Polypeptides for inducible and repressible binding to targets of interest and methods of use

Abstract

Provided are inducible and repressible binding proteins based on α-helical repeat binding proteins. In certain embodiments, provided are polypeptides comprising an α-helical binding domain comprising a concave paratope, where the termini of the α-helical binding domain comprise a cross-paratope topology. The α-helical binding domain is flanked via flexible linkers by a caging domain pair switchable between an associated state in which the paratope is caged and a dissociated state in which the paratope is uncaged. In some instances, the caging domain pair is photoswitchable or chemoswitchable between the associated state and the dissociated state. Also provided are methods of using the polypeptides for conditional binding to a target molecule.

Description

[0001] POLYPEPTIDES FOR INDUCIBLE AND REPRESSIBLE BINDING TO TARGETS OF INTEREST AND METHODS OF USE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 535,491 , filed August 30, 2023, which application is incorporated herein by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with Government support under contracts GM132687 and GM141677 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] INTRODUCTION

[0007] A remaining challenge in biology is the direct control of unmodified natural proteins with spatiotemporal precision. Thus, tools for fine control of where and when endogenous proteins are active would have widespread utility in interrogating the roles of specific proteins in biological responses. Furthermore, control of native proteins would greatly expand the options for exogenous regulation of cellular therapies. While genetic tools exist for manipulating endogenous protein abundance over long time courses via gene editing or modulation of mRNA levels1 2; generalizable methods for rapidly controlling endogenous protein activity are lacking. Small molecule inhibitors can provide a degree of temporal control, but they are unavailable for many targets and their effects cannot be restricted to genetically defined cells.

[0008] Synthetic binding domains (SBDs) are compact protein folds that have been developed as antibodies alternatives for efficient recognition of both extra- and intracellular target proteins3. SBDs can be rapidly engineered to bind a target by in vitro mutagenesis and selection4 7. Specifically, a defined paratope region of the SBD is diversified to bind targets while the rest of the scaffold is kept constant to preserve the fold. High-affinity SBDs are versatile tools for investigating cell signaling, constructing diagnostic probes, and developing therapeutic biologies8 15. By binding to a functionally important site of a target, SBDs can directly inhibit biochemical signaling14 15. Additionally, SBDs can be fused to membrane- or organelle-targeting sequences to relocalize target proteins11, or to enzymes to install posttranslational modifications on the target by proximity16. However, when expressed, the binding activity of SBDs is permanently active. This limits the ability to study dynamic signaling mechanisms and may lead to compensatory adaptation of the system under study. Thus, acute and reversible activation of SBD binding could provide new opportunities for precision perturbation of endogenous targets to study their function in dynamic processes in health and disease.17

[0009] Controllable SBDs that only bind to endogenous targets upon specified conditions would enable genetically-encodable and temporally precise perturbation of protein function. Examples of photoinducible SBDs have been reported18 21but they are limited in either regulatability or generalizability. Photoreconstitution of SBDs from split fragments can provide robust control under controlled conditions18, but basal and induced concentrations of reconstituted SBDs will depend on fragment concentrations, which may vary from cell to cell, and the approach showed negligible binding reversibility. Single-chain designs reduce concentration effects, and have been created by inserting LOV or PYP photodomains into nanobodies, monobodies, and affibodies for allosteric modulation of binding activity19 21. However, their utility is hampered by lack of a generalizable photodomain insertion site with predicable effects on binding19’20, variable levels of baseline binding and light-inducibility across targets19 21, or the need for exogenous chromophore supply.21 22

[0010] SUMMARY

[0011] Provided are inducible and repressible binding proteins based on a-helical repeat binding proteins. In certain embodiments, provided are polypeptides comprising an a-helical binding domain comprising a concave paratope, where the termini of the a-helical binding domain comprise a cross-paratope topology. The a-helical binding domain is flanked via flexible linkers by a caging domain pair switchable between an associated state in which the paratope is caged and a dissociated state in which the paratope is uncaged. In some instances, the caging domain pair is photoswitchable or chemoswitchable between the associated state and the dissociated state. Also provided are methods of using the polypeptides for conditional binding to a target molecule.

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1 : Schematic illustrations of exemplary psBinder and csBinder types, a, A photoinducible psBinder is caged in its basal state defined by the absence of excitation light for the specific photodomain. Thus, target binding is occluded in the basal state. Excitation of the photodissociable domain leads to uncaging and binding of the target to the paratope, b, A photorepressible psBinder is uncaged in its basal state defined by the absence of excitation light for the specific photodomain. Thus, the target can bind to the paratope in the basal state. Light excitation of the photodomain causes association of the photodomain and target occlusion, c, A chemoinducible csBinder is caged in its basal state defined by the absence of its specific ligand. Thus, target binding is occluded in the basal state. Addition of the ligand leads to dissociation of the chemodissociable domain and binding of the target to the paratope, d, A chemorepressible csBinder is uncaged it the basal state defined by the absence of its specific ligand. Thus, the target can bind to the paratope in the basal state. Ligand addition causes association of the chemoassociable domain and target occlusion. For all schematics, several additional intermediate states and equilibria exist but for simplicity these are not shown. Only the major states that are favored under the 7+ light or - / + ligand conditions are shown for simplicity to define the four types of photo- or chemoswitchable binders.

[0014] FIG. 2: Concept for a generalizable synthetic binding protein with photoinducible target recognition, a, Single-chain enzymes with photoinducible macromolecular substrate recognition can be engineered by attaching photodissociable dimeric Dronpa (pdDronpa) domains at locations flanking the active site. The autocatalytically-maturating and cofactorindependent pdDronpa domains are dimeric and brightly fluorescent at baseline, photoswitched into a monomeric and non-fluorescent state by absorption of cyan (~500-nm) light, and redimerized by violet (~400-nm) light. Flanking the active site, pdDronpa domains can sterically occlude substrate binding, causing the enzyme to exist in a caged form, while their photodissociation effectively uncages the enzyme to allow substrate binding. This concept of caging a protein-biomolecular interaction site by attachment of pdDronpa domains and then uncaging with light can be generalized to various target interactions by fusing pdDronpa to synthetic binding domains (SBDs). b, DARPins are cysteine-free SBDs with a centrally located concave binding surface (paratope) that has been mutagenized and selected for high-affinity binding to a variety of targets. DARPins are a-helical repeat proteins consisting of at least 1 , and typically 2-3, internal repeat units, flanked by a N- and a C-terminal capping repeat (N- and C- cap). The paratope is defined by diversifying select residues within the loops and concave face of the DARPin (magenta spheres). However, fusing pdDronpa to the termini (blue spheres) of the canonical DARPin scaffold is not expected to effectively occlude the paratope, c, An extended DARPin (exDARPin) with an additional a-helix appended to the C-cap is proposed to create a new C-terminal attachment point and thereby a cross-paratope topology of the termini so pdDronpa dimerization will occur in front of the paratope, effectively caging the exDARPin. d, Examples of two RosettaRemodel-designed exDARPins with extension, ex13 and ex17. Gold is the RosettaRemodel-predicted structure while gray is the experimentally determined crystal structure. Note, the added third C-cap a-helix is packed against the preceding C-cap helices at different angles in the two designs with ex17 C-terminus closer to the paratope, e, Model of how the pdDronpa-linker-exDARPin-linker-pdDronpa architecture creates a photoswitchable DARPin (psDARPin) that can bind and inhibit endogenous protein targets upon illumination with cyan light.

[0015] FIG. 3: Exploring cageability of extended DARPins. a, Domain organization of constructs used for testing DARPin caging by combining DARPin extensions and pdDronpa. Linkers are not explicitly shown in these diagrams. DARPins were localized in the cytosol by fusion of a nuclear export sequence (NES), and freely diffusing mTurquoise2 (mTurq2) was expressed as a target of the 3G124 DARPin. Cytosolic localization of the DARPin fusions was validated using a mScarlet tag. b, Representative images of HeLa cells expressing test constructs. Brief one-frame 488-nm excitation of pdDronpa immediately preceded 445-nm excitation to image mTurq2. The constructs are: 0, an extension-free DARPin recognizing E. coli maltose-binding protein with terminal pdDronpa1 .2 (pdD1.2) fusions, as a negative control not expected to show mTurq2 binding; 1 , the extension-free GFP-binding 3G124 (3G124.exO) without pdD1 .2 attachments, as a DARPin expected to constitutively sequester mTurq2 in the cytosol; 2, 3G124.exO with pdD1 .2 attached to both termini, to test the caging ability of a canonical DARPin without C-terminal extensions; 3, 3G124 with designed extension ex13 and terminal pdD1 .2 attachments (ps3G124.ex13), to test the caging improvement; 4, 3G124 with designed extension ex17 and terminal pdD1.2 attachments (ps3G124.ex17), to test the caging improvement; 5, an open pdD1.2 N145K 1158V mutant of ps3G124.ex13, is expected to constitutively bind mTurq2; and similarly 6, an open mutant of ps3G124.ex17, is also expected to constitutively bind mTurq2. The linkers were 7 and 5 amino acids long at the N- and C-terminus of all DARPins tested here, c, Quantification of the mTurq2 cytosolic / nuclear (C / N) ratio from b, that is positive correlated with mTurq2 binding to the NES-tagged DARPins. C / N ratios of ~1 and ~4.8 were observed with the negative and positive binding controls, construct 1 and 2, respectively. Mean C / N ratios are reported (n,N = 18 independent cells, 3 independent experiments per construct), and eror bars are standard deviation (SD) calculated using n. d, Top, a NES-tagged photoswitchable mTurq2-binding 3G124.ex17 (ps3G124.ex17) was used to test optical regulation of target engagement. Below, expected relationship between pdD1 .2 fluorescence and mTurq2 cytosolic localization. Illumination by 488-nm light switches pdD1 .2 into a dim monomeric off-state. The resulting uncaged psDARPin is expected to sequester mTurq2 in the cytosol, increasing the C / N ratio. Subsequent excitation by 405-nm light restores pdD1 .2 to a dimeric form emitting bright fluorescence upon 488 nm excitation. Free ps3G124.ex17 molecules are recaged, and the pdD1 .2 domains in ps3G124.ex17-mTurq2 complexes become primed for redimerization once mTurq2 disscociates. Following recaging and mTurq2 dissociation, mTurq2 will diffuse freely throughout the cell, including back into the nucleus, e, Example of bidirectional photoactivation of ps3G124.ex17 binding to mTurq2 in HeLa cells. Photoswitching of pdD1 .2 to its off-state by 488-nm confocal illumination for 10 s at 0.10 mW was correlated with depletion of mT urq2 from the nucleus, indicating photoinduction of ps3G124.ex17 binding. Confocal illumination at 405 nm for 5 s of 0.56 mW restored 488-nm excited pdD1 .2 fluorescence, and mTurq2 reaccumulated in the nucleus, indicating photorecaging of ps3G124.ex17. The reversible process was then repeated, f, Quantification of pdDronpa fluorescence and mTurq2 C / N ratios of e, plotted over time for n = 3 individual cells (thin lines) and their mean (bold line). All scale bars are 10 pm.

[0016] FIG. 4: Photoactivated inhibition of endogenous ERK and Ras with psDARPins. a, Top, schematic description of Ras-Raf-MEK-ERK signaling pathway in cells expressing the constitutively active EGFRvll I variant. The enzymes are shown in their active form. The canonical DARPins K27.ex0 and E40.ex0 are expected to inhibit respectively the formation of active GTP- bound Ras and phosphorylated ERK, RasGTP and pERK, by binding to the inactive GDP-bound Ras and unphosphorylated ERK. Middle, NES-tagged psE40.ex# and psK27.ex# were prepared and tested in Ras-ERK hyperactive U87-EGFRvlll cells. Bottom, the activity of ERK and upstream Ras was monitored using a mScarlet-tagged ERK-KTR kinase activity reporter. Active pERK causes ERK-KTR phosphorylation and accumulation in the cytosol, while the reporter is imported into the nucleus following phosphatase-mediated dephosphorylation. Thus, the C / N ratio is positively correlated with ERK activity, b, Representative mScarlet images from U87- EGFRvlll cells expressing test constructs under basal dark conditions and quantification of the associated ERK-KTR C / N ratios (n,N = 18 independent cells, 3 independent experiments per construct). The negative control Off.exO provides a baseline ERK-KTR C / N ratio of ~2.7 representing full Ras-ERK activity. Expression of E40.ex0 caused ERK inhibition and dropped the C / N ratio to ~0.4. By Comparing to these baselines, the caging ability of psE40.ex13 and psE40.ex17, and the inhibitory efficacy of the respective open (pdD1.2 N145K 1158V) mutants thereof were tested. Similarly, K27.ex0 was used to establish the baseline for Ras inhibition to allow testing the caging and binding ability of psK27.ex13 and psK27.ex17 and their open variants, respectively, c and d, Photoactivation of psE40.ex13 and psK27.ex17 with 0.21 mW 488-nm light for 10 s led to gradual inhibition of ERK and Ras with the C / N ratios (n,N = 12 independent cells, 3 independent experiments per construct) settling at -0.4 similar to the positive inhibition controls (E40.ex0 and K27.ex0 in b). In all instances, scale bars are 10 pm, mean C / N ratios are reported, and error bars are standard deviation (SD) calculated using n.

[0017] FIG. 5: Photoactivated inhibition of endogenous ERK and Ras with psDARPins. a, Schematic description of Ras-ERK signaling following stimulation with EGF. In the early burst phase EGF-complexed EGFR will activate the Ras-ERK pathway. Subsequently, EGFR endocytosis and negative feedback in the Ras-ERK pathway reduce the ERK activity, but low- level ERK activity is sustained in the late signaling phase. However, the precise role of Ras in late phase signaling is not confirmed, b, Representative images of Alexa Fluor 647-labelled EGF (EGF-AF647) used to monitor the dynamics of EGF endocytosis in serum-starved HeLa cells. EGF-AF647 was added at 10 ng / mL to cells co-expressing ERK-KTR-mScarlet and either the negative control psDARPin ps3G124.ex17 or the Ras-targeted psDARPin psK27.ex17. Similar behavior was seen under all conditions, and endocytosis was unaffected by illumination with 488 nm light. Clear endocytosis is seen from 20 min and onwards, c, Representative images of the ERK-KTR reporter in serum-starved HeLa cells stimulated with 10 ng / mL unlabeled EGF. Immediately after stimulation the reporter moves to the cytosol indicating ERK activation. Photoswitching with 488 nm light at 20 min post EGF addition immediately caused ERK-KTR to move back into the nucleus in the psK27.ex17 expressing cells but not in the ps3G214.ex17 expressing control cells, d, Quantification of ERK-KTR C / N ratios from c. In accordance with previous reports, HeLa cells expressing ps3G124.ex17 showed the expected peak in ERK activity at 30 min post EGF stimulation. In contrast, uncaging of psK27.ex17 caused an immediate drop in the ERK activity indicating that Ras activity is required to sustain late phase ERK activity. In b-c, uncaging was performed with 0.21 mW 488-nm light for 10 s. Scale bars are 10 pm, mean C / N ratios are reported (n,N = 20 independent cells, 3 independent experiments per construct), and error bars are standard deviation (SD) calculated using n.

[0018] FIG. 6: Designing exDARPin while preserving the diversified paratope positions of canonical DARPin libraries, a, Sequence alignments comparing the exDARPin designs to a canonical DARPin sequence. DARPin. lib represents an optimized canonical DARPin library sequence that is diversified on the positions marked with X (any amino acid except G / C / P) and Z (any of H / N / Y). The residue positions marked with a magenta X make up the primary paratope positions highlight in b as magenta spheres. However, random mutagenesis and affinity maturation may lead to DARPins that contain sporadic mutations of conserved residues beyond the initial diversified positions of the library. Over time, several groups have reported optimization of the C-cap. In this study, the mut5 cap, here exO, reported by Interlandi et al. (Interlandi, Gianluca, et al. "Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments." Journal of molecular biology 375.3 (2008): 837-854) was used as a representative canonical C-cap. Thus, exO has been tested in all the DARPins used in this study. Although DARPins 3G124, E40, and K27 were originally reported with older C-cap versions that were slightly different from exO, the experiments showed that these DARPins still recognize their targets when combined with exO. In the original DARPin libraries (Binz, H. Kaspar, et al. "Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins." Journal of Molecular Biology 332(2) (2003): 489-503; Binz, H. Kaspar, et al. "High-affinity binders selected from designed ankyrin repeat protein libraries." Nature Biotechnology (2004) 22:575-582), no position in the N-cap or C-cap were diversified. However, more recent libraries incorporate diversification of cap positions that structurally correspond to the canonically diversified positions of the internal repeats (Schilling, Johannes, Jendrik Schbppe, and Andreas Pluckthun. "From DARPins to LoopDARPins: novel LoopDARPin design allows the selection of low picomolar binders in a single round of ribosome display." Journal of molecular biology 426.3 (2014): 691 -721 ; Seeger, Markus A., et al. "Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity." Protein science 22.9 (2013): 1239-1257; Pluckthun, Andreas. "Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy." Annual review of pharmacology and toxicology 55 (2015): 489-51 1.). Furthermore, deviating slightly from the canonical paratope pattern (Seeger, Markus A., et al. "Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity." Protein science 22.9 (2013): 1239-1257), positions 2 and 1 1 of the internal repeats and C-cap (blue) have also been suggested as sites for library diversification. Thus, all of these positions were considered as potential paratope residues during the process of computationally designing the extended C-caps of exDARPins. Specifically, only underlined residues were computationally designed with RosettaRemodel. In addition to redesigning the C- cap, the residues of positions 30-32 of internal repeat n were also allowed to vary to facilitate interactions that could stabilize the new loop leading into the added helix. Otherwise, all the computationally optimized residues are positioned within, or as an extension of, the C-cap. Sequences alignments of exDARPins DARPin.ex13 and DARPin.ex17 that were used throughout this study show that the extended C-cap designs do not affect any of the library position (magenta and blue). This was the case for all designs. Extensions ex13 and ex17 are non-limiting examples. Optimization of the N-cap and internal repeats have previously been reported by mutating the residues highlighted with grey. Particularly, a D15L (Schilling, Johannes, et al. "Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs." Journal of Biological Chemistry 298.1 (2022)) mutation in the N-cap as well as E22D (Seeger, Markus A., et al. "Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity." Protein science 22.9 (2013): 1239-1257) and K26A (Seeger, Markus A., et al. "Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity." Protein science 22.9 (2013): 1239-1257) mutations in the internal repeats have been reported to optimize DARPin stability. None of these mutations were incorporated in this study, however, as is seen from the sequence alignments, exDARPins are fully compatible with these mutations, b, Crystal structure of 3G124.ex13 and Off7.ex17 with the computationally optimized positions highlighted in gold (i.e., corresponding to residues underlined in a) and library position marked with magenta spheres (i.e., corresponding to residues marked with X in a).

[0019] FIG. 7: Basal state caging of chemoinducible csBinders. a, A model of FKBP(F36M) fused via flexible linkers to an exDARPin thus caging the paratope in absence of the FKBP(F36M) ligand, Shieldl . Addition of Shieldl is expected to cause uncaging and allow the exDARPin to bind its target, b, Chemical structure of the FKBP(F36M) ligand Shieldl . c, Domain organization of constructs used for testing csDARPin caging by combining DARPin extensions and FKBP(F36M). Linkers are not explicitly shown in these diagrams. DARPins were localized in the cytosol by fusion of a nuclear export sequence (NES), and freely diffusing mTurquoise2 (mTurq2) was expressed as a target of the 3G124 DARPin. Cytosolic localization of the DARPin fusions was validated using a mScarlet tag. d, Left, representative images of HeLa cells expressing test constructs. The constructs are: 0, 3G124 with designed extension 17 and terminal monomeric FKBP(WT) attachments as a positive binding control; 1 , 3G124 with designed extension 17 and terminal FKBP(F36M) attachments (cs3G124.ex17) to test caging efficiency of the dimeric FKBP(F36M) domains; and 2, Off7, an extension-free DARPin recognizing E. coli maltose- binding protein and with FKBP(F36M) attachment as a negative control that is not expected to bind mTurq2. All three constructs had 7,5 (DARPin N,C-terminal) amino acids linkers. Right, quantification of the mTurq2 cytosol-to-nuclear fluorescence ratio of the images. The C / N ratio of construct 1 is similar to construct 2 indicating efficient caging of the csBinder cs3G124.ex17. e, Screening the effect of linker lengths on the caging ability of FKBP(F36M) fused to exDARPin 3G124.ex17 or 3G124.ex13. All constructs showed caging relative to the positive binding control of panel d (construct 1 ). For the tested set, the best caging (lowest C / N ratio) is observed for the 7,5 linker combination in FKBP(F36M)-linker-3G124.ex17-linker-FKBP(F36M). Bars represent mean C / N ratios from 12 biologically independent cells in 2 independent experiments per construct. Error bars represent standard deviation. Scale bar is 10 pm.

[0020] FIG. 8: Reversible ligand control of chemoinducible csBinders. a, Chemical structure of the bivalent AP20187 that is effectively two Shieldl ligands linked together allowing simultaneously bind to two FKBP(F36M) or FKBP(F36V) domains. Thus, AP20187 can be utilized to cage or recage FKBP(F36V) and FKBP(F36M) domains, respectively b, A chemoinducible NES-tagged csBinder was constructed by fusing FKBP(F36M) to 3G124.ex17 and co-expressed with its target mTurq2 in HeLa cells. The addition of 10 nM Shieldl switched the csBinder from a basal state with low target binding (mTurq2 C / N ~1 .2) to an uncaged state with efficient target binding as seen from the recruitment of mTurq2 to the cytosol within a few minutes. The C / N ratio increased to -3.5 at 6 min post ligand addition. By out-competing Shieldl , the addition of 0.5 pM AP20187 caused csDARPin recaging and an immediate drop in the C / N ratio. Alternatively, recaging may also be performed by extensive washing to dilute Shieldl . However, this process is expected to be relatively slow compared to AP20187-mediated recaging, c, A chemoinducible NES-tagged csBinder was constructed by fusing FKBP(F36M) to E40.ex13 to allow chemocontrolled inhibition of endogenous ERK in Ras-ERK hyperactive U87-EGFRvlll cells. ERK kinase activity was monitored using a mScarlet-tagged ERK-KTR translocation reporter. When ERK is active the C / N ratio of the ERK-KTR is high. Addition of 10 nM Shieldl caused a gradual drop in the C / N ratio indicating ERK inhibition. After 45 min 0.5 pM AP20187 was added and shown to reverse ERK inhibition indicating successful recaging of the csBinder. Linkers were 7,5 for both constructs presented in this figure. Scale bar is 10 pm.

[0021] FIG. 9: Reversible ligand control of chemoinducible csBinders. a, A model chemorepressible csDARPin with FKBP(F36V) fused via flexible linkers to an exDARPin allowing target binding in the basal state. Addition of AP20187 is expected to cause caging and thus repress target binding, b, A chemoinducible NES-tagged csBinder was constructed by fusing FKBP(F36V) to 3G124.ex17 and co-expressed with its target mTurq2 in HeLa cells. The addition of 10 nM AP20817 switched the csBinder from a basal state with high target binding (mTurq2 C / N -3.4) to a caged state with low target binding as seen from the movement of mTurq2 to the nucleus and decreasing C / N ratio. By out-competing AP20187, the addition of 1 pM Shieldl caused reversible FKBP(F36V) monomerization, csDARPin uncaging, and target binding as seen from the increasing C / N ratio. Alternatively, uncaging may also be performed without addition of Shieldl but by extensive washing to dilute AP20187. However, this process is expected to be relatively slow compared to Shieldl -mediated uncaging, c, A chemorepressible NES-tagged csBinder was constructed by fusing FKBP(F36V) to E40.ex13 to allow chemocontrol of endogenous ERK in Ras-ERK hyperactive U87-EGFRvlll cells. ERK kinase activity was monitored using a mScarlet-tagged ERK-KTR translocation reporter. When ERK is active the C / N ratio of the ERK-KTR is high. In the basal state the csBinder binds to and inhibits ERK causing a low C / N value of ~0.4. Addition of 10 AP20187 cause a gradual increase in the C / N ratio indicating ERK activation by csDARPin caging and release of inhibitory binding of ERK. After 45 min, 1 pM AP20187 was added and shown to reverse the ERK activity indicating successful monomerization of the FKBP(F36V) domains, paratope uncaging, and ERK binding. Linkers were 7,5 for both constructs presented in this figure. Scale bar is 10 pm.

[0022] FIG. 10: Testing basal caging and photoinduced uncaging of psaREPs. a, Domain organization of constructs used for testing psaREPs by adding the extension aex3 to the bGFP aREP and combining it with fusions to pdDronpal .2. The flexible linkers were 5,5 residues long. The psaREP was localized in the cytosol by fusion of a nuclear export sequence (NES), and freely diffusing mTurquoise2 (mTurq2) was expressed as a target of the bGFP aREP. Cytosolic localization of the aREP fusion was validated using a mScarlet tag. b, Representative images of HeLa cells expressing the test construct. In the basal state (i.e., no light), psbGFP.aex3 is efficiently caged as seen from the C / N ratio of -1 . Illumination with 488 nm light leads to psbGFP.aex3 uncaging and mTurq2 binding in the cytosol. Thus, the C / N ration increased to -4.8. Scale bar is 10 pm.

[0023] FIG. 11 : Testing basal caging of ps(RsLOV)DARPins. a, Domain organization of constructs used for testing ps(RsLOV)DARPin caging by adding the extension ex3 to the 3G124 DARPin and combining it with fusions to RsLOV via flexible linkers of different length. ps(RsLOV)DARPins were localized in the cytosol by fusion of a nuclear export sequence (NES), and freely diffusing mTurquoise2 (mTurq2) was expressed as a target of the 3G124 DARPin. Cytosolic localization of the DARPin fusions was validated using a mScarlet tag. b, Representative images of HeLa cells expressing the test constructs. In the basal state (no light), ps(RsLOV)3G124.ex17 is most efficiently caged in the 7,5 linker variant as seen from the low C / N ratio -1 . In contrast, some mTurq2 binding to ps(RsLOV)3G124.ex17 is seen when using linkers of 6,4 or 9,7. Scale bar is 10 pm.

[0024] FIG. 12: Photoinducible psDARPin can bind their target at membranes with subcellular precision, a, ps3G124.ex17 was localized to the outer mitochondrial membrane and facing the cytosol using an OMP-tag. In the basal state, the target, mTurq2, is distributed throughout the cell. Following uncaging with 488 nm light, mT urq2 is enriched at the mitochondrial membrane. Illumination with 405 nm light leads to ps3G124.ex17 recaging and release of mTurq2. b, ps3G124.ex17 was localized to the plasma membrane and facing the cytosol using a CAAX-tag. In the basal state, the target, mTurq2, is distributed throughout the cell. Following uncaging with 488 nm light, mTurq2 is enriched at the plasma membrane. Illumination with 405 nm light leads to ps3G124.ex17 recaging and release of mTurq2.

[0025] FIG. 13: Designing exaReps while preserving the diversified paratope positions of canonical aReps libraries, a, Extension of the aRep scaffold was explored to achieve a crossparatope topology of the SBD termini. The canonical aRep sequence (Urvoas, Agathe, et al. "Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (aRep) based on thermostable HEAT-like repeats." Journal of molecular biology 404.2 (2010): 307-327; Valerio-Lepiniec, Marie, et al. "The aRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications." Biochemical Society Transactions 43.5 (2015): 819-824) is described by the aRep. lib sequence and include a N-cap, n internal repeats with n > 1 , and a C-cap. The diversified positions of typical aRep libraries are annotated with a bold X in magenta and are present at equivalent positions in both the N-cap and internal repeats. In the N- cap, the variable position may be kept fixed in some aRep libraries, for example, residue 23 is in some instances fixed to alanine in some (Urvoas, Agathe, et al. "Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (aRep) based on thermostable HEAT-like repeats." Journal of molecular biology 404.2 (2010): 307-327). The diversified residues generally define the concave paratope that binds to the target. The underlined residues were allowed to mutate while creating extended exaReps using the RosettaRemodel software to redesign the aRep N-cap. The yellow highlight indicates positions that were allowed to repack during the computations. This aex3 design serves only as a nonlimiting example, b, Comparison of an AlphaFold2 prediction of the bGFPC.aex3 structure and an experimentally obtained crystal structure, c, The redesigned residues in the N-cap shown in gold on the AlphaFold2 model. The canonically diversified positions are shown as magenta spheres. Residue 18 of the N-cap is diversified in the canonical DARPin. However, inspection of aRep crystal structures indicated this position doesn’t generally contribute to binding the target. Thus, this position was also allowed to mutate during the computational redesign but ultimately fixed to proline as often found at this position (Valerio-Lepiniec, Marie, et al. "The aRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications." Biochemical Society Transactions 43.5 (2015): 819-824). This choice can be varied as necessary for other targets. Furthermore, the C-cap of the aRep scaffold may in some instances be truncated with 1- 6 residues.

[0026] FIG. 14: Additional exDARPin design examples. In addition to ex13 and ex17, other DARPin C-terminal extension designs were explored to create the cross-paratope topology of exDARPins. The designs were created using RosettaRemodel as described for FIG. 6. The N- cap and internal repeats 1 to n-1 of the designs are equivalent to FIG. 6 and not shown here for brevity. Thus, only the amino acid sequences of the modified internal repeat n and the C-cap are shown. For ex32 and ex42 residues 17 and 19 of the C-cap were optimized during the design process, while they were kept constant during the computational design of the other extensions.

[0027] DETAILED DESCRIPTION

[0028] Before the polypeptides and methods of the present disclosure are described in greater detail, it is to be understood that the polypeptides and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the polypeptides and methods will be limited only by the appended claims.

[0029] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the polypeptides and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the polypeptides and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the polypeptides and methods.

[0030] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the polypeptides and methods belong. Although any polypeptides and methods similar or equivalent to those described herein can also be used in the practice or testing of the polypeptides and methods, representative illustrative polypeptides and methods are now described.

[0032] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present polypeptides and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0033] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0034] It is appreciated that certain features of the polypeptides and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the polypeptides and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present polypeptides and methods and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0035] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0036] POLYPEPTIDES FOR INDUCIBLE AND REPRESSIBLE BINDING TO TARGET MOLECULES

[0037] Aspects of the present disclosure include polypeptides for inducible and repressible binding to target molecules. The polypeptides are based in part on the development of a redesigned a-helical repeat binding protein topology. The canonical topology consists of the bland C-terminal a-helixes pointing in the same direction. In some instances, the redesigned topology involves extending the N- or C-terminus (e.g., the C-terminal cap) with an additional a- helix thus creating a topology with cross-paratope placement of the termini. The cross-paratope placement of the termini, upon flanking of the paratope with associable domains via linkage to the termini, results in occlusion of the paratope when the associable domains are associated

[0038] Demonstrated herein is that the redesigned topology is applicable to multiple different a- helical repeat binding proteins, multiple different associable domains (often referred to herein as “caging domain pairs”), and multiple different modes of switchability (e.g., photoswitchability and chemoswitchability), thus establishing the generalizability of the redesigned topology. The polypeptides of the present disclosure constitute a marked improvement relative to previous single-chain binders suffering from less predictable allosteric control of target binding. Accordingly, the redesigned architecture provides a modular and generalizable approach to controllable target binding (e.g., binding endogenous proteins), and constitutes a new platform for investigating cell signaling, testing therapeutic hypotheses, administering regulatable therapies (e.g., regulatable cell therapies, and the like), etc. with temporal and spatial precision, e.g., for targets without high-quality small-molecule inhibitors. Accordingly, in certain aspects, a polypeptide of the present disclosure comprises an a- helical binding domain comprising a concave paratope, where the termini of the a-helical binding domain comprise a cross-paratope topology, and where the a-helical binding domain is flanked via flexible linkers by a caging domain pair (cagel :cage2) switchable between an associated state in which the paratope is caged and a dissociated state in which the paratope is uncaged. The polypeptide is generally described by the fusions order cagel -linker1 -SBD-linker2-cage2, where it is to be understood that the termini of the SBD must have a cross-paratope topology.

[0039] The terms “polypeptide”, “peptide”, or “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 “standard” genetically encodable amino acids, amino acid analogs, or a combination thereof. The term “amino acid” includes, but is not limited to, naturally-occurring amino acids and their stereoisomers. “Stereoisomers” of amino acids refer to mirror image isomers of the amino acids, such as L-amino acids or D-amino acids. For example, a stereoisomer of a naturally- occurring amino acid refers to the mirror image isomer of the naturally-occurring amino acid ( / .e., the D-amino acid). A polypeptide of the present disclosure may comprise only of L-amino acids, may comprise only of D-amino acids, or may comprise a mixture of L-amino acids and D-amino acids. Polypeptides comprised of D-amino acids (e.g., all-D amino acids) may be advantageous compared to their all-L counterparts in terms of PK, reduced immunogenicity, and / or the like. See Mandal et al. (2012) PNAS 109:14779-14784. Moreover, any of the polypeptides of the present disclosure may comprise one or more beta amino acids, one or more gamma amino acids, and / or one or more of any other amino acid types that suitably replace alpha-amino acids. See, e.g, Sang et al. Acc. Chem. Res. 53, 10, 2425-2442.

[0040] In certain embodiments, a polypeptide of the present disclosure comprise an a-helical binding domain comprising a concave paratope. The a-helical binding domain may be from a concave a-helical repeat protein. Concave a-helical repeat proteins consist of n+2 repeats given as n internal repeats capped by an amino-terminal N-cap and a carboxy-terminal C-cap: [N-Cap]- [(Internal Repeat)r]-[C-cap]. Each internal repeat consists of two a-helices connected by a short loop (loop-a-loop-a unit). The repeats are then interconnected by a loop of varying length. In some instances, the repeats are defined by internally conserved and repetitive sequences that ensure stable packing between the repeats. The N- and C-cap units seal and stabilize the scaffold by burying the hydrophobic edges of the internal repeat units. In canonical a-helical repeat proteins, the N- and C-cap also contain two a-helices. Libraries of a-helical repeat proteins can be prepared with amino acid diversification at position within, for example, the concave face and the repeat connecting loops or turns. The diversified positions exclude positions that are crucial for packing of the repeats against each other. Thus, libraries of a-helical repeat proteins can be used to select synthetic binding domains that bind to a target of interest using a concave binding surface without perturbing the overall protein fold, thus, preserving the concave topology as well as the position of the termini of the scaffold. Affinity maturation may introduce mutations outside the library defined positions of diversification to optimize the binding affinity between the target and the synthetic binding domain while preserving the overall fold and topology of the a-helical repeat protein. Furthermore, computational design algorithms (for example, ProteinMPNN as described in Dauparas, Justas, et al. "Robust deep learning-based protein sequence design using ProteinMPNN." Science 378.6615 (2022): 49-56; Bennett, Nathaniel R., et al. "Improving de novo protein binder design with deep learning." Nature Communications 14.1 (2023): 2625; and Yang, Wei, et al. "Design of High Affinity Binders to Convex Protein Target Sites." bioRxiv (2024) DOI: 10.1101 / 2024.05.01 .592114 may also be used to introduce sequence variations and mutations in both internal repeats units and capping units to improve, for example, stability, immunogenicity, and binding affinity, while preserving the overall fold and topology of the a-helical repeat protein. Such variations and mutations may account for 0-2%, 2-5 %, 5-10 %, 10-20 %, 30-40%, or 40-50% of the amino acids in polypeptide sequence. In addition, insertions and deletions are also possible.

[0041] When the a-helical binding domain is from a concave a-helical repeat protein, in certain embodiments, the a-helical repeat protein is a DARPin, an a-Rep protein, or a de novo designed concave a-helical repeat protein.

[0042] Designed ankyrin repeat proteins (DARPins) are versatile synthetic binding scaffolds having high thermostability and have been engineered to bind an array of targets with pico- to nanomolar affinities. Details regarding DARPins may be found, e.g., in Binz, H. Kaspar, et al. "Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins." Journal of molecular biology 332.2 (2003): 489- 503; High-affinity binders selected from designed ankyrin repeat protein libraries; Pluckthun, Andreas. "Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy." Annual review of pharmacology and toxicology 55 (2015): 489-51 1 ; Seeger, Markus A., et al. "Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity." Protein science 22.9 (2013): 1239-1257; Wild, Philipp, et al. (2022) "The DARPin Encyclopedia: from Basic Research towards Therapeutics."; WO 2002 / 020565.

[0043] Non-limiting examples of DARPins which can be turned into exDARPins (e.g., using the methodology shown in Fig. 6 to obtain a cross-paratope topology) include those in Table 1 below (all DARPin sequences in the references are incorporated herein by reference). Table 1 - Examples of DARPin-based SBDs aRep proteins (“alpha repeat proteins”; “alpha-helicoidal repeat proteins”; “alpha- helicoidal HEAT-like repeat proteins”) are derived from a natural family of modular proteins constituted of alpha-helical repeats, related to HEAT repeats, named after Huntingtin, the elongation factor 3 (EF3), the protein phosphatase 2A (PP2A), and the yeast kinase TOR1. The association of several HEAT repeats forms alpha-solenoids of various lengths, which are naturally found in a number of cellular proteins involved in intracellular transport and proteinprotein interaction. The biophysical properties of aRep proteins make them amenable to biological and medical applications. First, aRep proteins are easily expressed in bacteria as soluble proteins, implying a properly folded protein. Second, they are functional in reducing and oxidative environments due to their disulfide independent folding, and can be active inside living cells as well as in the extracellular milieu. Moreover, aRep proteins are thermostable, with Tm >70 °C. In addition, heat denaturation is reversible and aRep proteins refold properly when cooled to 25 °C; aggregation of aRep proteins does not occur at high concentrations; and their small size allows the recognition of epitopes of low accessibility, thus increasing the diversity of binders for one target. Further details regarding aFtep proteins can be found, e.g., in Urvoas, Agathe, et al. "Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (aRep) based on thermostable HEAT-like repeats." Journal of molecular biology 404.2 (2010): 307-327; Guellouz, Asma, et al. "Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep)." PLoS One 8.8 (2013): e71512; Valerio-Lepiniec, Marie, et al. "The aRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications." Biochemical Society Transactions 43.5 (2015): 819-824; and Chevrel, Anne, et al. "Specific GFP-binding artificial proteins (aRep): a new tool for in vitro to live cell applications." Bioscience reports 35.4 (2015): e00223.

[0044] Non-limiting examples of aRep proteins which can be turned into exaReps (e.g., using the methodology shown in Fig. 13 to obtain a cross-paratope topology) include those in Table 2 below (all aRep sequences in the references are incorporated herein by reference).

[0045] Table 2 - Examples of aRep-based SBDs

[0046] Details regarding de novo designed concave o-helical repeat proteins may be found, e g., in Brunette, T. J., et al. "Exploring the repeat protein universe through computational protein design." Nature 528.7583 (2015): 580-584 ; Parmeggiani, Fabio, and Po-Ssu Huang "Designing repeat proteins: a modular approach to protein design." Current Opinion in Structural Biology 45 (2017): 116-123; Jiang, Hanlun, et al. "De novo design of buttressed loops for sculpting protein functions." Nature Chemical Biology (2024): 1-7.; and Yang, Wei, et al. "Design of High Affinity Binders to Convex Protein Target Sites." bioRxiv (2024) DOI: 10.1101 / 2024.05.01.592114.

[0047] In certain embodiments, the o-helical binding domain comprises an extended c-helical repeat protein. The extension may involve redesigning either a new N- or C-cap to add an extra helix. The extension is typically only provided at one of the caps to ensure that the total number of o-helices is an uneven number, thereby ensuring that the N- and C- termini will take a cross- paratope geometry. In some instances, one extra helix is added to either of the termini. However, two or more helices may be incorporated so long as an uneven number of helices are incorporated in a way that ensures a cross-paratope topology of the termini. When adding a helix, the spatially adjacent residues of the starting SBD should be chosen to create favorable interactions. In some embodiments, the number of internal repeats is increased by adding inert internal repeats (i.e. internal repeats that do not participate or disturb binding) directly adjacent to either the N-cap or C-cap while ensuring that the cross-paratope topology is maintained.

[0048] The a-helical binding domain is flanked via flexible linkers by a caging domain pair. The caging domain pair may be a homodimeric pair or a heterodimeric pair. A heterodimeric pair may be referred to herein as “partnerl :partner2” or “cagel :cage2”. If only a single domain is listed it is to be understood that the caging domain pair is homodimeric. The exception may be if a unique name has been assigned to the heterodimeric pair or the heterodimeric pair is a split protein. In this case, either the pair or the systematic name may be used and are to be understood to represent the same caging domains. Caging domains may be linked in tandem using flexible linkers to increase the cage binding affinity by avidity. Heterodimers caging pairs may be fused in both orientations, such that cagel and cage 2 can be either of the two domains.

[0049] According to some embodiments, the caging domain pair is photoswitchable (“ps”) between the associated state and the dissociated state. Photoswitchable polypeptides may be photoinducible. By “photoinducible” is meant the polypeptide is caged and occludes target binding to the paratope in the absence of dissociating light (basal state) and uncaged upon irradiation with light sufficient to dissociate the caging domain pair and induce target binding. That is, the caging domain pair comprises photodissociable domains. As will be appreciated with the benefit of the present disclosure, the light with which the polypeptide is irradiated to induce target binding should match the action spectrum of photoswitching / photodissociation of the photodissociable domains. In some embodiments, the photodissociable domain can be reassociated (redimerized) by illumination with a different color of light that matched the action spectrum of reverse-photoswitching / photoassociation, or by gradual thermal decay to the basal state.

[0050] When the polypeptide is photoinducible, examples of photodissociable domains that may be employed as the caging domain pair include, but are not limited to, pdDronpa, UVR8, RsLOV, LOVTRAP, cpLOVTRAP, TtCBD, MxCBD, BICYCL-red, and photodissociable variants thereof. Details regarding such domains may be found in, e.g.: pdDronpa (Zhou, Xin X., et al. "Optical control of protein activity by fluorescent protein domains." Science 338.6108 (2012): 810-814; Zhou, Xin X., et al. "Optical control of cell signaling by single-chain photoswitchable kinases." Science 355.6327 (2017): 836-842); UVR8 (Chen, Daniel, Emily S. Gibson, and Matthew J. Kennedy. "A light-triggered protein secretion system." Journal of Cell Biology 201 .4 (2013): 631 -640.); RsLOV (Conrad, Karen S., Alexandrine M. Bilwes, and Brian R. Crane. "Light- induced subunit dissociation by a light-oxygen-voltage domain photoreceptor from Rhodobacter sphaeroides." Biochemistry 52.2 (2013): 378-391 ; Richter, Florian, et al. "Engineering of temperature-and light-switchable Cas9 variants." Nucleic acids research 44.20 (2013): gkw930); LOVTRAP (LOV2:ZDK1 / 2) (Wang, Hui, et al. "LOVTRAP: an optogenetic system for photoinduced protein dissociation." Nature methods 13.9 (2016): 755-758); cpLOVTRAP (cpLOV2:ZDK1 / 2) (He, Lian, et al. "Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering." Nature Chemical Biology 17.8 (2021 ): 915-923); TtCBD (Kainrath, Stephanie, et al. "Green-light-induced inactivation of receptor signaling using cobalamin-binding domains." Angewandte Chemie International Edition 56.16 (2017): 4608-461 1 ); and MxCBD (Kainrath, Stephanie, et al. "Green-light-induced inactivation of receptor signaling using cobalamin-binding domains." Angewandte Chemie International Edition 56.16 (2017): 4608- 4611 ; Mansouri, Maysam, et al. "Smart-watch-programmed green-light-operated percutaneous control of therapeutic transgenes." Nature Communications 12.1 (2021): 3388.); and BICYCL- red (Am1 :BAm red) (Jang, Jaewan, et al. "Engineering of bidirectional, cyanobacteriochrome- based light-inducible dimers (BICYCL)s." Nature Methods 20.3 (2023): 432-441 ). The disclosures of such references are incorporated herein by reference in their entireties for all purposes.

[0051] In other embodiments, a photoswitchable polypeptide of the present disclosure is photorepressible. As used herein, a “photorepressible” polypeptide is uncaged and binds to the target in the absence of associating light (basal state) and caged upon irradiation with light sufficient to associate the caging domain pair. That is, the caging domain pair comprises photoassociable domains. The light with which the polypeptide is irradiated to cage the paratope and occlude target binding should match the action spectrum of photoswitching / photoassociation of the photoassociable domains. In some embodiments, the photoassociable domain can be redissociated (remonomerized) by illumination with a different color of light that matches the action spectrum of reverse-photoswitching / photodissociation, or by gradual thermal decay to the basal state.

[0052] When the polypeptide is photorepressible, examples of photoassociable domains that may be employed as the caging domain pair include, but are not limited to, Cry2, Cry2:CIB1 , iLid, cpLID, TULIP, VVD, Magnets, EL222, FKF1 :GI, VfAU1 -LOV, PtAU1 -LOV, YtvA, BICYCL-green, Cph1 , PhyA:FHY1 , PhyA:FHL, PhyB:PIF3, PhyB:PIF6, BphP1 , BphP1 :PpsR2, BphP1 :Q-PAS1 , MagRed, and photoassociable variants thereof. Details regarding such domains may be found in, e.g.: Cry2 (Bugaj, Lukasz J., et al. "Optogenetic protein clustering and signaling activation in mammalian cells." Nature methods 10.3 (2013): 249-252); Cry2:CIB1 (Kennedy, Matthew J., et al. "Rapid blue-light-mediated induction of protein interactions in living cells." Nature methods 7 2 (2010): 973-975; Taslimi, Amir, et al. "Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase." Nature chemical biology 12.6 (2016): 425- 430.); iLid (LOV-ssrA:SspB_nano / micro / milli) (Guntas, Gurkan, et al. "Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins." Proceedings of the National Academy of Sciences 1 12.1 (2015): 1 12-1 17; Zimmerman, Seth P., et al. "Tuning the binding affinities and reversion kinetics of a light inducible dimer allows control of transmembrane protein localization." Biochemistry 55.37 (2016): 5264- 5271 ); cpLID (ssrA-cpLOV:SspB_nano / micro / milli) (He, Lian, et al. "Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering." Nature Chemical Biology 17.8 (2021 ): 915- 923); TULIP (LOVpep:ePDZ) (Strickland, Devin, et al. "TULIPs: tunable, light-controlled interacting protein tags for cell biology." Nature methods 9.4 (2012): 379-384): VVD (Wang, Xue, Xianjun Chen, and Yi Yang. "Spatiotemporal control of gene expression by a light-switchable transgene system." Nature methods 9.3 (2012): 266-269); Magnets (Kawano, Fuun, et al. "Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins." Nature communications 6.1 (2015): 6256; Benedetti, Lorena, et al. "Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells." Elife 9 (2020): e63230): EL222 (Motta-Mena, Laura B., et al. "An optogenetic gene expression system with rapid activation and deactivation kinetics." Nature chemical biology 10.3 (2014): 196-202.); FKF1 :GI (Yazawa, Masayuki, et al. "Induction of protein-protein interactions in live cells using light." Nature biotechnology 27.10 (2009): 941 -945.); VfAU1 -LOV (Grusch, Michael, et al. "Spatio-temporally precise activation of engineered receptor tyrosine kinases by light." The EMBO journal 33.15 (2014): 1713-1726.); PtAU1 -LOV (Heintz, Udo, and lime Schlichting. "Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1 a for its target DNA sequence." Elife 5 (2016): e11860.); BICYCL-green (Am1 :BAm green) (Jang, Jaewan, et al. "Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s." Nature Methods 20.3 (2023): 432-441 ); Cph1 (Reichhart. Eva, et al. "A phytochrome sensory domain permits receptor activation by red light." Angewandte Chemie International Edition 55.21 (2016): 6339-6342); PhyA:FHY1 or PhyA:FHL (Rausenberger, Julia, et al. "Photoconversion and nuclear trafficking cycles determine phytochrome A's response profile to far-red light." Cell 146.5 (201 1 ): 813-825.); PhyB:PIF3 or PhyB:PIF6 (Levskaya, Anselm, et al. "Spatiotemporal control of cell signalling using a light-switchable protein interaction." Nature 461 .7266 (2009): 997-1001 .); BphP1 (Leopold. Anna V., et al. "Neurotrophin receptor tyrosine kinases regulated with near-infrared light." Nature communications 10.1 (2019): 1129.); BphP1 :PpsR2 or BphP1 :Q-PAS1 (Kaberniuk, Andrii A., Anton A. Shemetov, and Vladislav V. Verkhusha. "A bacterial phytochrome-based optogenetic system controllable with near-infrared light." Nature methods 13.7 (2016): 591 -597.); and MagRed (DrBphP:Pfr) (Kuwasaki, Yuto, et al. "A red light-responsive photoswitch for deep tissue optogenetics." Nature Biotechnology 40.1 1 (2022): 1672-1679.). The disclosures of such references are incorporated herein by reference in their entireties for all purposes.

[0053] Information on photodissociable and photoassociable domains are described in, e.g., an online database (www.optobase.org), review papers (Seong, Jihye, and Michael Z. Lin. "Optobiochemistry: Genetically encoded control of protein activity by light." Annual Review of Biochemistry 90 (2021 ): 475-501 ; Tan, Peng, et al. "Optophysiology: Illuminating cell physiology with optogenetics." Physiological Reviews 102.3 (2022): 1263-1325.), and elsewhere.

[0054] According to some embodiments, the caging domain pair is chemoswitchable (“cs”) between the associated state and the dissociated state. Chemoswitchable polypeptides may be chemoinducible. By “chemoinducible” is meant the polypeptide is caged and occludes target binding to the paratope in the absence of dissociating ligand (e.g., small molecule or other ligand) (basal state) and uncaged upon contacting the polypeptide with a dissociating ligand in sufficient concentrations to dissociate the caging domain pair and induce target binding. That is, the caging domain pair comprises chemodissociable domains. The effect may be reversed by diminishing the concentration of the ligand. This can be performed in vitro, e.g., by removing the ligand from the supernatant (e.g., washing). In vivo, metabolism will generally gradually clear the ligand from circulation in the absence of repeated or continuous administration. In some instances, the ligand is an approved drug (e.g., small molecule drug).

[0055] When the polypeptide is chemoinducible, examples of chemodissociable domains that may be employed as the caging domain pair include, but are not limited to, FKBP-F36M, FKBP- F36L, Bcl-XL:BH3, Bcl-XL:LD3, Bcl2:LD3, NS3a:ANR, TetRB:TIP, aMinoC1 -sdAb:CX7C, and chemodissociable variants thereof. Details regarding such domains may be found in, e.g.: FKBP- F36M (Rivera, Victor M., et al. "Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum." Science 287.5454 (2000): 826-830; Rollins, Carl T., et al. "A ligand- reversible dimerization system for controlling protein-protein interactions." Proceedings of the National Academy of Sciences 97.13 (2000): 7096-7101 ); FKBP-F36L (Barrero, Juan J., et al. "An improved reversibly dimerizing mutant of the FK506-binding protein FKBP." Cellular Logistics 6.3 (2016): e1204848); Bcl-XL:BH3 (Goreshnik, Inna, and Dustin J. Maly. "A small molecule-regulated guanine nucleotide exchange factor." Journal of the American Chemical Society 132.3 (2010): 938-940; Rose, John C., et al. "Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics." Nature methods 14.9 (2017): 891 -896); Bcl-XL:LD3 v0 / v1 / v2 / v3 (Giordano-Attianese, Greta, et al. "A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy." Nature biotechnology 38.4 (2020): 426-432; Shui, Sailan, et al. "A rational blueprint for the design of chemically-controlled protein switches." Nature communications 12.1 (2021 ): 5754); Bcl2:LD3 v0 / v1 / v2 / v3 (Shui, Sailan, et al. "A rational blueprint for the design of chemically-controlled protein switches." Nature communications 12.1 (2021 ): 5754); NS3a:ANR (Cunningham-Bryant, Daniel, et al. "A chemically disrupted proximity system for controlling dynamic cellular processes." Journal of the American Chemical Society 141 .8 (2019): 3352-3355.); TetRB:TIP (Hotblack, Alastair, et al. "Tunable control of CAR T cell activity through tetracycline mediated disruption of protein-protein interaction." Scientific Reports 11.1 (2021 ): 21902); and aMinoC1 -sdAb:CX7C (Jha. Ram, et al. "Designer Small-Molecule Control System Based on Minocycline-Induced Disruption of Protein- Protein Interaction." ACS Chemical Biology 19.2 (2024): 308-324.). The disclosures of such references are incorporated herein by reference in their entireties for all purposes.

[0056] In other embodiments, a chemoswitchable polypeptide of the present disclosure is chemorepressible. As used herein, a “chemorepressible” polypeptide is uncaged and able to bind the target in the absence of an associating ligand (e.g., small molecule or other ligand) (basal state) and caged upon contacting the polypeptide with an associating ligand in sufficient concentration to associate the caging domain pair thereby caging / occluding the paratope. That is, the caging domain pair comprises chemoassociable domains. The effect may be reversed by diminishing the concentration of the ligand. This can be performed in vitro, e.g., by removing the ligand from the supernatant (e.g., washing). In vivo, metabolism will generally gradually clear the ligand from circulation in the absence of repeated or continuous administration. In some instances, the associating ligand may be referred to as a molecular glue. In some instances, the ligand is an approved drug (e.g., small molecule drug).

[0057] When the polypeptide is chemorepressible, examples of chemoassociable domains that may be employed as the caging domain pair include, but are not limited to, FKBP:FRB, iFKBP:FRB, FKBP, FKBP-F36V, GID1 :GAI, PYL:ABI, PYR:ABI, PYR:HAB1 , NS3a:GNCR1 , NS3a:DNCR2, NS3a:PRSIM_23, Bcl-XL:AZ1 , Bcl-XL:AZ21 , aCaffVHH, COSMO, split-Lyn, split- DHFR, split-GR2, split-TR , hRBP4:RS3, hRBP4:RS5, hRBP4:RF2, CRBN:IKZF3, CRBNA3JKZF3, CRBNA3:ZFP91 -IKZF3, CEL:ZIF, GntR:GntR, CA-14:DB-18, CA-14:DB-21 , FIREtag:FIREmate, hcy129.1 :miniH1 1 , split-buttressed-CHD_r1 , VHHAS2, split-mAb-Nic12, AR:MBP, and chemoassociable variants thereof. Details regarding such domains may be found in, e.g. : FKBP:FRB (Banaszynski, Laura A., Corey W. Liu, and Thomas J. Wandless. "Characterization of the FKBP Rapamycin FRB ternary complex." Journal of the American Chemical Society 127.13 (2005): 4715-4721 ); iFKBP:FRB (Karginov, Andrei V., et al. "Engineered allosteric activation of kinases in living cells." Nature biotechnology 28.7 (2010): 743-747); FKBP (Amara, Jane F., et al. "A versatile synthetic dimerizer for the regulation of protein-protein interactions." Proceedings of the national academy of sciences 94.20 (1997): 10618-10623; Spencer, David M., et al. "Controlling signal transduction with synthetic ligands." Science 262.5136 (1993): 1019-1024); FKBP-F36V (Clackson, Tim, et al. "Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity." Proceedings of the National Academy of Sciences 95.18 (1998): 10437-10442); GID1 :GAI (Miyamoto, Takafumi, et al. "Rapid and orthogonal logic gating with a gibberellin-induced dimerization system." Nature chemical biology 8.5 (2012): 465-470); PYL:ABI (Liang, Fu-Sen, Wen Qi Ho, and Gerald R. Crabtree. "Engineering the ABA plant stress pathway for regulation of induced proximity." Science signaling 4.164 (201 1): rs2-rs2); PYR:ABI (Ziegler, Michael J., et al. "Mandipropamid as a chemical inducer of proximity for in vivo applications." Nature chemical biology 18.1 (2022): 64-69); PYR:HAB1 (Beltran, Jesus, et al. "Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds." Nature Biotechnology 40.12 (2022): 1855-1861); NS3a:GNCR1 (Foight, Glenna Wink, et al. "Multi-input chemical control of protein dimerization for programming graded cellular responses." Nature biotechnology 37.10 (2019): 1209-1216); NS3a:DNCR2 (Foight, Glenna Wink, et al. "Multi-input chemical control of protein dimerization for programming graded cellular responses." Nature biotechnology 37.10 (2019): 1209-1216); NS3 / 4a:PRSIM 23 (Chin, Stacey E., et al. "A simeprevir-inducible molecular switch for the control of cell and gene therapies." Nature Communications 14.1 (2023): 7753); Bcl-XL:AZ1 or Bcl-XL:AZ21 (Hill, Zachary B., et al. "Human antibody-based chemically induced dimerizers for cell therapeutic applications." Nature chemical biology 14.2 (2018): 112-117; Martinko, Alexander J., et al. "Switchable assembly and function of antibody complexes in vivo using a small molecule." Proceedings of the National Academy of Sciences 119.9 (2022): e2117402119.); aCaffVHH (Bojar, Daniel, et al. "Caffeine-inducible gene switches controlling experimental diabetes." Nature communications 9.1 (2018): 2318.); COSMO (acVHH-Y104W) (Wang, Tianlu, et al. "Caffeine-Operated Synthetic Modules for Chemogenetic Control of Protein Activities by Life Style." Advanced Science 8.3 (2021 ): 2002148.); split-LYN (Rihtar, Erik, et al. "Chemically inducible split protein regulators for mammalian cells." Nature Chemical Biology 19.1 (2023): 64-71 ); split-DHFR (Rihtar, Erik, et al. "Chemically inducible split protein regulators for mammalian cells." Nature Chemical Biology 19.1 (2023): 64-71); split-GR2 (Rihtar, Erik, et al. "Chemically inducible split protein regulators for mammalian cells." Nature Chemical Biology 19.1 (2023): 64-71 ); split-TRB (Rihtar, Erik, et al. "Chemically inducible split protein regulators for mammalian cells." Nature Chemical Biology 19.1 (2023): 64-71 ); hRBP4:RS3 or hRBP4:RS5 or hRBP4:RF2 (Zajc, Charlotte U., et al. "A conformation-specific ON-switch for controlling CAR T cells with an orally available drug." Proceedings of the National Academy of Sciences 117.26 (2020): 14926-14935.); CRBN:IKZF3 (Jan, Max, et al. "Reversible ON-and OFF-switch chimeric antigen receptors controlled by lenalidomide." Science translational medicine 13.575 (2021 ): eabb6295.); CRBNA3:IKZF3 or CRBNA3:ZFP91-IKZF3 (Jan, Max, et al. "Reversible ON-and OFF-switch chimeric antigen receptors controlled by lenalidomide." Science translational medicine 13.575 (2021 ): eabb6295.); CEL:ZIF (Chung, Chan-I., Junjiao Yang, and Xiaokun Shu. "Chemogenetic Minitool for Dissecting the Roles of Protein Phase Separation." ACS Central Sc / ence (2023)); GntR:GntR (Teixeira, Ana Palma, et al. "Evolution of molecular switches for regulation of transgene expression by clinically licensed gluconate." Nucleic Acids Research (2023)'. gkad600.); and CA-14:DB-18 or CA-14:DB-21 (Kang, Shoukai, et al. "COMBINES-CID: An efficient method for de novo engineering of highly specific chemically induced protein dimerization systems." Journal of the American Chemical Society 141 .28 (2019): 10948-10952.); CATCHFIRE (FIREtag:FIREmate) (Bottone, Sara, et al. "A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity." Nature Methods 20.10 (2023): 1553-1562; Kumar, Pratik, et al. "Transforming chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers using chemistry." bioRxiv (2024) DOI: 10.1101 / 2023.12.30.573644): hcy129.1 :miniH11 (Lee. Gyu Rie, et al. "Small-molecule binding and sensing with a designed protein family." bioRxiv (2023) DOI: 10.1 101 / 2023.1 1 .01.565201 ); split-buttressed-CHD r1 (domain A:B) (An, Linna, et al. "Binding and sensing diverse small molecules using shape-complementary pseudocycles." Science 385.6706 (2024): 276-282.); VHHAS? (Scheller, Leo, et al. "Generalized extracellular molecule sensor platform for programming cellular behavior." Nature chemical biology 14.7 (2018): 723-729 and Spinelli, Silvia, et al. "Lateral recognition of a dye hapten by a llama VHH domain." Journal of molecular biology 31 1.1 (2001 ): 123-129); split-mAb-Nic12 (VH:V ) (Scheller, Leo, et al. "Generalized extracellular molecule sensor platform for programming cellular behavior." Nature chemical biology 14.7 (2018): 723-729); and AR:MBP (Glasgow, Anum A., et al. "Computational design of a modular protein sense-response system." Science 366.6468 (2019): 1024-1028). The disclosures of such references are incorporated herein by reference in their entireties for all purposes.

[0058] The a-helical binding domain is flanked by the caging domain pair via flexible linkers. By “flexible” is meant the linkers are of sufficient length and flexibility to freely permit cross-paratope association of the members of the caging domain pair under the desired conditions (e.g., absence of dissociating light, presence of associating light, absence of a dissociating chemical environment, or presence of an associating chemical environment. Flexible linkers are often rich in small or polar amino acids such as Gly and Ser to provide good flexibility and solubility. In certain embodiments, a Gly-Ser (GS) linker is employed, non-limiting examples of which are those comprising one or more GGGGS units, i.e. , (GGGGS)nwhere n is an integer of 1 or greater, e.g., 1 to 10. In some embodiments, a flexible linker comprising Gly and Ser, and one or more threonine (Thr) residues is employed. According to some embodiments, a flexible linker comprising Gly or Ser residues, and one or more Thr residues is employed. The amino acid sequences of non-limiting examples of such linkers that find use in operably flanking a caging domain pair to exDARPin and exaRep binding domains are provided in the sequence table herein. In some instances, the first member of a caging domain pair and the second member of a caging domain pair are fused to the termini of the binding domain via a linker having a length independently selected from 2 to 20 amino acids, 3 to 15 amino acids, 4 to 10 amino acids, or 5 to 8 amino acids. Suitable linker lengths will vary, e.g., based on the number of internal repeats of the binding domain. For binding domains with few to many internal repeats, in some instances, the optimal range will be 3 to 10 (e.g., 3 to 6, 5 to 8, or 7 to 10), 6 to 12 (e.g., 9 to 12), 8 to 14 (e.g., 11 to 14), 10 to 16 (e.g., 1 1 to 14, or 13 to 16), or 12 to 18 (e.g., 13 to 16, or 15 to 18) amino acids in length. Accordingly, linkers may be optimized as desired for the particular binding domain and caging pair employed in the polypeptide. Linker lengths may be adjusted for each combination of caging domain pair and binding domain using structural information in the form of, crystal structures, computationally predicted structures, or the like. Linked and Iinker2 can be different lengths. Linker lengths that allow for caging of the paratope can be predicted using computational simulations, manual structural modeling, etc. The dissociation constant, Kd, of a photoswitchable and chemoswitchable binder to its target is lower in the uncaged state compared to the caged state (Kd(caged)> Kd(uncaged)). In the uncaged state, the Kdof a given photoswitchable or chemoswitchable binder may take the values of <1 pM, 1 - 10 pM, 10 - 100 pM, 0.1 - 1 nM, 1 - 10 nM, 10 - 100 nM, 0.1 - 1 pM, 1 - 10 pM with lower values being generally preferable. The ratio Kd(caged) / Kd(uncaged) may take the values 1 - 3, 3 - 10, 10 - 30 , 30 - 100, 100 - 300, 300 - 1000, >1000 with higher values being generally preferable.

[0059] The amino acid sequences of domains of non-limiting examples of inducible polypeptides and repressible polypeptides according to embodiments of the present disclosure are provided in Table 3 below.

[0060] Table 3 - Amino acid sequences of exemplary polypeptide domains

[0061] The present disclosure provides each of the polypeptides provided in Table 3, and each of the individual domains therein, as well as nucleic acids that encode such polypeptides and individual domains. Cells comprising such polypeptides and nucleic acids are also provided. As will be appreciated, the present disclosure also provides variants of any of the polypeptides and individual domains therein, where in some instances a variant polypeptide or domain thereof comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91 % or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to the parental / reference sequence, or a functional fragment thereof, where the variant retains the functionality (e.g., target binding, photodissociability, photoassociability, chemodissociability, chemoassociability, and / or the like) of the parental / reference sequence.

[0062] In certain embodiments, polypeptides having one or more amino acid substitutions are provided. Conservative substitutions are shown in Table 4 under the heading of "preferred substitutions." More substantial changes are provided in Table 4 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into a polypeptide and the products screened for a desired activity, e.g., retained / improved target binding, photodissociability, photoassociability, chemodissociability, chemoassociability, and / or the like.

[0063] Table 4 - Amino Acid Substitutions

[0064]

[0065] Amino acids may be grouped according to common side-chain properties:

[0066] (1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0067] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu;

[0068] (4) basic: His, Lys, Arg;

[0069] (5) residues that influence chain orientation: Gly, Pro;

[0070] (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0071] Fusion Proteins

[0072] Aspects of the present disclosure also include fusion proteins. For example, provided are any of the polypeptides of the present disclosure fused to a heterologous protein domain, e.g., a heterologous functional protein domain (FPD). The FPD may for example be another SBD; an antibody or domains of an antibody; a half-life extending protein such as a serum albumin binding domain; or an enzyme that affects the function of the target or other biomolecules proximal to the target.

[0073] In certain embodiments, the photoswitchable or chemoswitchable binding proteins may be fused to one or several additional functional protein domains (FPDs) at either the N-terminus or the C-terminus. In some embodiments, the terminal fusions are achieved via flexible linkers. According to some embodiments, these fusion proteins are described by the fusion order:

[0074] FPD1 -linkerO-cagel -linkerl -SBD-linker2-cage2; cagel -linkerl -SBD-linker2-cage2-linkerO-FBD1 ;

[0075] FPD1 -linkerO-cagel -linkerl -SBD-linker2-cage2-linker3-FPD;

[0076] FPD1 -linkerO-FPD2-linker3-cage1 -linkerl -SBD-linker2-cage2; or cagel -linkerl -SBD-linker2-cage2-linkerO-FPD1 -Iinker3-FPD2.

[0077] FPDs may be conjugated to the polypeptide via a peptide linker. If present, linkerO and linkers may be of sufficient length to permit the polypeptide and the linked FPD to allow some flexible movement between the polypeptide and the linked FPD. Linker molecules may be, e.g., about 6-50 atoms long. The linkers can be of any suitable length, such as from 1 amino acid (e.g, Gly) to 20 or more amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1 , 2, 3, 4, 5, 6, or 7 amino acids in length.

[0078] Flexible linkers include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used where relatively unstructured amino acids are of interest, and may serve as a neutral tether between components. The ordinarily skilled artisan will recognize that design of a polypeptide conjugated to any FPD described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer a less flexible structure. Conjugates

[0079] Aspects of the present disclosure also include conjugates comprising any of the polypeptides of the present disclosure conjugated to an agent. The term “conjugated” generally refers to a chemical linkage, either covalent or non-covalent, usually covalent, that proximally associates one molecule of interest with a second molecule of interest. The site of conjugation generally should not overlap with the SBD paratope or the binding interfaces of the photoswitchable or chemoswitchable pairs including the ligand-binding pocket for the chemoswitchable pairs. Furthermore, the conjugate should not disturb the photoswitching or chemoswitching mechanism. In certain embodiments, the agent conjugated to the polypeptide is chosen from a chemotherapeutic agent, a toxin, a radiation-sensitizing agent, a radioactive isotope (e.g., a therapeutic radioactive isotope), a detectable label, and a half-life extending moiety.

[0080] According to some embodiments, the agent is a therapeutic agent, e.g., a chemotherapeutic agent. Therapeutic agents of interest include agents capable of affecting the function of a cell / tissue to which the conjugate binds via specific and inducible binding of the polypeptide portion of the conjugate to a target molecule. When the function of the cell / tissue is pathological, an agent that reduces the function of the cell / tissue may be employed. In certain embodiments, a conjugate of the present disclosure includes an agent that reduces the function of a target cell / tissue by inhibiting cell proliferation and / or killing the cell / tissue. Such agents may vary and include cytostatic agents and cytotoxic agents, e.g., an agent capable of killing a target cell tissue with or without being internalized into a target cell.

[0081] In certain embodiments, the therapeutic agent is a cytotoxic agent selected from an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. In some embodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065, CPT-11 (SN-38), topotecan, doxorubicin, morpholino-doxorubicin, rhizoxin, cyanomorpholinodoxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, maytansine DM1 , maytansine DM4, DM-1 , an auristatin or other dolastatin derivatives, such as auristatin E or auristatin F, AEB (AEB-071 ), AEVB (5-benzoylvaleric acid-AE ester), AEFP (antibody- endostatin fusion protein), MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), pyrrolobenzodiazepines (PBDs), eleutherobin, netropsin, or any combination thereof.

[0082] According to some embodiments, the agent is a toxin, such as a protein toxin selected from hemiasterlin and hemiasterlin analogs such as HTI-286 (e.g., see USPN 7,579,323; WO 2004 / 026293; and USPN 8,129,407, the full disclosures of which are incorporated herein by reference), abrin, brucine, cicutoxin, diphtheria toxin, batrachotoxin, botulism toxin, shiga toxin, endotoxin, Pseudomonas exotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin, falcarinol, fumonisin Bl, fumonisin B2, afla toxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcicludine, geldanamycin, gelonin, lotaustralin, ocratoxin A, patulin, ricin, strychnine, trichothecene, zearlenone, and tetradotoxin. Enzymatically active toxins and fragments thereof which may be employed include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin , restrictocin, phenomycin, enomycin and the tricothecenes.

[0083] In certain embodiments, the agent is a radiation-sensitizing agent. As used herein, a “radiation-sensitizing agent” is an agent that enhances the ability of radiation to kill tumor cells. Non-limiting examples of radiation-sensitizing agents that may be conjugated to the antibody or fusion protein include cisplatin, 5-fluorouracil (5-FU), AZD7762, selumetinib, and the like.

[0084] In certain embodiments, the agent is a radioisotope, e.g., useful for therapy and / or detection (e.g., imaging). Non-limiting examples of radioisotopes that may be conjugated to the antibody or fusion protein include but are not limited to225Ac,111Ag,114Ag,71As,72As,77As,211At,198Au,199Au,212Bi,213Bi,75Br,76Br,11C,13C,55Co,62Cu,64Cu,67Cu,165Dy,166Dy,169Er,18F,19F,52Fe,59Fe,66Ga,67Ga,68Ga,72Ga,154 158Gd,157Gd,159Gd,166Ho,120l,1211,123l,124l,125l,1311,110ln,111ln,113mln,194lr,81 mKr,177Lu,51Mn,52Mn,99Mo,13N,15N,15O,17O,32P,33P,211Pb,212Pb,109Pd,149Pm,151Pm,142Pr,143Pr,191PT,193mPT,195mPt,223Ra,142Rb,186Re,188Re,189Re,105Rh,47Sc,75Se,153Sm,117mSn,121Sn,83Sr,89Sr,161Tb,94Tc, "Tc,99mTc,227Th,201TI,172Tm,127Te,90Y,169Yb,175Yb,133X, and89Zr.

[0085] In certain embodiments, a radioisotope is conjugated to the polypeptide via a chelator, for example, a bifunctional chelator. A bifunctional chelator may contain a metal chelating moiety that binds the radioisotope in a stable coordination complex and a reactive functional group that is covalently linked to a targeting moiety, such as any of the polypeptides of the present disclosure, so that the radioisotope may be properly directed to the desirable molecular target in vivo. Non-limiting examples of bifunctional chelators that may be employed to conjugate a polypeptide of the present disclosure to a radioisotope include p-SCN-Bn-DOTA and p-SCN-Bn- deferoxamine. Additional examples of bifunctional chelators that may be employed to conjugate a polypeptide of the present disclosure to a radioisotope include those described in Price & Orvig (2014) Chem. Soc. Rev. 43:260; and Brechbiel (2008) Q J Nucl Med Mol Imaging 52(2)' 66- 73.

[0086] According to some embodiments, the radioisotope is a therapeutic radioisotope. In certain embodiments, the radioisotope is an alpha emitting radioisotope, e.g.,225Ac,211At,212Bi / 212Pb,213Bi,223Ra, or227Th. In other embodiments, the radioisotope is a beta minus emitting radioisotope, e.g.,32P,33P,67Cu,90Y,1311 or177Lu.

[0087] According to some embodiments, the agent is a labeling agent. By “labeling agent” (or “detectable label”) is meant the agent detectably labels the polypeptide, such that the polypeptide may be detected in an application of interest (e.g., in vitro and / or in vivo research and / or clinical applications). Detectable labels of interest include radioisotopes (e.g., gamma or positron emitters), enzymes that generate a detectable product (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, etc.), fluorescent proteins, paramagnetic atoms, and the like. In certain embodiments, the polypeptide is conjugated to a specific binding partner of detectable label, e.g., conjugated to biotin such that detection may occur via a detectable label that includes avidin / streptavidin.

[0088] In certain embodiments, the agent is a labeling agent that finds use in in vivo imaging, such as near-infrared (NIR) optical imaging, single-photon emission computed tomography (SPECT) ± CT imaging, positron emission tomography (PET) ± CT imaging, nuclear magnetic resonance (NMR) spectroscopy, or the like. Labeling agents that find use in such applications include, but are not limited to, fluorescent labels, radioisotopes, and the like. In certain aspects, the labeling agent is a multi-modal in vivo imaging agent that permits in vivo imaging using two or more imaging approaches (e.g., see Thorp-Greenwood and Coogan (201 1 ) Dalton Trans. 40:6129-6143).

[0089] In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in near-infrared (NIR) imaging applications. Such agents include, but are not limited to, a Kodak X- SIGHT dye, Pz 247, DyLight 750 and 800 Fluors, Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, IRDye 680 and 800CW Fluors. According to some embodiments, the labeling agent is an in vivo imaging agent that finds use in SPECT imaging applications, non-limiting examples of which include99mTc,111ln,123l,201TI, and133Xe. In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in PET imaging applications, e.g.,11C,13N,15O,18F,64Cu,62Cu,124l,76Br,82Rb,68Ga, or the like.

[0090] For half-life extension, the polypeptides of the present disclosure may be conjugated to an agent that provides for an improved pharmacokinetic profile (e.g., by PEGylation, hyperglycosylation, and the like). Modifications that can enhance serum half-life are of interest. A subject polypeptide may be “PEGylated”, as containing one or more polyethylene glycol) (PEG) moieties. Methods and reagents suitable for PEGylation of a protein are well known in the art and may be found, e.g., in US Pat. No. 5,849,860. PEG suitable for conjugation to a protein is generally soluble in water at room temperature and has the general formula R(O-CH2-CH2)nO- R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. Where R is a protective group, it generally has from 1 to 8 carbons. The PEG conjugated to the subject polypeptide can be linear. The PEG conjugated to the subject polypeptide may also be branched. Branched PEG derivatives such as those described in U.S. Pat. No. 5,643,575, “star-PEGs” and multi-armed PEGs. Star PEGs are described in the art including, e.g., in U.S. Patent No. 6,046,305.

[0091] Where the subject polypeptide is to be isolated from a source, the polypeptide may be conjugated to one or more moieties that facilitate purification, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), a lectin, and the like. The polypeptide can also be bound to (e.g., immobilized onto) a solid support, including, but not limited to, polystyrene plates or beads, magnetic beads, test strips, membranes, and the like.

[0092] Where the polypeptides are to be detected in an assay, the polypeptides may contain a detectable label, e.g., a radioisotope (e.g.,89Zr;111In, and the like), an enzyme which generates a detectable product (e.g., luciferase, p-galactosidase, horse radish peroxidase, alkaline phosphatase, and the like), a fluorescent protein, a chromogenic protein, dye (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g.,152Eu, or others of the lanthanide series, attached to the protein through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., luciferin; fluorescent proteins; and the like.

[0093] Any of the above agents may be conjugated to the polypeptide via a linker. If present, the linker molecule(s) may be of sufficient length to permit the polypeptide and the linked agent to allow some flexible movement between the polypeptide and the linked agent. Linker molecules may be, e.g., about 6-50 atoms long. Linker molecules may also be, e.g., aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.

[0094] Where the linkers are peptides, the linkers can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 or more amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1 , 2, 3, 4, 5, 6, or 7 amino acids in length.

[0095] Flexible linkers include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used where relatively unstructured amino acids are of interest, and may serve as a neutral tether between components. The ordinarily skilled artisan will recognize that design of a polypeptide conjugated to any agents described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer a less flexible structure.

[0096] According to some embodiments, the polypeptide is conjugated to the agent via a non- cleavable linker. Non-cleavable linkers of interest include, but are not limited to, thioether linkers. An example of a thioether linker that may be employed includes a succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) linker.

[0097] In certain embodiments, the polypeptide is conjugated to the agent via a cleavable linker. According to some embodiments, the linker is a chemically-labile linker, such as an acid- cleavable linker that is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the mildly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of a target cell (e.g., a cancer cell). Chemically-labile linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, etc. In certain embodiments, the linker is an enzyme-labile linker, such as an enzyme-labile linker that is stable in the bloodstream but undergoes enzymatic cleavage upon internalization into a target cell, e.g., by a lysosomal protease (such as cathepsin or plasmin) in a lysosome of the target cell (e.g., a cancer cell). Enzyme-labile linkers include, but are not limited to, linkers that include peptidic bonds, e.g., dipeptide-based linkers such as valine-citrulline (VC) linkers, such as a maleimidocaproyl-valine-citruline-p-aminobenzyl (MC-vc-PAB) linker, a valyl-alanyl-para- aminobenzyloxy (Val-Ala-PAB) linker, and the like. Chemically-labile linkers, enzyme-labile, and non-cleavable linkers are known and described in detail, e.g., in Ducry & Stump (2010) Bioconjugate Chem. 21 :5-13; Nolting, B. (2013) Methods Mol Biol. 1045:71 -100; Tsuchikama and An (2018) Protein & Ce / / 9(1 ):33-46; and elsewhere.

[0098] Numerous strategies are available for linking agents to a polypeptide directly, or indirectly via a linker. For example, the agent may be derivatized by covalently attaching a linker to the agent, where the linker has a functional group capable of reacting with a “chemical handle” on the polypeptide. The functional group on the linker may vary and may be selected based on compatibility with the chemical handle on the polypeptide. According to one embodiment, the chemical handle on the polypeptide is provided by incorporation of an unnatural amino acid having the chemical handle into the polypeptide. Unnatural amino acids which find use for preparing the conjugates of the present disclosure include those having a functional group selected from an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde (e.g., formylglycine, e.g, SMARTag™ technology from Catalent Pharma Solutions), nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, and boronic acid functional group. Unnatural amino acids which may be incorporated into a polypeptide of the present disclosure, which unnatural amino acid may be selected to provide a functional group of interest, are known and described in, e.g., Maza et al. (2015) Bioconjug. Chem. 26(9) :1884-9; Patterson et al. (2014) ACS Chem. Biol. 9:592-605; Adumeau et al. (2016) Mol. Imaging Biol. (2):153-65; and elsewhere. An unnatural amino acid may be incorporated into a polypeptide via chemical synthesis or recombinant approaches, e.g., using a suitable orthogonal amino acyl tRNA synthetase-tRNA pair for incorporation of the unnatural amino acid during translation of the polypeptide in a host cell.

[0099] The functional group of an unnatural amino acid present in the polypeptide may be an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde, asaldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, boronic acid, diazo, tetrazine, tetrazole, quadrocyclane, iodobenzene, or other suitable functional group, and the functional group on the linker is selected to react with the functional group of the unnatural amino acid (or vice versa). As just one example, an azide-bearing unnatural amino acid (e.g., 5-azido-L-norvaline, or the like) may be incorporated into the polypeptide and the linker portion of a linker-agent moiety may include an alkyne functional group, such that the polypeptide and linker-agent moiety are covalently conjugated via azide-alkyne cycloaddition. Conjugation may be carried out using, e.g., a copper-catalyzed azide-alkyne cycloaddition reaction.

[0100] In certain embodiments, the chemical handle on the polypeptide does not involve an unnatural amino acid. A polypeptide containing no unnatural amino acids may be conjugated to the agent by utilizing, e.g., nucleophilic functional groups of the polypeptide (such as the N- terminal amine or the primary amine of lysine, or any other nucleophilic amino acid residue) as a nucleophile in a substitution reaction with a moiety bearing a reactive leaving group or other electrophilic group. An example would be to prepare an agent-linker moiety bearing an N- hydroxysuccinimidyl (NHS) ester and allow it to react with the polypeptide under aqueous conditions at elevated pH (-10) or in polar organic solvents such as DMSO with an added non- nucleophi lie base, such as N,N-diisopropylethylamine.

[0101] It will be appreciated that the particular approach for attaching a linker, agent and / or polypeptide to each other may vary depending upon the particular linker, agent and / or polypeptide and functional groups selected and employed for conjugating the various components to each other.

[0102] Methods of Making Polypeptides

[0103] A polypeptide of the present disclosure may be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis). Where the polypeptide is produced using recombinant techniques, a polypeptide may be produced as an intracellular protein or as a secreted protein, using any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E. coli, including but not limited to E. coli strains engineered for incorporation of non-natural amino acids) or a yeast host cell, respectively. Other examples of eukaryotic cells that may be used as host cells include insect cells, mammalian cells, and / or plant cells. Where mammalian host cells are used, the cells may include one or more of the following: human cells (e.g. HeLa, 293, H9, and Jurkat cells); mouse cells (e.g., X3, NIH3T3, pancreatic ductal adenocarcinoma 2.1 , L cells, and C127 cells); primate cells (e.g. Cos 1 , Cos 7, and CV1 ) and hamster cells (e.g., Chinese hamster ovary (CHO) cells).

[0104] A wide range of host-vector systems suitable for the expression of the polypeptide may be employed according to standard procedures known in the art. See, e.g., Sambrook et al. 1989 Current Protocols in Molecular Biology Cold Spring Harbor Press, New York and Ausubel et al. 1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The method for transfer can be selected so as to provide for stable expression of the introduced polypeptide-encoding nucleic acid. The polypeptide-encoding nucleic acid can be provided as an inheritable episomal element (e.g., a plasmid) or can be genomically integrated. A variety of appropriate vectors for use in production of a polypeptide of interest are available commercially.

[0105] Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome. The expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7, and the like). Expression constructs generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest. A selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector. In addition, the expression construct may include additional elements. For example, the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. In addition the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selectable genes are well known in the art and will vary with the host cell used.

[0106] Isolation and purification of the polypeptide can be accomplished according to methods known in the art. For example, a polypeptide can be isolated from a lysate of cells genetically modified to express the protein constitutively and / or upon induction, or from a synthetic reaction mixture, by immunoaffinity purification, which generally involves contacting the sample with an anti-polypeptide antibody (or in the case of a tagged polypeptide, an anti-tag antibody), washing to remove non-specifically bound material, and eluting the specifically bound polypeptide. The isolated polypeptide can be further purified by dialysis and other methods normally employed in protein purification methods. In one embodiment, the polypeptide may be isolated using metal chelate chromatography methods. Polypeptides of the present disclosure may contain modifications (e.g., affinity tags or the like) to facilitate isolation.

[0107] The polypeptides may be prepared in substantially pure or isolated form (e.g., free from other polypeptides). The polypeptide can be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). Purified polypeptide may be provided such that the polypeptide is present in a composition that is substantially free of other expressed proteins, e.g., less than 98%, less than 95%, less than 90%, less than 80%, less than 60%, or less than 50%, of the composition is made up of other expressed proteins.

[0108] Where a polypeptide is chemically synthesized, the synthesis may proceed via liquidphase or solid-phase. Solid-phase synthesis (SPPS) allows the incorporation of unnatural amino acids, peptide / protein backbone modification. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing pro-peptides of the present disclosure. Details of the chemical synthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8). Briefly, small insoluble, porous beads are treated with functional units on which peptide chains are built. After repeated cycling of coupling / deprotection, the free N-terminal amine of a solid-phase attached peptide or amino acid is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached. The polypeptide remains immobilized on the solid-phase and may undergo a filtration process before being cleaved off.

[0109] NUCLEIC ACIDS, EXPRESSION CONSTRUCTS, CELLS AND COMPOSITIONS

[0110] Nucleic Acids and Expression Constructs

[0111] Aspects of the present disclosure further include nucleic acids and expression constructs. For example, provided are nucleic acids encoding any of the polypeptides of the present disclosure. Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of a polypeptide of interest provides a description of all the polynucleotides capable of encoding the polypeptide of interest. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the polypeptides and domains disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the polypeptide of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences set forth in Table 3.

[0112] The nucleotide sequences of the nucleic acids of the present may be codon-optimized. “Codon-optimized” refers to changes in the codons of the polynucleotide encoding a polypeptide to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, a nucleic acid of the present disclosure encoding a polypeptide may be codon-optimized for optimal production from the host organism selected for expression, e.g., human cells, such as human immune cells (e.g., human T cells).

[0113] Also provided are expression constructs comprising any of the nucleic acids of the present disclosure. As used herein, an “expression construct” is a circular or linear polynucleotide (a polymer composed of naturally occurring and / or non-naturally occurring nucleotides) comprising a region that encodes a polypeptide of the present disclosure, operably linked to a suitable promoter, e.g., a constitutive or inducible promoter. In some embodiments, expression of the polypeptide is under the control of one or more exogenous (including heterologous) regulatory elements, e.g., promoter, enhancer, etc., present in the expression construct. In some embodiments, expression of the polypeptide may be controlled by one or more endogenous regulatory elements, e.g., promoter, enhancer, etc., at or near a genomic locus into which the expression construct is inserted.

[0114] The expression constructs (e.g., vectors) can be suitable for replication and integration in prokaryotes, eukaryotes, or both. The expression constructs may contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the first subunit, the second subunit, or both. The expression constructs optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0115] To obtain high levels of expression of a cloned nucleic acid it is common to construct expression constructs which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription / translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) operon. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing the polypeptide are available using, for example, E. coli, Bacillus sp. and Salmonella. E. coli systems may also be used. Transducing cells with nucleic acids (e.g., expression constructs) can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells within the host range of the vector. In certain embodiments, upon delivery of an expression construct to cells, one or more of the expression constructs are episomal (e.g., extra-chromosomal), where by “episome” or “episomal” is meant a polynucleotide that replicates independently of the cell’s chromosomal DNA. A non-limiting example of an episome that may be employed is a plasmid.

[0116] According to some embodiments, upon delivery of an expression construct to cells, the expression construct integrates into the genome of the cell. In certain embodiments, the expression construct is adapted for site-specific integration into the genome. For example, an expression construct may be adapted for site-specific integration into the genome, where the sitespecific integration inactivates a target gene within the genome of the cell. Functional integration of an expression construct may be achieved through various means, including through the use of integrating vectors, including viral and non-viral vectors. In some instances, a retroviral vector, e.g., a lentiviral vector, may be employed. In some instances, a non-retroviral integrating vector may be employed. An integrating vector may be contacted with the cells in a suitable transduction medium, at a suitable concentration (or multiplicity of infection), and for a suitable time for the vector to infect the target cells, facilitating functional integration of the expression construct. Nonlimiting examples of useful viral vectors include retroviral vectors, lentiviral vectors, adenoviral (Ad) vectors, adeno-associated virus (AAV) vectors, hybrid Ad-AAV vector systems, and the like.

[0117] Also provided by the present disclosure are messenger RNAs (mRNAs) encoding any of the polypeptides of the present disclosure. In some instances, the mRNAs are encapsulated within particles, e.g., lipid nanoparticles. Such mRNAs and particles find use, e.g., for expression of a polypeptide in vivo (e.g., for switchable binding in a subject for therapeutic purposes) upon delivery of the mRNA to cells of a subject. Strategies for delivering mRNA-based therapeutics are described, e.g., in Kowalski et al. (2019) Mol Then 27(4)710-728; Hou et al. (2021 ) Nature Reviews Materials 6:1078-1094; Metkar, Mihir, Christopher S. Pepin, and Melissa J. Moore. "Tailor made: the art of therapeutic mRNA design." Nature Reviews Drug Discovery 23.1 (2024): 67-83; and Zhang, He, et al. "Algorithm for optimized mRNA design improves stability and immunogenicity." Nature 621 .7978 (2023): 396-403.

[0118] Cells

[0119] Aspects of the present disclosure further include cells comprising a nucleic acid of the present disclosure, as well as cells comprising an expression construct of the present disclosure. In certain embodiments, the cells are prokaryotic cells (e.g., bacteria), a yeast cells, insect (e.g, drosophila) cells, amphibian (e.g., frog, e.g., Xenopus) cells, plant cells, etc. According to some embodiments, the cells are mammalian cells. Mammalian cells of interest include human cells, rodent cells, and the like.

[0120] In some embodiments, the cells are immune cells. Non-limiting examples of immune cells include T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In certain embodiments, the cells are T cells. When the immune cells comprise T cells, the T cells may comprise one or any combination of naive T cells (TN), cytotoxic T cells (TCTL), memory T cells (TMEM), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), tissue resident memory T cells (TRM), effector T cells (TEFF), regulatory T cells (TREGS), helper T cells, CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha beta T cells (Tap), gamma delta T cells (TYB).

[0121] According to some embodiments, the cells are stem cells, e.g., mammalian (e.g., human) stem cells. For example, the population of cells may comprise embryonic stem (ES) cells, adult stem cells, hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), or any combination thereof.

[0122] Approaches for introducing the nucleic acid or expression construct into cells of interest are known and may include contacting a population of cells with the nucleic acid or expression construct under conditions in which the nucleic acid or expression construct is delivered to cells of the population of cells. The contacting step may comprise contacting the population of cells with the nucleic acid or expression construct, e.g., by combining the cells and the nucleic acid or expression construct in a single mixture under conditions suitable for delivery (e.g., transfection, transduction, etc.) of the nucleic acid or expression construct into cells of the population of cells.

[0123] A variety of suitable approaches and conditions for the delivery of nucleic acids and expression constructs to cells are known. According to some embodiments, delivery is carried out by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, DEAE-dextran-mediated transfer, and / or the like. In certain embodiments, the nucleic acid or expression construct is introduced into cells of the population of cells by AAV transduction. The AAV vector may comprise ITRs from AAV2, and a serotype from any one of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV 10. According to some embodiments, the AAV vector comprises ITRs from AAV2 and a serotype from AAV6. In certain embodiments, the nucleic acid or expression construct is introduced into the cells (e.g., T cells) by lentiviral or retroviral transduction. The lentiviral vector backbone may be derived from HIV-1 , HIV-2, visna- maedi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (Fl V), bovine immune deficiency virus (BIV), or simian immunodeficiency virus (SIV). The lentiviral vector may be integration competent or an integrase deficient lentiviral vector (TDLV). In one embodiment, IDLV vectors including an HIV-based vector backbone (i.e. , HIV cis-acting sequence elements) are employed.

[0124] In certain embodiments, a cell of the present disclosure expresses the polypeptide. In some instances, the polypeptide is an intracellular polypeptide. The intracellular may be soluble (e.g., present in the cytoplasm or the lumen of an organelle) or insoluble, e.g., associated with a membrane / envelope of an organelle.

[0125] According to some embodiments, a cell of the present disclosure expresses the polypeptide on the surface of the cell. By way of example, in some instances, the polypeptide is an extracellular antigen binding domain of an antigen receptor. Non-limiting examples of antigen receptors include a chimeric antigen receptor (CAR), a T cell receptor (TCR) such as a recombinant TCR, a synthetic notch receptor (synNotch), a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, or a generalized extracellular molecule sensor (GEMS) receptor.

[0126] As used herein, with respect to a protein, the term “recombinant” means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein, and cells or organisms that express the protein. With respect to a nucleic acid, the term “recombinant” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant.

[0127] In some embodiments, when the polypeptide is an extracellular antigen binding domain of an antigen receptor (e.g., a CAR), the polypeptide switchably binds to a tumor antigen. Nonlimiting examples of tumor antigens to which the polypeptide may switchably bind include 5T4, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1 , delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), EpCAM, ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvlll, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1 ), GLUT3, glycoprotein non-metastatic B (GPNMB), guanylate cyclase 2 C (GUCY2C), HCAM, human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Integrin alpha, lysosomal-associated membrane protein 1 (LAMP-1 ), Lewis Y, LIV-1 , leucine rich repeat containing 15 (LRRC15), mesothelin (MSLN), sodium-dependent phosphate transport protein 2B (NaPi2b), Nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT like family member 6 (SLITRK6), STEAP family member 1 (STEAP1 ), tissue factor (TF), T cell immunoglobulin and mucin protein-1 (TIM-1), trophoblast cell-surface antigen (TROP-2), and VEGF-A.

[0128] When a cell of the present disclosure expresses an antigen receptor (e.g., a CAR) comprising a polypeptide of the present disclosure as the extracellular antigen binding domain of the CAR, the antigen binding domain of the receptor (e.g., CAR) may be followed by one or more spacer domains that moves the antigen binding domain away from the cell surface expressing the receptor to enable proper cell / cell contact, antigen binding and / or activation. The spacer domain (and any other spacer domains, linkers, and / or the like described herein) may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In some embodiments, the spacer domain includes the CH2 and / or CH3 of lgG1 , lgG4, or IgD. Illustrative spacer domains suitable for use in the receptors (e.g., CARs) described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a and CD4, which may be wild-type hinge regions from these molecules or variants thereof. In certain embodiments, the hinge domain includes a CD8a hinge region. According to some embodiments, the hinge is a PD-1 hinge or CD152 hinge. In certain embodiments, the hinge is an lgG4 hinge.

[0129] The “transmembrane domain” (Tm domain) is the portion of the receptor (e.g., CAR) that fuses the extracellular binding portion and intracellular signaling domain and anchors the receptor to the plasma membrane of the cell (e.g., T-cell, NK cell or the like). The Tm domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, the Tm domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of the T-cell receptor, CD35, CD3 , CD3y, CD30, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1.

[0130] In one embodiment, a receptor (e.g., CAR) includes a Tm domain derived from CD28. In certain embodiments, a receptor includes a Tm domain derived from CD28 and a short oligo- or polypeptide linker, e.g., between 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the Tm domain and the intracellular signaling domain of the receptor. A glycine-serine linker may be employed as such a linker, for example.

[0131] The “intracellular signaling” domain of a receptor (e.g., a CAR) refers to the part of the receptor that participates in transducing the signal from binding to a target molecule / antigen into the interior of the cell to elicit cell function. Accordingly, the term “intracellular signaling domain” refers to the portion of a protein which transduces the signal and that directs the cell to perform a specialized function. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of a full-length intracellular signaling domain as long as it transduces the signal. The term intracellular signaling domain is meant to include any truncated portion of an intracellular signaling domain sufficient for transducing signal. The polypeptide of the precent disclosure may in some embodiments be fused to or bind to the intracellular signaling domain thereby allowing switchable control of intracellular signaling. Signals generated through the T cell receptor (TCR) alone are insufficient for full activation of the T cell, and a secondary or costimulatory signal is also required. Thus, T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR / CD3 complex) and costimulatory signaling domains that act in an antigen-independent manner to provide a secondary or costimulatory signal. As such, a receptor (e.g., CAR) expressed by a genetically modified cell may include an intracellular signaling domain that includes one or more (e.g., 1 , 2, or more) “costimulatory signaling domains” and a “primary signaling domain.”

[0132] Primary signaling domains regulate primary activation of the TCR complex either in a stimulatory manner, or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”). Non-limiting examples of ITAM-containing primary signaling domains suitable for use in a receptor of the present disclosure include those derived from FcRy, FcRp, CD3y, CD35, CD3E, CD3^, CD22, CD79a, CD79p, and CD665. In certain embodiments, a receptor includes a CD3^ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain.

[0133] In some embodiments, the receptor (e.g., CAR) includes one or more costimulatory signaling domains to enhance the efficacy and expansion of immune effector cells (e.g., T cells) expressing the receptor. As used herein, the term “costimulatory signaling domain” or “costimulatory domain” refers to an intracellular signaling domain of a costimulatory molecule or an active fragment thereof. Example costimulatory molecules suitable for use in receptors contemplated in particular embodiments include TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1 BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70. In some embodiments, the receptor (e.g., CAR) includes one or more costimulatory signaling domains selected from the group consisting of 4-1 BB (CD137), CD28, and CD134, and a CD3^ primary signaling domain.

[0134] A receptor (e.g., CAR) expressed by a cell genetically modified according to the methods of the present disclosure may include any variety of suitable domains including but not limited to a leader sequence; hinge, spacer and / or linker domain(s); transmembrane domain(s); costimulatory domain(s); signaling domain(s) (e.g., CD3^ domain(s)); ribosomal skip element(s); restriction enzyme sequence(s); reporter protein domains; and / or the like.

[0135] The cells of the present disclosure (e.g., T cells) may be expanded during a therapeutic cell manufacturing process. By “expanding” or “expanded” is meant the cells are cultured under conditions in which the cells proliferate. Suitable conditions may vary depending upon, e.g., the type of cells (e.g., the type of T cells) being expanded. Such conditions may include culturing the cells (e.g., T cells) in a suitable container (e.g., a cell culture plate or well thereof, a cassette, tube, bottle or bag suitable for use in an automated therapeutic cell manufacturing system, e.g., a closed automated therapeutic cell manufacturing system such as the CliniMACS Prodigy® system by Miltenyi Biotec, the Xuri® cell expansion system by Cytiva, the G-Rex® cell expansion system by Wilson Wolf, the Quantum® cell expansion system from Terumo, the Cocoon® system by Lonza, or the like), in suitable medium (e.g., cell culture medium, such as RPMI, DMEM, IMDM, MEM, DMEM / F-12, or the like) at a suitable temperature (e.g., 32°C - 42°C, such as 37°C) and pH (e.g., pH 7.0 - 7.7, such as pH 7.4) in an environment having a suitable percentage of CO2, e.g., 3% to 10%, such as 5%.

[0136] Methods for activating and expanding cells for therapy (e.g., therapeutic T cells and the like) are known in the art and are described, e.g., in U.S. Patent Nos. 6,905,874; 6,867,041 ; and 6,797,514; and PCT Publication No. WO 2012 / 079000, the contents of which are hereby incorporated by reference in their entirety. In the example of T cells, such methods may include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other embodiments, the T cells are activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012 / 129514, the contents of which are hereby incorporated by reference in their entirety.

[0137] In certain embodiments, cells of the present disclosure are expanded using an automated system designed for the manufacture of therapeutic cells. Non-limiting examples of such systems include the CliniMACS Prodigy® system by Miltenyi Biotec, the Xuri® cell expansion system by Cytiva, the G-Rex® cell expansion system by Wilson Wolf, the Quantum® cell expansion system from Terumo, the Cocoon® system by Lonza, etc. Detailed guidance and protocols for manufacturing therapeutic cells on such systems are available from the providers of such systems.

[0138] Compositions

[0139] Also provided by the present disclosure are compositions. According to some embodiments, provided are compositions comprising any of the polypeptides, nucleic acids, expression constructs, and / or cells of the present disclosure.

[0140] Such compositions may comprise the polypeptides, nucleic acids, expression constructs, and / or cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCL, KCI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions. In certain embodiments, the liquid medium is a cell culture medium. Non-limiting examples of cell culture media include Minimal Essential Media, DMEM, a-MEM, RPMI Media, Clicks, F-12, X-Vivo 15, X-Vivo 20, Optimizer, and the like.

[0141] In certain embodiments, provided are compositions comprising polypeptides or cells of the present disclosure, wherein the compositions are suitable for administration to a subject, e.g., a human subject. Such compositions may comprise the polypeptides or cells and a pharmaceutically acceptable carrier. The compositions generally include a therapeutically effective amount of the polypeptides or cells. By “therapeutically effective amount” is meant an amount sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease (e.g., cancer) or disorder associated, e.g., with a target cell or a population thereof (e.g., cancer cells), as compared to a control. An effective amount can be administered in one or more administrations. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject, e.g., a human subject. When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual). In some embodiments, a composition of the present disclosure includes from 1 x106to 5x1010of the cells of the present disclosure.

[0142] The polypeptides or cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. For example, the cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents. Formulations of the polypeptides or cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0143] The polypeptides or cells may be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration. An aqueous formulation of the polypeptides or cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0144] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0145] A surfactant may also be added to the formulation to reduce aggregation and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w / v.

[0146] In some embodiments, the composition comprises polypeptides or cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v).

[0147] METHODS OF USE

[0148] Aspects of the present disclosure further include methods of using the polypeptides and cells of the present disclosure. The methods utilize the switchability of the polypeptides of the present disclosure for regulatable binding of the polypeptides to targets of interest. Non-limiting inducible and repressible formats for regulatable binding are described elsewhere herein and schematically illustrated in FIG. 1 .

[0149] In certain embodiments, methods of photoswitchably binding a target molecule are provided, the methods employing a polypeptide for which binding to the target of interest is photoinducible. In some instances, the methods comprise combining the target molecule and a photoinducible polypeptide of the present disclosure (that is, in which the caging domain pair comprises photodissociable domains), where the paratope binds the target molecule when uncaged. Such methods further comprise irradiating the polypeptide with light sufficient to dissociate the caging domain pair when binding of the polypeptide to the target molecule is desired. The light with which the polypeptide is irradiated to induce target binding is selected to match the action spectrum of photodissociation of the photodissociable domains. In some instances, the irradiating is local irradiation to dissociate the caging domain pairs of only a subset of a population of the polypeptides. According to some embodiments, the methods further comprise ceasing the irradiating when binding of the polypeptide to the target molecule is no longer desired. Alternatively, or additionally, the methods may further comprise irradiating the polypeptide with sufficient light of a different wavelength to associate the caging domain pair when binding of the polypeptide to the target molecule is no longer desired.

[0150] According to some embodiments, methods of photoswitchably binding a target molecule are provided, the methods employing a polypeptide for which binding to the target of interest is photorepressible. For example, provided are methods comprising combining the target molecule and a photorepressible polypeptide of the present disclosure (that is, in which the caging domain pair comprises photoassociable domains), where the paratope binds the target molecule when uncaged. Such methods further comprise irradiating the polypeptide with light sufficient to associate the caging domain pair when binding of the polypeptide to the target molecule is not desired. According to some embodiments, the irradiating is local irradiation to associate the caging domain pairs of only a subset of a population of the polypeptides. In certain embodiments, the methods further comprise ceasing the irradiating when binding of the polypeptide to the target molecule is desired. Alternatively, or additionally, the methods may further comprise irradiating the polypeptide with sufficient light of another wavelength to dissociate the caging domain pair when binding of the polypeptide to the target molecule is desired.

[0151] In certain embodiments, methods of chemoswitchably binding a target molecule are provided, the methods employing a polypeptide for which binding to the target of interest is chemoinducible. In some instances, the methods comprise combining the target molecule and a chemoinducible polypeptide of the present disclosure (that is, in which the caging domain pair comprises chemodissociable domains), where the paratope binds the target molecule when uncaged. Such methods further comprise contacting the polypeptide with a compound that dissociates the caging domain pair when binding of the polypeptide to the target molecule is desired. In some instances, the methods further comprise ceasing the contacting of the polypeptide with the compound when binding of the polypeptide to the target molecule is no longer desired.

[0152] According to some embodiments, methods of chemoswitchably binding a target molecule are provided, the methods employing a polypeptide for which binding to the target of interest is chemorepressible. For example, provided are methods comprising combining the target molecule and a chemorepressible polypeptide of the present disclosure (that is, in which the caging domain pair comprises chemoassociable domains), where the paratope binds the target molecule when uncaged. The methods further comprise contacting the polypeptide with a compound that associates the caging domain pair when binding of the polypeptide to the target molecule is not desired. In some instances, the methods further comprise ceasing the contacting of the polypeptide with the compound when binding of the polypeptide to the target molecule is desired.

[0153] Any of the above-described methods may be performed in vitro, in vivo, or ex vivo. In some instances, when the methods are performed in vitro, the combining step occurs in a cell- free environment, e.g., in a cell-free liquid medium present in a tube, well, channel, or the like. According to some embodiments, when the methods are performed in vitro, the combining step occurs in a living cell (e.g., present in a tube, well, channel, or the like), where the cell expresses the polypeptide and comprises a cellular target to which switchable binding by the polypeptide is desired. Cellular targets (e.g., endogenous cellular targets) of interest include membrane- associated proteins, soluble proteins (e.g., soluble signaling proteins), etc.

[0154] In certain embodiments, the methods are performed in vivo. Such in vivo methods may comprise administering to a subject in need thereof the polypeptide or a population of cells expressing the polypeptide for photoswitchable or chemoswitchable binding of the polypeptide to an endogenous target molecule in the subject. According to the methods, photoswitchable or chemoswitchable binding is achieved by further administration to the subject of light or a compound sufficient for photoswitchable or chemoswitchable binding, respectively, of the polypeptide to the endogenous target molecule in the subject. As will be appreciated with the benefit of the present disclosure, temporal control of the binding of the polypeptide to the endogenous target is advantageous, e.g., for controlling the level of binding of the polypeptide to the endogenous target according to the response of the subject. Such control may be used, e.g., to increase potency or reduce side-effect on-demand in combination with real-time monitoring of the response of the subject to the treatment.

[0155] Photoswitching binding polypeptides of the present disclosure may be dissociated / associated in vivo by the application of light at a responsive wavelength of the selected photoswitchable caging partners by implanted or externally-introduced light sources, such as implantable LEDs, fiber-optic LED catheters, and other in vivo light sources, for example, as described in Mondello et al. (2021 ) A micro-LED implant and technique for optogenetic stimulation of the rat spinal cord, Exp. Neur. 335: 1 13480; Izadi et al, Wide Area Uniform Illumination Scheme Using LED Matrix for Optogenetic Cardiac Pacing; Photonics 8: 499; and Rajalingham et al., (2021 ) Chronically implantable LED arrays for behavioral optogenetics in primates; Nature Methods 18:1 1 12-1 1 16.

[0156] In some instances, when the methods are performed in vivo, the switchable binding is site-specific in the subject. For example, in the case of photoswitchable binding, only a particular site of the subject (e.g., tissue, compartment, or the like) is irradiated, thereby achieving photoswitchable binding at the particular location. As will be appreciated with the benefit of the present disclosure, site-specific binding of the polypeptide to the endogenous target is advantageous, e.g., for targeted binding of the polypeptide to the endogenous target at the desired site, where systemic (non-targeted) binding could result in undesirable side effects.

[0157] According to some embodiments, the in vivo methods comprise administering the polypeptide to the subject. As will also be appreciated with the benefit of the present disclosure, the administered polypeptide may include an “off switch” (photorepressible or chemorepressible so that the polypeptide no longer binds the target molecule), which is advantageous in the event of adverse side effects resulting from binding of the polypeptide to the target molecule in the subject. As will also be appreciated with the benefit of the present disclosure, the administered polypeptide may alternatively include an “on switch” (photoinducible or chemoinducible so that the polypeptide initiates binding to the target molecule), which is advantageous for testing increasing active levels of the polypeptide while for example monitoring for adverse side effects resulting from binding of the polypeptide to the target molecule in the subject. This may for example be used as way to control the dosing of polypeptides with long half-lives. In some embodiments, the “on switch” and “off switch” modalities may also be used to control the half-life of the polypeptide, fusion protein, or conjugate. This may, for example, be achieved, if the photoswitchable or chemoswitchable binder contains a serum albumin binding SBD.

[0158] In certain embodiments, the in vivo methods comprise administering a population of cells expressing the polypeptide (e.g., on the surface thereof) to the subject. For example, the present disclosure provides methods of administering a cell therapy to a subject in need thereof. “Cell therapy” refers to the transfer of autologous or allogeneic cellular material into a subject for medical purposes. In some instances, the cells are therapeutic T cells or therapeutic NK cells, e.g., CAR-T cells or CAR-NK cells in which the extracellular antigen binding domain of the CAR is a switchable polypeptide of the present disclosure, enabling regulatable binding of the cells to the target antigen.

[0159] The constant antigenic burden during chronic viral infections, cancer, and the like, causes CD8+T cells to develop a state of dysfunction often referred to as T cell exhaustion, characterized by a loss of some effector functions and up-regulation of multiple inhibitory receptors. The result is a population of antigen-specific CD8+T cells that are unable to successfully clear or stabilize an infection or tumor. CAR-T cells also develop exhaustion, e.g., from tonic signaling or persistent antigen stimulation. As will be appreciated with the benefit of the present disclosure, the therapeutic cells (e.g., CAR-T cells) of the present disclosure comprising the switchable polypeptides of the present disclosure as the antigen-binding domain may be regulated (e.g., photo- or chemically regulated) to prevent or reduce exhaustion of the CAR-T cells, thus providing a novel approach for enhancing the efficacy of cell therapies such as CAR-T cell therapy. As will also be appreciated with the benefit of the present disclosure, the therapeutic cells (e.g., CAR-T cells) of the present disclosure include an “on switch” (photoinducible or chemoinducible so that the receptor initiates binding the antigen) or “off switch” (photorepressible or chemorepressible so that the receptor no longer binds the antigen), which may be advantageous to proactively or acutely control the level of active CAR-T cells prior to after the onset of circumstances such as cytokine storm or cytokine release syndrome as an adverse event of the cell therapy. Accordingly, provided by the present disclosure are methods of administering a cell therapy in which binding of the receptor to the target antigen is switched on or off (temporarily (e.g., intermittently) or permanently) to control exhaustion of the therapeutic cells (i.e. therapy performance) and / or to control the activity of the therapeutic cells to proactively avoid or acutely address a cytokine storm or cytokine release syndrome (i.e. therapy safety).

[0160] In some instances, the in vivo methods comprise administering a virus particle, liposome or nanoparticle to the subject, e.g., any of the virus particles, liposomes or nanoparticles (e.g., comprising an mRNA encoding a polypeptide of the present disclosure) described herein. Details regarding viral-based delivery of therapeutic nucleic acids is found, e.g., in Bulcha et al. (2021 ) Viral vector platforms within the gene therapy landscape. Sig Transduct Target Ther 6, 53; and Li et al (2023) Viral Vector-Based Gene Therapy. Int. J. Mol. Sci. 24, 7736. Strategies for delivering mRNA-based therapeutics are described, e.g., in Kowalski et al. (2019) Mol Ther. 27(4):710-728; and Hou et al. (2021 ) Nature Reviews Materials 6:1078-1094.

[0161] The following examples are offered by way of illustration and not by way of limitation.

[0162] EXPERIMENTAL

[0163] Example 1 - A redesigned DARPin topology

[0164] General methods for spatiotemporal control of specific endogenous proteins would be broadly useful for probing protein function in living cells. Synthetic protein binders that bind and inhibit endogenous protein targets can be obtained from nanobodies, designed ankyrin repeat proteins (DARPins), and other small protein scaffolds, but generalizable methods to control their binding activity are lacking. Reported in Examples 1 -6 herein are robust single-chain photoswitchable DARPins (psDARPins) for bidirectional optical control of endogenous proteins. Topological variants of the DARPin scaffold were created by computer-aided design so fusion of photodissociable dimeric Dronpa (pdDronpa) results in occlusion of target binding at baseline. Cyan light induces pdDronpa dissociation to expose the binding surface (paratope), while violet light restores pdDronpa dimerization and paratope caging. Since the DARPin redesign leaves the paratope intact, the approach was easily applied to existing DARPins for GFP, ERK, and Ras, as demonstrated by relocalizing GFP-family proteins and inhibiting endogenous ERK and Ras with optical control. Finally, a Ras-targeted psDARPin was used to determine that, following pulsed activation of EGFR, Ras is required for sustained EFGR to ERK signaling. In summary, psDARPins provide a generalizable strategy for precise spatiotemporal dissection of endogenous protein function, including those that lack direct pharmacological antagonists.

[0165] Photodissociable dimeric Dronpa (pdDronpa) has been used to control protein-protein interactions including enzymes that utilize protein substrates23 29. In these studies, two pdDronpa domains were attached at positions flanking an interaction site so that pdDronpa dimerization at baseline would cage the site. Upon illumination with cyan (-500 nm) light, pdDronpa switches from a fluorescent dimeric ON-state to a non-fluorescent monomeric OFF-state, thereby exposing the site for protein or substrate binding. The process is reversible, as pdDronpa can be redimerized by reverse photoswitching with violet (~400-nm) light or by slow (ti / 2~1 h) thermal relaxation25, resulting in protein recaging (Fig. 2a).

[0166] It was set out to apply this general strategy for the design of photoswitchable proteins to DARPins. DARPins were selected since they are stable, small, and disulfide-free a-helical repeat proteins that contain a single rigid and central paratope and fold robustly in fusion proteins14’1530. They can be selected to bind and inactivate protein targets of interest, and function in both reducing intracellular environments and oxidizing extracellular environments. Importantly, the concave paratope is flanked by constant N- and C-terminal cap regions for fusion of the photoswitchable domains. Finally, numerous DARPins have already been discovered and characterized against a diverse array of targets, and several DARPin-based therapies have entered clinical trials14 1530. Thus, it was expected that the successful discovery of a photoswitchable DARPins design would enable optical control over a variety of endogenous proteins.

[0167] Canonical DARPins are repeat proteins consisting of at least 1 , and typically 2-3, internal repeat units15containing the primary paratope, plus N- and C-cap (amino- and carboxy-terminal cap) units that seal and stabilize the scaffold by burying the hydrophobic edges of the internal repeat units (Fig. 2b)1531 32. In this canonical topology, the N- and C-terminal helixes of DARPins point in the same direction, but this geometry cannot support paratope caging by fusion to pdDronpa (Fig. 2b). To enable target occlusion, the DARPin scaffold was redesigned by extending the C-terminal cap with an additional a-helix, thus creating a topology with crossparatope placement of the termini allowing pdDronpa dimerization in front of the paratope (Fig. 2c). Importantly, the redesigned segments of the C-cap do not overlap with the diversified residues of DARPin libraries that are generally responsible for target binding. Thus, the new topology is not expected to affect target recognition. It was hypothesized that this extended DARPin (exDARPin) scaffold would allow paratope caging by pdDronpa and modular combination with a large set of existing and future DARPins.15 Novel exDARPins were generated using computer-aided protein design. Starting from the crystal structures of a model DARPin, GFP-targeted 3G12433, RosettaRemodel34was used to generate the novel C-cap of the exDARPins with various loop and helix lengths. In solution, exDARPins had high thermal stability in differential scanning fluorimetry with Tm> 90 °C. C-cap unfolding marks the first event of DARPin denaturating35, and the data shows that the extended C-caps generally had higher thermal stability than previously optimized C-caps3536. Crystal structures were obtained for exDARPins with the designed C-cap inserted in either 3G124 or Off7, a maltose binding protein (MBP) targeted DARPin32. Two designs, ex13 and ex17 (Fig. 2d), were studied further. These differed in the positioning of the additional C-terminal helix with the ex17 C-terminus angled closer to the paratope.

[0168] Next, fusions of pdDronpa1.2 (pdD1.2) and exDARPin domains were computationally screened to predict optimal linker lengths for paratope caging using RosettaRemodel37. To define the various tested fusions, the exDARPins are referred to by the version of their C-cap variant, for example, the exDARPin based on DARPin 3G124 combined with ex13 is 3G124.ex13. Finally, when pdD1 .2 is fused to both the N- and C-termini of an exDARPin, a ps- prefix is added to denote photoswitchability, e.g. ps3G124.ex13. RosettaRemodel was used to identify linker lengths that would allow pdDronpal .2 to dimerize in the intended location in front of the paratope. These predictions were used to design the constructs for experimental validation of the psDARPin architecture (Fig. 2e).

[0169] Example 2 - Intracellular validation of the psDARPin architecture

[0170] To test the intracellular function of the psDARPins, a live-cell nucleus-cytosol partition assay was developed using the cyan fluorescent protein mTurquoise2 (mTurq2) as a model target (Fig. 3a). As mTurq2 is derived from GFP3839, it was expected the anti-GFP DARPin 3G12440fused to a nuclear export sequence (NES) could bind mTurq2 and sequester it in the cytosol. To establish the baseline behavior of the assay, a negative binding control psOff7.exO- NES was prepared, which does not target mTurq2 but that contains pdDronpal .2 domains, and a positive binding control 3G124.exO-NES. Here exO refers to the canonical C-cap. When expressed in HeLa cells, 3G124.exO-NES recruited mTurq2 to the cytosol, while mTurq2 was homogenously distributed in the cells expressing psOff7.exO (Fig. 3b). These controls demonstrate that mTurq2 is not recruited to the cytosol by interactions with either a random DARPin domain or pdD1 .2 itself, and establish cytosol-to-nuclear (C / N) fluorescence ratios of ~ 4.8 for full mTurq2 sequestration and ~1 .0 for no mTurq2 binding (Fig. 3c).

[0171] Tested next was the candidate photoswitchable DARPins based on 3G124.ex13 and 3G124.ex17 with computationally suggested linker lengths for caging efficiency in the dark (Fig. 3b). The green pdDronpa fluorescence of NES-fused ps3G124 variants was localized to the cytosol, and mTurq2 fluorescence was homogenously distributed throughout the cell. With mTurq2 C / N values of 0.97-1 .47 all tested linker variants showed caging and target binding occlusion. In contrast, ps3G124.exO, which lacks the extended C-cap, was not efficiently caged by pdDronpa1.2, producing a C / N ratio of 3.2 (Fig. 3c). Thus, as expected, the exDARPin topology is crucial for supporting paratope caging. For both ps3G124.ex13 and ps3G124.ex17, the 7,5 linker combination performed the best with C / N ratios of 0.97 and 1 .28, respectively. For subsequent experiments this 7,5 linker combination was used. Further analysis showed that ps3G124.ex17 variants were efficiently caged in cells even at increasing target or psDARPin levels, while the caging ability of ps3G124.ex13 variants decreased slightly with increasing protein levels.

[0172] Next, to validate that the fusion of pdDronpa did not in itself interfere with mTurq2 binding, the monomeric pdDronpal .2 N145K 1158V mutant25was fused to 3G124.ex13 and 3G124.ex17 to open the pdDronpa cage under dark basal conditions. The open state variants of ps3G124.ex13 and ps3G124.ex17 efficiently bound mTurq2 with C / N ratios of ~4.8 matching the positive binding control.

[0173] Finally, tested was whether the psDARPins could be bidirectionally and reversibility controlled with light. Focusing on ps3G124.ex17 that had showed slightly better dark-state caging than ps3G124.ex13 (Fig. 3c), this construct was tested for bidirectional photocontrol of target binding (Fig. 3d). Cyan 488 nm light cause photoswitching of pdDronpal .2 into its non- fluorescent OFF-state followed by accumulation of mTurq2 in the cytosol indicating successful photoactivation of target binding (Fig. 3e). In contrast, violet 405 nm light cause reverse photoswitching of pdDronpa into its fluorescent ON-state and movement of mTurq2 back into the nucleus. Repeating the procedure saw mTurq2 shuttling back-and-forth between the cytosol and the nucleus and the C / N ratio increasing and decreasing, respectively (Fig. 3f). Thus, cyan and violet light reversibly activates and deactivates target binding of the psDARPins.

[0174] Example 3 - Subcellular target recruitment

[0175] By anchoring psDARPins at organelle membranes, subcellular accumulation of the target can be performed for proximity induced reactions or to sequester it away from the native site of function. This mechanism of action can be used to regulate DARPin targets when binding itself does not inhibit target function. To test this idea, ps3G124.ex17 was fused to the outer mitochondrial membrane or the plasma membrane (Fig. 12). The psDARPins labeled the organelles with the expected localization pattern. In the dark, mTurq2 was homogenously distributed in the cells indicating robust caging. Following cyan light uncaging of the psDARPins, mTurq2 was enriched at the location of the psDARPin (Fig. 12). Upon illumination with violet light, mTurq2 was released to the cytosol (Fig. 12), confirming reversible bidirectional photocontrol with the psDARPins. These experiments show that a subcellularly anchored psDARPins can mediate photoinduced relocalization of a target protein. In this photoinducible psDARPin, the target protein is unbound before illumination, assuring normal distribution and function. Example 4 - Regulating endogenous kinase activity

[0176] The preceding experiments used mTurq2 as a convenient exogenous target to validate the psDARPin function. However, the major feature of psDARPins would be the ability to directly control endogenous proteins. To demonstrate control of endogenous signaling, the initial focus was on ERK1 / 2 of Ras-Raf-MEK-ERK pathway (Fig. 4a, hereafter the ERK and Ras-ERK pathway) that is involved in regulating cell differentiation and proliferation under both normal and pathological conditions41 44.

[0177] Previous studies showed that the E40 DARPin binds to unphosphorylated ERK4546and prevents phosphorylation and activation by MEK45. However, as E40 had previously been shown only to prevent phosphorylation of chimeric exogenous ERK45, it was first established that E40 can inhibit endogenous ERK activity in a live-cell assay. As a real-time kinase activity reporter, mScarlet-labelled ERK-KTR47which moves from the nucleus to the cytosol upon phosphorylation by active ERK (i.e., pERK, Fig. 4a), was used. Thus, the cytosol-to-nuclear (C / N) mScarlet fluorescence ratio increases with ERK activity47. When expressed in Ras-ERK hyperactive human glioma U87-EGFRvlll (U87E, expressing the constitutively active EGFR variant III48) cells together with the non-binding Off7.exO control, the ERK-KTR was predominantly localized in the cytosol with a C / N ratio of 2.72 (Fig. 4b). In contrast, the C / N ratio was 0.42 in E40.ex0 expressing cells (Fig. 4b), indicating that E40 is indeed capable of inhibiting activation of endogenous ERK.

[0178] To design a photoswitchable ERK-inhibiting DARPin, the paratope-containing internal repeats of 3G124.ex13 and 3G124.ex17 were exchanged with the homologous region of E40. Assessed next was how well the resulting psE40.ex13 and psE40.ex17 were caged in the dark, or were activated by light. In U87E cells, non-illuminated psE40.ex13- and psE40.ex17- expressing cells exhibited C / N values of 2.65 and 2.26, respectively (Fig. 4b). The C / N ratio for nonilluminated psE40.ex13 is similar to that of Off7.exO negative control cells, indicating effective caging of the paratope in this design. After illumination of psE40.ex13, the C / N ratio dropped to 0.42 at 100 min post illumination (Fig. 4c). This is comparable to E40.ex0 (Fig. 4b) and suggests efficient uncaging of the paratope and preservation of binding activity given enough time for pERK dephosphorylation. Finally, illumination with violet light led to an increase in Erk activity confirming that the photoactivated inhibition was reversible.

[0179] Example 5 - Regulating endogenous wild-type RAS

[0180] Ras GTPases are notoriously difficult to inhibit using small molecules42,49. With a few exceptions5052, recent progress primarily stems from the design of inhibitors targeting specific Ras mutants42,49, and few pan-isoform inhibitors of WT Ras has been reported50,51. Thus, there is a shortage of specific and potent probes for acute inhibition of endogenous WT Ras. SBDs often have larger binding surface areas than small molecule inhibitors, which can facilitate the inhibition of proteins with shallow pockets and flat surfaces. Indeed, a range of low nM affinity WT Ras inhibiting monobodies and DARPins have been reported53 56. The focus turned to DARPin K27 that binds all Ras isoforms (i.e., HRas, KRas, and NRas) in the inactive GDP-bound state and blocks interactions with SOS and RAF, thereby inhibiting RAS activation and subsequent signaling5457.

[0181] K27 contains three internal repeat units allowing modular replacement in the already validated psDARPin designs creating psK27.ex13 and psK27.ex17. Since ERK is activated downstream of Ras (Fig. 4a), co-expression with ERK-KTR (Fig. 4a) in U87E cells was also used to monitor Ras activity. Expression of K27.ex0 established a C / N peak of 0.38 for constitutively inhibited Ras (Fig. 4b). In the dark, psK27.ex13 (C / N 2.49) and psK27.ex17 (C / N 2.68) both match the C / N value of the non-binding Off7.exO control indicating efficient caging (Fig. 4b). Constitutively open variants of psK27.ex13 and psK27.ex17 efficiently bound and inhibited Ras with C / N ratios similar to K27.ex0 indicating the new caps did not interfere with binding to Ras (Fig. 4b). Light-initiated uncaging of the anti-Ras psDARPins resulted in gradual decreasing C / N values. At 100 min post-illumination, psK27.ex17 reached a C / N value of 0.37 matching the K27.ex0 control (Fig. 4d). Within the experimental error, the C / N ratio decayed at the same rate following Ras inhibition with psK27.ex17 or ERK inhibition with psE40.ex13 indicating that the rate determining step of the assay is likely ERK or ERK-KTR dephosphorylation in the U87E cells.

[0182] Example 6 - Ras is required for sustained EGFR to ERK signaling

[0183] Finally, psK27.ex17 was used to investigate the function of endogenous Ras at specific steps in cellular signaling. Following stimulation with EGF, dimerization dependent activation of EGFR at the plasma membrane leads to a transient burst of Ras-ERK activity. Following the initial burst, endocytosis and deactivation of ligand-bound EGFR and negative feedback in the Ras-ERK signaling pathway leads to a down-regulation of ERK activity for a sustained low level (~5-10% of maximum burst activity)58,59, referred to herein as the late phase signaling. Recent studies with endogenous levels of Ras expression suggested that late phase ERK activity is sustained by a small fraction of active EGFR residing at the plasma membrane triggering low but sustained levels of Ras activity58,59, however, unequivocal evidence for the role of Ras activity in late phase ERK signaling has not been reported. Untagged Ras signaling can be study by observing Ras activation via biosensors60,61, performing RAF-mediated RasGTP pull-downs62, chemogenetic or optogenetic activation of Ras37,63 65, or chronically depleting it, but the undruggable nature of wild-type endogenous Ras has previously made temporally precise loss- of-function experiments unachievable. The unique ability of psK27.ex17 to inactivate endogenous Ras at precise times may finally allow us to directly address if Ras activity is required for late phase ERK signaling. Conveniently, the pan-isoform RAS inhibiting K27 DARPin avoids the known complication of compensatory induction of Ras-ERK signaling by Ras isoform upregulation56 69.

[0184] It was first confirmed that EGFR activation in HeLa cells has a distinct late phase. Application of 10 ng / mL labelled EGF to serum-starved HeLa cells was followed by clear internalization within 20 min (Fig. 5b), replicating earlier observations58,59. The dynamics of Ras- ERK signaling were then monitored in serum-starved HeLa cells using the ERK-KTR sensor. Serum-starved HeLa cells have low basal ERK activity supported by a C / N ratio of ~0.5 (Fig. 5c- d) relative to -2.7 for U87E cells (Fig. 4b-d). Cells transfected with GFP-binding ps3G124.ex17 were used to establish a negative control for non-specific effects of psDARPin expression and cyan light illumination. Following stimulation with 10 ng / mL EGF, the C / N ratio saw a transient increase peaking after 30 min (Fig. 5d) in accordance with earlier studies in serum-starved HeLa cells using a ERK-KTR reporter70. It should be stressed that the response of the ERK-KTR reported is expected to be slightly delayed relative to pERK levels59and ERK nuclear translocation71.

[0185] Next, the experiments were repeated in cells transfected with the photoswitchable Ras- binding DARPin psK27.ex17. Photoactivation of psK27.ex17-transfected cells caused an immediate decay in the C / N ratio that reached baseline level at -50 min (Fig. 5c-d). These results clearly indicate that Ras activity is required for late phase signaling from EGFR to ERK ruling out the possibility for alternative mechanisms of EGF-induced EGFR to ERK signaling in HeLa cells. Thus, no buffering capacity appears to present in the Raf-ERK pathway.

[0186] Example 7 - Chemoinducible csDARPins

[0187] Chemoinducible csBinders were created by fusing the homodimer FKBP(F36M) cage to the termini of an exDARPin (Fig. 7a). Upon administration of the small-molecule ligand Shieldl , the cage dissociates due to Shieldl binding to the caging subunits (Fig. 7a-b). A csDARPin construct with linker lengths at the N- and C-terminus of 7 and 5 amino acids, respectively, shows good caging of the extended DARPin ex3G124.ex17 since the mTurq2 target was distributed throughout the cell with a C / N ratio close to the Off.exO-based negative control (Fig. 7c-d). A linker length screen showed that linkers of various lengths were able to support FKBP(F36M)- mediated caging of the csDARPin (Fig. 7e) with C / N ratios lower than the positive binding control, FKBP(WT)-3G124.ex17-FKBP(WT) (Fig. 7d). The best performing construct in this experiment was FKBP(F36M)-3G124.d17-FKBP(F36M){7,5) (i.e. with the 7,5 linker combination). To reversible uncage and cage of FKBP(F36M)-based csDARPins, Shieldl can be added for uncaging (Fig. 7b) while the bivalent AP20187 (Fig. 8a) can be used for subsequent re-caging (Fig. 8a). After the addition of 10 nM Shield 1 , uncaging of FKBP(F36M)-3G124.d17- FKBP(F36M){7,5} was observed as recruitment of the mTurq2 target to the cytosol and an increase in the C / N ratio (Fig. 8b). The binding activity could subsequently be repressed by the addition of 0.5 pM AP20187 which led to decreasing C / N ratio (Fig. 8b). The caging domain FKBP(F36M) also allowed reversible ligand-gated uncaging and caging of the ERK-inhibiting E40.ex13 DARPin (Fig. 8c). Example 8 - Chemorepressible csDARPins

[0188] A chemorepressible csBinder can be created by fusing the FKBP(F36V) chemoassociable domain to the termini of an exDARPin (Fig. 9a). Upon administration of the ligand AP20187 (Fig. 8a), the cage is closed by bridging of the caging domains via the bivalent ligand (Fig. 9a). In the basal state the binding activity of the chemorepressible FKBP(F36V)- 3G124.d17-FKBP(F36V){7,5} is active leading to recruitment of mTurq2 to the cytosol (Fig. 9b). Upon addition of 10 nM AP20187, the cage is closed and target binding reduced as seen from the decreasing C / N ratio (Fig. 9b). The binding activity was reactivated by outcompeting AB20187 with the addition of 1 pM Shieldl (Fig. 9b). Similarly, the anti-ERK DARPin E40.ex13 also becomes chemorepressible by the fusion to FKBP(F36V) (Fig. 9c). At baseline, ERK is inactive as seen from the low C / N ratio indicating efficient paratope engagement with the target (Fig. 9c). The C / N value increases following the addition of AP20187 indicating ligand-induced repression of target binding and increase in kinase activity (Fig. 9c). The binding activity and ERK inhibition could be reinstated by the addition of Shieldl (Fig. 9c).

[0189] Example 9 - A photoinducible psaRep

[0190] Photoinducible psBinders can be created with a-helical repeat proteins containing a concave paratope and a cross-paratope topology of the N- and C-termini. A aRep scaffold was computationally redesigned by adding a a-helical extension at the N-terminus. By fusing the homodimer pdDronpa1.2 to the termini of an extended aRep.aex3 (Fig. 10a) a photoinducible psaRep was created. Like photoinducible ps3G124.ex13 or ps3G124.ex17, the GFP-targeted photoinducible psaRep based on bGFPC.aex3 could be used to reversibly bind to mTurq2. In the basal state, pdDronpal .2-aRep_bGFPC.aex3-pdDronpa1 .2{5,5} is caged and the C / N ratio ~1 (Fig. 10b). Illumination with cyan light results in uncaging a recruitment of mTurq2 to the cytosol and an increase in the C / N ratio.

[0191] Example 10 - A photoinducible psDARPin using RsLOV as the caging domain

[0192] Photoinducible psBinders can be prepared using photodissociable cages beyond pdDronpa. Fusing the homodimeric RsLOV cage to the extended anti-GFP DARPin 3G124.ex17 (Fig. 1 1 a) allows caging of the paratope in the basal state. Particularly, the RsLOV-3G124.ex17- RsLOV fusion with 7,5 amino acid long linkers show minimal mTurq2 binding with a C / N ratio of ~1 .1 in the basal state (Fig. 1 1 b).

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[0261] (2020).

[0262] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:1 . A polypeptide comprising an a-helical binding domain comprising a concave paratope, wherein the termini of the a-helical binding domain comprise a cross-paratope topology, and wherein the a-helical binding domain is flanked via flexible linkers by a caging domain pair switchable between an associated state in which the paratope is caged and a dissociated state in which the paratope is uncaged.

2. The polypeptide of claim 1 , wherein the a-helical binding domain comprises from 2 to 6 internal repeats.

3. The polypeptide of claim 2, wherein the a-helical binding domain comprises 3 internal repeats.

4. The polypeptide of any one of claim 1 to 3, wherein the a-helical binding domain is of an extended designed ankyrin repeat protein (exDARPin), an extended a repeat protein (exaRep), or a de novo designed concave a-helical repeat protein.

5. The polypeptide of claim 4, wherein the a-helical binding domain is an exDARPin.

6. The polypeptide of claim 4, wherein the a-helical binding domain is an exaRep.

7. The polypeptide of claim 4, wherein the a-helical binding domain is a de novo designed concave a-helical repeat protein.

8. The polypeptide of any one of claims 1 to 7, wherein the caging domain pair is photoswitchable between the associated state and the dissociated state.

9. The polypeptide of claim 8, wherein the polypeptide is photoinducible, and wherein the caging domain pair comprises photodissociable domains.

10. The polypeptide of claim 9, wherein the photodissociable domains are selected from the group consisting of: pdDronpa, UVR8, RsLOV, LOVTRAP, cpLOVTRAP, TtCBD, MxCBD, BICYCL-red, and photodissociable variants thereof.11 . The polypeptide of claim 8, wherein the polypeptide is photorepressible, and wherein the caging domain pair comprises photoassociable domains.

12. The polypeptide of claim 11 , wherein the photoassociable domains are selected from the group consisting of: Cry2, Cry2:CIB1 , ibid, cpLID, TULIP, VVD, Magnets, EL222, FKF1 :GI, VfAU1 -LOV, PtAU1 -LOV, YtvA, BICYCL-green, Cph1 , PhyA:FHY1 , PhyA:FHL, PhyB:PIF3, PhyB:PIF6, BphP1 , BphP1 :PpsR2, BphP1 :Q-PAS1 , MagRed, and photoassociable variants thereof.

13. The polypeptide of any one of claims 1 to 7, wherein the caging domain pair is chemoswitchable between the associated state and the dissociated state.

14. The polypeptide of claim 13, wherein the polypeptide is chemoinducible, and wherein the caging domain pair comprises chemodissociable domains.

15. The polypeptide of claim 14, wherein the chemodissociable domains are selected from the group consisting of: FKBP-F36M, FKBP-F36L, Bcl-XL:BH3, Bcl-XL:LD3, Bcl2:LD3, NS3a:ANR, TetRB:TIP, aMinoC1 -sdAb:CX7C, and chemodissociable variants thereof.

16. The polypeptide of claim 13, wherein the polypeptide is chemorepressible, and wherein the caging domain pair comprises chemoassociable domains.

17. The polypeptide of claim 16, wherein the chemoassociable domains are selected from the group consisting of: FKBP:FRB, iFKBP:FRB, FKBP, FKBP-F36V, GID1 :GAI, PYL:ABI, PYR:ABI, PYR:HAB1 , NS3a:GNCR1 , NS3a:DNCR2, NS3a:PRSIM_23, Bcl-XL:AZ1 , Bcl- XLAZ21 , aCaffVHH, COSMO, split-Lyn, split-DHFR, split-GR2, split-TR , hRBP4:RS3, hRBP4:RS5, hRBP4:RF2, CRBNJKZF3, CRBNA3:IKZF3, CRBNA3:ZFP91 -IKZF3, CEL:ZIF, GntR:GntR, CA-14:DB-18, CA-14:DB-21 , FIREtag:FIREmate, hcy129.1 :miniH1 1 ; split- buttressed-CHD_r1 ; VHHA52; split-mAb-Nic12; AR:MBP, and chemoassociable variants thereof.

18. The polypeptide of any one of claims 1 to 17 fused to a heterologous protein domain.

19. The polypeptide of claim 18, wherein the heterologous protein domain interacts with a target molecule different than a target molecule bound by the a-helical binding domain of the polypeptide.

20. The polypeptide of claim 19, wherein the heterologous protein domain is a second polypeptide of any one of claims 1 to 17.21 . A conjugate comprising the polypeptide of any one of claims 1 to 20 conjugated to an agent.

22. The conjugate of claim 21 , wherein the agent is chosen from a chemotherapeutic agent, a toxin, a radiation sensitizing agent, a radioactive isotope, a detectable label, and a half-life extending moiety.

23. A nucleic acid encoding the polypeptide of any one of claims 1 to 20.

24. The nucleic acid of claim 23, wherein the nucleic acid encodes the polypeptide fused to a second polypeptide.

25. An expression construct comprising the nucleic acid of claim 23 or claim 24 operably linked to a promoter.

26. A cell comprising the expression construct of claim 25.

27. The cell of claim 26, wherein the cell expresses the polypeptide.

28. The cell of claim 27, wherein the polypeptide is an intracellular polypeptide.

29. The cell of claim 28, wherein the polypeptide is a soluble intracellular polypeptide.

30. The cell of claim 27, wherein the polypeptide is expressed on the intracellular surface of the cell plasma membrane.31 . The cell of claim 27, wherein the polypeptide is expressed on the extracellular surface of the cell plasma membrane.

32. The cell of claim 31 , wherein the polypeptide is an extracellular antigen binding domain of an antigen receptor.

33. The cell of claim 32, wherein the antigen receptor is a chimeric antigen receptor (CAR).

34. The cell of claim 32, wherein the antigen receptor is a recombinant T cell receptor(TCR).

35. The cell of any one of claims 26 to 34, wherein the cell is an immune cell.

36. The cell of claim 35, wherein the cell is a T cell or a natural killer (NK) cell.

37. The cell of any one of claims 26 to 36, wherein the cell is a human cell.

38. A virus particle comprising the nucleic acid or expression construct of any one of claims23 to 25.

39. A liposome or a nanoparticle comprising the nucleic acid or expression construct of any one of claims 23 to 25.

40. The liposome or nanoparticle of claim 39, wherein the nucleic acid is a messenger RNA (mRNA) encoding the polypeptide.41 . The liposome or nanoparticle of claim 39, comprising an expression construct comprising the nucleic acid.

42. The liposome or nanoparticle of any one of claims 39 to 41 , wherein the nanoparticle is a lipid nanoparticle.

43. A method of photoswitchably binding a target molecule, the method comprising: combining the target molecule and the polypeptide of claim 9 or 10, wherein the paratope binds the target molecule when uncaged; and irradiating the polypeptide with light sufficient to dissociate the caging domain pair when binding of the polypeptide to the target molecule is desired.

44. The method according to claim 43, wherein the irradiating is local irradiation to dissociate the caging domain pairs of only a subset of a population of the polypeptides.

45. The method according to claim 43 or 44, further comprising ceasing the irradiating when binding of the polypeptide to the target molecule is no longer desired.

46. The method according to any one of claims 43 to 45, further comprising irradiating the polypeptide with sufficient light of another wavelength to associate the caging domain pair when binding of the polypeptide to the target molecule is no longer desired.

47. A method of photoswitchably binding a target molecule, the method comprising: combining the target molecule and the polypeptide of claim 11 or 12, wherein the paratope binds the target molecule when uncaged; andirradiating the polypeptide with light sufficient to associate the caging domain pair when binding of the polypeptide to the target molecule is not desired.

48. The method according to claim 47, wherein the irradiating is local irradiation to associate the caging domain pairs of only a subset of a population of the polypeptides.

49. The method according to claim 47 or 48, further comprising ceasing the irradiating when binding of the polypeptide to the target molecule is desired.

50. The method according to any one of claims 47 to 49, further comprising irradiating the polypeptide with sufficient light of another wavelength to dissociate the caging domain pair when binding of the polypeptide to the target molecule is desired.51 . A method of chemoswitchably binding a target molecule, the method comprising: combining the target molecule and the polypeptide of claim 14 or 15, wherein the paratope binds the target molecule when uncaged; and contacting the polypeptide with a compound that dissociates the caging domain pair when binding of the polypeptide to the target molecule is desired.

52. The method according to claim 51 , further comprising ceasing the contacting of the polypeptide with the compound when binding of the polypeptide to the target molecule is no longer desired.

53. A method of chemoswitchably binding a target molecule, the method comprising: combining the target molecule and the polypeptide of claim 16 or 17, wherein the paratope binds the target molecule when uncaged; and contacting the polypeptide with a compound that associates the caging domain pair when binding of the polypeptide to the target molecule is not desired.

54. The method according to claim 47, further comprising ceasing the contacting of the polypeptide with the compound when binding of the polypeptide to the target molecule is desired.

55. The method according to any one of claims 43 to 54, wherein the method is performed in vitro.

56. The method according to any one of claims 43 to 54, wherein the method is performed in vivo.

57. The method according to claim 56, wherein the method comprises administering to a subject in need thereof the polypeptide of any one of claims 1 to 20, a population of cells of any one of claims 27 to 37, the virus particle of claim 38, or the liposome or nanoparticle of any one of claims 39 to 42, for photoswitchable or chemoswitchable binding of the polypeptide to a target molecule in the subject, wherein the photoswitchable or chemoswitchable binding is achieved by further administration to the subject of light or a compound sufficient for photoswitchable or chemoswitchable binding, respectively, to the target molecule in the subject.

58. The method according to claim 57, wherein the switchable binding is site-specific in the subject.

59. The method according to claim 57 or 58, wherein the target molecule is endogenous to the subject.

60. The method according to any one of claims 57 to 59, comprising administering to the subject the polypeptide.61 . The method according to any one of claims 57 to 59, which is a cell therapy method comprising administering to the subject a population of cells expressing the polypeptide.

62. The method according to claim 61 , wherein the population of cells comprises the cells of any one of claims 31 to 37.

63. The method according to claim 62, wherein the polypeptide is an extracellular antigen binding domain of an antigen receptor.

64. The method according to claim 63, wherein the population of cells is a population of CAR-T cells.

65. A method of delivering an expression construct encoding the polypeptide of any one of claims 1 to 20 to a target cell, the method comprising contacting the target cell with the virus particle of claim 38 or the liposome or nanoparticle of claim 41 or 42 under conditions in which the expression construct is delivered to the target cell.

66. The method according to claim 65, wherein the method is performed in vivo and comprises administering the virus particle, liposome or nanoparticle to a subject in need thereof, wherein the expression construct is delivered to a target cell in the subject.

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