominant-negative p38 MAPK Mutant and Novel Selective Inhibitors of p38 MAPK Reduce Insulin-stimulated Glucose Uptake in 3T3-L1 Adipocytes without Affecting GLUT4 Translocation,” J Biol Chem, 277(52), 50386-50395 (2002)), MEK1 (Bastow, E., et al., “Selective Activation of the MEK-ERK Pathway Is Regulated by Mechanical Stimuli in Forming Joints and Promotes Pericellular Matrix Formation,” 280(12), 11749-11758 (2005)), and JNK1 and c-Raf (Chen, Y., et al., “The Role of c-Jun N-terminal Kinase (JNK) in Apoptosis Induced by Ultraviolet C and γ Radiation,” J Biol Chem, 271(50), 31929-31936 (1996)). In some embodiments, a dominant negative signaling moiety of a signaling entity may or may not correspond to an entire signaling entity. In other words, a dominant negative signaling moiety of a signaling entity may correspond to an entire signaling entity or a portion of a signaling entity (e.g., a fragment, a domain, a moiety, etc.). For example, a dominant negative signaling moiety of an enzymatic signaling entity may correspond to an entire enzymatic signaling entity, a fragment of an enzymatic signaling entity or a portion of an enzymatic signaling entity (e.g., a moiety of an enzymatic signaling entity (e.g., including an enzymatic domain) or an enzymatic domain).
[0183] In some embodiments, a dominant negative signaling moiety of a signaling entity may be produced by mutating a sequence (e.g., amino acid or nucleic acid sequence) of a signaling entity. Exemplary mutations include point mutations, additions and/or truncations. Mutations can be made in portions of a signaling entity associated with an activity (e.g., an enzymatic domain, such as a kinase domain); however, mutations are not limited to those portions of a signaling entity and may be made in a portion of a signaling entity that impacts, e.g., the conformation or cellular localization of a signaling entity.
[0184] In some embodiments, a dominant negative signaling moiety of a signaling entity may be produced by making post-translational modifications. Post-translational modifications can include, but are not limited to, ubiquitination, phosphorylation, acetylation, glycosylation (N- and O-linked), glycation, myristolyation, palmitoylation, prenylation, amidation, akylation, hydroxylation, biotinylation, pegylation, methylation, sulfation, SUMOylation, dephosphorylation, deacetylation, deglycosylation, deamidation, dihydroxylation, demethylation, deubiquitination, and/or desulfation. Post-translational modifications can be made in portions of a signaling entity associated with an activity (e.g., an enzymatic domain, such as a kinase domain); however, post-translational modifications may also be made in a portion of a signaling entity that impacts, e.g., the conformation or cellular localization of a signaling entity.
[0185] In some embodiments, a constitutively active signaling moiety based on a signaling entity in a T cell activation pathway (e.g., as listed in Table 4) can be used in a trigger-responsive constitutively active signaling polypeptide described here. Constitutively active moieties based on phosphatases within a T cell activation pathway are available and/or can be readily generated. SHP1 is a tyrosine phosphatase that dephosphorylates and deactivates both Zap70 and LCK.
[0186] In some embodiments, a constitutively active SHP1 moiety can be encoded by DNA having a nucleotide sequence according to SEQ ID NO: 25. As disclosed herein, SEQ ID NO: 25 represents an exemplary nucleotide sequence encoding a constitutively active SHP1 moiety. In some embodiments, a constitutively active SHP1 moiety can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 25. In some embodiments, a constitutively active SHP1 moiety can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 25.
TABLE-US-00009 SEQ ID NO: 25 CGGCAGCCGTACTATGCCACGAGGGTGAATGCGGCTGACATTGAGAACCG AGTGTTGGAACTGAACAAGAAGCAGGAGTCCGAGGATACAGCCAAGGCTG GCTTCTGGGAGGAGTTTGAGAGTTTGCAGAAGCAGGAGGTGAAGAACTTG CACCAGCGTCTGGAAGGGCAGCGGCCAGAGAACAAGGGCAAGAACCGCTA CAAGAACATTCTCCCCTTTGACCACAGCCGAGTGATCCTGCAGGGACGGG ACAGTAACATCCCCGGGTCCGACTACATCAATGCCAACTACATCAAGAAC CAGCTGCTAGGCCCTGATGAGAACGCTAAGACCTACATCGCCAGCCAGGG CTGTCTGGAGGCCACGGTCAATGACTTCTGGCAGATGGCGTGGCAGGAGA ACAGCCGTGTCATCGTCATGACCACCCGAGAGGTGGAGAAAGGCCGGAAC AAATGCGTCCCATACTGGCCCGAGGTGGGCATGCAGCGTGCTTATGGGCC CTACTCTGTGACCAACTGCGGGGAGCATGACACAACCGAATACAAACTCC GTACCTTACAGGTCTCCCCGCTGGACAATGGAGACCTGATTCGGGAGATC TGGCATTACCAGTACCTGAGCTGGCCCGACCATGGGGTCCCCAGTGAGCC TGGGGGTGTCCTCAGCTTCCTGGACCAGATCAACCAGCGGCAGGAAAGTC TGCCTCACGCAGGGCCCATCATCGTGCACTGCAGCGCCGGCATCGGCCGC ACAGGCACCATCATTGTCATCGACATGCTCATGGAGAACATCTCCACCAA GGGCCTGGACTGTGACATTGACATCCAGAAGACCATCCAGATGGTGCGGG CGCAGCGCTCGGGCATGGTGCAGACGGAGGCGCAGTACAAGTTCATCTAC GTGGCCATCGCCCAGTTCATTGAAACCACTAAGAAGAAGCTGGAGGTCCT GCAGTCGCAGAAGGGCCAGGAGTCGGAGTACGGGAACATCACCTATCCCC CAGCCATGAAGAATGCCCATGCCAAGGCCTCCCGCACCTCGTCCAAACAC AAGGAGGATGTGTATGAGAACCTGCACACTAAGAACAAGAGGGAGGAGAA AGTGAAGAAGCAGCGGTCAGCAGACAAGGAGAAGAGCAAGGGTTCCCTCA AGAGGAAG
[0187] In some embodiments, a constitutively active SHP1 can have an amino acid sequence according to SEQ ID NO: 23. As disclosed herein, SEQ ID NO: 23 represents an exemplary amino acid sequence of a constitutively active SHP1 moiety. In some embodiments, a constitutively active SHP1 moiety can have an amino acid sequence substantially similar to SEQ ID NO: 23. In some embodiments, a constitutively active SHP1 moiety can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 23.
TABLE-US-00010 SEQ ID NO: 23 RQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNL HQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKN QLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRN KCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREI WHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGR TGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIY VAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKH KEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRK
[0188] In some embodiments, a constitutively active signaling moiety of a signaling entity may or may not correspond to an entire signaling entity. In other words, a constitutively active signaling moiety of a signaling entity may correspond to an entire signaling entity or a portion of a signaling entity (e.g., a fragment, a domain, a moiety, etc.). For example, a constitutively active signaling moiety of an enzymatic signaling entity may correspond to an entire enzymatic signaling entity, a fragment of an enzymatic signaling entity or a portion of an enzymatic signaling entity (e.g., a moiety of an enzymatic signaling entity (e.g., including an enzymatic domain) or an enzymatic domain).
[0189] In some embodiments, a constitutively active signaling moiety of a signaling entity may be produced by mutating a sequence (e.g., amino acid or nucleic acid sequence) of a signaling entity. Exemplary mutations include point mutations, additions and/or truncations. Mutations can be made in portions of a signaling entity associated with an activity (e.g., an enzymatic domain, such as a phosphatase domain); however, mutations are not limited to those portions of a signaling entity and may be made in a portion of a signaling entity that impacts, e.g., the conformation or cellular localization of a signaling entity.
[0190] In some embodiments, a constitutively active signaling moiety of a signaling entity may be produced by making post-translational modifications. Post-translational modifications can include, but are not limited to, ubiquitination, phosphorylation, acetylation, glycosylation (N- and O-linked), glycation, myristolyation, palmitoylation, prenylation, amidation, akylation, hydroxylation, biotinylation, pegylation, methylation, sulfation, SUMOylation, dephosphorylation, deacetylation, deglycosylation, deamidation, dihydroxylation, demethylation, deubiquitination, and/or desulfation. Post-translational modifications can be made in portions of a signaling entity associated with an activity (e.g., an enzymatic domain, such as a phosphatase domain); however, post-translational modifications may also be made in a portion of a signaling entity that impacts, e.g., the conformation or cellular localization of a signaling entity.
Trigger-Responsive Immune-Inactivating Signaling Polypeptides
[0191] Among other things, the present disclosure provides a trigger-responsive immune-inactivating signaling polypeptide, which can adopt at least first and second state (e.g., conformations), and can switch from one to the other in response to a particular trigger. In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide is inhibited in one state relative to the other state. In some embodiments, when a trigger-responsive immune-inactivating signaling polypeptide is in its second state, the inhibition is relieved. A trigger-responsive immune-inactivating signaling polypeptide can transition between the first state and the second state when exposed to a trigger.
[0192] In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide (which can be, for example, a fusion polypeptide) can include a modulating domain. A modulating domain can be characterized by an ability to adopt a first state and a second state. In some embodiments, a modulating domain can transition between the first state and the second state when exposed to a trigger. A modulating domain can be a portion of the trigger-responsive immune-inactivating signaling polypeptide that can change conformations, e.g., between a first and second conformation, preferably in response to a particular trigger. The present disclosure recognizes that a modulating domain can be utilized to inhibit, mask and/or inactivate, in a trigger responsive manner, an immune-inactivating moiety.
[0193] Among other things, the present disclosure provides a trigger-responsive dominant negative signaling polypeptide, which can adopt at least first and second state (e.g., conformations), and can switch from one to the other in response to a particular trigger. In some embodiments, a trigger-responsive dominant negative signaling polypeptide is inhibited in one state relative to the other state. In some embodiments, when a trigger-responsive dominant negative signaling polypeptide is in its second state, the inhibition is relieved. A trigger-responsive dominant negative signaling polypeptide can transition between the first state and the second state when exposed to a trigger.
[0194] In some embodiments, a trigger-responsive dominant negative signaling polypeptide (which can be, for example, a fusion polypeptide) can include a modulating domain. A modulating domain can be characterized by an ability to adopt a first state and a second state. In some embodiments, a modulating domain can transition between the first state and the second state when exposed to a trigger. A modulating domain can be a portion of the trigger-responsive dominant negative signaling polypeptide that can change conformations, e.g., between a first and second conformation, preferably in response to a particular trigger. The present disclosure recognizes that a modulating domain can be utilized to inhibit, mask and/or inactivate, in a trigger responsive manner, a dominant negative signaling moiety.
[0195] Among other things, the present disclosure also provides a trigger-responsive constitutively active signaling polypeptide, which can adopt at least first and second state (e.g., conformations), and can switch from one to the other in response to a particular trigger. In some embodiments, a trigger-responsive constitutively active signaling polypeptide is inhibited in one state relative to the other state. In some embodiments, when a trigger-responsive constitutively active signaling polypeptide is in its second state, the inhibition is relieved. A trigger-responsive constitutively active signaling polypeptide can transition between the first state and the second state when exposed to a trigger.
[0196] In some embodiments, a trigger-responsive constitutively active signaling polypeptide (which can be, for example, a fusion polypeptide) can include a modulating domain. A modulating domain can be characterized by an ability to adopt a first state and a second state. In some embodiments, a modulating domain can transition between the first state and the second state when exposed to a trigger. A modulating domain can be a portion of the trigger-responsive constitutively active signaling polypeptide that can change conformations, e.g., between a first and second conformation, preferably in response to a particular trigger. The present disclosure recognizes that a modulating domain can be utilized to inhibit, mask and/or inactivate, in a trigger responsive manner, a constitutively active signaling moiety.
Modulating Domain
[0197] The present disclosure utilizes the insight that ligand binding domains of certain nuclear receptors have been demonstrated to effectively mask or inactivate, in a ligand-binding-dependent-manner, activity of polypeptide agents with which they are operatively associated. For example, Feil, et al. demonstrated the use of an ER(T2) mutated ligand binding domain fragment of human estrogen receptor-α to control the activity of a fusion protein that also included CRE recombinase. (Fiel, et al., Regulation of Cre Recombinase Activity by Mutated Estrogen Receptor Ligand-Binding Domains, Biochem and Biophys Research Comms, 752-757 (1997)). The fusion protein of Feil, et al. has been used to perform tamoxifen mediated excision of target genes in mice and other organisms. There has also been a report of the use of ER(T2) to allow for tamoxifen control the activity of a protein that is located in the cytoplasm, the BRAF kinase (see, for example, Ortiz et al. Genesis 51:448, June 2013; epub Mar. 28, 2013).
[0198] The present disclosure encompasses the recognition that association of such a modulating domain with a dominant negative signaling moiety (e.g., a dominant negative kinase moiety) as described herein can create a trigger-responsive dominant negative signaling polypeptide (e.g., a trigger-responsive dominant negative kinase polypeptide) useful, e.g., to allow for trigger (e.g., ligand) mediated control of activity of a dominant negative signaling moiety (e.g., such as in modulating T cell activity) either in the nucleus or the cytoplasm. Such an application requires a large dynamic range of regulation. For example, it may be necessary for a dominant negative signaling moiety to be mostly or completely inactive in the absence of a trigger and be highly active in the presence of a trigger, e.g., to overcome the activity of a corresponding signaling entity (e.g., a wild-type or endogenous signaling entity). In certain embodiments, an activity of a dominant negative signaling moiety in a trigger-responsive dominant negative signaling polypeptide is regulated in a trigger dose-dependent manner. Among other things, present disclosure utilizes the discovery that trigger-responsive dominant negative signaling polypeptide described herein (e.g., including a dominant negative Zap70 moiety operatively linked to an ER(T2) or ER(T12) domain) has a dynamic range needed to effectively regulate T cells activated by antigen in a finely-tuned manner.
[0199] The present disclosure also encompasses the recognition that association of such a modulating domain with a constitutively active signaling moiety (e.g., a constitutively active phosphatase moiety) as described herein can create a trigger-responsive constitutively active signaling polypeptide (e.g., a trigger-responsive constitutively active phosphatase polypeptide) useful, e.g., to allow for trigger (e.g., ligand) mediated control of activity of a constitutively active signaling moiety (e.g., such as in modulating T cell activity) either in the nucleus or the cytoplasm. Such an application requires a large dynamic range of regulation. For example, it may be necessary for a constitutively active signaling moiety to be mostly or completely inactive in the absence of a trigger and be highly active in the presence of a trigger. In certain embodiments, an activity of a constitutively active signaling moiety in a trigger-responsive constitutively active signaling polypeptide is regulated in a trigger dose-dependent manner. Among other things, present disclosure utilizes the discovery that trigger-responsive constitutively active signaling polypeptide described herein (e.g., including a constitutively active SHP1 moiety operatively linked to an ER(T2) or ER(T12) domain) has a dynamic range needed to effectively regulate T cells activated by antigen in a finely-tuned manner.
[0200] In some embodiments, a modulating domain for use in accordance with the present disclosure comprises a nuclear receptor or a portion thereof. In some embodiments, a nuclear receptor can include a thyroid hormone receptor (e.g. a thyroid hormone receptor-α or a thyroid hormone receptor-ß), a retinoic acid receptor (e.g., a retinoic acid receptor-α, a retinoic acid receptor-ß, or a retinoic acid receptor-γ), a peroxisome proliferator-activated receptor (e.g., a peroxisome proliferator-activated receptor-α, a peroxisome proliferator-activated receptor-ß, or a peroxisome proliferator-activated receptor-γ), a Rev-ErbA receptor, a RAR-related orphan receptor (e.g., a RAR-related orphan receptor-α, a RAR-related orphan receptor-ß, or a RAR-related orphan receptor-γ), a liver X receptor (e.g., a liver X receptor-α or a liver X receptor-ß), a farnesoid X receptor (e.g., a farnesoid X receptor-α or a farnesoid X receptor-ß), a vitamin D receptor, a pregnane X receptor, an androstane receptor, a hepatocyte nuclear factor-4 receptor (e.g., hepatocyte nuclear factor-4-α receptor or hepatocyte nuclear factor-4-γ receptor), a retinoid X receptor (e.g., a retinoid X receptor-α, a retinoid X receptor-ß, or a retinoid X receptor-γ), a testicular receptor (e.g., a testicular receptor 2 or a testicular receptor 4), an estrogen receptor (e.g., an estrogen receptor-α or an estrogen receptor-ß), an estrogen-related receptor (e.g., an estrogen-related receptor-α, an estrogen-related receptor-ß, or an estrogen-related receptor-γ), a glucocorticoid receptor, a mineralocorticoid receptor, a progesterone receptor, or an androgen receptor. In some embodiments, a modulating domain includes a steroid hormone receptor or a portion thereof. In certain embodiments, a modulating domain includes an estrogen receptor or portion thereof; in some such embodiments, a modulating domain includes an estrogen receptor-α or portion thereof.
[0201] In some embodiments, a nuclear receptor is a mammalian nuclear receptor, preferably, a human nuclear receptor. In some embodiments, a nuclear receptor can be a mammalian wild-type nuclear receptor, for example, a human wild-type nuclear receptor. In some embodiments, a nuclear receptor is a homolog of a human nuclear receptor. In some embodiments, a nuclear receptor can be a nuclear receptor variant.
[0202] Canonical nucleotide sequences that encode for nuclear receptors (e.g., wild-type nuclear receptors) are known to those of skill in the art. In some embodiments, a nuclear receptor can be encoded by DNA having a nucleotide sequence substantially similar to a canonical nucleotide sequence encoding for the nuclear receptor. In some embodiments, a nuclear receptor can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a canonical nucleotide sequence for the nuclear receptor.
[0203] In some embodiments, a nuclear receptor can be a hormone receptor. In some embodiments, a hormone receptor can be an estrogen receptor-α, e.g., a human estrogen receptor-α. In some embodiments, an estrogen receptor-α can be encoded by DNA having a nucleotide sequence according to SEQ ID NO: 11. As disclosed herein, SEQ ID NO: 11 represents an exemplary nucleotide sequence encoding an estrogen receptor-α. In some embodiments, an estrogen receptor-α can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 11. In some embodiments, an estrogen receptor-α can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
TABLE-US-00011 SEQ ID NO: 11 ATGACCATGACCCTCCACACCAAAGCATCTGGGATGGCCCTACTGCATCA GATCCAAGGGAACGAGCTGGAGCCCCTGAACCGTCCGCAGCTCAAGATCC CCCTGGAGCGGCCCCTGGGCGAGGTGTACCTGGACAGCAGCAAGCCCGCC GTGTACAACTACCCCGAGGGCGCCGCCTACGAGTTCAACGCCGCGGCCGC CGCCAACGCGCAGGTCTACGGTCAGACCGGCCTCCCCTACGGCCCCGGGT CTGAGGCTGCGGCGTTCGGCTCCAACGGCCTGGGGGGTTTCCCCCCACTC AACAGCGTGTCTCCGAGCCCGCTGATGCTACTGCACCCGCCGCCGCAGCT GTCGCCTTTCCTGCAGCCCCACGGCCAGCAGGTGCCCTACTACCTGGAGA ACGAGCCCAGCGGCTACACGGTGCGCGAGGCCGGCCCGCCGGCATTCTAC AGGCCAAATTCAGATAATCGACGCCAGGGTGGCAGAGAAAGATTGGCCAG TACCAATGACAAGGGAAGTATGGCTATGGAATCTGCCAAGGAGACTCGCT ACTGTGCAGTGTGCAATGACTATGCTTCAGGCTACCATTATGGAGTCTGG TCCTGTGAGGGCTGCAAGGCCTTCTTCAAGAGAAGTATTCAAGGACATAA CGACTATATGTGTCCAGCCACCAACCAGTGCACCATTGATAAAAACAGGA GGAAGAGCTGCCAGGCCTGCCGGCTCCGCAAATGCTACGAAGTGGGAATG ATGAAAGGTGGGATACGAAAAGACCGAAGAGGAGGGAGAATGTTGAAACA CAAGCGCCAGAGAGATGATGGGGAGGGCAGGGGTGAAGTGGGGTCTGCTG GAGACATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGATCAAACGC TCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTCAG TGCCTTGTTGGATGCTGAGCCCCCCATACTCTATTCCGAGTATGATCCTA CCAGACCCTTCAGTGAAGCTTCGATGATGGGCTTACTGACCAACCTGGCA GACAGGGAGCTGGTTCACATGATCAACTGGGCGAAGAGGGTGCCAGGCTT TGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGC TAGAGATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCCAGGG AAGCTACTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCAGGGAAAATG TGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTC GGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAAATCT ATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAA GTCTCTGGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAG ACACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAG CACCAGCGGCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCAGGCACAT GAGTAACAAAGGCATGGAGCATCTGTACAGCATGAAGTGCAAGAACGTGG TGCCCCTCTATGACCTGCTGCTGGAGATGCTGGACGCCCACCGCCTACAT GCGCCCACTAGCCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAAGCCA CTTGGCCACTGCGGGCTCTACTTCATCGCATTCCTTGCAAAAGTATTACA TCACGGGGGAGGCAGAGGGTTTCCCTGCCACGGTCTGA
[0204] Canonical amino acid sequences for nuclear receptors (e.g., wild-type nuclear receptors) are known to those of skill in the art. In some embodiments, a nuclear receptor can have an amino acid sequence substantially similar to a canonical amino acid sequence for the nuclear receptor. In some embodiments, a nuclear receptor can have an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a canonical amino acid sequence for the nuclear receptor.
[0205] In some embodiments, a nuclear receptor can be a hormone receptor. In some embodiments, a hormone receptor can be an estrogen receptor-α, e.g., a human estrogen receptor-α. In some embodiments, an estrogen receptor-α can have an amino acid sequence according to SEQ ID NO: 12. As disclosed herein, SEQ ID NO: 12 represents an exemplary amino acid sequence of an estrogen receptor-α. In some embodiments, an estrogen receptor-α can have an amino acid sequence substantially similar to SEQ ID NO: 12. In some embodiments, an estrogen receptor-α can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12.
TABLE-US-00012 SEQ ID NO: 12 MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYLDSSKPA VYNYPEGAAYEFNAAAAANAQVYGQTGLPYGPGSEAAAFGSNGLGGFPPL NSVSPSPLMLLHPPPQLSPFLQPHGQQVPYYLENEPSGYTVREAGPPAFY RPNSDNRRQGGRERLASTNDKGSMAMESAKETRYCAVCNDYASGYHYGVW SCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGM MKGGIRKDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKR SKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLA DRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPG KLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKS IILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQ HQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLH APTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV
[0206] In some embodiments, a modulating domain is a portion of a nuclear receptor. In some embodiments, a modulating domain can comprise one or more domains of a nuclear receptor. Generally, nuclear receptors are characterized as including five domains: an activation function 1 domain, a DNA binding domain, a hinge domain, a ligand binding domain, and an activation function 2 domain, as shown in
[0207] In some embodiments, a modulating domain can include a ligand binding domain of a nuclear receptor. In certain embodiments, a modulating domain includes an estrogen receptor ligand binding domain, preferably, an estrogen receptor-α ligand binding domain.
[0208] Canonical nucleotide sequences that encode for ligand binding domains of nuclear receptors (e.g., wild-type nuclear receptors) are known to those of skill in the art. In some embodiments, a ligand binding domain of a nuclear receptor can be encoded by DNA having a nucleotide sequence substantially similar to a canonical nucleotide sequence encoding for a ligand binding domain of the nuclear receptor. For example, a ligand binding domain of an estrogen receptor-α of the present disclosure can be encoded by DNA having a nucleotide sequence substantially similar to a canonical nucleotide sequence encoding for a ligand binding domain of an estrogen receptor-α. In some embodiments, a nuclear receptor can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a canonical nucleotide sequence for a ligand binding domain of the nuclear receptor. In some embodiments, a ligand binding domain of an estrogen receptor-α of the present disclosure can be encoded by DNA having a nucleotide sequence substantially similar to a nucleotide sequence comprising or consisting essentially of nucleotides 984 to 1784 of SEQ ID NO: 11. In some embodiments, a nuclear receptor can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleotide sequence comprising or consisting essentially of nucleotides 984 to 1784 of SEQ ID NO: 11.
[0209] Canonical amino acid sequences for ligand binding domains of nuclear receptors (e.g., wild-type nuclear receptors) are known to those of skill in the art. In some embodiments, a ligand binding domain of a nuclear receptor can have an amino acid sequence substantially similar to a canonical amino acid sequence for a ligand binding domain of the nuclear receptor. For example, a ligand binding domain of an estrogen receptor-α of the present disclosure can have an amino acid sequence substantially similar to a canonical amino acid sequence of a ligand binding domain of an estrogen receptor-α. In some embodiments, a nuclear receptor can have an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a canonical amino acid sequence for a ligand binding domain of the nuclear receptor. In some embodiments, a ligand binding domain of an estrogen receptor-α of the present disclosure can have an amino acid sequence substantially similar to an amino acid sequence comprising or consisting essentially of amino acids 302 to 595 of SEQ ID NO: 12. In some embodiments, a nuclear receptor can have an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence comprising or consisting essentially of amino acids 302 to 595 of SEQ ID NO: 12.
[0210] In some embodiments, a modulating domain includes an estrogen receptor ligand binding domain variant. In some embodiments, a modulating domain includes an estrogen receptor-α ligand binding domain variant, such as ER(T2) or ER(T12).
[0211] The present disclosure provides insight that estrogen receptor variants or fragments thereof are effective modulating domains. Furthermore, the present disclosure provides the insight that modulating domains that include estrogen receptor or fragment thereof with a mutation at residue 400 in SEQ ID NO: 12 (which corresponds, e.g., to residue 119 in SEQ ID NO: 4 and residue 415 in SEQ ID NO: 8) may be particularly useful. In some embodiments, a modulating domain includes an estrogen receptor or fragment thereof comprising an amino acid substitution at position G400 of SEQ ID NO: 12. In some embodiments, a modulating domain includes an estrogen receptor or fragment thereof comprising an amino acid substitution at position G400 of SEQ ID NO: 12 with V, M, A, L, or I.
[0212] In some embodiments, a modulating domain includes an estrogen receptor or fragment thereof comprising at least one mutation at a residue corresponding to residue 400, residue 521, residue 539, residue 540, residue 543, and/or residue 544 of SEQ ID NO: 12. Residue 521 of SEQ ID NO: 12 corresponds to residue 240 in SEQ ID NO: 4 and residue 536 in SEQ ID NO: 8. Residue 539 of SEQ ID NO: 12 corresponds to residue 258 in SEQ ID NO: 4 and residue 554 in SEQ ID NO: 8. Residue 540 of SEQ ID NO: 12 corresponds to residue 259 in SEQ ID NO: 4 and residue 555 in SEQ ID NO: 8. Residue 543 of SEQ ID NO: 12 corresponds to residue 262 in SEQ ID NO: 4 and residue 558 in SEQ ID NO: 8, and residue 544 of SEQ ID NO: 12 corresponds to residue 263 in SEQ ID NO: 4 and residue 559 in SEQ ID NO: 8. In some embodiments, a modulating domain includes an estrogen receptor or fragment thereof comprising at least one mutation selected from the group consisting of G400V, G400M, G400A, G400L, G400I, G521R, G521T, L539A, L540A, M543A and L544A, wherein the residue numbering is based on SEQ ID NO: 12. In some embodiments, a modulating domain includes an estrogen receptor or fragment thereof comprising at least one mutation that is either G400V or G400L, wherein the residue numbering is based on SEQ ID NO: 12.
[0213] The present disclosure provides the insight that certain combinations of mutations in an estrogen receptor or fragment thereof are particularly advantageous. For example, in some embodiments, a modulating domain includes an estrogen receptor or fragment thereof comprising at least one mutation selected from the group consisting of G400V, G400M, G400A, G400L, G400I, G521R, and G521T, wherein the residue numbering is based on SEQ ID NO: 12. In certain embodiments, a modulating domain includes an estrogen receptor or fragment thereof comprising at least one mutation that is either G400V or G400L, wherein the residue numbering is based on SEQ ID NO: 12. Without wishing to be bound to any particular theory, mutations at residues corresponding to residues 400 and/or 521 of SEQ ID NO: 12 can facilitate an interaction with heat shock proteins, such as, Hsp90. In some embodiments, a modulating domain includes an estrogen receptor or fragment thereof comprising a second mutation selected from L539A and L540A, wherein the residue numbering is based on SEQ ID NO: 12. In some embodiments, the estrogen receptor or fragment thereof of the modulating domain comprises a second or additional mutation selected from M543A and L544A, wherein the residue numbering is based on SEQ ID NO: 12. Without wishing to be bound to any particular theory, mutations at residues corresponding to residues 539, 540, 543, and/or 544 of SEQ ID NO: 12 can abolish or diminish binding of the binding between estradiol (e.g., 17-beta estradiol) and a ligand binding domain of an estrogen receptor, without affecting or minimally affecting binding between endoxifen or other tamoxifen metabolites and a ligand binding domain of an estrogen receptor.
[0214] In some embodiments, mutation(s) in an estrogen receptor or fragment thereof confer increased affinity for at least one chaperone protein, e.g., Hsp27, Hsp70, and Hsp90. In some embodiments, an estrogen receptor ligand binding domain variant includes mutations that confer on the estrogen receptor ligand binding domain a reduced affinity to at least one naturally occurring estrogen, e.g., estradiol (e.g., 17-beta estradiol), estrone, or estriol. In some embodiments, an estrogen receptor ligand binding domain variant includes mutations that confer on the estrogen receptor ligand binding domain preferential binding to at least one synthetic estrogen receptor ligand, e.g., tamoxifen, endoxifen, or 4-hydroxytamoxifen.
[0215] In some embodiments, an ER(T2) domain can be encoded by DNA having a nucleotide sequence according to SEQ ID NO: 3. As disclosed herein, SEQ ID NO: 3 represents an exemplary nucleotide sequence encoding an ER(T2) domain. In some embodiments, an ER(T2) domain can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 3. In some embodiments, an ER(T2) domain can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3.
TABLE-US-00013 SEQ ID NO: 3 TCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCC AAGCCCGCTCATGATCAAACGCTCTAAGAAGAACA GCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTC AGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTA TTCCGAGTATGATCCTACCAGACCCTTCAGTGAAG CTTCGATGATGGGCTTACTGACCAACCTGGCAGAC AGGGAGCTGGTTCACATGATCAACTGGGCGAAGAG GGTGCCAGGCTTTGTGGATTTGACCCTCCATGATC AGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATC CTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCA CCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCT TGGACAGGAACCAGGGAAAATGTGTAGAGGGCATG GTGGAGATCTTCGACATGCTGCTGGCTACATCATC TCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGT TTGTGTGCCTCAAATCTATTATTTTGCTTAATTCT GGAGTGTACACATTTCTGTCCAGCACCCTGAAGTC TCTGGAAGAGAAGGACCATATCCACCGAGTCCTGG ACAAGATCACAGACACTTTGATCCACCTGATGGCC AAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCG GCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCA GGCACATGAGTAACAAAGGCATGGAGCATCTGTAC AGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGA CCTGCTGCTGGAGGCGGCGGACGCCCACCGCCTAC ATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAG GAGACGGACCAAAGCCACTTGGCCACTGCGGGCTC TACTTCATCGCATTCCTTGCAAAAGTATTACATCA CGGGGGAGGCAGAGGGTTTCCCTGCCACGGTC
[0216] In some embodiments, an ER(T2) domain can have an amino acid sequence according to SEQ ID NO: 4. As disclosed herein, SEQ ID NO: 4 represents an exemplary amino acid sequence of an ER(T2) domain. In some embodiments, an ER(T2) domain can have an amino acid sequence substantially similar to SEQ ID NO: 4. In some embodiments, an ER(T2) domain can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.
TABLE-US-00014 SEQ ID NO: 4 S A G D M R A A N L W P S P L M I K R S K K N S L A L S L T A D Q M V S A L L D A E P P I L Y S E Y D P T R P F S E A S M M G L L T N L A D R E L V H M I N W A K R V P G F V D L T L H D Q V H L L E C A W L E I L M I G L V W R S M E H P V K L L F A P N L L L D R N Q G K C V E G M V E I F D M L L A T S S R F R M M N L Q G E E F V C L K S I I L L N S G V Y T F L S S T L K S L E E K D H I H R V L D K I T D T L I H L M A K A G L T L Q Q Q H Q R L A Q L L L I L S H I R H M S N K G M E H L Y S M K C K N V V P L Y D L L L E A A D A H R L H A P T S R G G A S V E E T D Q S H L A T A G S T S S H S L Q K Y Y I T G E A E G F P A T V
[0217] In some embodiments, an ER(T12) domain can be encoded by DNA having a nucleotide sequence according to SEQ ID NO: 14. As disclosed herein, SEQ ID NO: 14 represents an exemplary nucleotide sequence encoding an ER(T12) domain. In some embodiments, an ER(T12) domain can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 14. In some embodiments, an ER(T12) domain can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 14.
TABLE-US-00015 SEQ ID NO: 14 TCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCC AAGCCCGCTCATGATCAAACGCTCTAAGAAGAACA GCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTC AGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTA TTCCGAGTATGATCCTACCAGACCCTTCAGTGAAG CTTCGATGATGGGCTTACTGACCAACCTGGCAGAC AGGGAGCTGGTTCACATGATCAACTGGGCGAAGAG GGTGCCAGGCTTTGTGGATTTGACCCTCCATGATC AGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATC CTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCA CCCActgAAGCTACTGTTTGCTCCTAACTTGCTCT TGGACAGGAACCAGGGAAAATGTGTAGAGGGCATG GTGGAGATCTTCGACATGCTGCTGGCTACATCATC TCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGT TTGTGTGCCTCAAATCTATTATTTTGCTTAATTCT GGAGTGTACACATTTCTGTCCAGCACCCTGAAGTC TCTGGAAGAGAAGGACCATATCCACCGAGTCCTGG ACAAGATCACAGACACTTTGATCCACCTGATGGCC AAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCG GCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCA GGCACATGAGTAACAAAGGCATGGAGCATCTGTAC AGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGA CCTGCTGCTGGAGgcggcgGACGCCCACCGCCTAC ATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAG GAGACGGACCAAAGCCACTTGGCCACTGCGGGCTC TACTTCATCGCATTCCTTGCAAAAGTATTACATCA CGGGGGAGGCAGAGGGTTTCCCTGCCACGGTC
[0218] In some embodiments, an ER(T12) domain can have an amino acid sequence according to SEQ ID NO: 13. As disclosed herein, SEQ ID NO: 13 represents an exemplary amino acid sequence of an ER(T12) domain. In some embodiments, an ER(T12) domain can have an amino acid sequence substantially similar to SEQ ID NO: 13. In some embodiments, an ER(T12) domain can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13.
TABLE-US-00016 SEQ ID NO: 13 SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMV SALLDAEPPILYSEYDPTRPFSEASMMGLLTNLAD RELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI LMIGLVWRSMEHPLKLLFAPNLLLDRNQGKCVEGM VEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNS GVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMA KAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLY SMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV
[0219] In some embodiments, a modulating domain can include an amino acid sequence that starts at residue 251, 282, or 305 of SEQ ID NO: 12 and ends at residue 545 or 595 of SEQ ID NO: 12. In some embodiments, a modulating domain can have an amino acid sequence substantially similar to an amino acid sequence that starts at residue 251, 282, or 305 of SEQ ID NO: 12 and ends at residue 545 or 595 of SEQ ID NO: 12. In some embodiments, a modulating domain can have an amino acid sequence that is least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence that starts at residue 251, 282, or 305 of SEQ ID NO: 12 and ends at residue 545 or 595 of SEQ ID NO: 12.
[0220] In some embodiments, a modulating domain does not include a hinge domain of a nuclear receptor (see, e.g.,
Arrangement
[0221] The present disclosure recognizes that there are multiple configurations in which an immune-inactivating moiety (such as a dominant negative signaling moiety or constitutively active signaling moiety) can be operatively associated with a modulating domain to form a trigger-responsive immune-inactivating signaling polypeptide. As one example, a trigger-responsive immune-inactivating signaling polypeptide can have an N-terminus and a C-terminus. If a first entity (e.g., a variant, portion, domain or moiety) is “upstream” of a second entity, the first entity is closer to the N-terminus than the second entity. Conversely, if a first entity is “downstream” of a second entity, the first entity is closer to the C-terminus than the second entity. In some embodiments, an immune-inactivating signaling moiety can be upstream of a modulating domain in a trigger-responsive immune-inactivating signaling polypeptide of the present disclosure (see, e.g.,
[0222] In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide can include one or more immune-inactivating signaling moieties. For example, a trigger-responsive immune-inactivating signaling polypeptide can include one, two, or three immune-inactivating signaling moieties. In some embodiments, the one or more immune-inactivating signaling moieties of a trigger-responsive immune-inactivating signaling polypeptide are the same immune-inactivating signaling moiety or are different immune-inactivating signaling moieties.
[0223] In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide can include one or more modulating domains (see, e.g.,
[0224] In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide can include an immune-inactivating signaling moiety between modulating domains (see, e.g.,
[0225] In some embodiments, an immune-inactivating signaling moiety can be operatively linked to a modulating domain directly (see, e.g.,
[0226] In some embodiments, one or more immune-inactivating signaling moieties can be operatively linked to one another directly. In other embodiments, one or more immune-inactivating signaling moieties can be operatively linked to one another indirectly, e.g., via a linker. In some embodiments, a linker can comprise a polyalanine (including, e.g., 1-10 alanines).
[0227] In some embodiments, one or more modulating domains can be operatively linked to one another directly (see, e.g.,
Additional Moieties
[0228] In addition to an immune-inactivating signaling moiety and/or a modulating domain, a trigger-responsive immune-inactivating signaling polypeptide can include additional moieties, such as regulatory elements, signal sequences, and tags. In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide includes a nuclear export signal (NES). A nuclear export signal can be a short amino acid sequence that targets an associated polypeptide for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. In some embodiments, a nuclear export signal includes at least four hydrophobic residues. SEQ ID NO: 5 includes a nucleotide sequence encoding an exemplary nuclear export signal. In some embodiments, a nuclear export signal can have an amino acid sequence according to SEQ ID NO: 6.
TABLE-US-00017 SEQ ID NO: 5 AATGAATTAGCCTTGAAATTAGCAGGTCTTGATA TCAACAAGACA SEQ ID NO: 6 N E L A L K L A G L D I N K T
Triggers
[0229] The present disclosure encompasses trigger-responsive immune-inactivating signaling polypeptides that can adopt at least first and second state, and can switch from one to the other in response to a particular trigger. The present disclosure utilizes a trigger as mechanism to tightly control the activity of an immune-inactivating signaling moiety in a trigger-responsive immune-inactivating signaling polypeptide via a modulating domain. In some embodiments, the present disclosure provides technologies in which a trigger-responsive immune-inactivating signaling polypeptide, is exposed to a trigger for a limited period of time (e.g., due to removal, expiration, inactivation, and/or destruction of the trigger). The present disclosure provides an insight that reversibility of immune-inactivating activity according to such technologies offers unique advantages for regulation of T cell activity, among other things avoiding difficulties associated with alternative approaches for regulating T cells where T cell activity, once triggered, cannot readily be shut back off Indeed, in some embodiments, the present disclosure provides systems that permit not simply “on-off” control of T cell activity, but potentially adjustable “dial-up/dial-down” control (e.g., based on concentration, intensity, or frequency of trigger).
[0230] In some embodiments, a trigger can be a condition, e.g., a local condition. For example, a trigger can be a particular pH range, temperature range, range of oxygen levels, etc. In some embodiments, a trigger can be an entity, such as a molecule, e.g., a small molecule or a macromolecule (e.g., a polypeptide, nucleic acid or carbohydrate).
[0231] In some embodiments, when a trigger is not present or is present at a level below a threshold value, a modulating domain can be in a first state. In some embodiments, when a trigger is present or is present at a level above a threshold value, a modulating domain can be in a second state. In some embodiments, a trigger can be introduced, for example, by adding a trigger to a sample (e.g., cells) or administering a trigger to a subject (e.g., a human). In certain embodiments, a trigger-responsive immune-inactivating signaling polypeptide is only exposed to or in the presence of a trigger when its switch between a first state and a second state is desired.
[0232] In some embodiments, a trigger has a temporal nature. In some embodiments, a trigger can have a relatively short-half life in a system (e.g., cells, tissue, subject (e.g., human)) to which the trigger has been introduced. For example, a trigger can have a half-life of no more than 1 hour, no more than 2 hours, no more than 5 hours, no more than 12 hours, no more than 24 hours, or no more than two days.
[0233] In some embodiments, a trigger can have a relatively rapid clearance from a system (e.g., cells, tissue, subject (e.g., human)) to which the trigger has been introduced. For example, a trigger can have 95% clearance from a system in less than 30 min, in less than an hour, in less than 2 hours, in less than 5 hours, in less than 12 hours, in less than 24 hours, or in less than two days.
[0234] As discussed above, a trigger-responsive immune-inactivating signaling polypeptide can include a modulating domain, which can be the portion of the trigger-responsive immune-inactivating signaling polypeptide that adopts at least a first and a second state, and can switch from one to the other in response to a particular trigger. In some embodiments, a modulating domain can include a nuclear receptor or a portion thereof. In embodiments in which a trigger-responsive immune-inactivating signaling polypeptide includes a modulating domain comprising a ligand binding domain of a nuclear receptor, a trigger can be a ligand or other agent that binds to the ligand binding domain. In some embodiments, a ligand can be a natural ligand of a ligand binding domain. In some embodiments, a ligand can be a synthetic ligand designed to bind a ligand binding domain. Exemplary ligands that bind to ligand binding domains of select nuclear receptors are shown in Table 5 below.
TABLE-US-00018 TABLE 5 Nuclear Receptor Exemplary Ligands thyroid hormone receptor (e.g. a thyroid Thyroid hormone hormone receptor-α or a thyroid hormone Thyroid hormone analogs receptor-β) Thyroid hormone derivatives retinoic acid receptor (e.g., a retinoic acid Vitamin A receptor-α, a retinoic acid receptor-β, or a Vitamin A analogs retinoic acid receptor-γ) Vitamin A derivatives, including retinoic acid peroxisome proliferator-activated receptor Prostaglandin (e.g., a peroxisome proliferator-activated Fatty Acids receptor-α, a peroxisome proliferator-activated Eicosanoids receptor-β, or a peroxisome proliferator- 5-oxo-15(S)-HETE activated receptor-γ) 5-oxo-ETE 15(S)-HETE 15(R)-HETE 15-HpETE Leukotriene B4 Rev-ErbA receptor Heme RAR-related orphan receptor (e.g., a RAR- Cholesterol related orphan receptor-α, a RAR-related Cholesterol derivatives orphan receptor-β, or a RAR-related orphan Tretinoin receptor-γ) Melatonin liver X receptor Oxysterols, including 22(R)- (e.g., a liver X receptor-α or a liver hydroxycholesterol, 24(S)-hydroxycholesterol, X receptor-β) 27-hydroxycholesterol, and cholestenoic acid farnesoid X receptor Oxysterols (e.g., a farnesoid X receptor-α or a Chenodeoxycholic acid farnesoid X receptor-β) vitamin D receptor Vitamin D androstane receptor Androstane hepatocyte nuclear factor-4 receptor (e.g., Fatty Acids hepatocyte nuclear factor-4-α receptor or Linoleic acid hepatocyte nuclear factor-4-γ receptor) retinoid X receptor (e.g., a retinoid X Retinoids, including 9-cis retinoic acid receptor-α, a retinoid X receptor-β, or a and 9-cis-13,14-dihydro-retinoic acid retinoid X receptor-γ) estrogen receptor (e.g., an estrogen Estradiol (e.g., 17-beta estradiol) receptor-α or an estrogen receptor-β) Estrone Estriol Raloxifene Genistein Endoxifen Tamoxifen 4-hydroxytamoxifen Fulvestrant OP-1250 OP-1124 OP-1074 AZD-9496 ARN-810 SRN-927 SERMs and SERDs Estrogen analogs glucocorticoid receptor Glucocorticoids, including cortisol mineralocorticoid receptor Mineralocorticoids, including aldosterone and deoxycorticosterone Glucocorticoids, including cortisol Sprionolactone Eplerenone progesterone receptor Progesterone Progesterone analogs Progesterone derivatives androgen receptor Testosterone Testosterone analogs Testosterone derivatives 2-Quinolones Phthalamides Bicalutamides Coumarins Nonsteroidal SARMS
Pharmaceutical Compositions
[0235] In some embodiments, a trigger can be included in a pharmaceutical composition. In some embodiments, a pharmaceutical composition can include physiologically acceptable carrier or excipient. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc., as well as combinations thereof. A pharmaceutical composition can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like), which do not deleteriously react with the active compounds or interfere with their activity. In certain embodiments, a water-soluble carrier suitable for intravenous administration is used. In some embodiments, a pharmaceutical composition can be sterile.
[0236] A suitable pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. A pharmaceutical composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. A pharmaceutical composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral pharmaceutical compositions can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharine, cellulose, magnesium carbonate, etc.
[0237] A pharmaceutical composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. The formulation of a pharmaceutical composition should suit the mode of administration. For example, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where a pharmaceutical composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where a pharmaceutical composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[0238] A trigger described herein can be formulated as neutral or salt forms in a pharmaceutical composition. A trigger can include pharmaceutical composition that has received regulatory approval.
Nucleic Acids
[0239] Among other things, the present disclosure provides nucleic acids encoding a trigger-responsive immune-inactivating signaling polypeptide described herein. Such nucleic acids can be DNA or RNA.
[0240] In a certain embodiment, a trigger-responsive dominant negative signaling polypeptide is an endoxifen-responsive dominant negative Zap-70 polypeptide. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide is encoded by a nucleotide sequence according to SEQ ID NO: 7 or SEQ ID NO: 29. As disclosed herein, SEQ ID NO: 7 and SEQ ID NO: 29 represent exemplary nucleotide sequences encoding endoxifen-responsive dominant negative Zap-70 polypeptides. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 7 or SEQ ID NO: 29. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 7 or the nucleotide sequence of SEQ ID NO: 29.
TABLE-US-00019 SEQ ID NO: 7 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACAATGCCAGACCCCGCGGCGCACC TGCCCTTCTTCTACGGCAGCATCTCGCGTGCCGAG GCCGAGGAGCACCTGAAGCTGGCGGGCATGGCGGA CGGGCTCTTCCTGCTGCGCCAGTGCCTGCGCTCGC TGGGCGGCTATGTGCTGTCGCTCGTGCACGATGTG CGCTTCCACCACTTTCCCATCGAGCGCCAGCTCAA CGGCACCTACGCCATTGCCGGCGGCAAAGCGCACT GTGGACCGGCAGAGCTCTGCGAGTTCTACTCGCGC GACCCCGACGGGCTGCCCTGCAACCTGCGCAAGCC GTGCAACCGGCCGTCGGGCCTCGAGCCGCAGCCGG GGGTCTTCGACTGCCTGCGAGACGCCATGGTGCGT GACTACGTGCGCCAGACGTGGAAGCTGGAGGGCGA GGCCCTGGAGCAGGCCATCATCAGCCAGGCCCCGC AGGTGGAGAAGCTCATTGCTACGACGGCCCACGAG CGGATGCCCTGGTACCACAGCAGCCTGACGCGTGA GGAGGCCGAGCGCAAACTTTACTCTGGGGCGCAGA CCGACGGCAAGTTCCTGCTGAGGCCGCGGAAGGAG CAGGGCACATACGCCCTGTCCCTCATCTATGGGAA GACGGTGTACCACTACCTCATCAGCCAAGACAAGG CGGGCAAGTACTGCATTCCCGAGGGCACCAAGTTT GACACGCTCTGGCAGCTGGTGGAGTATCTGAAGCT GAAGGCGGACGGGCTCATCTACTGCCTGAAGGAGG CCTGCCCCAACAGCAGTGCCAGCAACGCCTCAGGG GCTGCTGCTCCCACACTCCCAGCCCACCCATCCAC GTTGACGGGATCCTCTGCTGGAGACATGAGAGCTG CCAACCTTTGGCCAAGCCCGCTCATGATCAAACGC TCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGC CGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGC CCCCCATACTCTATTCCGAGTATGATCCTACCAGA CCCTTCAGTGAAGCTTCGATGATGGGCTTACTGAC CAACCTGGCAGACAGGGAGCTGGTTCACATGATCA ACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTG ACCCTCCATGATCAGGTCCACCTTCTAGAATGTGC CTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGC GCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCT CCTAACTTGCTCTTGGACAGGAACCAGGGAAAATG TGTAGAGGGCATGGTGGAGATCTTCGACATGCTGC TGGCTACATCATCTCGGTTCCGCATGATGAATCTG CAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTAT TTTGCTTAATTCTGGAGTGTACACATTTCTGTCCA GCACCCTGAAGTCTCTGGAAGAGAAGGACCATATC CACCGAGTCCTGGACAAGATCACAGACACTTTGAT CCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGC AGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATC CTCTCCCACATCAGGCACATGAGTAACAAAGGCAT GGAGCATCTGTACAGCATGAAGTGCAAGAACGTGG TGCCCCTCTATGACCTGCTGCTGGAGGCGGCGGAC GCCCACCGCCTACATGCGCCCACTAGCCGTGGAGG GGCATCCGTGGAGGAGACGGACCAAAGCCACTTGG CCACTGCGGGCTCTACTTCATCGCATTCCTTGCAA AAGTATTACATCACGGGGGAGGCAGAGGGTTTCCC TGCCACGGTC
[0241] In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide is encoded by a nucleotide sequence according to SEQ ID NO: 16 or SEQ ID NO: 32. As disclosed herein, SEQ ID NO: 16 and SEQ ID NO: 32 represent exemplary nucleotide sequences encoding endoxifen-responsive dominant negative Zap-70 polypeptides. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 16 or SEQ ID NO: 32. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 16 or the nucleotide sequence of SEQ ID NO: 32.
TABLE-US-00020 SEQ ID NO: 16 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACAATGCCAGACCCCGCGGCGCACC TGCCCTTCTTCTACGGCAGCATCTCGCGTGCCGAG GCCGAGGAGCACCTGAAGCTGGCGGGCATGGCGGA CGGGCTCTTCCTGCTGCGCCAGTGCCTGCGCTCGC TGGGCGGCTATGTGCTGTCGCTCGTGCACGATGTG CGCTTCCACCACTTTCCCATCGAGCGCCAGCTCAA CGGCACCTACGCCATTGCCGGCGGCAAAGCGCACT GTGGACCGGCAGAGCTCTGCGAGTTCTACTCGCGC GACCCCGACGGGCTGCCCTGCAACCTGCGCAAGCC GTGCAACCGGCCGTCGGGCCTCGAGCCGCAGCCGG GGGTCTTCGACTGCCTGCGAGACGCCATGGTGCGT GACTACGTGCGCCAGACGTGGAAGCTGGAGGGCGA GGCCCTGGAGCAGGCCATCATCAGCCAGGCCCCGC AGGTGGAGAAGCTCATTGCTACGACGGCCCACGAG CGGATGCCCTGGTACCACAGCAGCCTGACGCGTGA GGAGGCCGAGCGCAAACTTTACTCTGGGGCGCAGA CCGACGGCAAGTTCCTGCTGAGGCCGCGGAAGGAG CAGGGCACATACGCCCTGTCCCTCATCTATGGGAA GACGGTGTACCACTACCTCATCAGCCAAGACAAGG CGGGCAAGTACTGCATTCCCGAGGGCACCAAGTTT GACACGCTCTGGCAGCTGGTGGAGTATCTGAAGCT GAAGGCGGACGGGCTCATCTACTGCCTGAAGGAGG CCTGCCCCAACAGCAGTGCCAGCAACGCCTCAGGG GCTGCTGCTCCCACACTCCCAGCCCACCCATCCAC GTTGACGGGATCCTCTGCTGGAGACATGAGAGCTG CCAACCTTTGGCCAAGCCCGCTCATGATCAAACGC TCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGC CGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGC CCCCCATACTCTATTCCGAGTATGATCCTACCAGA CCCTTCAGTGAAGCTTCGATGATGGGCTTACTGAC CAACCTGGCAGACAGGGAGCTGGTTCACATGATCA ACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTG ACCCTCCATGATCAGGTCCACCTTCTAGAATGTGC CTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGC GCTCCATGGAGCACCCACTGAAGCTACTGTTTGCT CCTAACTTGCTCTTGGACAGGAACCAGGGAAAATG TGTAGAGGGCATGGTGGAGATCTTCGACATGCTGC TGGCTACATCATCTCGGTTCCGCATGATGAATCTG CAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTAT TTTGCTTAATTCTGGAGTGTACACATTTCTGTCCA GCACCCTGAAGTCTCTGGAAGAGAAGGACCATATC CACCGAGTCCTGGACAAGATCACAGACACTTTGAT CCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGC AGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATC CTCTCCCACATCAGGCACATGAGTAACAAAGGCAT GGAGCATCTGTACAGCATGAAGTGCAAGAACGTGG TGCCCCTCTATGACCTGCTGCTGGAGGCGGCGGAC GCCCACCGCCTACATGCGCCCACTAGCCGTGGAGG GGCATCCGTGGAGGAGACGGACCAAAGCCACTTGG CCACTGCGGGCTCTACTTCATCGCATTCCTTGCAA AAGTATTACATCACGGGGGAGGCAGAGGGTTTCCC TGCCACGGTCTGA
[0242] In a certain embodiment, a trigger-responsive dominant negative signaling polypeptide is an endoxifen-responsive dominant negative LCK polypeptide. In some embodiments, an endoxifen-responsive dominant negative LCK polypeptide is encoded by a nucleotide sequence according to SEQ ID NO: 22 or SEQ ID NO: 34. As disclosed herein, SEQ ID NO: 22 and SEQ ID NO: 34 represent exemplary nucleotide sequences encoding endoxifen-responsive dominant negative LCK polypeptides. In some embodiments, an endoxifen-responsive dominant negative LCK polypeptide can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 22 or SEQ ID NO: 34. In some embodiments, an endoxifen-responsive dominant negative LCK polypeptide can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 22 or the nucleotide sequence of SEQ ID NO: 34.
TABLE-US-00021 SEQ ID NO: 22 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACAATGGGCTGTGGCTGCAGCTCAC ACCCGGAAGATGACTGGATGGAAAACATCGATGTG TGTGAGAACTGCCATTATCCCATAGTCCCACTGGA TGGCAAGGGCACGCTGCTCATCCGAAATGGCTCTG AGGTGCGGGACCCACTGGTTACCTACGAAGGCTCC AATCCGCCGGCTTCCCCACTGCAAGACAACCTGGT TATCGCTCTGCACAGCTATGAGCCCTCTCACGACG GAGATCTGGGCTTTGAGAAGGGGGAACAGCTCCGC ATCCTGGAGCAGAGCGGCGAGTGGTGGAAGGCGCA GTCCCTGACCACGGGCCAGGAAGGCTTCATCCCCT TCAATTTTGTGGCCAAAGCGAACAGCCTGGAGCCC GAACCCTGGTTCTTCAAGAACCTGAGCCGCAAGGA CGCGGAGCGGCAGCTCCTGGCGCCCGGGAACACTC ACGGCTCCTTCCTCATCCGGGAGAGCGAGAGCACC GCGGGATCGTTTTCACTGTCGGTCCGGGACTTCGA CCAGAACCAGGGAGAGGTGGTGAAACATTACAAGA TCCGTAATCTGGACAACGGTGGCTTCTACATCTCC CCTCGAATCACTTTTCCCGGCCTGCATGAACTGGT CCGCCATTACACCAATGCTTCAGATGGGCTGTGCA CACGGTTGAGCCGCCCCTGCCAGACCCAGAAGCCC CAGAAGCCGTGGTGGGAGGACGAGTGGGAGGTTCC CAGGGAGACGCTGAAGCTGGTGGAGCGGCTGGGGG CTGGACAGTTCGGGGAGGTGTGGATGGGGTACTAC AACGGGGGATCCTCTGCTGGAGACATGAGAGCTGC CAACCTTTGGCCAAGCCCGCTCATGATCAAACGCT CTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCC GACCAGATGGTCAGTGCCTTGTTGGATGCTGAGCC CCCCATACTCTATTCCGAGTATGATCCTACCAGAC CCTTCAGTGAAGCTTCGATGATGGGCTTACTGACC AACCTGGCAGACAGGGAGCTGGTTCACATGATCAA CTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTGA CCCTCCATGATCAGGTCCACCTTCTAGAATGTGCC TGGCTAGAGATCCTGATGATTGGTCTCGTCTGGCG CTCCATGGAGCACCCACTGAAGCTACTGTTTGCTC CTAACTTGCTCTTGGACAGGAACCAGGGAAAATGT GTAGAGGGCATGGTGGAGATCTTCGACATGCTGCT GGCTACATCATCTCGGTTCCGCATGATGAATCTGC AGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATT TTGCTTAATTCTGGAGTGTACACATTTCTGTCCAG CACCCTGAAGTCTCTGGAAGAGAAGGACCATATCC ACCGAGTCCTGGACAAGATCACAGACACTTTGATC CACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCA GCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATCC TCTCCCACATCAGGCACATGAGTAACAAAGGCATG GAGCATCTGTACAGCATGAAGTGCAAGAACGTGGT GCCCCTCTATGACCTGCTGCTGGAGGCGGCGGACG CCCACCGCCTACATGCGCCCACTAGCCGTGGAGGG GCATCCGTGGAGGAGACGGACCAAAGCCACTTGGC CACTGCGGGCTCTACTTCATCGCATTCCTTGCAAA AGTATTACATCACGGGGGAGGCAGAGGGTTTCCCT GCCACGGTCTGA
[0243] In a certain embodiment, a trigger-responsive constitutively active signaling polypeptide is an endoxifen-responsive constitutively active SHP1 polypeptide. In some embodiments, an endoxifen-responsive constitutively active SHP1 polypeptide is encoded by a nucleotide sequence according to SEQ ID NO: 26 or SEQ ID NO: 36. As disclosed herein, SEQ ID NO: 26 and or SEQ ID NO: 36 represent exemplary nucleotide sequences encoding endoxifen-responsive constitutively active SHP1 polypeptides. In some embodiments, an endoxifen-responsive constitutively active SHP1 polypeptide can be encoded by DNA having a nucleotide sequence substantially similar to SEQ ID NO: 26 or SEQ ID NO: 36. In some embodiments, an endoxifen-responsive constitutively active SHP1 polypeptide can be encoded by DNA having a nucleotide sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 26 or the nucleotide sequence of SEQ ID NO: 36.
TABLE-US-00022 SEQ ID NO: 26 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACACGGCAGCCGTACTATGCCACGA GGGTGAATGCGGCTGACATTGAGAACCGAGTGTTG GAACTGAACAAGAAGCAGGAGTCCGAGGATACAGC CAAGGCTGGCTTCTGGGAGGAGTTTGAGAGTTTGC AGAAGCAGGAGGTGAAGAACTTGCACCAGCGTCTG GAAGGGCAGCGGCCAGAGAACAAGGGCAAGAACCG CTACAAGAACATTCTCCCCTTTGACCACAGCCGAG TGATCCTGCAGGGACGGGACAGTAACATCCCCGGG TCCGACTACATCAATGCCAACTACATCAAGAACCA GCTGCTAGGCCCTGATGAGAACGCTAAGACCTACA TCGCCAGCCAGGGCTGTCTGGAGGCCACGGTCAAT GACTTCTGGCAGATGGCGTGGCAGGAGAACAGCCG TGTCATCGTCATGACCACCCGAGAGGTGGAGAAAG GCCGGAACAAATGCGTCCCATACTGGCCCGAGGTG GGCATGCAGCGTGCTTATGGGCCCTACTCTGTGAC CAACTGCGGGGAGCATGACACAACCGAATACAAAC TCCGTACCTTACAGGTCTCCCCGCTGGACAATGGA GACCTGATTCGGGAGATCTGGCATTACCAGTACCT GAGCTGGCCCGACCATGGGGTCCCCAGTGAGCCTG GGGGTGTCCTCAGCTTCCTGGACCAGATCAACCAG CGGCAGGAAAGTCTGCCTCACGCAGGGCCCATCAT CGTGCACTGCAGCGCCGGCATCGGCCGCACAGGCA CCATCATTGTCATCGACATGCTCATGGAGAACATC TCCACCAAGGGCCTGGACTGTGACATTGACATCCA GAAGACCATCCAGATGGTGCGGGCGCAGCGCTCGG GCATGGTGCAGACGGAGGCGCAGTACAAGTTCATC TACGTGGCCATCGCCCAGTTCATTGAAACCACTAA GAAGAAGCTGGAGGTCCTGCAGTCGCAGAAGGGCC AGGAGTCGGAGTACGGGAACATCACCTATCCCCCA GCCATGAAGAATGCCCATGCCAAGGCCTCCCGCAC CTCGTCCAAACACAAGGAGGATGTGTATGAGAACC TGCACACTAAGAACAAGAGGGAGGAGAAAGTGAAG AAGCAGCGGTCAGCAGACAAGGAGAAGAGCAAGGG TTCCCTCAAGAGGAAGGGATCCTCTGCTGGAGACA TGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATG ATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTC CCTGACGGCCGACCAGATGGTCAGTGCCTTGTTGG ATGCTGAGCCCCCCATACTCTATTCCGAGTATGAT CCTACCAGACCCTTCAGTGAAGCTTCGATGATGGG CTTACTGACCAACCTGGCAGACAGGGAGCTGGTTC ACATGATCAACTGGGCGAAGAGGGTGCCAGGCTTT GTGGATTTGACCCTCCATGATCAGGTCCACCTTCT AGAATGTGCCTGGCTAGAGATCCTGATGATTGGTC TCGTCTGGCGCTCCATGGAGCACCCACTGAAGCTA CTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCA GGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCG ACATGCTGCTGGCTACATCATCTCGGTTCCGCATG ATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAA ATCTATTATTTTGCTTAATTCTGGAGTGTACACAT TTCTGTCCAGCACCCTGAAGTCTCTGGAAGAGAAG GACCATATCCACCGAGTCCTGGACAAGATCACAGA CACTTTGATCCACCTGATGGCCAAGGCAGGCCTGA CCCTGCAGCAGCAGCACCAGCGGCTGGCCCAGCTC CTCCTCATCCTCTCCCACATCAGGCACATGAGTAA CAAAGGCATGGAGCATCTGTACAGCATGAAGTGCA AGAACGTGGTGCCCCTCTATGACCTGCTGCTGGAG GCGGCGGACGCCCACCGCCTACATGCGCCCACTAG CCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAA GCCACTTGGCCACTGCGGGCTCTACTTCATCGCAT TCCTTGCAAAAGTATTACATCACGGGGGAGGCAGA GGGTTTCCCTGCCACGGTCTGA
[0244] In some embodiments, RNA encoding a trigger-responsive immune-inactivating signaling polypeptide described herein can be transcribed from one of the nucleic acid sequences described herein.
[0245] In certain instances, recombinant DNA techniques can be used to produce a trigger-responsive immune-inactivating signaling polypeptide. The process of cloning DNA (e.g., cDNA) segments and sequences that encode the respective polypeptides, polypeptide fragments, domains, and/or moieties (e.g., a modulating domain and an immune-inactivating signaling moiety), the production of DNA sequences encoding any of various peptide linkers, the ligation of different DNA (e.g., cDNA sequences), the construction of the expression vectors (e.g., plasmid, bacteriophage, phagemid, or viral vector), and the protein expression and purification of a resulting recombinant polypeptide (e.g., a fusion polypeptide) can be performed by conventional recombinant molecular biology and protein biochemistry techniques such as those described in Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons (2014) (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc. (2005).
[0246] Expression of a trigger-responsive immune-inactivating signaling polypeptide can include construction of an expression vector containing a polynucleotide that encodes a trigger-responsive immune-inactivating signaling polypeptide described herein. An expression vector polynucleotide can further include sequences that encode additional amino acids for the purpose of protein purification, or identifying or locating a trigger-responsive immune-inactivating signaling polypeptide in the expression system or during the protein purification process. Once a polynucleotide encoding a trigger-responsive immune-inactivating signaling polypeptide has been obtained, the vector for the production of a trigger-responsive immune-inactivatingsignaling polypeptide can be produced by recombinant DNA technology using techniques well known in the art. In addition to including a polynucleotide that encodes a trigger-responsive immune-inactivatingsignaling polypeptide, an expression vector can also include, e.g., appropriate replication, transcriptional and translational control signals.
[0247] In one aspect, provided herein is a vector comprising a nucleic acid sequence encoding a trigger-responsive immune-inactivatingsignaling polypeptide described herein. In some embodiments, a vector can include one or more regulatory elements (e.g., viral arms, origins of replication, integration elements, etc.) that permit transfer from one context to another and/or delivery to a particular context of interest. In some embodiments, the vector can be a plasmid, a bacteriophage, a phagemid, a cosmid, a viral vector, or a viral particle. These vectors are known in the art. In one embodiment, provided herein is a plasmid comprising a nucleic acid sequence encoding a trigger-responsive immune-inactivatingsignaling polypeptide described herein. For example, the plasmid is a bacterial plasmid. In one embodiment of a vector described, the vector is an expression vector. For example, the plasmid (vector) is an expression plasmid for the recombinant protein expression in a bacteria, e.g., Escherichia coli. In one embodiment of an expression vector described, the expression vector is a bacterial expression vector. In one embodiment of an expression vector described, the expression vector is a prokaryotic expression vector. In one embodiment of an expression vector described, the expression vector is an eukaryotic expression vector. In one embodiment of an expression vector described, the expression vector is a mammalian expression vector. In one embodiment, the expression vector is a yeast expression vector.
[0248] The expression vector can be transferred to a host cell by conventional techniques and the transfected cells can then be cultured by conventional techniques to produce a trigger-responsive immune-inactivatingsignaling polypeptide of the present disclosure. Thus, the present disclosure encompasses host cells containing a polynucleotide encoding a trigger-responsive immune-inactivatingsignaling polypeptide, operably linked to a promoter. Various regulatory sequences or elements may be incorporated in a vector suitable for the present invention. Exemplary regulatory sequences or elements include, but are not limited to, promoters, enhancers, repressors or suppressors, 5′ untranslated (or noncoding) sequences, introns, 3′ untranslated (or non-coding) sequences, terminators, and splice elements.
[0249] As used herein, a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter bound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bound at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. The promoter may be operably associated with or operably linked to the expression control sequences, including enhancer and repressor sequences or with a nucleic acid to be expressed. In some embodiments, the promoter may be inducible. In some embodiments, the inducible promoter may be unidirectional or bio-directional. In some embodiments, the promoter may be a constitutive promoter. In some embodiments, the promoter can be a hybrid promoter, in which the sequence containing the transcriptional regulatory region is obtained from one source and the sequence containing the transcription initiation region is obtained from a second source. Systems for linking control elements to coding sequence within a transgene are well known in the art (general molecular biological and recombinant DNA techniques are described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Commercial vectors suitable for inserting a transgene for expression in various host cells under a variety of growth and induction conditions are also well known in the art.
[0250] In some embodiments, a specific promoter may be used to control expression of a nucleic acid encoding a trigger-responsive immune-inactivatingsignaling polypeptide in a mammalian host cell such as, but are not limited to, SRα-promoter (Takebe, et al., Molec. and Cell. Bio. 8:466-472 (1988)), the human CMV immediate early promoter (Boshart, et al., Cell 41:521-530 (1985); Foecking, et al., Gene 45:101-105 (1986)), human CMV promoter, the human CMV5 promoter, the murine CMV immediate early promoter, the EF1-α-promoter, a hybrid CMV promoter for liver specific expression (e.g., made by conjugating CMV immediate early promoter with the transcriptional promoter elements of either human α-1-antitrypsin (HAT) or albumin (HAL) promoter), or promoters for hepatoma specific expression (e.g., wherein the transcriptional promoter elements of either human albumin (HAL; about 1000 bp) or human α-1-antitrypsin (HAT, about 2000 bp) are combined with a 145 long enhancer element of human α-1-microglobulin and bikunin precursor gene (AMBP); HAL-AMBP and HAT-AMBP); the SV40 early promoter region (Benoist, et al., Nature 290:304-310 (1981)), the Orgyia pseudotsugata immediate early promoter, the herpes thymidine kinase promoter (Wagner, et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)); or the regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)). In some embodiments, the mammalian promoter is a is a constitutive promoter such as, but not limited to, the hypoxanthine phosphoribosyl transferase (HPTR) promoter, the adenosine deaminase promoter, the pyruvate kinase promoter, the beta-actin promoter as well as other constitutive promoters known to those of ordinary skill in the art.
[0251] In some embodiments, a specific promoter may be used to control expression of a nucleic acid encoding a trigger-responsive immune-inactivatingsignaling polypeptide in a prokaryotic host cell such as, but are not limited to, the β-lactamase promoter (Villa-Komaroff, et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)); the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); the T7 promoter, the T3 promoter, the M13 promoter or the M16 promoter; in a yeast host cell such as, but are not limited to, the GAL1, GAL4 or GAL10 promoter, the ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, glyceraldehyde-3-phosphate dehydrogenase III (TDH3) promoter, glyceraldehyde-3-phosphate dehydrogenase II (TDH2) promoter, glyceraldehyde-3-phosphate dehydrogenase I (TDH1) promoter, pyruvate kinase (PYK), enolase (ENO), or triose phosphate isomerase (TPI).
[0252] In some embodiments, the promoter may be a viral promoter, many of which are able to regulate expression of a nucleic acid encoding a trigger-responsive immune-inactivatingsignaling polypeptide in several host cell types, including mammalian cells. Viral promoters that have been shown to drive constitutive expression of coding sequences in eukaryotic cells include, for example, simian virus promoters, herpes simplex virus promoters, papilloma virus promoters, adenovirus promoters, human immunodeficiency virus (HIV) promoters, Rous sarcoma virus promoters, cytomegalovirus (CMV) promoters, the long terminal repeats (LTRs) of Moloney murine leukemia virus and other retroviruses, the thymidine kinase promoter of herpes simplex virus as well as other viral promoters known to those of ordinary skill in the art.
[0253] In some embodiments, the gene control elements of an expression vector may also include 5′ non-transcribing and 5′ non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, Kozak sequence and the like. Enhancer elements can optionally be used to increase expression levels of a polypeptide or protein to be expressed. Examples of enhancer elements that have been shown to function in mammalian cells include the SV40 early gene enhancer, as described in Dijkema, et al., EMBO J. (1985) 4: 761 and the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (RSV), as described in Gorman, et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and human cytomegalovirus, as described in Boshart, et al., Cell (1985) 41:521. Genetic control elements of an expression vector will also include 3′ non-transcribing and 3′ non-translating sequences involved with the termination of transcription and translation. Respectively, such as a poly polyadenylation (polyA) signal for stabilization and processing of the 3′ end of an mRNA transcribed from the promoter. Poly A signals included, for example, the rabbit beta globin polyA signal, bovine growth hormone polyA signal, chicken beta globin terminator/polyA signal, or SV40 late polyA region.
[0254] Expression vectors will preferably, but optionally, be include at least one selectable marker. In some embodiments, the selectable maker is a nucleic acid sequence encoding a resistance gene operably linked to one or more genetic regulatory elements, to bestow upon a host cell the ability to maintain viability when grown in the presence of a cytotoxic chemical and/or drug. In some embodiments, a selectable agent may be used to maintain retention of the expression vector within the host cell. In some embodiments, the selectable agent is may be used to prevent modification (i.e. methylation) and/or silencing of the transgene sequence within the expression vector. In some embodiments, a selectable agent is used to maintain episomal expression of the vector within the host cell. In some embodiments, the selectable agent is used to promote stable integration of the transgene sequence into the host cell genome. In some embodiments, an agent and/or resistance gene may include, but is not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017, ampicillin, neomycin (G418), zeomycin, mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) for eukaryotic host cell; tetracycline, ampicillin, kanamycin or chloramphenicol for a prokaryotic host cell; and URA3, LEU2, HIS3, LYS2, HIS4, ADE8, CUP1 or TRP1 for a yeast host cell.
[0255] Expression vectors may be transfected, transformed or transduced into a host cell. As used herein, the terms “transfection,” “transformation” and “transduction” all refer to the introduction of an exogenous nucleic acid sequence into a host cell. In some embodiments, expression vectors containing nucleic acid sequences encoding for I2S and/or FGE are transfected, transformed or transduced into a host cell at the same time. In some embodiments, expression vectors containing nucleic acid sequences encoding for I2S and/or FGE are transfected, transformed or transduced into a host cell sequentially. For example, a vector encoding an I2S protein may be transfected, transformed or transduced into a host cell first, followed by the transfection, transformation or transduction of a vector encoding an FGE protein, and vice versa. Examples of transformation, transfection and transduction methods, which are well known in the art, include liposome delivery, i.e., Lipofectamine™ (Gibco BRL) Method of Hawley-Nelson, Focus 15:73 (1193), electroporation, CaPO4 delivery method of Graham and van der Erb, Virology, 52:456-457 (1978), DEAE-Dextran medicated delivery, microinjection, biolistic particle delivery, polybrene mediated delivery, cationic mediated lipid delivery, transduction, and viral infection, such as, e.g., retrovirus, lentivirus, adenovirus adenoassociated virus and Baculovirus (Insect cells). General aspects of cell host transformations have been described in the art, such as by Axel in U.S. Pat. No. 4,399,216; Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, chapters 1, 9, 13, 15, and 16. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology (1989), Keown et al., Methods in Enzymology, 185:527-537 (1990), and Mansour et al., Nature, 336:348-352 (1988).
[0256] For long-term, high-yield production of recombinant proteins, stable expression is often preferred. For example, cell lines which stably express a trigger-responsive immune-inactivatingsignaling polypeptide can be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express a trigger-responsive immune-inactivatingsignaling polypeptide. Such engineered cell lines can be particularly useful in screening and evaluation of compounds that interact directly or indirectly with a trigger-responsive immune-inactivatingsignaling polypeptide.
[0257] A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., Cell, 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy, et al., Cell, 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sci. USA, 77:357 (1980); O'Hare, et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418; Wu and Wu, Biotherapy, 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 32:573-596 (1993); Mulligan, Science, 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem., 62:191-217 (1993); Can, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Current Protocols in Molecular Biology, Ausubel, et al., eds. (John Wiley & Sons, N Y 1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual (Stockton Press, N Y 1990); and Current Protocols in Human Genetics, Dracopoli, et al., eds. (John Wiley & Sons, N Y 1994), Chapters 12 and 13; Colberre-Garapin, et al., J. Mol. Biol., 150:1 (1981).
[0258] The expression levels of a trigger-responsive immune-inactivatingsignaling polypeptide described herein can be increased by vector amplification (for a review, see Bebbington and Hentschel, “The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning,” Vol. 3. (Academic Press, New York (1987)). When a marker in the vector system expressing a trigger-responsive immune-inactivatingsignaling polypeptide is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleic acid sequence encoding a trigger-responsive immune-inactivatingsignaling polypeptide described herein, production of a trigger-responsive immune-inactivatingsignaling polypeptide can also increase (Crouse, et al., Mol. Cell. Biol., 3:257 (1983)).
Polypeptides
[0259] Among other things, the present disclosure provides a trigger-responsive dominant negative signaling polypeptide described herein.
[0260] In certain embodiments, a trigger-responsive dominant negative signaling polypeptide is an endoxifen-responsive dominant negative Zap-70 polypeptide. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide has an amino acid sequence according to SEQ ID NO: 8 or SEQ ID NO: 30. As disclosed herein, SEQ ID NO: 8 and SEQ ID NO: 30 represent exemplary amino acid sequences of endoxifen-responsive dominant negative Zap-70 polypeptides. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide can have an amino acid sequence substantially similar to SEQ ID NO: 8 or SEQ ID NO: 30. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide can have an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 30.
TABLE-US-00023 SEQ ID NO: 8 M N E L A L K L A G L D I N K T M P D P A A H L P F F Y G S I S R A E A E E H L K L A G M A D G L F L L R Q C L R S L G G Y V L S L V H D V R F H H F P I E R Q L N G T Y A I A G G K A H C G P A E L C E F Y S R D P D G L P C N L R K P C N R P S G L E P Q P G V F D C L R D A M V R D Y V R Q T W K L E G E A L E Q A I I S Q A P Q V E K L I A T T A H E R M P W Y H S S L T R E E A E R K L Y S G A Q T D G K F L L R P R K E Q G T Y A L S L I Y G K T V Y H Y L I S Q D K A G K Y C I P E G T K F D T L W Q L V E Y L K L K A D G L I Y C L K E A C P N S S A S N A S G A A A P T L P A H P S T L T G S S A G D M R A A N L W P S P L M I K R S K K N S L A L S L T A D Q M V S A L L D A E P P I L Y S E Y D P T R P F S E A S M M G L L T N L A D R E L V H M I N W A K R V P G F V D L T L H D Q V H L L E C A W L E I L M I G L V W R S M E H P V K L L F A P N L L L D R N Q G K C V E G M V E I F D M L L A T S S R F R M M N L Q G E E F V C L K S I I L L N S G V Y T F L S S T L K S L E E K D H I H R V L D K I T D T L I H L M A K A G L T L Q Q Q H Q R L A Q L L L I L S H I R H M S N K G M E H L Y S M K C K N V V P L Y D L L L E A A D A H R L H A P T S R G G A S V E E T D Q S H L A T A G S T S S H S L Q K Y Y I T G E A E G F P A T V
[0261] In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide has an amino acid sequence according to SEQ ID NO: 15 or SEQ ID NO: 31. As disclosed herein, SEQ ID NO: 15 and SEQ ID NO: 31 represent exemplary amino acid sequences of endoxifen-responsive dominant negative Zap-70 polypeptides. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide can have an amino acid sequence substantially similar to SEQ ID NO: 15 or SEQ ID NO: 31. In some embodiments, an endoxifen-responsive dominant negative Zap-70 polypeptide can have an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 15 or the nucleotide sequence of SEQ ID NO: 31.
TABLE-US-00024 SEQ ID NO: 15 MNELALKLAGLDINKTMPDPAAHLPFFYGSISRAE AEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDV RFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSR DPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVR DYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHE RMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKE QGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKF DTLWQLVEYLKLKADGLIYCLKEACPNSSASNASG AAAPTLPAHPSTLTGSSAGDMRAANLWPSPLMIKR SKKNSLALSLTADQMVSALLDAEPPILYSEYDPTR PFSEASMMGLLTNLADRELVHMINWAKRVPGFVDL TLHDQVHLLECAWLEILMIGLVWRSMEHPLKLLFA PNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNL QGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHI HRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLI LSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAAD AHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQ KYYITGEAEGFPATV
[0262] In certain embodiments, a trigger-responsive dominant negative signaling polypeptide is an endoxifen-responsive dominant negative LCK polypeptide. In some embodiments, an endoxifen-responsive dominant negative LCK polypeptide has an amino acid sequence according to SEQ ID NO: 18 or SEQ ID NO: 33. As disclosed herein, SEQ ID NO: 18 and SEQ ID NO: 33 represent exemplary amino acid sequences of endoxifen-responsive dominant negative LCK polypeptides. In some embodiments, an endoxifen-responsive dominant negative LCK polypeptide can have an amino acid sequence substantially similar to SEQ ID NO: 18 or SEQ ID NO: 33. In some embodiments, an endoxifen-responsive dominant negative LCK polypeptide can have an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 18 or the nucleotide sequence of SEQ ID NO: 33.
TABLE-US-00025 SEQ ID NO: 18 MNELALKLAGLDINKTMGCGCSSHPEDDWMENIDV CENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGS NPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLR ILEQSGEWWKAQSLTTGQEGFIPFNFVAKANSLEP EPWFFKNLSRKDAERQLLAPGNTHGSFLIRESEST AGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYIS PRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKP QKPWWEDEWEVPRETLKLVERLGAGQFGEVWMGYY NGGSSAGDMRAANLWPSPLMIKRSKKNSLALSLTA DQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLT NLADRELVHMINWAKRVPGFVDLTLHDQVHLLECA WLEILMIGLVWRSMEHPLKLLFAPNLLLDRNQGKC VEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSII LLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLI HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM EHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGG ASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFP ATV
[0263] In certain embodiments, a trigger-responsive constitutively active signaling polypeptide is an endoxifen-responsive constitutively active SHP1 polypeptide. In some embodiments, an endoxifen-responsive constitutively active SHP1 polypeptide has an amino acid sequence according to SEQ ID NO: 24 or SEQ ID NO: 35. As disclosed herein, SEQ ID NO: 24 and SEQ ID NO: 35 represent exemplary amino acid sequences of endoxifen-responsive constitutively active SHP1 polypeptides. In some embodiments, an endoxifen-responsive constitutively active SHP1 polypeptide can have an amino acid sequence substantially similar to SEQ ID NO: 24 or SEQ ID NO: 35. In some embodiments, an endoxifen-responsive constitutively active SHP1 polypeptide can have an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 24 or the nucleotide sequence of SEQ ID NO: 35.
TABLE-US-00026 SEQ ID NO: 24 MNELALKLAGLDINKTRQPYYATRVNAADIENRVL ELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRL EGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPG SDYINANYIKNQLLGPDENAKTYIASQGCLEATVN DFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEV GMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNG DLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQ RQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENI STKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFI YVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPP AMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVK KQRSADKEKSKGSLKRKGSSAGDMRAANLWPSPLM IKRSKKNSLALSLTADQMVSALLDAEPPILYSEYD PTRPFSEASMMGLLTNLADRELVHMINWAKRVPGF VDLTLHDQVHLLECAWLEILMIGLVWRSMEHPLKL LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRM MNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEK DHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQL LLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLE AADAHRLHAPTSRGGASVEETDQSHLATAGSTSSH SLQKYYITGEAEGFPATV
[0264] Once a trigger-responsive immune-inactivating signaling polypeptide of the invention has been expressed, it can be purified by any method known in the art for protein purification for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, a trigger-responsive immune-inactivating signaling polypeptide described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
[0265] For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins can be used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpress™ system (Qiagen®) useful with histidine-tagged proteins. Tags can also facilitate the detection of a trigger-responsive immune-inactivating signaling polypeptide. Examples of such tags can include the various fluorescent proteins (e.g., GFP), as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagluttinin (HA), and c-myc tags.
[0266] In view of the above, one aspect provided herein is a method of producing or manufacturing a trigger-responsive immune-inactivating signaling polypeptide described herein. In some embodiments, a method of producing or manufacturing a trigger-responsive immune-inactivating signaling polypeptide described herein includes expressing the trigger-responsive immune-inactivating signaling polypeptide from a nucleic acid or a vector that encodes the trigger-responsive immune-inactivating signaling polypeptide in a cell (e.g., a host cell). In some embodiments, a method further comprises recovering the trigger-responsive immune-inactivating signaling polypeptide.
[0267] In some embodiments, a method can include (a) culturing a cell comprising a nucleic acid sequence encoding a trigger-responsive immune-inactivating signaling polypeptide described herein, or a vector (e.g., a plasmid) comprising a nucleic acid sequence encoding a trigger-responsive immune-inactivating signaling polypeptide described herein, or a viral particle comprising such a nucleic acid or a vector, where the culturing is performed under conditions such that the trigger-responsive immune-inactivating signaling polypeptide is expressed; and (b) recovering the trigger-responsive immune-inactivating signaling polypeptide.
[0268] In some embodiments, a method of manufacturing a trigger-responsive immune-inactivating signaling polypeptide can include expressing the trigger-responsive immune-inactivating signaling polypeptide from the nucleic acid or the vector described herein in a host cell. In some embodiments, a method of manufacturing a trigger-responsive immune-inactivating signaling polypeptide can include recovering an expressed trigger-responsive immune-inactivating signaling polypeptide from a host cell.
[0269] In some embodiments, a method of manufacture can include introducing a nucleic acid or a vector described herein into a T cell.
Viral Particle
[0270] Among other things, the present disclosure provides a viral particle comprising a nucleic acid sequence encoding a trigger-responsive immune-inactivating signaling polypeptide described herein or a trigger-responsive immune-inactivating signaling polypeptide described herein. In some embodiments, a viral particle can include an adenoviral particle, retroviral particle, lentiviral particle, and/or combinations thereof.
Cells
[0271] Among other things, the present disclosure provides a cell comprising a nucleic acid sequence (such as a vector) encoding a trigger-responsive immune-inactivating signaling polypeptide described herein or a viral particle described herein. A variety of technologies are known to those skilled in the art for engineering any of a variety of cells to contain and/or express a nucleic acid (e.g., a nucleic acid vector) encoding a trigger-responsive immune-inactivating signaling polypeptide as described herein (see, for example, Green & Sambrook Molecular Cloning, Cold Spring Harbor Laboratory Press). To give but a few examples, available technologies for introducing nucleic acids into mammalian cells include transfection (e.g., mediated by cationic lipid reagents, by calcium phosphate, by DEAE-Dextran, by DOTMA/DOGS, by electroporation, and/or by combinations thereof) and use of viral vectors (e.g., adenoviral vectors, retroviral vectors, lentiviral vectors, and/or combinations thereof).
[0272] In some embodiments, a provided cell may (e.g., may be engineered to) transiently contain and/or express a nucleic acid that encodes trigger-responsive immune-inactivating signaling polypeptide; in some embodiments, a provided cell may (e.g., may be engineered to) stably contain and/or express a nucleic acid that encodes trigger-responsive immune-inactivating signaling polypeptide. In some embodiments, a provided cell may (e.g., may be engineered to) contain and/or express multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more copies or instances of a nucleic acid that encodes trigger-responsive immune-inactivating signaling polypeptide; in some embodiments, a provided cell may (e.g., may be engineered to) contain and/or express only a single copy of a nucleic acid that encodes trigger-responsive immune-inactivating signaling polypeptide.
[0273] In some embodiments, a cell as provided herein may be designed, engineered and/or utilized for production and/or secretion of a trigger-responsive immune-inactivating signaling polypeptide as described herein. A variety of host-expression vector systems can be utilized to express a trigger-responsive immune-inactivating signaling polypeptide described herein. Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express a trigger-responsive immune-inactivating signaling polypeptide described herein in situ. These include but are not limited to microorganisms such as prokaryotic bacteria (e.g., attenuated Bacillus anthracis strains, E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a trigger-responsive immune-inactivating signaling polypeptide coding sequence; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing a trigger-responsive immune-inactivating signaling polypeptide coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a trigger-responsive immune-inactivating signaling polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a trigger-responsive immune-inactivating signaling polypeptide coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, NS0, 293, or 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In some embodiments, a cell may be or comprise a human cell (e.g., a T cell such as a CAR-T or TCR-T cell as described herein).
[0274] A host cell can be chosen that modulates the expression of an inserted sequence, or modifies and processes a gene product in the specific fashion desired. In some embodiments, modifications (e.g., glycosylation) and processing (e.g., cleavage) of polypeptide products (e.g., a trigger-responsive immune-inactivating signaling polypeptide) can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
[0275] In some embodiments, mammalian host cells can include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells. In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the coding sequence of trigger-responsive immune-inactivating signaling polypeptide can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) can result in a recombinant virus that is viable and capable of expressing a trigger-responsive immune-inactivating signaling polypeptide in infected hosts. (See, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984)). Specific initiation signals can also be required for efficient translation of inserted trigger-responsive immune-inactivating signaling polypeptide coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner, et al., Methods in Enzymol., 153:51-544 (1987)).
[0276] In some embodiments, a host cell can be a cell of the immune system (e.g., a monocyte, eosinophil, neutrophil, basophil, macrophage, dendritic cell, natural killer cell, T cell (e.g., helper T cell and cytotoxic T cell), T regulatory cell, or B cell). In some embodiments, a host cell can be a T cell, e.g., primary T cell or an immortal T cell line. An immortal T cell line can be a Jurkat cell line, for example, Neo Jurkat cells, BCL2 Jurkat cells, Jurkat E6.1 cells, J.RT3-T3.5 cells, Daudi cells, HuT78 cells, I9.2 cells, or Loucy cells. In some embodiments, a T cell can be a wild-type T cell. In some embodiments, a T cell can be an engineered T cell, e.g., a CAR-T cell.
[0277] CAR-T cells are T cells that have been engineered to express a chimeric antigen receptor (a “CAR”). Typically, CARs are composed of an extracellular antigen-recognition moiety that is linked, via spacer/hinge and transmembrane domains, to an intracellular signaling domain that can include costimulatory domains and T cell activation moieties. In some embodiments, CARs can recognize unprocessed antigens independently of their expression of major histocompatibility antigens, which is one example of how CARs can differ from wild-type TCRs. In some embodiments, a CAR can be characterized by its ability to bind to a protein, a polypeptide, a carbohydrate, a ganglioside, a proteoglycan, and or a glycosylated protein.
[0278] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for a trigger-responsive immune-inactivating signaling polypeptide being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of a trigger-responsive immune-inactivating signaling polypeptide, vectors which direct the expression of high levels of a trigger-responsive immune-inactivating signaling polypeptide product that can be readily purified can be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther, et al., EMBO J., 2:1791 (1983)), in which a trigger-responsive immune-inactivating signaling polypeptide coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res., 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem., 24:5503-5509 (1989)); and the like pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such trigger-responsive immune-inactivating signaling polypeptides are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. Alternately, the pET expression vectors can be used for producing histidine-tagged recombinant proteins, where the histidine-tagged recombinant proteins can be affinity purified by a nickel column. Expression of recombinant proteins in Pichia pastoris is described by Holliger, P., Meth. Mol. Biol., 178:349-57 (2002). In some embodiments, expression of a trigger-responsive immune-inactivating signaling polypeptide can be under the control of an inducible expression system, e.g., IPTG-inducible expression in E. coli, baculovirus expression, or methanol-inducible AOX1-directed expression in P. pastoris.
[0279] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. A virus can grow in Spodoptera frugiperda cells. A trigger-responsive immune-inactivating signaling polypeptide coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
[0280] Large scale expression of heterologous proteins in the algae Chlamydomonas reinhardtii are described by Griesbeck, C., et al., Mol. Biotechnol. 34:213-33 (2006); Manuell, A L, et al. Plant Biotechnol J. Eprint (2007); Franklin S E and Mayfield S P, Expert Opin Biol Ther. 5(2):225-35 (2007); Mayfield S P and Franklin S E, Vaccine, 23(15):1828-32 (2005); and Fuhrmann M., Methods Mol Med. 94:191-5 (2004). Foreign heterologous coding sequences can be inserted into the genome of the nucleus, chloroplast and mitochondria by homologous recombination. The chloroplast expression vector p64 carrying the most versatile chloroplast selectable marker aminoglycoside adenyl transferase (aadA), which confers resistance to spectinomycin or streptomycin, can be used to express foreign protein in the chloroplast. A biolistic gene gun method can be used to introduce the vector in the algae. Upon its entry into chloroplasts, the foreign DNA can be released from the gene gun particles and integrates into the chloroplast genome through homologous recombination.
Compositions that Deliver a Trigger-Responsive Immune-Inactivating Signaling Polypeptide
[0281] In accordance with the present disclosure, any of a variety of modalities may be utilized to deliver a trigger-responsive immune-inactivating signaling polypeptide described herein. To give but a few examples, in some embodiments, an immune-inactivating signaling polypeptide as described herein is administered (i.e., to a subject or system). In some embodiments, a nucleic acid that encodes an immune-inactivating signaling polypeptide may be administered; in some such embodiments, the encoding nucleic acid may be associated with one or more elements that directs its expression. In some embodiments, a cell containing and/or expressing an immune-inactivating signaling polypeptide and/or a nucleic acid that encodes it is administered; in some such embodiments, the cell is an immune system cell, e.g., a monocyte, eosinophil, neutrophil, basophil, macrophage, dendritic cell, natural killer cell, T cell (e.g., helper T cell and cytotoxic T cell), T regulatory cell, or B cell. In some embodiments, the cell is a T cell (e.g., a CAR-T or TCR T cell). In some embodiments, a T cell (e.g., a CAR-T or TCR T cell) that has been engineered to contain (e.g., to express) a trigger-responsive immune-inactivating signaling polypeptide, and/or a nucleic acid that encodes it, is administered. In some embodiments, a viral particle containing an immune-inactivating signaling polypeptide and/or a nucleic acid that encodes and/or expresses it is administered.
[0282] Thus some embodiments, a trigger-responsive immune-inactivating signaling polypeptide described herein can be directly administered. As such, in some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein includes a trigger-responsive immune-inactivating signaling polypeptide described herein.
[0283] In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide described herein can be delivered by delivering a nucleic acid that encodes a trigger-responsive immune-inactivating signaling polypeptide described herein, a vector that includes such a nucleic acid, a cell that includes a nucleic acid that encodes a trigger-responsive immune-inactivating signaling polypeptide described herein, a cell that includes a vector comprising a nucleic acid that encodes a trigger-responsive immune-inactivating signaling polypeptide described herein, and/or a cell that includes a trigger-responsive immune-inactivating signaling polypeptide described herein. As such, in some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein includes a nucleic acid that encodes a trigger-responsive immune-inactivating signaling polypeptide described herein, a vector that includes such a nucleic acid, a cell that includes a nucleic acid that encodes a trigger-responsive immune-inactivating signaling polypeptide described herein, a cell that includes a vector comprising a nucleic acid that encodes a trigger-responsive immune-inactivating signaling polypeptide described herein, and/or a cell that includes a trigger-responsive immune-inactivating signaling polypeptide described herein.
[0284] In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide described herein can be delivered by delivering a viral particle that comprises a nucleic acid that encodes a trigger-responsive immune-inactivating signaling polypeptide described herein, a vector that includes such a nucleic acid, and/or a trigger-responsive immune-inactivating signaling polypeptide described herein. As such, in some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein includes a viral particle that comprises a nucleic acid that encodes a trigger-responsive immune-inactivating signaling polypeptide described herein, a vector that includes such a nucleic acid, and/or a trigger-responsive immune-inactivating signaling polypeptide described herein. Exemplary nucleic acids, vectors, cells and viral particles are described herein.
Pharmaceutical Compositions
[0285] In some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide can be a pharmaceutical composition. In some embodiments, a pharmaceutical composition can include physiologically acceptable carrier or excipient. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc., as well as combinations thereof. A pharmaceutical composition can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like), which do not deleteriously react with the active compounds or interfere with their activity. In certain embodiments, a water-soluble carrier suitable for intravenous administration is used. In some embodiments, a pharmaceutical composition can be sterile.
[0286] A suitable pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. A pharmaceutical composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. A pharmaceutical composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral pharmaceutical compositions can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharine, cellulose, magnesium carbonate, etc.
[0287] A pharmaceutical composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. The formulation of a pharmaceutical composition should suit the mode of administration. For example, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where a pharmaceutical composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where a pharmaceutical composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[0288] A trigger-responsive immune-inactivating signaling polypeptide described herein can be formulated as neutral or salt forms in a pharmaceutical composition. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
Uses
Methods of Regulating the Activity of T Cells
[0289] The present disclosure recognizes that therapies involving the activation of T cells (e.g., ATCT) show promise in treating various conditions and/or diseases (e.g., cancer). However, the present disclosure also recognizes that, while activated T cells can be a powerful tool in treating various conditions and/or diseases, controlling T cell activation presents a significant challenge and a risk to patient health. For example, uncontrolled T cell activation can result in a “cytokine storm,” a potential lethal outcome. Therefore, there remains a need in the field for methods of treating subjects (e.g., human patients) that utilizes activated T cells, but also able to “dial back” T cell activity, if and when desired. The present disclosure addresses this need and provides methods by which activity of a T cell population (which may be a maintained T cell population) can be reversibly decreased and increased through application and removal of a trigger. Combining trigger-responsiveness with maintenance of T cell levels (and, in at least some embodiments, reversibility and/or tunability through adjustment of trigger “intensity”—e.g., concentration, level and/or frequency of application, etc) provides a remarkably sophisticated and effective system that, moreover, is applicable to any of a variety of T cell populations including, for example, existing ATCT (e.g., CAR-T and/or TCR) T cell populations. In many embodiments, provided methods allow for reversible inhibition of T cell activity.
[0290] In addition, the present disclosure provides a variety of other advantages relative to available method for regulating T cell activity including, for example, that methods utilizing a trigger-responsive immune-inactivating signaling peptide described herein can inhibit T cell activity without destroying T cells. This advantage allows for a substantial improvement in patient care. As discussed above, adoptive T Cell Therapy (ATCT) is one current approach that shows promise in treating various conditions and/or diseases (e.g., cancer). ATCT entails collection and isolation of T cells from a subject (e.g., a patient). Isolated T cells are then clonally enriched, modified, and/or engineered to achieve a T cell population having desired properties and/or characteristics. The T cell population can then be expanded through ex-vivo growth and reintroduced into the subject to allow the enriched, modified, and/or engineered T cells to specifically attack cells of interest. Using current methodologies, the reintroduced T cells (e.g., genetically modified T cells, e.g., CAR-T cells) are destroyed if a decrease in T cell activity is necessitated. Consequently, in order for a patient to continue with T cell therapy or undergo a subsequent round of T cell therapy, the patient may need to go through painful, expensive and/or time intensive procedures, to allow for the isolation of T cells, enrichment of T cells, modification and/or engineering of a T cell population, and reintroduction of T cells into the patient. The methods provided herein allow for control of T cell activity without destroying T cells. The provided methods represent a significant improvement in patient care because they reduce the risk of an adverse event involving increased and undesired T cell activity (e.g., a cytokine storm). Regardless, if such an adverse event were to occur, the provided methods eliminate or reduce the need for subsequent procedures needed to continue T cell therapy, which can significantly improve the patient experience and/or patient accessibility to T cell therapies.
[0291] In some embodiments, a method of regulating activity of T cells includes introducing a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide as described herein. In some embodiments, introducing a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide as described herein can include introducing the composition that delivers the trigger-responsive immune-inactivating signaling polypeptide to a cell, which can be performed, e.g., in vitro or ex vivo. In some embodiments, such a cell can be a primary T cell, a modified and/or engineered T cell (e.g., a CAR-T cell), or a T cell line (e.g., a Jurkat cell line).
[0292] In some embodiments, a primary T cell is obtained from a subject (e.g., a patient, e.g., a human patient). In some embodiments, a primary T cell is modified and/or engineered, e.g., to express a chimeric antigen receptor (CAR).
[0293] In some embodiments, a provided method can include introducing the composition that delivers the trigger-responsive immune-inactivating signaling polypeptide to a primary T cell or modified and/or engineered T cell. In some embodiments, following the introduction of the composition that delivers the trigger-responsive immune-inactivating signaling polypeptide into the primary T cell or modified and/or engineered T cell, the resulting T cell is introduced into a subject. In some embodiments, the subject into which the resulting T cell is introduced is the same subject the primary T cell is obtained from. In some embodiments, the subject into which the resulting T cell is introduced is a different subject than the primary T cell is obtained from.
[0294] In some embodiments, introducing a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide as described herein can include administering the composition that delivers the trigger-responsive immune-inactivating signaling polypeptide to a subject (e.g., a patient, e.g., a human patient). A composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein can be administered by any appropriate route. In some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein is administered systemically. Systemic administration may be intravenous, intradermal, intracranial, intrathecal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, oral, and/or transmucosal administration. In some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein is administered subcutaneously. As used herein, the term “subcutaneous tissue,” is defined as a layer of loose, irregular connective tissue immediately beneath the skin. For example, the subcutaneous administration may be performed by injecting a composition into areas including, but not limited to, the thigh region, abdominal region, gluteal region, or scapular region. In some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein is administered intravenously. In some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein is administered orally. In some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein is administered intracranially. In some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein is administered intrathecally. As used herein, the term “intrathecal administration” or “intrathecal injection” refers to an injection into the spinal canal (intrathecal space surrounding the spinal cord). Various techniques may be used including, without limitation, lateral cerebroventricular injection through a burrhole or cisternal or lumbar puncture or the like. More than one route can be used concurrently, if desired.
[0295] The present disclosure contemplates single, as well as, multiple administrations of a therapeutically effective amount of a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein. In some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition.
[0296] In many embodiments, a method of regulating activity of T cells includes introducing a trigger described herein. In some embodiments, introducing a trigger as described herein can include introducing the trigger to a cell, which can be performed, e.g., in vitro or ex vivo. In some embodiments, such a cell can be a primary T cell, a modified and/or engineered T cell (e.g., a CAR-T cell), or a T cell line (e.g., a Jurkat cell line).
[0297] In some embodiments, introducing a trigger as described herein can include administering the trigger to a subject (e.g., a patient, e.g., a human patient). A trigger described herein can be administered by any appropriate route. In some embodiments, a trigger described herein is administered systemically. Systemic administration may be intravenous, intradermal, intracranial, intrathecal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, oral, and/or transmucosal administration. In some embodiments, a trigger described herein is administered subcutaneously. As used herein, the term “subcutaneous tissue,” is defined as a layer of loose, irregular connective tissue immediately beneath the skin. For example, the subcutaneous administration may be performed by injecting a composition into areas including, but not limited to, the thigh region, abdominal region, gluteal region, or scapular region. In some embodiments, a trigger described herein is administered intravenously. In some embodiments, a trigger is administered orally. In some embodiments, a trigger is administered intracranially. In some embodiments, a trigger is administered intrathecally. As used herein, the term “intrathecal administration” or “intrathecal injection” refers to an injection into the spinal canal (intrathecal space surrounding the spinal cord). Various techniques may be used including, without limitation, lateral cerebroventricular injection through a burrhole or cisternal or lumbar puncture or the like. More than one route of administering a trigger can be used concurrently, if desired.
[0298] In some embodiments, a trigger is present in the blood of a subject at a free concentration of greater than 1 picomolar, greater than 10 picomolar, greater than 100 picomolar, greater than 1 nanomolar, greater than 10 nanomolar, greater than 100 nanomolar, or greater than 1 micromolar. In some embodiments, a trigger is present in the blood of a subject at a free concentration of less than 1 micromolar, less than 100 nanomolar, less than 10 nanomolar, less than 1 nanomolar, less than 100 picomolar, or less than 10 picomolar. In some embodiments, a trigger is present in the blood of a subject at a free concentration of between 1 picomolar and 1 nanomolar, between 1 picomolar and 100 picomolar, or between 1 picomolar and 10 picomolar. In some embodiments, a trigger is present in the blood of a subject at a total concentration of greater than 1 picomolar, greater than 10 picomolar, greater than 100 picomolar, greater than 1 nanomolar, greater than 10 nanomolar, greater than 100 nanomolar, greater than 1 micromolar, or greater than 10 micromolar. In some embodiments, a trigger is present in the blood of a subject at a total concentration of less than 100 micromolar, less than 10 micromolar, less than 1 micromolar, less than 100 nanomolar, less than 10 nanomolar, less than 1 nanomolar, less than 100 picomolar, or less than 10 picomolar. In some embodiments, a trigger is present in the blood of a subject at a total concentration of between 10 nanomolar and 100 micromolar, between 100 nanomolar and 10 micromolar, or between 1 micromolar and 10 micromolar.
[0299] The present disclosure contemplates single, as well as, multiple administrations of a therapeutically effective amount of a composition that delivers a trigger described herein. In some embodiments, a trigger described herein can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition. In some embodiments, a composition that delivers a trigger-responsive immune-inactivating signaling polypeptide described herein can be administered when T cell activity (as determined by a level of, e.g., a cytokine, e.g., IL-2) exceeds a threshold.
[0300] The present disclosure encompasses methods of regulating activity of T cells, which can include endogenous T cells, and/or engineered and/or modified T cells (e.g., CAR-T cells). In some embodiments, provided methods of regulating T cells can include regulating the activity of an endogenous TCR (e.g., a wild-type TCR or an endogenous TCR variant) or the activity of an engineered and/or modified TCR or a CAR. In some embodiments, provided methods of regulating T cells can include regulating the activity of CARs that target CD19, CD20, CD22, Igk light chain, CD30, CD138, BCMA, CD33, CD123, NKG2D ligands, ROR1, EGFR, EFGRvIII, GD2, IL13Ra2, HER2, Mesotheli, PSMA, FAP, GPC3, MET, MUC16, CEA, Lewis-Y, or MUC1. In some embodiments, provided methods of regulating T cells can include regulating the activity of CARs that target various neoantigens.
[0301] The present disclosure encompasses methods of regulating activity of T cells, which can include endogenous T cells present in a subject, engineered and/or modified T cells present in a subject (e.g., having been previously administered or introduced), or engineered and/or modified T cells being administered to a subject. In some embodiments, a method of regulating activity of T cells includes administering a modified and/or engineered T cell (e.g., a genetically modified T cell, e.g., a CAR-T cell) to the subject.
Therapeutic Uses
[0302] The present disclosure recognizes that methods of regulating activity of T cells described herein can be useful as methods of treating various conditions (e.g., T cell exhaustion or cytokine dysregulation, e.g., hypercytokinemia) and/or diseases (e.g., cancer).
[0303] Among other things, the present disclosure provides a method of preventing or treating cytokine dysregulation. In some embodiments, a method of preventing or treating cytokine dysregulation includes administering a composition that delivers the trigger-responsive immune-inactivating signaling polypeptide as described herein to a subject (e.g., a patient, e.g., a human patient). In some embodiments, a method of preventing or treating cytokine dysregulation includes administering a trigger as described herein. In some embodiments, a composition that delivers the trigger-responsive immune-inactivating signaling polypeptide and/or the trigger are included in pharmaceutical compositions.
[0304] In some embodiments, cytokine dysregulation can include hypercytokinemia (e.g., a cytokine storm). In some embodiments, hypercytokinemia can associated with graft-versus-host disease.
[0305] T cell exhaustion is a state of T cell dysfunction that can, in some instances, result from stimulation. For instance, chronic or persistent exposure to a T cell pathway activating antigen and/or inflammatory signals can exhaust a T cell. T cell exhaustion can be characterized by, in at least some instances, one or more of poor effector function, elevated or sustained expression of inhibitory receptors, and a transcriptional state distinct from equivalent non-exhausted cells. Exhausted T cells are less effective in interacting with targets. T cell exhaustion may entail exhaustion of a subset of T cells present in a subject. T cell exhaustion can entail partial or complete, exhaustion of one or more T cells, such as a subset of T cells present in a subject. For instance, effector function can be progressively repressed during the development of T cell exhaustion, e.g., leading to a heterogeneous population of T cells at various levels of exhaustion. In some instances, partially exhausted T cells can display sustained expression of a minimal number of immune inhibitory receptors (IRs) and differential expression of T-bet and Eomes (T-bet.sup.highEomes.sup.low T cells), whereas, e.g., fully exhausted T cells can be marked by coexpression of multiple IRs in some instances. T cell exhaustion has been reviewed, e.g., in Wherry (2015 Nat. Rev. Immunol. 15(8): 486-499), which is incorporated herein by reference. Various means of detecting, identifying, and/or predicting T cell exhaustion are known in the art. Modulation of one or more immune pathways can result in rejuvenation of partially exhausted T cells.
[0306] Among other things, the present disclosure provides a method of treating T cell exhaustion. The present disclosure encompasses the insight that a condition of T cell exhaustion in a T cell, or a subject including an exhausted T cell, can be treated by partial or complete inactivation of an immune activity, signal, or pathway, e.g., by physical or other regulatory interaction with the immune activity pathway (e.g., immune inactivation). The present disclosure encompasses the further insight that such partial or complete inactivation of an immune activity, signal, or pathway can be achieved in a T cell that includes, expresses, or encodes a trigger-responsive immune-inactivating signaling polypeptide, e.g., upon exposure to trigger. Accordingly, the present insight includes the application of methods and compositions described herein for the treatment of T cell exhaustion, e.g., by administration of a trigger to a subject including, identified as including, or identified as at risk of including exhausted T cells including, expressing, or encoding a trigger-responsive immune-inactivating signaling polypeptide.
[0307] In certain embodiments, a method of treating T cell exhaustion is a method of treating T cell exhaustion in a subject having been administered an engineered and/or modified T cell (e.g. CAR-T cell) including or encoding a trigger-responsive immune-inactivating signaling polypeptide. In certain embodiments, a method of treating T cell exhaustion is a method of treating T cell exhaustion in a subject having been administered an adoptive T cell therapy regimen, which regimen included administration to the subject of an engineered and/or modified T cell (e.g. CAR-T cell) including or encoding a trigger-responsive immune-inactivating signaling polypeptide. In various embodiments, T cell exhaustion or a risk thereof has been identified with respect to T cells (e.g., engineered and/or modified T cells (e.g. CAR-T cells)) administered to, present in, or to be administered to the subject.
[0308] In various instances of treating T cell exhaustion, trigger may be administered in any manner or regimen, or for any duration, in accordance with the present disclosure. In various instances, administration of trigger may be limited to a specified period of time or in limited in accordance with the evaluation of a medical practitioner. For instance, a regimen for administration of a trigger in the treatment of T cell exhaustion may include administration of trigger to a subject in one or more same or different doses over a period such as 6 hours, 12 hours, one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, two weeks, three weeks, one month, two months, three months, four months, five months, or six months.
[0309] In some embodiments, a method of treating T cell exhaustion can include administering a composition that delivers the trigger-responsive immune-inactivating signaling polypeptide as described herein to a subject (e.g., a patient, e.g., a human patient). In some embodiments, a method of treating T cell exhaustion includes administering a trigger as described herein. In some embodiments, a composition that delivers the trigger-responsive immune-inactivating signaling polypeptide and/or the trigger are included in pharmaceutical compositions. In some embodiments, a method of treating T cell exhaustion can include administering an engineered and/or modified T cell (e.g. CAR-T cell) to the subject.
[0310] Among other things, the present disclosure provides a method of treating cancer. In some embodiments, a method of treating cancer can include administering a composition that delivers the trigger-responsive immune-inactivating signaling polypeptide as described herein to a subject (e.g., a patient, e.g., a human patient). In some embodiments, a method of treating cancer includes administering a trigger as described herein. In some embodiments, a composition that delivers the trigger-responsive immune-inactivating signaling polypeptide and/or the trigger are included in pharmaceutical compositions.
[0311] In some embodiments, a method of treating cancer can include administering an engineered and/or modified T cell (e.g. CAR-T cell) to the subject.
[0312] In some embodiments, a cancer can be carcinoma, sarcoma, melanoma, lymphoma, leukemia, or blastoma. In some embodiments, a carcinoma can be a basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ, invasive ductal carcinoma, and/or adenocarcinoma. In some embodiments, a carcinoma can be a prostate cancer, an ovarian cancer, a uterine cancer, a cervical cancer, a colorectal cancer, a breast cancer, a bladder cancer, a pancreatic cancer, an esophageal cancer, a gastrointestinal cancer, a hepatocellular cancer, a thyroid cancer, or a lung cancer. In some embodiments, a sarcoma can be a angiosarcoma, a chondrosarcoma, an Ewing's sarcoma, fibrosarcoma, a gastrointestinal stromal cancer, a Leiomyosarcoma, a liposarcoma, an osteosarcoma, a pleomorphic sarcoma, a rhabdomyosarcoma, or a synovial sarcoma. In some embodiments, a melanoma can be a superficial spreading melanoma, a nodular melanoma, a lentigo maligna melanoma, or an acral melanoma. In some embodiments, a lymphoma can be a B-cell lymphoma, a T cell lymphoma, or an NK-cell lymphoma. In some embodiments, a leukemia can be an acute myeloid leukemia, a chronic myeloid leukemia, acute lymphocytic leukemia, or a chronic lymphocytic leukemia. In some embodiments, a blastoma can be a hepatoblastoma, a medulloblastoma, a nephroblastoma, a neuroblastoma, a pancreatoblastoma, a pleuropulmonary blastoma, retinoblastoma, or a glioblastoma. In some embodiments, a cancer can be a Stage I, Stage II, Stage III, or Stage IV cancer. In some embodiments, a cancer can be metastatic.
[0313] In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide is administered in combination with one or more therapeutic agents (e.g., an anti-cancer agent). In some embodiments, the one or more therapeutic agents have received regulatory approval and are currently used for treatment of a condition and/or disease. In some embodiments, therapeutic agent(s) is/are administered according to its standard or approved dosing regimen and/or schedule. In some embodiments, therapeutic agent(s) is/are administered according to a regimen that is altered as compared with its standard or approved dosing regimen and/or schedule. In some embodiments, such an altered regimen differs from the standard or approved dosing regimen in that one or more unit doses is altered (e.g., reduced or increased) in amount, and/or in that dosing is altered in frequency (e.g., in that one or more intervals between unit doses is expanded, resulting in lower frequency, or is reduced, resulting in higher frequency). In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide is administered at the same time as with one or more therapeutic agents (e.g., an anti-cancer agent). In some embodiments, a trigger-responsive immune-inactivating signaling polypeptide and one or more therapeutic agents (e.g., an anti-cancer agent) are administered as part of the same course of treatment but are not administered together.
[0314] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.
EXAMPLES
[0315] Other features, objects, and advantages of the present invention are apparent in the examples that follow. It should be understood, however, that the examples, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the examples.
Example 1: Construction of a Vector Encoding a Trigger-Responsive Dominant Negative Signaling Polypeptide
[0316] This example illustrates an exemplary DNA construct engineered for delivery and expression of a trigger-responsive dominant negative signaling polypeptide. It will be clear to one skilled in the art that a number of alternative approaches, expression vectors and cloning techniques are available.
[0317] As shown in
[0318] The DNA encoding the endoxifen-responsive dominant negative Zap-70 polypeptide had a nucleotide sequence according to SEQ ID NO: 7, and the endoxifen-responsive dominant negative Zap-70 polypeptide had an amino acid sequence according to SEQ ID NO: 8.
Example 2: Construction of a Vector Encoding a Dominant Negative Signaling Moiety
[0319] This example illustrates an exemplary DNA construct engineered for delivery and expression of a dominant negative signaling moiety, which can be useful as, among other things, as a control. It will be clear to one skilled in the art that a number of alternative approaches, expression vectors and cloning techniques are available.
[0320] As shown in
[0321] The DNA encoding the polypeptide had a nucleotide sequence according to SEQ ID NO: 9, and the polypeptide had an amino acid sequence according to SEQ ID NO: 10.
TABLE-US-00027 SEQ ID NO: 9 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACAATGCCAGACCCCGCGGCGCACC TGCCCTTCTTCTACGGCAGCATCTCGCGTGCCGAG GCCGAGGAGCACCTGAAGCTGGCGGGCATGGCGGA CGGGCTCTTCCTGCTGCGCCAGTGCCTGCGCTCGC TGGGCGGCTATGTGCTGTCGCTCGTGCACGATGTG CGCTTCCACCACTTTCCCATCGAGCGCCAGCTCAA CGGCACCTACGCCATTGCCGGCGGCAAAGCGCACT GTGGACCGGCAGAGCTCTGCGAGTTCTACTCGCGC GACCCCGACGGGCTGCCCTGCAACCTGCGCAAGCC GTGCAACCGGCCGTCGGGCCTCGAGCCGCAGCCGG GGGTCTTCGACTGCCTGCGAGACGCCATGGTGCGT GACTACGTGCGCCAGACGTGGAAGCTGGAGGGCGA GGCCCTGGAGCAGGCCATCATCAGCCAGGCCCCGC AGGTGGAGAAGCTCATTGCTACGACGGCCCACGAG CGGATGCCCTGGTACCACAGCAGCCTGACGCGTGA GGAGGCCGAGCGCAAACTTTACTCTGGGGCGCAGA CCGACGGCAAGTTCCTGCTGAGGCCGCGGAAGGAG CAGGGCACATACGCCCTGTCCCTCATCTATGGGAA GACGGTGTACCACTACCTCATCAGCCAAGACAAGG CGGGCAAGTACTGCATTCCCGAGGGCACCAAGTTT GACACGCTCTGGCAGCTGGTGGAGTATCTGAAGCT GAAGGCGGACGGGCTCATCTACTGCCTGAAGGAGG CCTGCCCCAACAGCAGTGCCAGCAACGCCTCAGGG GCTGCTGCTCCCACACTCCCAGCCCACCCATCCAC GTTGACG SEQ ID NO: 10 M N E L A L K L A G L D I N K T M P D P A A H L P F F Y G S I S R A E A E E H L K L A G M A D G L F L L R Q C L R S L G G Y V L S L V H D V R F H H F P I E R Q L N G T Y A I A G G K A H C G P A E L C E F Y S R D P D G L P C N L R K P C N R P S G L E P Q P G V F D C L R D A M V R D Y V R Q T W K L E G E A L E Q A I I S Q A P Q V E K L I A T T A H E R M P W Y H S S L T R E E A E R K L Y S G A Q T D G K F L L R P R K E Q G T Y A L S L I Y G K T V Y H Y L I S Q D K A G K Y C I P E G T K F D T L W Q L V E Y L K L K A D G L I Y C L K E A C P N S S A S N A S G A A A P T L P A H P S T L T
Example 3: Inhibition of NFAT-Luciferase Expression by Endoxifen-Responsive Dominant Negative Zap-70 Polypeptide in Jurkat E6.1 Cells
[0322] This example demonstrates that a modulating domain of a trigger-responsive dominant can inhibit the activity of an operatively linked dominant negative moiety, and that the inhibition of the dominant negative signaling moiety by the modulating domain can be relieved in the presence of the trigger. This example further shows that, when the inhibition of the dominant negative signaling moiety is relieved by the interaction of the trigger with the modulating domain, the dominant negative signaling moiety can inhibit a T cell activation cascade.
[0323] One outcome of T cell activation is the expression of Nuclear Factor of Activated T cells (NFAT) transcription factors. Accordingly, a construct containing a reporter gene, such as firefly luciferase (Luc), under the control of an NFAT response element can be used to assess T cell activity. Such a construct can either be transiently transfected into cells (e.g., Jurkat cells, which are an immortalized line of human T cells) or stably integrated into a cell line (e.g., a derivative Jurkat cell line). These reporter genes can be used to measure activation, or inhibition, of T cell activation pathways.
[0324] To examine whether an endoxifen-responsive dominant negative Zap-70 polypeptide could inhibit T cell activity resulting in NFAT-luciferase expression, Jurkat E6.1 cells (purchased from ATCC (Manassas, Va.)) were transiently transfected with an NFAT-Luciferase reporter construct and a test construct of (1) a control vector (“vector”), (2) a vector encoding a dominant negative Zap70 moiety, as described in Example 2 (“Zap70dnm”), or (3) a vector encoding an endoxifen-responsive dominant negative Zap70 polypeptide, including an ER(T2) domain operatively linked to a dominant negative Zap70 moiety, as described in Example 1 (“Zap70dnm-ER(T2)”). The transient transfections were performed using LTX and PLUS transfection reagent (ThermoFisher Scientific) according to manufacturer's protocols.
[0325] Transfected cells were treated in the absence or presence of 1:30 Immunocult™ CD3/CD28/CD2 tetrameric antibody mixture (StemCell Technologies, Canada) (“α-TCR”), which stimulates T cell activity. Stimulated cells were then further treated with either RPMI 1640 media plus 4.4% FBS stripped with charcoal dextran (ThermoFisher Scientific) or 100 nM endoxifen in RPMI 1640 media plus 4.4% FBS stripped with charcoal dextran (ThermoFisher Scientific).
[0326] A summary of the Jurkat cells samples tested is included in Table 6 below.
TABLE-US-00028 TABLE 6 1 2 3 4 5 6 7 8 9 NFAT- NFAT- NFAT- NFAT- NFAT- NFAT- NFAT- NFAT- NFAT- luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase Vector Vector Vector Zap70dnm Zap70dnm Zap70dnm Zap70dnm- Zap70dnm- Zap70dnm- ER(T2) ER(T2) ER(T2) Vehicle α-TCR α-TCR Vehicle α-TCR α-TCR Vehicle α-TCR α-TCR Vehicle Vehicle Endoxifen Vehicle Vehicle Endoxifen Vehicle Vehicle Endoxifen
[0327] After 19 hours, luciferase activity was assayed with Bright-Glo substrate according to manufacturer's protocols (Promega). Luminescence was detected on the Varioskan Lux (ThermoFisher Scientific) plate reader at a 1 second interval. The luminescence was measured and reported in relative light units (RLU). The results are graphically represented in
[0328] The control vector was co-transfected into Jurkat cells as a negative control and demonstrated that any effects observed from the co-transfection of the Zap70dnm and Zap70dnm-ER(T2) constructs were result of the encoded dominant negative Zap70 moiety or the encoded endoxifen-responsive dominant negative Zap70 polypeptide, respectively. Accordingly, samples 1-3, which were co-transfected with the control vector, helped to establish that the system was working as expected. For example, as expression of a gene under the control of an NFAT response element should be driven by T cell activation and the subsequent activation of NFAT transcription factors, it was expected that no luciferase expression from the NFAT-luciferase construct would be detected in Jurkat cells that were co-transfected with a control vector and treated with vehicle (i.e., not with α-TCR). Indeed, that is what was observed for sample 1. As shown, no significant luciferase activity was observed when the T cell pathway was not activated by α-TCR.
[0329] On the flip side, it was expected that significant luciferase expression would be observed from Jurkat cells that were co-transfected with the control vector and treated with vehicle. Again, the control data matched what was expected. As shown for sample 2, nearly 70,000 RLUs were observed in Jurkat cells co-transfected with a control vector and treated with α-TCR.
[0330] It was hypothesized that endoxifen treatment, in the absence of the endoxifen-responsive dominant negative Zap70 polypeptide, would not impact the level of luciferase expression because it should neither promote or inhibit luciferase expression from the NFAT-luciferase construct on its own. As shown, the addition of endoxifen to Jurkat cells co-transfected with the control vector and treated with α-TCR (sample 3) showed similar levels luciferase activity to Jurkat cells co-transfected with the control vector and treated with α-TCR (sample 2). These control data demonstrate that endoxifen on its own was not affecting luciferase expression.
[0331] Samples 4-6 include Jurkat cells co-transfected with the NFAT-luciferase construct and Zap70dnm that encodes a dominant negative Zap70 moiety. As shown in the data for sample 4, no luciferase expression was detected when the cells were treated with vehicle (i.e., not treated with α-TCR or endoxifen). In sample 5, the cells were treated with α-TCR, which activated the T cells activation cascade (see, e.g., sample 2). However, the luciferase expression detected was low, which suggested that the dominant negative Zap70 moiety was expressed from the Zap70dnm construct and inhibited the T cell activation cascade. In sample 6, the Jurkat cells were treated with α-TCR, which activated the T cells activation cascade, and treated with endoxifen. As shown in
[0332] Samples 7-9 include Jurkat cells co-transfected with the NFAT-luciferase construct and Zap70dnm-ER(T2) that encodes an endoxifen-responsive dominant negative Zap70 polypeptide. As shown in the data for sample 7, no luciferase expression was detected when the cells were treated with vehicle (i.e., not treated with α-TCR or endoxifen). In sample 8, the cells were treated with α-TCR, which activated the T cells activation cascade (see, e.g., sample 2). A significant level of luciferase expression was detected from sample 8, particularly when compared to the luciferase levels observed from sample 5. As discussed above, in sample 5, the dominant negative Zap70 moiety inhibited the T cell activation pathway, and therefore, inhibited expression of luciferase from the NFAT-luciferase construct. In contrast, the data for sample 8 indicated that, in the absence of endoxifen, the dominant negative Zap70 moiety present in the endoxifen-responsive dominant negative Zap70 polypeptide was inhibited or masked by the ER(T2) modulating domain, and therefore, unable to inhibit the T cell activation cascade and resulting NFAT regulated luciferase expression. Finally, in sample 9, the Jurkat cells were treated with α-TCR, which activated the T cells activation cascade, and treated with endoxifen. As shown in
[0333] In sum, the data shown in
Example 4: Inhibition of NFAT-Luciferase by Endoxifen-Responsive Dominant Negative Zap-70 Polypeptide in Jurkat E6.1 Cells was Controlled by Endoxifen in a Dose Dependent Manner
[0334] This example demonstrates that the inhibition or masking of a dominant negative signaling moiety of a trigger-responsive dominant negative signaling polypeptide by a modulating domain can be relieved in a dose-dependent manner by a trigger. This example further shows that, when the inhibition of the dominant negative signaling moiety is relieved by the interaction of the trigger with the modulating domain, the dominant negative signaling moiety can inhibit a T cell activation cascade in a dose dependent manner.
[0335] To examine whether the concentration of endoxifen affected the level of inhibition of T cell activity by an endoxifen-responsive dominant negative Zap-70 polypeptide, Jurkat E6.1 cells (purchased from ATCC (Manassas, Va.)) were transiently transfected with an NFAT-Luciferase reporter construct and a test construct of (1) a vector encoding a dominant negative Zap70 moiety, as described in Example 2 (“Zap70dnm”), or (2) a vector encoding an endoxifen-responsive dominant negative Zap70 polypeptide, including an ER(T2) domain operatively linked to a dominant negative Zap70 moiety, as described in Example 1 (“Zap70dnm-ER(T2)”). The transient transfections were performed using LTX and PLUS transfection reagent (ThermoFisher Scientific) according to manufacturer's protocols.
[0336] Transfected cells were treated with an anti-TCR antibody, clone 305, which stimulated T cell activity. Stimulated cells were then further treated with 0 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, or 1000 nM of endoxifen in RPMI 1640 media plus 4.4% FBS stripped with charcoal dextran (ThermoFisher Scientific).
[0337] After 6 hours, luciferase activity was assayed with Bright-Glo substrate according to manufacturer's protocols (Promega). Luminescence was detected on the Varioskan Lux (ThermoFisher Scientific) plate reader at a 1 second interval. The luminescence was measured and reported in relative light units (RLU). Points represent mean activity from quadruplicate wells with error bars representing SEM.
[0338] As shown in
Example 5: Inhibition of NFAT-Luciferase Expression Increased with the Amount of Endoxifen-Responsive Dominant Negative Zap-70 Polypeptide in Jurkat E6.1 Cells
[0339] This example demonstrates that the activity of a dominant negative signaling moiety included in a trigger-responsive dominant negative signaling polypeptide, and in turn, the level of T cell activation cascade inhibition, can be regulated by the amount of a trigger-responsive dominant negative signaling polypeptide.
[0340] To examine whether the amount of Zap70dnm-ER(T2) construct affected the level of inhibition of T cell activity by an encoded endoxifen-responsive dominant negative Zap-70 polypeptide, Jurkat E6.1 cells (purchased from ATCC (Manassas, Va.)) that were either transiently transfected with an NFAT-Luciferase reporter construct (
[0341] Transfected cells were then treated with an anti-TCR antibody, clone 305, which stimulated T cell activity. Stimulated cells were then further treated with 100 nM endoxifen in RPMI 1640 media plus 4.4% FBS stripped with charcoal dextran (ThermoFisher Scientific). After incubation for 6 hours, luciferase activity was assayed with Bright-Glo substrate according to manufacturer's protocols (Promega). Luminescence was detected on the Varioskan Lux (ThermoFisher Scientific) plate reader at a 1 second interval. The luminescence was measured and reported in relative light units (RLU). The results are graphically represented in
[0342] For both the transient and stable transfections, the luciferase expression in Jurkat cells that were transiently transfected with 10 ng of Zap70dnm-ER(T2) construct was lower than the luciferase expression observed from Jurkat cells transiently transfected with 1 ng of Zap70dnm-ER(T2) construct. As a higher amount of Zap70dnm-ER(T2) construct is likely resulted in a higher amount of endoxifen-responsive dominant negative Zap-70 polypeptide, these data suggest that a higher amount of endoxifen-responsive dominant negative Zap-70 polypeptide was able to inhibit the T cell activation cascade and expression of luciferase from the NFAT-luciferase construct.
Example 6: Inhibition of NFAT-Luciferase Expression was Regulated by the Amount of Endoxifen-Responsive Dominant Negative Zap-70 Polypeptide in a Dose Dependent Manner
[0343] This example demonstrates that the activity of a dominant negative signaling moiety included in a trigger-responsive dominant negative signaling polypeptide, and in turn, the level of T cell activation cascade inhibition, can be regulated by the amount of a trigger-responsive dominant negative signaling polypeptide in a dose dependent manner.
[0344] To examine whether the amount of Zap70dnm-ER(T2) construct affected the level of inhibition of T cell activity by an encoded endoxifen-responsive dominant negative Zap-70 polypeptide in a dose dependent manner, Jurkat E6.1 cells (purchased from ATCC (Manassas, Va.)) were transiently transfected with an NFAT-Luciferase reporter construct and 0.05 ng, 0.1 ng, 0.5 ng, 1 ng, 5 ng, 10 ng, or 50 ng of a vector encoding an endoxifen-responsive dominant negative Zap70 polypeptide, including an ER(T2) domain operatively linked to a dominant negative Zap70 moiety, as described in Example 1 (“Zap70dnm-ER(T2)”). The transient transfections were performed using LTX and PLUS transfection reagent (ThermoFisher Scientific) according to manufacturer's protocols.
[0345] Transfected cells were then treated with an anti-TCR antibody, clone 305, which stimulated T cell activity. Stimulated cells were then further treated with 100 nM endoxifen in RPMI 1640 media plus 4.4% FBS stripped with charcoal dextran (ThermoFisher Scientific). After incubation for 6 hours, luciferase activity was assayed with Bright-Glo substrate according to manufacturer's protocols (Promega). Luminescence was detected on the Varioskan Lux (ThermoFisher Scientific) plate reader at a 1 second interval. The luminescence was measured and reported in relative light units (RLU). Points represent mean activity from quadruplicate wells with error bars representing SEM.
[0346] As shown in
Example 7: Inhibition of NFAT-Luciferase by Endoxifen-Responsive Dominant Negative Zap-70 Polypeptides Including a G400V or G400L Mutation were Controlled by Endoxifen in a Dose Dependent Manner
[0347] This example demonstrates that a modulating domain that includes either a G400V or a G400L mutation can inhibit or mask a dominant negative signaling moiety of a trigger-responsive dominant negative signaling polypeptide. This example further demonstrates that the inhibition or masking of a dominant negative signaling moiety of a trigger-responsive dominant negative signaling polypeptide by a modulating domain that includes either a G400V or a G400L mutation can be relieved in a dose-dependent manner by a trigger. Additionally, this example shows that, when the inhibition of the dominant negative signaling moiety is relieved by the interaction of the trigger with such a modulating domain, the dominant negative signaling moiety can inhibit a T cell activation cascade in a dose dependent manner.
[0348] To examine whether a modulating domain that includes either a G400V or a G400L mutation can inhibit or mask a dominant negative Zap-70 polypeptide of an endoxifen-responsive dominant negative Zap-70 polypeptide, Jurkat E6.1 cells (purchased from ATCC (Manassas, Va.)) were transiently transfected with an NFAT-Luciferase reporter construct and a test construct of (1) a vector encoding an endoxifen-responsive dominant negative Zap70 polypeptide, comprising an ER(T2) domain, which included a G400V mutation, operatively linked to a dominant negative Zap70 moiety (“G400V”) or (2) a vector encoding an endoxifen-responsive dominant negative Zap70 polypeptide, comprising an ER(T2) domain, which included a G400L mutation, operatively linked to a dominant negative Zap70 moiety (“G400L”). The transient transfections were performed using LTX and PLUS transfection reagent (ThermoFisher Scientific) according to manufacturer's protocols.
[0349] Transfected cells were treated in the absence or presence of 1:30 Immunocult™ CD3/CD28/CD2 tetrameric antibody mixture (StemCell Technologies, Canada) (“α-TCR”), which stimulates T cell activity. Stimulated cells were then further treated with either RPMI 1640 media plus 4.4% FBS stripped with charcoal dextran (ThermoFisher Scientific) or one of 0, 0.3 nM, 1 nM, 3.2 nM, 10 nM, 32 nM and 100 nM endoxifen in RPMI 1640 media plus 4.4% FBS stripped with charcoal dextran (ThermoFisher Scientific). After 19 hours, luciferase activity was assayed with Bright-Glo substrate according to manufacturer's protocols (Promega). Luminescence was detected on the Varioskan Lux (ThermoFisher Scientific) plate reader at a 1 second interval. The luminescence was measured and reported in relative light units (RLU). The results are graphically represented in
[0350] As shown in
Example 8: Inhibition of the Activity of Dominant Negative Zap70 in an Endoxifen-Responsive Dominant Negative Zap70 Polypeptide is Effectively Relieved by the Interaction of Endoxifen with an ER(T2) Domain Having Either a G400V or a G400L Mutation
[0351] This example demonstrates that the inhibition or masking of a dominant negative signaling moiety of a trigger-responsive dominant negative signaling polypeptide by an ER(T2) domain including either a G400V or a G400L mutation can be relieved by endoxifen at biologically relevant concentrations. This example further demonstrates that, while the inhibition or masking of a dominant negative signaling moiety of a trigger-responsive dominant negative signaling polypeptide by an ER(T2) domain including either a G400V or a G400L mutation can be relieved in a dose-dependent manner by a trigger, the dose curves obtained for trigger-responsive dominant negative signaling polypeptide that include an ER(T2) domain having a G400V or a G400L mutation are different from one another. Additionally, this example demonstrates that the IC50 or pIC50 of a trigger can vary based on a modulating domain (e.g., ER(T2) domain) present in a trigger-responsive dominant negative signaling polypeptide.
[0352] To examine how effective endoxifen is at relieving the inhibition or masking of a dominant negative signaling moiety of a trigger-responsive dominant negative signaling polypeptide by an ER(T2) domain including either a G400V or a G400L mutation, Jurkat E6.1 cells were transiently transfected with 90 ng NFAT-Luciferase reporter construct and 10 ng of a G400V or G400L construct. The transfected cells were stimulated the addition of 1:30 Immunocult CD3/CD28/CD2 tetrameric antibody mixture, and treated with or without endoxifen for 19 hours before assaying for luciferase activity. Results were normalized to maximum TCR stimulation with Immunocult alone, with 0% representing the activity of unstimulated transfected cells. Points represent mean activity from sextuplicate wells with error bars representing SEM. pIC50's were calculated using a variable slope sigmoidal dose-response model using GraphPad Prism.
[0353] As shown in
Example 9: Interleukin 2 Levels can be Measured to Determine T Cell Activation and the Activity of an Endoxifen-Responsive Dominant Negative Zap-70 Polypeptide
[0354] This example demonstrates that the level of T cell activity and the activity of a dominant negative signaling moiety included in a trigger-responsive dominant negative signaling polypeptide can be determined by examining the level of interleukin 2 (“IL-2”).
[0355] One hundred thousand Jurkat E6.1 cells (ATCC, Manassas, Va.) are plated into individual wells of a 96-well plate with RPMI medium plus 5% FBS stripped with charcoal dextran (ThermoFisher Scientific). Cells are pre-treated with 1:30 ImmunoCult CD3/CD28/CD2 tetrameric antibody mixture (STEMCELL Technologies, Vancouver, BC) for 2 days to pre-stimulate T cell activation. Cells are washed and rested for 2 days before the cells are transduced with a vector containing trigger-responsive dominant negative signaling polypeptide. Efficient stable transduction of this vector is achieved utilizing a lentiviral vector, or alternatively, by liposome-mediated transfection with co-expression of a selective marker containing a gene resistant to ouabain or antibiotic. At least 4 hours later cells are re-exposed to 1:30 Immunocult plus an appropriate ligand trigger in RPMI medium plus 5% FBS stripped with charcoal dextran. A 1×cocktail of phorbol myristate acetate and ionomycin are used as a positive control for IL-2 induction (BioLegend, San Diego, Calif.). At least 16 hours later, supernatants are collected for immediate use, or are stored at −80° C. Supernatants are diluted 1:10 and used in an ELISA for IL-2 using the manufacturer's recommended protocol (BD Bioscience, San Jose, Calif.). Absorbance is read using the Varioskan LUX plate reader (ThermoFisher Scientific).
Example 10: Modulation of Dominant-Negative Signaling Moiety Activity by an ER(T12) Modulating Domain
[0356] This example demonstrates that modulating domain ER(T12) can inhibit activity of a dominant-negative signaling moiety to which it is operatively linked, and that the inhibition of the dominant negative signaling moiety by the ER(T12) modulating domain can be relieved in the presence of a trigger. This example further shows that, when inhibition of the dominant negative signaling moiety by the ER(T12) modulating domain is relieved by the interaction of trigger with modulating domain, the dominant negative signaling moiety can inhibit a T cell activation cascade.
[0357] ER(T12) is an endoxifen-responsive modulating domain that is a variant of a ligand binding domain (LBD) fragment (amino acids 282-595) of human estrogen receptor-α having an amino acid sequence as set forth in SEQ ID NO: 13, including G400L (ctg), M543A (gcg), and L544A (gcg). This modulating domain can be encoded by the nucleic acid sequence set forth in SEQ ID NO: 14.
[0358] To examine the ability of ER(T12) to modulate activity of a dominant-negative signaling moiety to which it is operatively linked, a nucleic acid that can express a polypeptide including a LAC70dn domain operatively linked to ER(T12) (“ZAP70dn(1-278)-ER(T12)”) was generated (
[0359] To provide a means for assessing immune pathway activity, a construct containing a reporter gene, such as firefly luciferase (Luc), under the control of an NFAT response element can be used, as described, e.g., in Example 3. In the present Example, Jurkat E6.1 cells were transiently transfected with an NFAT-Luciferase reporter construct as well as one of (a) empty vector; (b) an expression construct encoding ZAP70dn(1-278); (c) an expression construct encoding ZAP70dn(1-278)-ER(T2); and (d) an expression construct encoding ZAP70dn(1-278)-ER(T12). Transient transfections were performed using LTX and PLUS transfection reagent (ThermoFisher Scientific) according to manufacturer's protocols.
[0360] A first set of transfected cells was treated in the absence or presence of 1:30 dilution of Immunocult CD3/CD28/CD2 tetrameric antibody mixture (StemCell Technologies, Canada), which stimulated T-cell activity. Stimulated cells were then further treated with RPMI 1640 media plus 4.8% FBS stripped with charcoal dextran (ThermoFisher Scientific), either without Z-endoxifen or with a designated amount of Z-endoxifen (Axon-Medchem). Luciferase activity (luminescence) was measured and reported as a percentage induction of NFAT-Luciferase.
[0361] In Jurkat E6.1 cells transiently transfected with NFAT-Luciferase reporter construct as well as either an expression construct encoding ZAP70dn(1-278)-ER(T2) or an expression construct encoding ZAP70dn(1-278)-ER(T12), luciferase induction was generally inverse to the exposure of cells to endoxifen trigger, at least in that both ZAP70dn(1-278)-ER(T2) and ZAP70dn(1-278)-ER(T12) demonstrated greater inhibition of luciferase induction at certain relatively higher concentrations of endoxifen than at certain relatively lower concentrations of endoxifen (
[0362] A further set of transfected cells was treated in the absence or presence of 0.25 μg/ml C305 (αTCR, Sigma-Aldrich) plus 2 μg/ml αCD28.2 (Biolegend), which stimulated T-cell activity. Stimulated cells were then further treated with RPMI 1640 media plus 4.8% FBS stripped with charcoal dextran (ThermoFisher Scientific), either without Z-endoxifen or with a designated amount of Z-endoxifen (Axon-Medchem; 0.05 to 50 nM). Luciferase activity (luminescence) was measured and reported in relative light units (RLU).
[0363] Results shown in
Example 11: Inhibition of Expression by Trigger-Responsive Dominant Negative LCK Polypeptides
[0364] This example demonstrates that modulating domain ER(T12) can inhibit activity of a dominant-negative signaling moiety to which it is operatively linked, and that the inhibition of the dominant negative signaling moiety by the ER(T12) modulating domain can be relieved in the presence of a trigger. This example further shows that, when inhibition of the dominant negative signaling moiety by the ER(T12) modulating domain is relieved by the interaction of trigger with modulating domain, the dominant negative signaling moiety can inhibit a T cell activation cascade. Moreover, this example shows that LCK(1-266) is a dominant negative signaling moiety that can inhibit a T cell activation cascade. Further, this example shows that LCK(1-266) is a dominant negative signaling moiety that can be trigger-responsive when operatively linked to a modulating domain in a trigger-responsive dominant negative signaling polypeptide.
[0365] To examine the ability of LCK(1-266) to modulate activity of an immune pathway in a dominant-negative manner, and further to show that LCK(1-266) is a dominant negative signaling moiety that can be trigger-responsive when operatively linked to a modulating domain, various constructs were prepared. These constructs included, among other constructs and components, a construct capable of expressing a LCK(1-266)-ER(T12) trigger-responsive dominant negative signaling polypeptide (
[0366] To provide a means for assessing T cell activity, a construct containing a reporter gene, such as firefly luciferase (Luc), under the control of an NFAT response element can be used, as described, e.g., in Example 3. In the present Example, Jurkat E6.1 cells were transiently transfected with an NFAT-Luciferase reporter construct, as well as one of (a) empty vector control; (b) an expression construct encoding LCK(1-266); and (c) an expression construct encoding LCK(1-266)-ER(T12). Transient transfections were performed using LTX and PLUS transfection reagent (ThermoFisher Scientific) according to manufacturer's protocols.
[0367] Transfected cells were treated in the absence or presence of 1:80 dilution of Immunocult CD3/CD28 tetrameric antibody mixture (StemCell Technologies, Canada), which stimulated T-cell activity. Stimulated cells were then further treated with RPMI 1640 media plus 4.8% FBS stripped with charcoal dextran (ThermoFisher Scientific), either without Z-endoxifen or with 100 nM Z-endoxifen (Axon-Medchem). Luciferase activity (luminescence) was measured and reported as percent induction of NFAT-Luciferase.
[0368]
Example 12: Inhibition of Expression by Trigger-Responsive Constitutively Active SHP1 Polypeptides
[0369] This example demonstrates that modulating domain ER(T12) can inhibit activity of a constitutively active signaling moiety to which it is operatively linked, and that the inhibition of the constitutively active signaling moiety by the ER(T12) modulating domain can be relieved in the presence of a trigger. This example further shows that, when inhibition of the constitutively active signaling moiety by the ER(T12) modulating domain is relieved by the interaction of trigger with modulating domain, the constitutively active signaling moiety can inhibit a T cell activation cascade. Moreover, this example shows that SHP1(210-595) is a constitutively active signaling moiety that can inhibit a T cell activation pathway. It is hypothesized, without wishing to be bound by any particular scientific theory, that deletion of SH2 domains renders SHP-1 constitutively active, in which state SHP1 inhibits T-cell activation, e.g., by removing phosphates from LCK, from Zeta chains, and from endogenous ZAP70. Further, this example shows that SHP1(210-595) is a constitutively active signaling moiety that can be trigger-responsive when operatively linked to a modulating domain in a trigger-responsive dominant negative signaling polypeptide.
[0370] To examine the ability of SHP1(210-595) to modulate activity of an immune pathway in a constitutive manner, and further to show that SHP1(210-595) is a constitutively active signaling moiety that can be trigger-responsive when operatively linked to a modulating domain, various constructs were prepared. These constructs included, among other constructs and components, a construct capable of expressing a SHP1(210-595)-ER(T12) trigger-responsive constitutively active signaling polypeptide (
[0371] To provide a means for assessing T cell activity, a construct containing a reporter gene, such as firefly luciferase (Luc), under the control of an IL2 response element can be used. In the present Example, Jurkat E6.1 cells cells were transiently transfected with an IL2-Luciferase reporter construct, as well as one of (a) empty vector; (b) an expression construct encoding SHP1(210-595); and (c) an expression construct encoding SHP1(210-595)-ER(T12). Transient transfections were performed using LTX and PLUS transfection reagent (ThermoFisher Scientific) according to manufacturer's protocols.
[0372] Transfected cells were treated in the absence or presence of 1:30 dilution of Immunocult CD3/CD28 tetrameric antibody mixture (StemCell Technologies, Canada), which stimulated T-cell activity. Stimulated cells were then further treated with RPMI 1640 media plus 4.8% FBS stripped with charcoal dextran (ThermoFisher Scientific), without Z-endoxifen or with 50 nM Z-endoxifen (Axon-Medchem). Luciferase activity (luminescence) was measured and reported as percent induction of IL2-Luciferase.
[0373]
[0374]
TABLE-US-00029 SEQUENCES Exemplary nucleotide sequence encoding a dominant negative Zap70 SEQ ID NO: 1 ATGCCAGACCCCGCGGCGCACCTGCCCTTCTTCTA CGGCAGCATCTCGCGTGCCGAGGCCGAGGAGCACC TGAAGCTGGCGGGCATGGCGGACGGGCTCTTCCTG CTGCGCCAGTGCCTGCGCTCGCTGGGCGGCTATGT GCTGTCGCTCGTGCACGATGTGCGCTTCCACCACT TTCCCATCGAGCGCCAGCTCAACGGCACCTACGCC ATTGCCGGCGGCAAAGCGCACTGTGGACCGGCAGA GCTCTGCGAGTTCTACTCGCGCGACCCCGACGGGC TGCCCTGCAACCTGCGCAAGCCGTGCAACCGGCCG TCGGGCCTCGAGCCGCAGCCGGGGGTCTTCGACTG CCTGCGAGACGCCATGGTGCGTGACTACGTGCGCC AGACGTGGAAGCTGGAGGGCGAGGCCCTGGAGCAG GCCATCATCAGCCAGGCCCCGCAGGTGGAGAAGCT CATTGCTACGACGGCCCACGAGCGGATGCCCTGGT ACCACAGCAGCCTGACGCGTGAGGAGGCCGAGCGC AAACTTTACTCTGGGGCGCAGACCGACGGCAAGTT CCTGCTGAGGCCGCGGAAGGAGCAGGGCACATACG CCCTGTCCCTCATCTATGGGAAGACGGTGTACCAC TACCTCATCAGCCAAGACAAGGCGGGCAAGTACTG CATTCCCGAGGGCACCAAGTTTGACACGCTCTGGC AGCTGGTGGAGTATCTGAAGCTGAAGGCGGACGGG CTCATCTACTGCCTGAAGGAGGCCTGCCCCAACAG CAGTGCCAGCAACGCCTCAGGGGCTGCTGCTCCCA CACTCCCAGCCCACCCATCCACGTTGACG Exemplary amino acid sequence of a dominant negative ZAP70 SEQ ID NO: 2 MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFL LRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA IAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRP SGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQ AIISQAPQVEKLIATTAHERMPWYHSSLTREEAER KLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADG LIYCLKEACPNSSASNASGAAAPTLPAHPSTLT Exemplary nucleotide sequence encoding ER(T2) polypeptide SEQ ID NO: 3 TCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCC AAGCCCGCTCATGATCAAACGCTCTAAGAAGAACA GCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTC AGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTA TTCCGAGTATGATCCTACCAGACCCTTCAGTGAAG CTTCGATGATGGGCTTACTGACCAACCTGGCAGAC AGGGAGCTGGTTCACATGATCAACTGGGCGAAGAG GGTGCCAGGCTTTGTGGATTTGACCCTCCATGATC AGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATC CTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCA CCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCT TGGACAGGAACCAGGGAAAATGTGTAGAGGGCATG GTGGAGATCTTCGACATGCTGCTGGCTACATCATC TCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGT TTGTGTGCCTCAAATCTATTATTTTGCTTAATTCT GGAGTGTACACATTTCTGTCCAGCACCCTGAAGTC TCTGGAAGAGAAGGACCATATCCACCGAGTCCTGG ACAAGATCACAGACACTTTGATCCACCTGATGGCC AAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCG GCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCA GGCACATGAGTAACAAAGGCATGGAGCATCTGTAC AGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGA CCTGCTGCTGGAGGCGGCGGACGCCCACCGCCTAC ATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAG GAGACGGACCAAAGCCACTTGGCCACTGCGGGCTC TACTTCATCGCATTCCTTGCAAAAGTATTACATCA CGGGGGAGGCAGAGGGTTTCCCTGCCACGGTC Exemplary amino acid sequence of ER(T2) polypeptide SEQ ID NO: 4 SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMV SALLDAEPPILYSEYDPTRPFSEASMMGLLTNLAD RELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGM VEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNS GVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMA KAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLY SMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV Exemplary nucleotide sequence of a Nuclear Export Signal SEQ ID NO: 5 AATGAATTAGCCTTGAAATTAGCAGGTCTTGAT ATCAACAAGACA Exemplary amino acid sequence of a Nuclear Export Signal SEQ ID NO: 6 NELALKLAGLDINKT Exemplary nucleotide sequence encoding an endoxifen-responsive dominant negative Zap-70 polypeptide (NES-ZAP70dn-ER(T2) SEQ ID NO: 7 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACAATGCCAGACCCCGCGGCGCACC TGCCCTTCTTCTACGGCAGCATCTCGCGTGCCGAG GCCGAGGAGCACCTGAAGCTGGCGGGCATGGCGGA CGGGCTCTTCCTGCTGCGCCAGTGCCTGCGCTCGC TGGGCGGCTATGTGCTGTCGCTCGTGCACGATGTG CGCTTCCACCACTTTCCCATCGAGCGCCAGCTCAA CGGCACCTACGCCATTGCCGGCGGCAAAGCGCACT GTGGACCGGCAGAGCTCTGCGAGTTCTACTCGCGC GACCCCGACGGGCTGCCCTGCAACCTGCGCAAGCC GTGCAACCGGCCGTCGGGCCTCGAGCCGCAGCCGG GGGTCTTCGACTGCCTGCGAGACGCCATGGTGCGT GACTACGTGCGCCAGACGTGGAAGCTGGAGGGCGA GGCCCTGGAGCAGGCCATCATCAGCCAGGCCCCGC AGGTGGAGAAGCTCATTGCTACGACGGCCCACGAG CGGATGCCCTGGTACCACAGCAGCCTGACGCGTGA GGAGGCCGAGCGCAAACTTTACTCTGGGGCGCAGA CCGACGGCAAGTTCCTGCTGAGGCCGCGGAAGGAG CAGGGCACATACGCCCTGTCCCTCATCTATGGGAA GACGGTGTACCACTACCTCATCAGCCAAGACAAGG CGGGCAAGTACTGCATTCCCGAGGGCACCAAGTTT GACACGCTCTGGCAGCTGGTGGAGTATCTGAAGCT GAAGGCGGACGGGCTCATCTACTGCCTGAAGGAGG CCTGCCCCAACAGCAGTGCCAGCAACGCCTCAGGG GCTGCTGCTCCCACACTCCCAGCCCACCCATCCAC GTTGACGGGATCCTCTGCTGGAGACATGAGAGCTG CCAACCTTTGGCCAAGCCCGCTCATGATCAAACGC TCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGC CGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGC CCCCCATACTCTATTCCGAGTATGATCCTACCAGA CCCTTCAGTGAAGCTTCGATGATGGGCTTACTGAC CAACCTGGCAGACAGGGAGCTGGTTCACATGATCA ACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTG ACCCTCCATGATCAGGTCCACCTTCTAGAATGTGC CTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGC GCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCT CCTAACTTGCTCTTGGACAGGAACCAGGGAAAATG TGTAGAGGGCATGGTGGAGATCTTCGACATGCTGC TGGCTACATCATCTCGGTTCCGCATGATGAATCTG CAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTAT TTTGCTTAATTCTGGAGTGTACACATTTCTGTCCA GCACCCTGAAGTCTCTGGAAGAGAAGGACCATATC CACCGAGTCCTGGACAAGATCACAGACACTTTGAT CCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGC AGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATC CTCTCCCACATCAGGCACATGAGTAACAAAGGCAT GGAGCATCTGTACAGCATGAAGTGCAAGAACGTGG TGCCCCTCTATGACCTGCTGCTGGAGGCGGCGGAC GCCCACCGCCTACATGCGCCCACTAGCCGTGGAGG GGCATCCGTGGAGGAGACGGACCAAAGCCACTTGG CCACTGCGGGCTCTACTTCATCGCATTCCTTGCAA AAGTATTACATCACGGGGGAGGCAGAGGGTTTCCC TGCCACGGTC Exemplary amino acid sequence of an endoxifen-responsive dominant negative Zap-70 polypeptide (NES-ZAP70dn-ER(T2) SEQ ID NO: 8 MNELALKLAGLDINKTMPDPAAHLPFFYGSISRAE AEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDV RFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSR DPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVR DYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHE RMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKE QGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKF DTLWQLVEYLKLKADGLIYCLKEACPNSSASNASG AAAPTLPAHPSTLTGSSAGDMRAANLWPSPLMIKR SKKNSLALSLTADQMVSALLDAEPPILYSEYDPTR PFSEASMMGLLTNLADRELVHMINWAKRVPGFVDL TLHDQVHLLECAWLEILMIGLVWRSMEHPVKLLFA PNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNL QGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHI HRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLI LSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAAD AHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQ KYYITGEAEGFPATV Exemplary nucleotide sequence encoding polypeptide including a nuclear export signal (NES) and a dominant negative Zap-70 moiety SEQ ID NO: 9 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACAATGCCAGACCCCGCGGCGCACC TGCCCTTCTTCTACGGCAGCATCTCGCGTGCCGAG GCCGAGGAGCACCTGAAGCTGGCGGGCATGGCGGA CGGGCTCTTCCTGCTGCGCCAGTGCCTGCGCTCGC TGGGCGGCTATGTGCTGTCGCTCGTGCACGATGTG CGCTTCCACCACTTTCCCATCGAGCGCCAGCTCAA CGGCACCTACGCCATTGCCGGCGGCAAAGCGCACT GTGGACCGGCAGAGCTCTGCGAGTTCTACTCGCGC GACCCCGACGGGCTGCCCTGCAACCTGCGCAAGCC GTGCAACCGGCCGTCGGGCCTCGAGCCGCAGCCGG GGGTCTTCGACTGCCTGCGAGACGCCATGGTGCGT GACTACGTGCGCCAGACGTGGAAGCTGGAGGGCGA GGCCCTGGAGCAGGCCATCATCAGCCAGGCCCCGC AGGTGGAGAAGCTCATTGCTACGACGGCCCACGAG CGGATGCCCTGGTACCACAGCAGCCTGACGCGTGA GGAGGCCGAGCGCAAACTTTACTCTGGGGCGCAGA CCGACGGCAAGTTCCTGCTGAGGCCGCGGAAGGAG CAGGGCACATACGCCCTGTCCCTCATCTATGGGAA GACGGTGTACCACTACCTCATCAGCCAAGACAAGG CGGGCAAGTACTGCATTCCCGAGGGCACCAAGTTT GACACGCTCTGGCAGCTGGTGGAGTATCTGAAGCT GAAGGCGGACGGGCTCATCTACTGCCTGAAGGAGG CCTGCCCCAACAGCAGTGCCAGCAACGCCTCAGGG GCTGCTGCTCCCACACTCCCAGCCCACCCATCCAC GTTGACG Exemplary peptide sequence of a polypeptide including a nuclear export signal (NES) and a dominant negative Zap-70 moiety SEQ ID NO: 10 MNELALKLAGLDINKTMPDPAAHLPFFYGSISRAE AEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDV RFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSR DPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVR DYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHE RMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKE QGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKF DTLWQLVEYLKLKADGLIYCLKEACPNSSASNASG AAAPTLPAHPSTLT Nucleotide sequence of the wild-type human Estrogen Receptor-alpha (ESRI) cDNA SEQ ID NO: 11 ATGACCATGACCCTCCACACCAAAGCATCTGGGAT GGCCCTACTGCATCAGATCCAAGGGAACGAGCTGG AGCCCCTGAACCGTCCGCAGCTCAAGATCCCCCTG GAGCGGCCCCTGGGCGAGGTGTACCTGGACAGCAG CAAGCCCGCCGTGTACAACTACCCCGAGGGCGCCG CCTACGAGTTCAACGCCGCGGCCGCCGCCAACGCG CAGGTCTACGGTCAGACCGGCCTCCCCTACGGCCC CGGGTCTGAGGCTGCGGCGTTCGGCTCCAACGGCC TGGGGGGTTTCCCCCCACTCAACAGCGTGTCTCCG AGCCCGCTGATGCTACTGCACCCGCCGCCGCAGCT GTCGCCTTTCCTGCAGCCCCACGGCCAGCAGGTGC CCTACTACCTGGAGAACGAGCCCAGCGGCTACACG GTGCGCGAGGCCGGCCCGCCGGCATTCTACAGGCC AAATTCAGATAATCGACGCCAGGGTGGCAGAGAAA GATTGGCCAGTACCAATGACAAGGGAAGTATGGCT ATGGAATCTGCCAAGGAGACTCGCTACTGTGCAGT GTGCAATGACTATGCTTCAGGCTACCATTATGGAG TCTGGTCCTGTGAGGGCTGCAAGGCCTTCTTCAAG AGAAGTATTCAAGGACATAACGACTATATGTGTCC AGCCACCAACCAGTGCACCATTGATAAAAACAGGA GGAAGAGCTGCCAGGCCTGCCGGCTCCGCAAATGC TACGAAGTGGGAATGATGAAAGGTGGGATACGAAA AGACCGAAGAGGAGGGAGAATGTTGAAACACAAGC GCCAGAGAGATGATGGGGAGGGCAGGGGTGAAGTG GGGTCTGCTGGAGACATGAGAGCTGCCAACCTTTG GCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGA ACAGCCTGGCCTTGTCCCTGACGGCCGACCAGATG GTCAGTGCCTTGTTGGATGCTGAGCCCCCCATACT CTATTCCGAGTATGATCCTACCAGACCCTTCAGTG AAGCTTCGATGATGGGCTTACTGACCAACCTGGCA GACAGGGAGCTGGTTCACATGATCAACTGGGCGAA GAGGGTGCCAGGCTTTGTGGATTTGACCCTCCATG ATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGAG ATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGA GCACCCAGGGAAGCTACTGTTTGCTCCTAACTTGC TCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGC ATGGTGGAGATCTTCGACATGCTGCTGGCTACATC ATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGG AGTTTGTGTGCCTCAAATCTATTATTTTGCTTAAT TCTGGAGTGTACACATTTCTGTCCAGCACCCTGAA GTCTCTGGAAGAGAAGGACCATATCCACCGAGTCC TGGACAAGATCACAGACACTTTGATCCACCTGATG GCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCA GCGGCTGGCCCAGCTCCTCCTCATCCTCTCCCACA TCAGGCACATGAGTAACAAAGGCATGGAGCATCTG TACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTA TGACCTGCTGCTGGAGATGCTGGACGCCCACCGCC TACATGCGCCCACTAGCCGTGGAGGGGCATCCGTG GAGGAGACGGACCAAAGCCACTTGGCCACTGCGGG CTCTACTTCATCGCATTCCTTGCAAAAGTATTACA TCACGGGGGAGGCAGAGGGTTTCCCTGCCACGGTC TGA Amino acid sequence of the wild-type human Estrogen Receptor-alpha (ESR1) SEQ ID NO: 12 MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPL ERPLGEVYLDSSKPAVYNYPEGAAYEFNAAAAANA QVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSP SPLMLLHPPPQLSPFLQPHGQQVPYYLENEPSGYT VREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMA MESAKETRYCAVCNDYASGYHYGVWSCEGCKAFFK RSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKC YEVGMMKGGIRKDRRGGRMLKHKRQRDDGEGRGEV GSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQM VSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLA DRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLE ILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEG MVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLN SGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLM AKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHL YSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASV EETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV Exemplary amino acid sequence of ER(T12) modulating domain SEQ ID NO: 13 SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMV SALLDAEPPILYSEYDPTRPFSEASMMGLLTNLAD RELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI LMIGLVWRSMEHPLKLLFAPNLLLDRNQGKCVEGM VEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNS GVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMA KAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLY SMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV Exemplary nucleic acid sequence encoding ER(T12) modulating domain SEQ ID NO: 14 TCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCC AAGCCCGCTCATGATCAAACGCTCTAAGAAGAACA GCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTC AGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTA TTCCGAGTATGATCCTACCAGACCCTTCAGTGAAG CTTCGATGATGGGCTTACTGACCAACCTGGCAGAC AGGGAGCTGGTTCACATGATCAACTGGGCGAAGAG GGTGCCAGGCTTTGTGGATTTGACCCTCCATGATC AGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATC CTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCA CCCActgAAGCTACTGTTTGCTCCTAACTTGCTCT TGGACAGGAACCAGGGAAAATGTGTAGAGGGCATG GTGGAGATCTTCGACATGCTGCTGGCTACATCATC TCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGT TTGTGTGCCTCAAATCTATTATTTTGCTTAATTCT GGAGTGTACACATTTCTGTCCAGCACCCTGAAGTC TCTGGAAGAGAAGGACCATATCCACCGAGTCCTGG ACAAGATCACAGACACTTTGATCCACCTGATGGCC AAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCG GCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCA GGCACATGAGTAACAAAGGCATGGAGCATCTGTAC AGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGA CCTGCTGCTGGAGgcggcgGACGCCCACCGCCTAC ATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAG GAGACGGACCAAAGCCACTTGGCCACTGCGGGCTC TACTTCATCGCATTCCTTGCAAAAGTATTACATCA CGGGGGAGGCAGAGGGTTTCCCTGCCACGGTC Exemplary amino acid sequence of NES-ZAP70dn(1-278)-ER(T12) SEQ ID NO: 15 MNELALKLAGLDINKTMPDPAAHLPFFYGSISRAE AEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDV RFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSR DPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVR DYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHE RMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKE QGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKF DTLWQLVEYLKLKADGLIYCLKEACPNSSASNASG AAAPTLPAHPSTLTGSSAGDMRAANLWPSPLMIKR SKKNSLALSLTADQMVSALLDAEPPILYSEYDPTR PFSEASMMGLLTNLADRELVHMINWAKRVPGFVDL TLHDQVHLLECAWLEILMIGLVWRSMEHPLKLLFA PNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNL QGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHI HRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLI LSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAAD AHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQ KYYITGEAEGFPATV Exemplary nucleic acid sequence encoding NES-ZAP70dn(1-278)-ER(T12) SEQ ID NO: 16 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACAATGCCAGACCCCGCGGCGCACC TGCCCTTCTTCTACGGCAGCATCTCGCGTGCCGAG GCCGAGGAGCACCTGAAGCTGGCGGGCATGGCGGA CGGGCTCTTCCTGCTGCGCCAGTGCCTGCGCTCGC TGGGCGGCTATGTGCTGTCGCTCGTGCACGATGTG CGCTTCCACCACTTTCCCATCGAGCGCCAGCTCAA CGGCACCTACGCCATTGCCGGCGGCAAAGCGCACT GTGGACCGGCAGAGCTCTGCGAGTTCTACTCGCGC GACCCCGACGGGCTGCCCTGCAACCTGCGCAAGCC GTGCAACCGGCCGTCGGGCCTCGAGCCGCAGCCGG GGGTCTTCGACTGCCTGCGAGACGCCATGGTGCGT GACTACGTGCGCCAGACGTGGAAGCTGGAGGGCGA GGCCCTGGAGCAGGCCATCATCAGCCAGGCCCCGC AGGTGGAGAAGCTCATTGCTACGACGGCCCACGAG CGGATGCCCTGGTACCACAGCAGCCTGACGCGTGA GGAGGCCGAGCGCAAACTTTACTCTGGGGCGCAGA CCGACGGCAAGTTCCTGCTGAGGCCGCGGAAGGAG CAGGGCACATACGCCCTGTCCCTCATCTATGGGAA GACGGTGTACCACTACCTCATCAGCCAAGACAAGG CGGGCAAGTACTGCATTCCCGAGGGCACCAAGTTT GACACGCTCTGGCAGCTGGTGGAGTATCTGAAGCT GAAGGCGGACGGGCTCATCTACTGCCTGAAGGAGG CCTGCCCCAACAGCAGTGCCAGCAACGCCTCAGGG GCTGCTGCTCCCACACTCCCAGCCCACCCATCCAC GTTGACGGGATCCTCTGCTGGAGACATGAGAGCTG CCAACCTTTGGCCAAGCCCGCTCATGATCAAACGC TCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGC CGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGC CCCCCATACTCTATTCCGAGTATGATCCTACCAGA CCCTTCAGTGAAGCTTCGATGATGGGCTTACTGAC CAACCTGGCAGACAGGGAGCTGGTTCACATGATCA ACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTG ACCCTCCATGATCAGGTCCACCTTCTAGAATGTGC CTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGC GCTCCATGGAGCACCCACTGAAGCTACTGTTTGCT CCTAACTTGCTCTTGGACAGGAACCAGGGAAAATG TGTAGAGGGCATGGTGGAGATCTTCGACATGCTGC TGGCTACATCATCTCGGTTCCGCATGATGAATCTG CAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTAT TTTGCTTAATTCTGGAGTGTACACATTTCTGTCCA GCACCCTGAAGTCTCTGGAAGAGAAGGACCATATC CACCGAGTCCTGGACAAGATCACAGACACTTTGAT CCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGC AGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATC CTCTCCCACATCAGGCACATGAGTAACAAAGGCAT GGAGCATCTGTACAGCATGAAGTGCAAGAACGTGG TGCCCCTCTATGACCTGCTGCTGGAGGCGGCGGAC GCCCACCGCCTACATGCGCCCACTAGCCGTGGAGG GGCATCCGTGGAGGAGACGGACCAAAGCCACTTGG CCACTGCGGGCTCTACTTCATCGCATTCCTTGCAA AAGTATTACATCACGGGGGAGGCAGAGGGTTTCCC TGCCACGGTCTGA Exemplary amino acid sequence of LCK(1-266) SEQ ID NO: 17 MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGT LLIRNGSEVRDPLVTYEGSNPPASPLQDNLVIALH SYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTT GQEGFIPFNFVAKANSLEPEPWFFKNLSRKDAERQ LLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQG EVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYT NASDGLCTRLSRPCQTQKPQKPWWEDEWEVPRETL KLVERLGAGQFGEVWMGYYNG Exemplary amino acid sequence of NES-LCK(1-266)-ER(T12) SEQ ID NO: 18 MNELALKLAGLDINKTMGCGCSSHPEDDWMENIDV CENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGS NPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLR ILEQSGEWWKAQSLTTGQEGFIPFNFVAKANSLEP EPWFFKNLSRKDAERQLLAPGNTHGSFLIRESEST AGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYIS PRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKP QKPWWEDEWEVPRETLKLVERLGAGQFGEVWMGYY NGGSSAGDMRAANLWPSPLMIKRSKKNSLALSLTA DQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLT NLADRELVHMINWAKRVPGFVDLTLHDQVHLLECA WLEILMIGLVWRSMEHPLKLLFAPNLLLDRNQGKC VEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSII LLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLI HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM EHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGG ASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFP ATV Exemplary nucleic acid sequence encoding LCK(1-266) SEQ ID NO: 19 ATGGGCTGTGGCTGCAGCTCACACCCGGAAGATGA CTGGATGGAAAACATCGATGTGTGTGAGAACTGCC ATTATCCCATAGTCCCACTGGATGGCAAGGGCACG CTGCTCATCCGAAATGGCTCTGAGGTGCGGGACCC ACTGGTTACCTACGAAGGCTCCAATCCGCCGGCTT CCCCACTGCAAGACAACCTGGTTATCGCTCTGCAC AGCTATGAGCCCTCTCACGACGGAGATCTGGGCTT TGAGAAGGGGGAACAGCTCCGCATCCTGGAGCAGA GCGGCGAGTGGTGGAAGGCGCAGTCCCTGACCACG GGCCAGGAAGGCTTCATCCCCTTCAATTTTGTGGC CAAAGCGAACAGCCTGGAGCCCGAACCCTGGTTCT TCAAGAACCTGAGCCGCAAGGACGCGGAGCGGCAG CTCCTGGCGCCCGGGAACACTCACGGCTCCTTCCT CATCCGGGAGAGCGAGAGCACCGCGGGATCGTTTT CACTGTCGGTCCGGGACTTCGACCAGAACCAGGGA GAGGTGGTGAAACATTACAAGATCCGTAATCTGGA CAACGGTGGCTTCTACATCTCCCCTCGAATCACTT TTCCCGGCCTGCATGAACTGGTCCGCCATTACACC AATGCTTCAGATGGGCTGTGCACACGGTTGAGCCG CCCCTGCCAGACCCAGAAGCCCCAGAAGCCGTGGT GGGAGGACGAGTGGGAGGTTCCCAGGGAGACGCTG AAGCTGGTGGAGCGGCTGGGGGCTGGACAGTTCGG GGAGGTGTGGATGGGGTACTACAACGGG Exemplary amino acid sequence encoding polypeptide including a nuclear export signal (NES) and a dominant negative LCK1(1-266) moiety SEQ ID NO: 20 MNELALKLAGLDINKTMGCGCSSHPEDDWMENIDV CENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGS NPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLR ILEQSGEWWKAQSLTTGQEGFIPFNFVAKANSLEP EPWFFKNLSRKDAERQLLAPGNTHGSFLIRESEST AGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYIS PRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKP QKPWWEDEWEVPRETLKLVERLGAGQFGEVWMGYY NG Exemplary nucleotide sequence encoding polypeptide including a nuclear export signal (NES) and a dominant negative LCK1(1-266) moiety SEQ ID NO: 21 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACAATGGGCTGTGGCTGCAGCTCAC ACCCGGAAGATGACTGGATGGAAAACATCGATGTG TGTGAGAACTGCCATTATCCCATAGTCCCACTGGA TGGCAAGGGCACGCTGCTCATCCGAAATGGCTCTG AGGTGCGGGACCCACTGGTTACCTACGAAGGCTCC AATCCGCCGGCTTCCCCACTGCAAGACAACCTGGT TATCGCTCTGCACAGCTATGAGCCCTCTCACGACG GAGATCTGGGCTTTGAGAAGGGGGAACAGCTCCGC ATCCTGGAGCAGAGCGGCGAGTGGTGGAAGGCGCA GTCCCTGACCACGGGCCAGGAAGGCTTCATCCCCT TCAATTTTGTGGCCAAAGCGAACAGCCTGGAGCCC GAACCCTGGTTCTTCAAGAACCTGAGCCGCAAGGA CGCGGAGCGGCAGCTCCTGGCGCCCGGGAACACTC ACGGCTCCTTCCTCATCCGGGAGAGCGAGAGCACC GCGGGATCGTTTTCACTGTCGGTCCGGGACTTCGA CCAGAACCAGGGAGAGGTGGTGAAACATTACAAGA TCCGTAATCTGGACAACGGTGGCTTCTACATCTCC CCTCGAATCACTTTTCCCGGCCTGCATGAACTGGT CCGCCATTACACCAATGCTTCAGATGGGCTGTGCA CACGGTTGAGCCGCCCCTGCCAGACCCAGAAGCCC CAGAAGCCGTGGTGGGAGGACGAGTGGGAGGTTCC CAGGGAGACGCTGAAGCTGGTGGAGCGGCTGGGGG CTGGACAGTTCGGGGAGGTGTGGATGGGGTACTAC AACGGG Exemplary nucleic acid sequence encoding NES-LCK(1-266)-ER(T12) SEQ ID NO: 22 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACAATGGGCTGTGGCTGCAGCTCAC ACCCGGAAGATGACTGGATGGAAAACATCGATGTG TGTGAGAACTGCCATTATCCCATAGTCCCACTGGA TGGCAAGGGCACGCTGCTCATCCGAAATGGCTCTG AGGTGCGGGACCCACTGGTTACCTACGAAGGCTCC AATCCGCCGGCTTCCCCACTGCAAGACAACCTGGT TATCGCTCTGCACAGCTATGAGCCCTCTCACGACG GAGATCTGGGCTTTGAGAAGGGGGAACAGCTCCGC ATCCTGGAGCAGAGCGGCGAGTGGTGGAAGGCGCA GTCCCTGACCACGGGCCAGGAAGGCTTCATCCCCT TCAATTTTGTGGCCAAAGCGAACAGCCTGGAGCCC GAACCCTGGTTCTTCAAGAACCTGAGCCGCAAGGA CGCGGAGCGGCAGCTCCTGGCGCCCGGGAACACTC ACGGCTCCTTCCTCATCCGGGAGAGCGAGAGCACC GCGGGATCGTTTTCACTGTCGGTCCGGGACTTCGA CCAGAACCAGGGAGAGGTGGTGAAACATTACAAGA TCCGTAATCTGGACAACGGTGGCTTCTACATCTCC CCTCGAATCACTTTTCCCGGCCTGCATGAACTGGT CCGCCATTACACCAATGCTTCAGATGGGCTGTGCA CACGGTTGAGCCGCCCCTGCCAGACCCAGAAGCCC CAGAAGCCGTGGTGGGAGGACGAGTGGGAGGTTCC CAGGGAGACGCTGAAGCTGGTGGAGCGGCTGGGGG CTGGACAGTTCGGGGAGGTGTGGATGGGGTACTAC AACGGGGGATCCTCTGCTGGAGACATGAGAGCTGC CAACCTTTGGCCAAGCCCGCTCATGATCAAACGCT CTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCC GACCAGATGGTCAGTGCCTTGTTGGATGCTGAGCC CCCCATACTCTATTCCGAGTATGATCCTACCAGAC CCTTCAGTGAAGCTTCGATGATGGGCTTACTGACC AACCTGGCAGACAGGGAGCTGGTTCACATGATCAA CTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTGA CCCTCCATGATCAGGTCCACCTTCTAGAATGTGCC TGGCTAGAGATCCTGATGATTGGTCTCGTCTGGCG CTCCATGGAGCACCCACTGAAGCTACTGTTTGCTC CTAACTTGCTCTTGGACAGGAACCAGGGAAAATGT GTAGAGGGCATGGTGGAGATCTTCGACATGCTGCT GGCTACATCATCTCGGTTCCGCATGATGAATCTGC AGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATT TTGCTTAATTCTGGAGTGTACACATTTCTGTCCAG CACCCTGAAGTCTCTGGAAGAGAAGGACCATATCC ACCGAGTCCTGGACAAGATCACAGACACTTTGATC CACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCA GCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATCC TCTCCCACATCAGGCACATGAGTAACAAAGGCATG GAGCATCTGTACAGCATGAAGTGCAAGAACGTGGT GCCCCTCTATGACCTGCTGCTGGAGGCGGCGGACG CCCACCGCCTACATGCGCCCACTAGCCGTGGAGGG GCATCCGTGGAGGAGACGGACCAAAGCCACTTGGC CACTGCGGGCTCTACTTCATCGCATTCCTTGCAAA AGTATTACATCACGGGGGAGGCAGAGGGTTTCCCT GCCACGGTCTGA Exemplary amino acid sequence of SHP1(210-595) SEQ ID NO: 23 RQPYYATRVNAADIENRVLELNKKQESEDTAKAGF WEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNI LPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGP DENAKTYIASQGCLEATVNDFWQMAWQENSRVIVM TTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGE HDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPD HGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCS AGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQ MVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLE VLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKH KEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKR K Exemplary amino acid sequence of NES-SHP1(210-595)-ER(T12) SEQ ID NO: 24 MNELALKLAGLDINKTRQPYYATRVNAADIENRVL ELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRL EGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPG SDYINANYIKNQLLGPDENAKTYIASQGCLEATVN DFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEV GMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNG DLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQ RQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENI STKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFI YVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPP AMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVK KQRSADKEKSKGSLKRKGSSAGDMRAANLWPSPLM IKRSKKNSLALSLTADQMVSALLDAEPPILYSEYD PTRPFSEASMMGLLTNLADRELVHMINWAKRVPGF VDLTLHDQVHLLECAWLEILMIGLVWRSMEHPLKL LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRM MNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEK DHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQL LLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLE AADAHRLHAPTSRGGASVEETDQSHLATAGSTSSH SLQKYYITGEAEGFPATV Exemplary nucleic acid sequence encoding SHP1(210-595) SEQ ID NO: 25 CGGCAGCCGTACTATGCCACGAGGGTGAATGCGGC TGACATTGAGAACCGAGTGTTGGAACTGAACAAGA AGCAGGAGTCCGAGGATACAGCCAAGGCTGGCTTC TGGGAGGAGTTTGAGAGTTTGCAGAAGCAGGAGGT GAAGAACTTGCACCAGCGTCTGGAAGGGCAGCGGC CAGAGAACAAGGGCAAGAACCGCTACAAGAACATT CTCCCCTTTGACCACAGCCGAGTGATCCTGCAGGG ACGGGACAGTAACATCCCCGGGTCCGACTACATCA ATGCCAACTACATCAAGAACCAGCTGCTAGGCCCT GATGAGAACGCTAAGACCTACATCGCCAGCCAGGG CTGTCTGGAGGCCACGGTCAATGACTTCTGGCAGA TGGCGTGGCAGGAGAACAGCCGTGTCATCGTCATG ACCACCCGAGAGGTGGAGAAAGGCCGGAACAAATG CGTCCCATACTGGCCCGAGGTGGGCATGCAGCGTG CTTATGGGCCCTACTCTGTGACCAACTGCGGGGAG CATGACACAACCGAATACAAACTCCGTACCTTACA GGTCTCCCCGCTGGACAATGGAGACCTGATTCGGG AGATCTGGCATTACCAGTACCTGAGCTGGCCCGAC CATGGGGTCCCCAGTGAGCCTGGGGGTGTCCTCAG CTTCCTGGACCAGATCAACCAGCGGCAGGAAAGTC TGCCTCACGCAGGGCCCATCATCGTGCACTGCAGC GCCGGCATCGGCCGCACAGGCACCATCATTGTCAT CGACATGCTCATGGAGAACATCTCCACCAAGGGCC TGGACTGTGACATTGACATCCAGAAGACCATCCAG ATGGTGCGGGCGCAGCGCTCGGGCATGGTGCAGAC GGAGGCGCAGTACAAGTTCATCTACGTGGCCATCG CCCAGTTCATTGAAACCACTAAGAAGAAGCTGGAG GTCCTGCAGTCGCAGAAGGGCCAGGAGTCGGAGTA CGGGAACATCACCTATCCCCCAGCCATGAAGAATG CCCATGCCAAGGCCTCCCGCACCTCGTCCAAACAC AAGGAGGATGTGTATGAGAACCTGCACACTAAGAA CAAGAGGGAGGAGAAAGTGAAGAAGCAGCGGTCAG CAGACAAGGAGAAGAGCAAGGGTTCCCTCAAGAGG AAG Exemplary nucleic acid sequence encoding NES-SHP1(210-595)-ER(T12) SEQ ID NO: 26 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACACGGCAGCCGTACTATGCCACGA GGGTGAATGCGGCTGACATTGAGAACCGAGTGTTG GAACTGAACAAGAAGCAGGAGTCCGAGGATACAGC CAAGGCTGGCTTCTGGGAGGAGTTTGAGAGTTTGC AGAAGCAGGAGGTGAAGAACTTGCACCAGCGTCTG GAAGGGCAGCGGCCAGAGAACAAGGGCAAGAACCG CTACAAGAACATTCTCCCCTTTGACCACAGCCGAG TGATCCTGCAGGGACGGGACAGTAACATCCCCGGG TCCGACTACATCAATGCCAACTACATCAAGAACCA GCTGCTAGGCCCTGATGAGAACGCTAAGACCTACA TCGCCAGCCAGGGCTGTCTGGAGGCCACGGTCAAT GACTTCTGGCAGATGGCGTGGCAGGAGAACAGCCG TGTCATCGTCATGACCACCCGAGAGGTGGAGAAAG GCCGGAACAAATGCGTCCCATACTGGCCCGAGGTG GGCATGCAGCGTGCTTATGGGCCCTACTCTGTGAC CAACTGCGGGGAGCATGACACAACCGAATACAAAC TCCGTACCTTACAGGTCTCCCCGCTGGACAATGGA GACCTGATTCGGGAGATCTGGCATTACCAGTACCT GAGCTGGCCCGACCATGGGGTCCCCAGTGAGCCTG GGGGTGTCCTCAGCTTCCTGGACCAGATCAACCAG CGGCAGGAAAGTCTGCCTCACGCAGGGCCCATCAT CGTGCACTGCAGCGCCGGCATCGGCCGCACAGGCA CCATCATTGTCATCGACATGCTCATGGAGAACATC TCCACCAAGGGCCTGGACTGTGACATTGACATCCA GAAGACCATCCAGATGGTGCGGGCGCAGCGCTCGG GCATGGTGCAGACGGAGGCGCAGTACAAGTTCATC TACGTGGCCATCGCCCAGTTCATTGAAACCACTAA GAAGAAGCTGGAGGTCCTGCAGTCGCAGAAGGGCC AGGAGTCGGAGTACGGGAACATCACCTATCCCCCA GCCATGAAGAATGCCCATGCCAAGGCCTCCCGCAC CTCGTCCAAACACAAGGAGGATGTGTATGAGAACC TGCACACTAAGAACAAGAGGGAGGAGAAAGTGAAG AAGCAGCGGTCAGCAGACAAGGAGAAGAGCAAGGG TTCCCTCAAGAGGAAGGGATCCTCTGCTGGAGACA TGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATG ATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTC CCTGACGGCCGACCAGATGGTCAGTGCCTTGTTGG ATGCTGAGCCCCCCATACTCTATTCCGAGTATGAT CCTACCAGACCCTTCAGTGAAGCTTCGATGATGGG CTTACTGACCAACCTGGCAGACAGGGAGCTGGTTC ACATGATCAACTGGGCGAAGAGGGTGCCAGGCTTT GTGGATTTGACCCTCCATGATCAGGTCCACCTTCT AGAATGTGCCTGGCTAGAGATCCTGATGATTGGTC TCGTCTGGCGCTCCATGGAGCACCCACTGAAGCTA CTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCA GGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCG ACATGCTGCTGGCTACATCATCTCGGTTCCGCATG ATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAA ATCTATTATTTTGCTTAATTCTGGAGTGTACACAT TTCTGTCCAGCACCCTGAAGTCTCTGGAAGAGAAG GACCATATCCACCGAGTCCTGGACAAGATCACAGA CACTTTGATCCACCTGATGGCCAAGGCAGGCCTGA CCCTGCAGCAGCAGCACCAGCGGCTGGCCCAGCTC CTCCTCATCCTCTCCCACATCAGGCACATGAGTAA CAAAGGCATGGAGCATCTGTACAGCATGAAGTGCA AGAACGTGGTGCCCCTCTATGACCTGCTGCTGGAG GCGGCGGACGCCCACCGCCTACATGCGCCCACTAG CCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAA GCCACTTGGCCACTGCGGGCTCTACTTCATCGCAT TCCTTGCAAAAGTATTACATCACGGGGGAGGCAGA GGGTTTCCCTGCCACGGTCTGA Exemplary amino acid sequence encoding polypeptide including a nuclear export signal (NES) and a dominant negative SHP1(210-595) moiety SEQ ID NO: 27 MNELALKLAGLDINKTRQPYYATRVNAADIENRVL ELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRL EGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPG SDYINANYIKNQLLGPDENAKTYIASQGCLEATVN DFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEV GMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNG DLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQ RQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENI STKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFI YVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPP AMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVK KQRSADKEKSKGSLKRK Exemplary nucleotide sequence encoding polypeptide including a nuclear export signal (NES) and a dominant negative LCK1(1-266) moiety SEQ ID NO: 28 ATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGA TATCAACAAGACACGGCAGCCGTACTATGCCACGA GGGTGAATGCGGCTGACATTGAGAACCGAGTGTTG GAACTGAACAAGAAGCAGGAGTCCGAGGATACAGC CAAGGCTGGCTTCTGGGAGGAGTTTGAGAGTTTGC AGAAGCAGGAGGTGAAGAACTTGCACCAGCGTCTG GAAGGGCAGCGGCCAGAGAACAAGGGCAAGAACCG CTACAAGAACATTCTCCCCTTTGACCACAGCCGAG TGATCCTGCAGGGACGGGACAGTAACATCCCCGGG TCCGACTACATCAATGCCAACTACATCAAGAACCA GCTGCTAGGCCCTGATGAGAACGCTAAGACCTACA TCGCCAGCCAGGGCTGTCTGGAGGCCACGGTCAAT GACTTCTGGCAGATGGCGTGGCAGGAGAACAGCCG TGTCATCGTCATGACCACCCGAGAGGTGGAGAAAG GCCGGAACAAATGCGTCCCATACTGGCCCGAGGTG GGCATGCAGCGTGCTTATGGGCCCTACTCTGTGAC CAACTGCGGGGAGCATGACACAACCGAATACAAAC TCCGTACCTTACAGGTCTCCCCGCTGGACAATGGA GACCTGATTCGGGAGATCTGGCATTACCAGTACCT GAGCTGGCCCGACCATGGGGTCCCCAGTGAGCCTG GGGGTGTCCTCAGCTTCCTGGACCAGATCAACCAG CGGCAGGAAAGTCTGCCTCACGCAGGGCCCATCAT CGTGCACTGCAGCGCCGGCATCGGCCGCACAGGCA CCATCATTGTCATCGACATGCTCATGGAGAACATC TCCACCAAGGGCCTGGACTGTGACATTGACATCCA GAAGACCATCCAGATGGTGCGGGCGCAGCGCTCGG GCATGGTGCAGACGGAGGCGCAGTACAAGTTCATC TACGTGGCCATCGCCCAGTTCATTGAAACCACTAA GAAGAAGCTGGAGGTCCTGCAGTCGCAGAAGGGCC AGGAGTCGGAGTACGGGAACATCACCTATCCCCCA GCCATGAAGAATGCCCATGCCAAGGCCTCCCGCAC CTCGTCCAAACACAAGGAGGATGTGTATGAGAACC TGCACACTAAGAACAAGAGGGAGGAGAAAGTGAAG AAGCAGCGGTCAGCAGACAAGGAGAAGAGCAAGGG TTCCCTCAAGAGGAAG Exemplary nucleotide sequence encoding an endoxifen-responsive dominant negative Zap-70 polypeptide (ZAP70dn-ER(T2) SEQ ID NO: 29 ATGCCAGACCCCGCGGCGCACCTGCCCTTCTTCTA CGGCAGCATCTCGCGTGCCGAGGCCGAGGAGCACC TGAAGCTGGCGGGCATGGCGGACGGGCTCTTCCTG CTGCGCCAGTGCCTGCGCTCGCTGGGCGGCTATGT GCTGTCGCTCGTGCACGATGTGCGCTTCCACCACT TTCCCATCGAGCGCCAGCTCAACGGCACCTACGCC ATTGCCGGCGGCAAAGCGCACTGTGGACCGGCAGA GCTCTGCGAGTTCTACTCGCGCGACCCCGACGGGC TGCCCTGCAACCTGCGCAAGCCGTGCAACCGGCCG TCGGGCCTCGAGCCGCAGCCGGGGGTCTTCGACTG CCTGCGAGACGCCATGGTGCGTGACTACGTGCGCC AGACGTGGAAGCTGGAGGGCGAGGCCCTGGAGCAG GCCATCATCAGCCAGGCCCCGCAGGTGGAGAAGCT CATTGCTACGACGGCCCACGAGCGGATGCCCTGGT ACCACAGCAGCCTGACGCGTGAGGAGGCCGAGCGC AAACTTTACTCTGGGGCGCAGACCGACGGCAAGTT CCTGCTGAGGCCGCGGAAGGAGCAGGGCACATACG CCCTGTCCCTCATCTATGGGAAGACGGTGTACCAC TACCTCATCAGCCAAGACAAGGCGGGCAAGTACTG CATTCCCGAGGGCACCAAGTTTGACACGCTCTGGC AGCTGGTGGAGTATCTGAAGCTGAAGGCGGACGGG CTCATCTACTGCCTGAAGGAGGCCTGCCCCAACAG CAGTGCCAGCAACGCCTCAGGGGCTGCTGCTCCCA CACTCCCAGCCCACCCATCCACGTTGACGGGATCC TCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCC AAGCCCGCTCATGATCAAACGCTCTAAGAAGAACA GCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTC AGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTA TTCCGAGTATGATCCTACCAGACCCTTCAGTGAAG CTTCGATGATGGGCTTACTGACCAACCTGGCAGAC AGGGAGCTGGTTCACATGATCAACTGGGCGAAGAG GGTGCCAGGCTTTGTGGATTTGACCCTCCATGATC AGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATC CTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCA CCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCT TGGACAGGAACCAGGGAAAATGTGTAGAGGGCATG GTGGAGATCTTCGACATGCTGCTGGCTACATCATC TCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGT TTGTGTGCCTCAAATCTATTATTTTGCTTAATTCT GGAGTGTACACATTTCTGTCCAGCACCCTGAAGTC TCTGGAAGAGAAGGACCATATCCACCGAGTCCTGG ACAAGATCACAGACACTTTGATCCACCTGATGGCC AAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCG GCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCA GGCACATGAGTAACAAAGGCATGGAGCATCTGTAC AGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGA CCTGCTGCTGGAGGCGGCGGACGCCCACCGCCTAC ATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAG GAGACGGACCAAAGCCACTTGGCCACTGCGGGCTC TACTTCATCGCATTCCTTGCAAAAGTATTACATCA CGGGGGAGGCAGAGGGTTTCCCTGCCACGGTC Exemplary amino acid sequence of an endoxifen-responsive dominant negative Zap-70 polypeptide (ZAP70dn-ER(T2) SEQ ID NO: 30 MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFL LRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA IAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRP SGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQ AIISQAPQVEKLIATTAHERMPWYHSSLTREEAER KLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADG LIYCLKEACPNSSASNASGAAAPTLPAHPSTLTGS SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMV SALLDAEPPILYSEYDPTRPFSEASMMGLLTNLAD RELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGM VEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNS GVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMA KAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLY SMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV Exemplary amino acid sequence of ZAP70dn(1-278)-ER(T12) SEQ ID NO: 31 MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFL LRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA IAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRP SGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQ AIISQAPQVEKLIATTAHERMPWYHSSLTREEAER KLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADG LIYCLKEACPNSSASNASGAAAPTLPAHPSTLTGS SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMV SALLDAEPPILYSEYDPTRPFSEASMMGLLTNLAD RELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI LMIGLVWRSMEHPLKLLFAPNLLLDRNQGKCVEGM VEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNS GVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMA KAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLY SMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV Exemplary nucleic acid sequence encoding ZAP70dn(1-278)-ER(T12) SEQ ID NO: 32 ATGCCAGACCCCGCGGCGCACCTGCCCTTCTTCTA CGGCAGCATCTCGCGTGCCGAGGCCGAGGAGCACC TGAAGCTGGCGGGCATGGCGGACGGGCTCTTCCTG CTGCGCCAGTGCCTGCGCTCGCTGGGCGGCTATGT GCTGTCGCTCGTGCACGATGTGCGCTTCCACCACT TTCCCATCGAGCGCCAGCTCAACGGCACCTACGCC ATTGCCGGCGGCAAAGCGCACTGTGGACCGGCAGA GCTCTGCGAGTTCTACTCGCGCGACCCCGACGGGC TGCCCTGCAACCTGCGCAAGCCGTGCAACCGGCCG TCGGGCCTCGAGCCGCAGCCGGGGGTCTTCGACTG CCTGCGAGACGCCATGGTGCGTGACTACGTGCGCC AGACGTGGAAGCTGGAGGGCGAGGCCCTGGAGCAG GCCATCATCAGCCAGGCCCCGCAGGTGGAGAAGCT CATTGCTACGACGGCCCACGAGCGGATGCCCTGGT ACCACAGCAGCCTGACGCGTGAGGAGGCCGAGCGC AAACTTTACTCTGGGGCGCAGACCGACGGCAAGTT CCTGCTGAGGCCGCGGAAGGAGCAGGGCACATACG CCCTGTCCCTCATCTATGGGAAGACGGTGTACCAC TACCTCATCAGCCAAGACAAGGCGGGCAAGTACTG CATTCCCGAGGGCACCAAGTTTGACACGCTCTGGC AGCTGGTGGAGTATCTGAAGCTGAAGGCGGACGGG CTCATCTACTGCCTGAAGGAGGCCTGCCCCAACAG CAGTGCCAGCAACGCCTCAGGGGCTGCTGCTCCCA CACTCCCAGCCCACCCATCCACGTTGACGGGATCC TCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCC AAGCCCGCTCATGATCAAACGCTCTAAGAAGAACA GCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTC AGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTA TTCCGAGTATGATCCTACCAGACCCTTCAGTGAAG CTTCGATGATGGGCTTACTGACCAACCTGGCAGAC AGGGAGCTGGTTCACATGATCAACTGGGCGAAGAG GGTGCCAGGCTTTGTGGATTTGACCCTCCATGATC AGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATC CTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCA CCCACTGAAGCTACTGTTTGCTCCTAACTTGCTCT TGGACAGGAACCAGGGAAAATGTGTAGAGGGCATG GTGGAGATCTTCGACATGCTGCTGGCTACATCATC TCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGT TTGTGTGCCTCAAATCTATTATTTTGCTTAATTCT GGAGTGTACACATTTCTGTCCAGCACCCTGAAGTC TCTGGAAGAGAAGGACCATATCCACCGAGTCCTGG ACAAGATCACAGACACTTTGATCCACCTGATGGCC AAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCG GCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCA GGCACATGAGTAACAAAGGCATGGAGCATCTGTAC AGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGA CCTGCTGCTGGAGGCGGCGGACGCCCACCGCCTAC ATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAG GAGACGGACCAAAGCCACTTGGCCACTGCGGGCTC TACTTCATCGCATTCCTTGCAAAAGTATTACATCA CGGGGGAGGCAGAGGGTTTCCCTGCCACGGTCTGA Exemplary amino acid sequence of LCK(1-266)-ER(T12) SEQ ID NO: 33 MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGT LLIRNGSEVRDPLVTYEGSNPPASPLQDNLVIALH SYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTT GQEGFIPFNFVAKANSLEPEPWFFKNLSRKDAERQ LLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQG EVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYT NASDGLCTRLSRPCQTQKPQKPWWEDEWEVPRETL KLVERLGAGQFGEVWMGYYNGGSSAGDMRAANLWP SPLMIKRSKKNSLALSLTADQMVSALLDAEPPILY SEYDPTRPFSEASMMGLLTNLADRELVHMINWAKR VPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEH PLKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSS RFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKS LEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQR LAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYD LLLEAADAHRLHAPTSRGGASVEETDQSHLATAGS TSSHSLQKYYITGEAEGFPATV Exemplary nucleic acid sequence encoding LCK(1-266)-ER(T12) SEQ ID NO: 34 ATGGGCTGTGGCTGCAGCTCACACCCGGAAGATGA CTGGATGGAAAACATCGATGTGTGTGAGAACTGCC ATTATCCCATAGTCCCACTGGATGGCAAGGGCACG CTGCTCATCCGAAATGGCTCTGAGGTGCGGGACCC ACTGGTTACCTACGAAGGCTCCAATCCGCCGGCTT CCCCACTGCAAGACAACCTGGTTATCGCTCTGCAC AGCTATGAGCCCTCTCACGACGGAGATCTGGGCTT TGAGAAGGGGGAACAGCTCCGCATCCTGGAGCAGA GCGGCGAGTGGTGGAAGGCGCAGTCCCTGACCACG GGCCAGGAAGGCTTCATCCCCTTCAATTTTGTGGC CAAAGCGAACAGCCTGGAGCCCGAACCCTGGTTCT TCAAGAACCTGAGCCGCAAGGACGCGGAGCGGCAG CTCCTGGCGCCCGGGAACACTCACGGCTCCTTCCT CATCCGGGAGAGCGAGAGCACCGCGGGATCGTTTT CACTGTCGGTCCGGGACTTCGACCAGAACCAGGGA GAGGTGGTGAAACATTACAAGATCCGTAATCTGGA CAACGGTGGCTTCTACATCTCCCCTCGAATCACTT TTCCCGGCCTGCATGAACTGGTCCGCCATTACACC AATGCTTCAGATGGGCTGTGCACACGGTTGAGCCG CCCCTGCCAGACCCAGAAGCCCCAGAAGCCGTGGT GGGAGGACGAGTGGGAGGTTCCCAGGGAGACGCTG AAGCTGGTGGAGCGGCTGGGGGCTGGACAGTTCGG GGAGGTGTGGATGGGGTACTACAACGGGGGATCCT CTGCTGGAGACATGAGAGCTGCCAACCTTTGGCCA AGCCCGCTCATGATCAAACGCTCTAAGAAGAACAG CCTGGCCTTGTCCCTGACGGCCGACCAGATGGTCA GTGCCTTGTTGGATGCTGAGCCCCCCATACTCTAT TCCGAGTATGATCCTACCAGACCCTTCAGTGAAGC TTCGATGATGGGCTTACTGACCAACCTGGCAGACA GGGAGCTGGTTCACATGATCAACTGGGCGAAGAGG GTGCCAGGCTTTGTGGATTTGACCCTCCATGATCA GGTCCACCTTCTAGAATGTGCCTGGCTAGAGATCC TGATGATTGGTCTCGTCTGGCGCTCCATGGAGCAC CCACTGAAGCTACTGTTTGCTCCTAACTTGCTCTT GGACAGGAACCAGGGAAAATGTGTAGAGGGCATGG TGGAGATCTTCGACATGCTGCTGGCTACATCATCT CGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTT TGTGTGCCTCAAATCTATTATTTTGCTTAATTCTG GAGTGTACACATTTCTGTCCAGCACCCTGAAGTCT CTGGAAGAGAAGGACCATATCCACCGAGTCCTGGA CAAGATCACAGACACTTTGATCCACCTGATGGCCA AGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGG CTGGCCCAGCTCCTCCTCATCCTCTCCCACATCAG GCACATGAGTAACAAAGGCATGGAGCATCTGTACA GCATGAAGTGCAAGAACGTGGTGCCCCTCTATGAC CTGCTGCTGGAGGCGGCGGACGCCCACCGCCTACA TGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAGG AGACGGACCAAAGCCACTTGGCCACTGCGGGCTCT ACTTCATCGCATTCCTTGCAAAAGTATTACATCAC GGGGGAGGCAGAGGGTTTCCCTGCCACGGTCTGA Exemplary amino acid sequence of SHP1(210-595)-ER(T12) SEQ ID NO: 35 RQPYYATRVNAADIENRVLELNKKQESEDTAKAGF WEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNI LPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGP DENAKTYIASQGCLEATVNDFWQMAWQENSRVIVM TTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGE HDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPD HGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCS AGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQ MVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLE VLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKH KEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKR KGSSAGDMRAANLWPSPLMIKRSKKNSLALSLTAD QMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTN LADRELVHMINWAKRVPGFVDLTLHDQVHLLECAW LEILMIGLVWRSMEHPLKLLFAPNLLLDRNQGKCV EGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIIL LNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIH LMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGME HLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGA SVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPA TV Exemplary nucleic acid sequence encoding SHP1(210-595)-ER(T12) SEQ ID NO: 36 CGGCAGCCGTACTATGCCACGAGGGTGAATGCGGC TGACATTGAGAACCGAGTGTTGGAACTGAACAAGA AGCAGGAGTCCGAGGATACAGCCAAGGCTGGCTTC TGGGAGGAGTTTGAGAGTTTGCAGAAGCAGGAGGT GAAGAACTTGCACCAGCGTCTGGAAGGGCAGCGGC CAGAGAACAAGGGCAAGAACCGCTACAAGAACATT CTCCCCTTTGACCACAGCCGAGTGATCCTGCAGGG ACGGGACAGTAACATCCCCGGGTCCGACTACATCA ATGCCAACTACATCAAGAACCAGCTGCTAGGCCCT GATGAGAACGCTAAGACCTACATCGCCAGCCAGGG CTGTCTGGAGGCCACGGTCAATGACTTCTGGCAGA TGGCGTGGCAGGAGAACAGCCGTGTCATCGTCATG ACCACCCGAGAGGTGGAGAAAGGCCGGAACAAATG CGTCCCATACTGGCCCGAGGTGGGCATGCAGCGTG CTTATGGGCCCTACTCTGTGACCAACTGCGGGGAG CATGACACAACCGAATACAAACTCCGTACCTTACA GGTCTCCCCGCTGGACAATGGAGACCTGATTCGGG AGATCTGGCATTACCAGTACCTGAGCTGGCCCGAC CATGGGGTCCCCAGTGAGCCTGGGGGTGTCCTCAG CTTCCTGGACCAGATCAACCAGCGGCAGGAAAGTC TGCCTCACGCAGGGCCCATCATCGTGCACTGCAGC GCCGGCATCGGCCGCACAGGCACCATCATTGTCAT CGACATGCTCATGGAGAACATCTCCACCAAGGGCC TGGACTGTGACATTGACATCCAGAAGACCATCCAG ATGGTGCGGGCGCAGCGCTCGGGCATGGTGCAGAC GGAGGCGCAGTACAAGTTCATCTACGTGGCCATCG CCCAGTTCATTGAAACCACTAAGAAGAAGCTGGAG GTCCTGCAGTCGCAGAAGGGCCAGGAGTCGGAGTA CGGGAACATCACCTATCCCCCAGCCATGAAGAATG CCCATGCCAAGGCCTCCCGCACCTCGTCCAAACAC AAGGAGGATGTGTATGAGAACCTGCACACTAAGAA CAAGAGGGAGGAGAAAGTGAAGAAGCAGCGGTCAG CAGACAAGGAGAAGAGCAAGGGTTCCCTCAAGAGG AAGGGATCCTCTGCTGGAGACATGAGAGCTGCCAA CCTTTGGCCAAGCCCGCTCATGATCAAACGCTCTA AGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGAC CAGATGGTCAGTGCCTTGTTGGATGCTGAGCCCCC CATACTCTATTCCGAGTATGATCCTACCAGACCCT TCAGTGAAGCTTCGATGATGGGCTTACTGACCAAC CTGGCAGACAGGGAGCTGGTTCACATGATCAACTG GGCGAAGAGGGTGCCAGGCTTTGTGGATTTGACCC TCCATGATCAGGTCCACCTTCTAGAATGTGCCTGG CTAGAGATCCTGATGATTGGTCTCGTCTGGCGCTC CATGGAGCACCCACTGAAGCTACTGTTTGCTCCTA ACTTGCTCTTGGACAGGAACCAGGGAAAATGTGTA GAGGGCATGGTGGAGATCTTCGACATGCTGCTGGC TACATCATCTCGGTTCCGCATGATGAATCTGCAGG GAGAGGAGTTTGTGTGCCTCAAATCTATTATTTTG CTTAATTCTGGAGTGTACACATTTCTGTCCAGCAC CCTGAAGTCTCTGGAAGAGAAGGACCATATCCACC GAGTCCTGGACAAGATCACAGACACTTTGATCCAC CTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCA GCACCAGCGGCTGGCCCAGCTCCTCCTCATCCTCT CCCACATCAGGCACATGAGTAACAAAGGCATGGAG CATCTGTACAGCATGAAGTGCAAGAACGTGGTGCC CCTCTATGACCTGCTGCTGGAGGCGGCGGACGCCC ACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCA TCCGTGGAGGAGACGGACCAAAGCCACTTGGCCAC TGCGGGCTCTACTTCATCGCATTCCTTGCAAAAGT ATTACATCACGGGGGAGGCAGAGGGTTTCCCTGCC ACGGTCTGA
EQUIVALENTS
[0375] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.