Ultraspecific cell targeting using de novo designed co-localization dependent protein switches

Abstract

To provide a method of increasing selectivity of a cell in vitro, ex vivo, or in vivo.SOLUTION: The invention provides: protein switches that can sequester bioactive peptides and / or binding domains, holding them in an inactive ("off") state, until combined with a second designed polypeptide called a key and inducing a conformational change that activates ("on") the bioactive peptide or binding domain only when the protein switch components are co-localized when bound to targets; components of such protein switches; and their use.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application Nos. 62 / 848,802, filed May 16, 2019, and 62 / 964,016, filed January 21, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Federal Funding Statement This invention was made with government support under Grant No. CHE-1629214 awarded by the National Science Foundation, Grant No. HDTRA1-18-1-0001 awarded by the Defense Threat Reduction Agency, and Grant No. R01CA114536 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Reference to sequence listings submitted electronically via EFS-Web This application contains a Sequence Listing, filed as an electronic text file entitled "19-851-PCT_Sequence-Listing_ST25.txt," having a size of 32 MB bytes, created on May 14, 2020. The information contained in this electronic file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5). [Background technology]

[0004] Biology is adept at integrating multiple signals to control function. However, natural systems are highly evolved, making it difficult to repurpose specific functions. Engineering systems that can integrate and predictively respond to combinations of binding events remains an open challenge. Such systems would be particularly useful for targeting cells based on the recognition of combinations of surface markers. Most mammalian cell types differ from other tissues only by the combination of markers present on their surface. Summary of the Invention

[0005] In one aspect, the present disclosure provides a method for enhancing cell selectivity in vitro, ex vivo, or in vivo, comprising: (a) contacting a cell with a first cage polypeptide fused to a first binding domain, the first cage polypeptide comprising (i) a structural region and (ii) a latch region further comprising one or more bioactive peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of co-localization with the key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present on or within the cell; (b) contacting the cell with a first key polypeptide fused to a second binding domain, wherein when co-localized with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region and activating one or more bioactive peptides, and the second binding domain is capable of binding to a second cellular moiety present on or within the cell; Methods are provided wherein the first cell portion and the second cell portion are different or identical.

[0006] In another aspect, the present disclosure provides methods for detecting and characterizing the interactions between proteins in vitro, ex vivo, or in vivo. A method for enhancing the selectivity of cells using the method, comprising: (a) contacting two or more cells with a first cage polypeptide fused to a first binding domain, the first cage polypeptide comprising (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides in the absence of co-localization with the key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present within a synapse between the two or more cells; (b) contacting two or more cells with a first key polypeptide fused to a second binding domain, wherein the first key polypeptide, upon co-localization with the first cage polypeptide, is capable of binding to a cage structural region and activating one or more biologically active peptides, and the second binding domain is capable of binding to a second cellular moiety present on a synapse between the two or more cells; Methods are provided wherein the first cell portion and the second cell portion are the same or different.

[0007] In a further aspect, the present disclosure provides a method of targeting heterologous cells (three or more different cell types) in vitro, ex vivo, or in vivo, wherein a first cell portion and a second cell portion are present on a first cell, and a first cell portion and a third cell portion are present on a second cell; (a) contacting two or more cells with a first cage polypeptide fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more bioactive peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of co-localization with the key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present on or within the two or more cells; (b) contacting two or more cells with a first key polypeptide fused to a second binding domain, wherein upon co-localization, the first key polypeptide is capable of binding to the cage structure region and activating one or more bioactive peptides, and the second binding domain is capable of binding to a second cellular moiety present on a cell that also includes the first cellular moiety; (c) contacting the two or more cells with a second key polypeptide fused to a third binding domain, wherein upon co-localization, the second key polypeptide is capable of binding to the cage structure region and activating one or more bioactive peptides, and the third binding domain is capable of binding to a third cellular moiety within the cell that contains the first cellular moiety; Methods are provided wherein the first cell portion, the second cell portion, and the third cell portion are different, and wherein the cells comprising the second cell portion and the cells comprising the third cell portion are different.

[0008] In one aspect, the present disclosure provides a method of reducing off-target activity in vitro, ex vivo, or in vivo, comprising: (a) contacting two or more cells with a first cage polypeptide fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more bioactive peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of co-localization with the key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present on the cells; (b) contacting two or more cells with a first key polypeptide fused to a second binding domain, wherein upon co-localization, the first key polypeptide is capable of binding to the cage structure region and activating one or more bioactive peptides, and the second binding domain is capable of binding to a second cellular moiety present on a cell that also contains the first cellular moiety. And, (c) contacting two or more cells with a decoy cage polypeptide fused to a third binding domain, wherein the decoy cage polypeptide comprises a decoy structural region and, when co-localized with the key polypeptide and the first cage polypeptide, is capable of preferentially binding to the first key polypeptide, and the third binding domain is capable of binding to a third cell portion within a cell comprising the first cell portion and the second cell portion.

[0009] In another aspect, the present disclosure provides a protein complex comprising: (i) a first cage polypeptide fused to a first binding domain; and (ii) a first key polypeptide fused to a second binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides; wherein the first key polypeptide binds to the cage structural region and the one or more biologically active peptides are activated; the first binding domain binds to a first cellular moiety present on or within a cell, or at a synapse of two interacting cells; and the second binding domain binds to a second cellular moiety present on or within a cell, or at a synapse of two interacting cells, wherein the first cellular moiety and the second cellular moiety are different or identical.

[0010] In a further aspect, the present disclosure provides a protein complex comprising: (i) a first key polypeptide fused to a first binding domain; and (ii) a decoy cage polypeptide fused to a second binding domain, wherein the first key polypeptide binds to the decoy cage polypeptide, the first binding domain binds to a first cellular moiety present on or within a cell, or at a synapse of two interacting cells, and the second binding domain binds to a second cellular moiety present on or within a cell, or at a synapse of two interacting cells, and wherein the first cellular moiety and the second cellular moiety are different or identical.

[0011] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) a first cage polypeptide or a polynucleotide encoding the same fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides in the absence of co-localization with the key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present on or within a cell; (b) a first key polypeptide or a polynucleotide encoding the same fused to a second binding domain, wherein when co-localized with the first cage polypeptide, the first key polypeptide can bind to a cage structural region and activate one or more bioactive peptides, and the second binding domain can bind to a second cellular moiety present on or within a cell, Compositions are provided wherein the first cell portion and the second cell portion are different or identical.

[0012] In another aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (a) a first cage polypeptide comprising: (i) a structural region; (ii) a latch region further comprising one or more biologically active peptides; and (iii) a first binding domain, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides; (b) capable of binding to the cage structure region and activating one or more biologically active peptides; a first key polypeptide capable of binding to a second binding domain; a first key polypeptide, wherein the first binding domain and the second binding domain bind to (i) different portions on the surface of the same cell, (ii) the same portion on the surface of the same cell, (iii) different portions at a synapse between two contacting cells, or (iv) the same portion at a synapse between two contacting cells; (c) optionally, one or more effectors that bind to the one or more bioactive peptides when the one or more bioactive peptides are activated.

[0013] In a further aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (a) (i) a first cage polypeptide comprising: (i) a structural region; (ii) a latch region further comprising one or more biologically active peptides; and (iii) a first binding domain, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides; and (ii) one or more expression vectors encoding and / or cells expressing a first key polypeptide capable of binding to a cage structure region and activating one or more biologically active peptides, wherein the first key polypeptide comprises a second binding domain; an expression vector and / or a cell, wherein the first binding domain and the second binding domain bind to (i) different portions on the surface of the same cell, (ii) the same portion on the surface of the same cell, (iii) different portions at a synapse between two contacting cells, or (iv) the same portion at a synapse between two contacting cells; (b) optionally, one or more effectors that bind to the one or more biologically active peptides when the one or more biologically active peptides are activated, and / or one or more nucleic acids encoding the one or more effectors.

[0014] In one aspect, the present disclosure provides a method for cell targeting, comprising: (a) a biological sample containing cells, (i) a cage polypeptide comprising: (i) a structural region; (ii) a latch region further comprising one or more bioactive peptides; and (iii) a first binding domain that targets a cell of interest, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides; and (ii) contacting the cell of interest with a key polypeptide comprising a second binding domain that targets the cell of interest, wherein the first binding domain and the second binding domain bind to (i) different portions on the surface of the same cell, (ii) the same portion on the surface of the same cell, (iii) different portions at a synapse between two contacting cells, or (iv) the same portion at a synapse between two contacting cells; contacting, wherein the contacting occurs for a time and under conditions to promote binding of the cage polypeptide and the key polypeptide to the cell of interest only if the cage polypeptide and the key polypeptide are co-localized in the cell of interest, promote binding of the key polypeptide to the cage structural region to displace the latch region and activate one or more biologically active peptides; (b) contacting the biological sample with one or more effectors under conditions that promote binding of the one or more effector molecules to the one or more activated biologically active peptides to form effector-bioactive peptide complexes; (c) optionally detecting the effector-bioactive peptide complex, wherein the effector-bioactive peptide complex provides a measure of a cell of interest in the biological sample.

[0015] In another aspect, the present disclosure provides a non-naturally occurring polypeptide, comprising: (a) a helical bundle containing 2 to 7 alpha-helices; (b) one or more binding domains; Non-naturally occurring polypeptides are provided in which the helix bundle and the one or more binding domains are both absent from naturally occurring polypeptides.

[0016] In a further aspect, the present disclosure provides a non-naturally occurring polypeptide, comprising: (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a caged polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27278-27321, or a caged polypeptide listed in Table 7, Table 8, or Table 9, excluding optional amino acid residues, wherein the N-terminal and / or C-terminal 60 amino acids of the polypeptide are optional; (b) one or more binding domains.

[0017] In one aspect, the present disclosure provides a non-naturally occurring polypeptide, comprising: (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, and 27,278-27,321, excluding amino acid residues within the latch region; (b) one or more binding domains.

[0018] In one aspect, the disclosure provides that the non-naturally occurring polypeptide comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27359-27392, including optional amino acid residues, or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27393-27398, including optional amino acid residues. [Brief explanation of the drawings]

[0019] [Figure 1a-g]A newly designed protein switch performs AND logic on the cell surface. a. The ability to compute logic operations on the cell surface may enhance targeting selectivity, provide flexibility to heterogeneous tissues, and avoid healthy tissue. b. Structure of the novel cage design used to create Co-LOCKR. The X-ray crystal structure (white) matches the computational design model (green) with a 1.1 Å RMSD across all backbone atoms. The cross-section shows the asymmetric packing of hydrophobic residues (red boxes) and the asymmetric hydrogen-bonding network (blue boxes). c. Schematic of a colocalization-dependent protein switch in which the cage and key domains do not interact in solution but strongly interact upon colocalization on the surface. Co-LOCKR subunits bind to the surface via the targeting domain. d. Flow cytometry identifies Her2+ / EGFR+ cells within a mixed population of K562 cells expressing Her2-eGFP, EGFR-iRFP, both, or neither. e. Schematic showing the "AND" logic by which effector protein recruitment occurs when the cage and key colocalize on the surface of the same cell. f. The mixed population of K562 cells from Figure 1c was incubated with 111 nM Her2-targeting cage, 111 nM EGFR-targeting key, and 50 nM Bcl2-AF594. Bcl2 binding was observed only in K562 / Her2 / EGFR cells. g. The mixed population of K562 cells from Figure 1c was incubated with a dilution series of Her2-targeting cage and EGFR-targeting key. In addition, 50 nM Bcl2-AF594 was either co-incubated with Co-LOCKR (solid line) or added after washing the cells (dashed line). The gray shaded area of ​​the plot represents colocalization-independent activation, where excess amounts of cage and key outcompete the cage-key-Bcl2 complex (formed in solution) from binding to target cells. Bcl2 binding is reported relative to K562 cells incubated with 3000 nM Her2 targeting cage, 3000 nM EGFR targeting key, and 50 nM Bcl2-AF594. [Figure 2a-d]Tuning Co-LOCKR sensitivity. a. Design model of Co-LOCKR using the Bim functional peptide (yellow). Three buried hydrophobic amino acids were mutated to either Ala or Ser to weaken cage-latch affinity and thereby promote cage-key binding. b. The tuned Co-LOCKR mutants demonstrate greater colocalization-dependent activation than the unmutated parent mutant. CL_CHKE mutants recruiting Bcl2-AF594 were assessed by flow cytometry using a mixed population of K562 cells from Figure 1c. Data shown represent 12.3 nM CL_CHKE, and Figure 8c shows a full dilution series of each mutant. c. Confocal microscopy of the HEK293T cell line demonstrates that the Co-LOCKR switch recruits the Bcl2-AF680 effector protein only when Her2 and EGFR are colocalized. Prior to imaging, each cell line was incubated with CL_CHKE (I269S cage) and Bcl2-AF680. NucBlue™ is a nuclear stain, eGFP indicates Her2 localization, mCherry™ indicates EGFR localization, AF680 indicates Bcl2 binding in response to Co-LOCKR activation, and white indicates intersection of Her2-eGFP and EGFR-mCherry™ signals. Scale bars are 10 μm. Uncropped versions of these images are included in Figures 15a-c. d. Heatmap showing AF680 signal intensity (Co-LOCKR activation) versus eGFP (Her2) and mCherry™ (EGFR) pixel intensity. Calculations are based on the uncropped 293T / Her2 / EGFR image in Figure 15a. [Figure 3a-d]Co-LOCKR performs two-input and three-input logic operations on mixed cell populations. a. Co-LOCKR was used to recruit Bcl2-AF594 to two populations of K562 cells expressing different combinations of Her2, EGFR, and EpCAM. Marker expression for each cell line and the identity of the Cage and Key targeting domains are shown below each bar. Red highlighting indicates the expected magnitude of Bcl2-AF594 signal based on relative antigen expression. b. Schematic of the [Her2 AND (EGFR OR EpCAM)] logic mechanism. c. [Ag1 AND (Ag2 OR Ag3)] combinatorial logic was used to recruit Bcl2-AF594. d. Schematic of the [Her2 AND EpCAM NOT EGFR] logic mechanism. The decoy acts as a sponge to sequester Key, thereby preventing Cage activation. e. CL_CHKEpDE was used to recruit BCL2-AF594. The parent cage (left) was compared with the I287A cage (right). Because the decoy may have compromised key binding in solution, the signal magnitude of CL_CHKEpDE is smaller than that of CL_CHKEp. However, sufficient signal remains to calculate the [Her2 AND EpCAM NOT EGFR] logic. In all panels, population 1 was [K562 / EpCAM low, K562 / EGFR / EpCAM low, K562 / EpCAM low / Her2, and K562 / EGFR / EpCAM low / Her2], and population 2 was [K562 / EpCAM low, K562 / EGFR / EpCAM low, K562 / EpCAM high / Her2, and K562 / EGFR / EpCAM high / Her2]. Error bars represent the SEM of six independent replicates of K562 and K562 / EGFR and three independent replicates of all others. [Figure 4a-c]Computational design of Co-LOCKR. a. Overview of how LOCKRa was designed by Langan et al. (9). The existing homotrimer (10) was connected to a single polypeptide chain, and the cage / latch interface was adjusted so that key binding induces activation. b. Computational design of Co-LOCKR. All side chains were removed from the LOCKRa backbone except for residues involved in the existing hydrogen-bonding network and the cage-latch interface. In a new Rosetta design run, asymmetric hydrogen-bonding networks were explored, and core and surface residues were designed asymmetrically. The resulting helical bundle was shortened to reduce aggregation, and the cage-latch and cage-key interfaces were adjusted to achieve colocalization dependence. Decoys were created by redesigning the Co-LOCKR cage to remove the Bim functional peptide and adjust affinity for the key. c. Cross-sections of LOCKRa and Co-LOCKR showing core redesign to replace the C3 symmetric hydrophobic packing with a new hydrogen-bonding network (left) or asymmetric hydrophobic packing (right). d. LOCKRa and Co-LOCKR share 60.8% sequence identity (pairwise sequence identity performed using Geneious software, global alignment with free end gaps). [Figure 5] Redesigning the LOCKR cage reduces aggregation. The LOCKRa cage and asymLOCKR (top) from Langan et al. (9), as well as three new mutants of the Co-LOCKR cage with 0, 7, or 10 residues deleted from the C-terminus of the latch (bottom), were evaluated by size-exclusion chromatography using a Superdex™ 75 Increase 10 / 300 GL column (GE). [Figure 6a-c]The Co-LOCKR system is controlled by a thermodynamic mechanism based on reversible protein-protein interactions. Colocalization of the cage and key on the same surface significantly increases their local concentrations, shifting the binding equilibrium. According to the thermodynamic mechanism, the complex can form in solution (a) or on a surface (b). Our flow cytometry data show that pre-complexed Co-LOCKR generated in solution does not cause appreciable staining of single-antigen target cells. c. Colocalization shifts the response curve to the left, allowing activation at lower concentrations of Co-LOCKR protein. [Figure 7a-b] The strength of the cage and decoy can be tuned by adjusting the cage-latch, cage-key, decoy-latch, and decoy-key interfaces. Residues involved in the cage-latch and cage-key interfaces are shown in orange. Bim is shown in magenta. The affinity of these interfaces was rationally reduced by replacing large hydrophobic amino acids with small hydrophobic amino acids or serine. a. Side view of the cage in the "off" conformation. b. Side view of the key. c. Cross section of the cage in the "off" conformation. [Figure 8a-e]Mutations at the cage-latch interface predictably tune the sensitivity of the Co-LOCKR switch. a. Design model of Co-LOCKR using the Bim functional peptide (yellow). Three buried hydrophobic amino acids were mutated to either Ala or Ser to weaken cage-latch affinity and thereby promote cage-key binding. This panel is replicated from Figure 2a. b. Colocalization-independent activation was assessed using BioLayer Interferometry (Octet). A dilution series of CL_CHKE was assessed for binding to biotinylated Bcl2 immobilized on a streptavidin Octet chip. More disruptive mutations increased the switch's sensitivity. c. The tuned Co-LOCKR mutants exhibit higher colocalization-dependent activation sensitivity and responsiveness than the parent Co-LOCKR mutant. A dilution series of CL_CHKE mutants was assessed by flow cytometry using a mixed population of K562 cells from Figure 1c. Bcl2-AF594 was recruited to K562 / Her2 / EGFR cells (solid line) with minimal binding to K562, K562 / Her2, and K562 / EGFR cells (dotted lines represent maximal off-target binding signals). More disruptive mutations increased switch sensitivity, with the I269S mutant exhibiting maximal switch activation. On-target binding peaked at approximately 37 nM for the parent mutant and approximately 12 nM for the mutated mutant. d. Switch activation of the I269S mutant was enhanced for low CL_CHKE concentrations by incubating cells in a larger volume before flow cytometry. e. On-target, but not off-target, switch activation was increased when 2 nM CL_CHKE I269S mutant was incubated with target cells in a larger incubation volume. [Figure 9a-c]Co-LOCKR mutants were evaluated for colocalization-dependent activation in mixed populations of K562 cells expressing Her2-eGFP, EGFR-iRFP, both, or neither. Co-LOCKR cage mutants and keys were mixed, serially diluted, and evaluated for on-target activation (a), off-target activation (b), and specificity (on-target / maximum off-target, c) as measured by Bcl2-AF594 binding. Mutant I269S had the highest on-target activation, the parent cage had the lowest off-target activation, and mutant I287A had the best fold targeting specificity. On-target binding peaked at approximately 37 nM cage and key for the parent mutant and approximately 12 nM cage and key for the adjusted mutants. Each bar represents a single data point. [Figure 10a-b] Expression levels of EGFR, EpCAM, and Her2 in K562 and Raji tumor cells. Flow cytometry analysis of EGFR (red), EpCAM (blue), and Her2 (green) expression in the indicated K562 (a) or Raji (b) cell lines. All antibodies were used in the PE channel to allow quantification of the number of surface molecules using Quantibrite beads. [Figure 11a-c]The "AND" logic of Co-LOCKR distinguishes cancer cell lines based on the combination of surface antigens. a. Using a targeting domain directly fused to Bim, we measured the relative expression of Her2, EGFR, and EpCAM via Bcl2-AF594. b. Co-LOCKR distinguished A431 (Her2-low / EGFR-high / EpCAM-low) from SKBR3 (Her2-high / EGFR-low / EpCAM-low) cells based on endogenous antigen expression levels. K562 / Her2 / EGFR / EpCAM KO cells were used as a specificity control. Co-LOCKR activation was measured by Bcl2-AF594 recruitment. c. Consistent with a stoichiometric activation mechanism, Co-LOCKR signal is limited by the amount of less expressed surface antigen. Furthermore, activation signal is higher when one antigen is expressed at a high level compared to when both antigens are expressed at a low level. This suggests that Co-LOCKR can function as a threshold gate to avoid cells with low antigen expression. Indeed, this may explain the preferential targeting of K562 cells expressing high levels of EpCAM in Figure 3a. The vertical axis is the recruitment of Bcl2-AF594 by Co-LOCKR, and the horizontal axis is the recruitment of Bcl2-AF594 by Bim-DARPin targeted to a less expressed antigen in the logic operation. [Figure 12] We used scFvs to target Co-LOCKR in a mixed population of K562 cells expressing Her2-eGFP, EGFR-iRFP, both, or neither. Cage_I269S, which targets Her2 via the anti-Her2 scFv, was combined with Key, which targets EGFR via the anti-EGFR scFv. This mixture was serially diluted and evaluated for its ability to specifically target K562 cells co-expressing Her2 and EGFR, as measured by Bcl2-AF594 binding. Solid lines indicate unwashed cells; dashed lines indicate cells washed within 30 minutes of analysis. [Figure 13a-b]Cage and decoy mutants were tuned to implement the [Her2 AND EpCAM NOT EGFR] logic. a. Cages with a strong cage-latch interface exhibit weak "AND" activation and tight "NOT" deactivation, while cages with a weak cage-latch interface exhibit strong "AND" activation and leaky "NOT" deactivation. These results demonstrate that cage activity can be tailored to the desired biological function. For example, mutants I287A, I287S, and I269S exhibit greater sensitivity to [Her2 AND EpCAM low] with minimal leakiness in the presence of EGFR, whereas the parent cage exhibits better deactivation of [Her2 AND EpCAM low NOT EGFR]. b. Decoys can be tuned to reduce leakiness of "NOT" deactivation. Decoy mutants with destabilizing mutations or truncations to weaken the latch were evaluated for their ability to execute the [Her2 AND EpCAM NOT EGFR] logic against mixed populations of cells: K562 / EpCAM low (gray), K562 / EGFR / EpCAM low (yellow), K562 / Her2 / EpCAM high (purple), and K562 / Her2 / EpCAM high / EGFR (brown). The strongest decoy (e.g., G24) exhibits minimal leakage but reduced targeting of K562 / Her2 / EpCAM high, likely due to colocalization-independent key binding. The weakest decoy (e.g., Box1C1) exhibits the highest targeting of K562 / Her2 / EpCAM high, with substantial leakage in K562 / Her2 / EpCAM high / EGFR. Each bar represents one sample. [Figure 14a-d]The cage and decoy variants were adjusted to implement the [Her2 AND EpCAM NOT EGFR] logic. Various Key and Cage concentrations were tested with either 0 nM, 5 nM, or 20 nM EGFR_Decoy1 or EGFR_Decoy_G31. The purple "on-target" line corresponds to the desired AND signal of K562 / EpCAM high / Her2 in the absence of decoy, while the brown "off-target" line corresponds to the undesired AND signal of K562 / EGFR / EpCAM high / Her2 that the decoy must block. Using 5 nM EGFR_Decoy_G31 as a NOT gate enhances the on-target binding signal while minimizing the undesired targeting of K562 / EGFR / EpCAM high / Her2. These results are consistent with the hypothesis that decoy-Key binding in solution must be minimized to preserve the Co-LOCKR signal. a. 5 nM Key_EpCAM, 5 nM Her2_Cage. b. 5nM Key_EpCAM, 5nM Her2_Cage_I287A. c. 20nM Key_EpCAM, 20nM Her2_Cage. Annotated to the original state described in Figure 3e. d. 20nM Key_EpCAM 20nM Her2_Cage_I287A. [Figure 15a-c] Uncropped confocal microscopy images of Co-LOCKR targeting HEK293T cells expressing Her2 and EGFR. a. Uncropped 293T / Her2 / EGFR image used to generate Figure 2c-d (green is Her2-eGFP, red is EGFR-mCherry, and blue is Bcl2-AF680). b. Uncropped 293T / Her2 / EGFR image pseudocolored as in Figure 2c (white is the intersection of Her2-eGFP and EGFR-mCherry™, blue is NucBlue™, and magenta is Bcl2-AF680). The scale bar in the top panel is 20 μm, and the scale bar in the bottom panel is 10 μm. c. Uncropped images of all cell lines and staining conditions evaluated by confocal microscopy. The scale bar is 20 μm. [Figure 16] DARPin binder affinity measured by flow cytometry. Anti-Her2 or anti-EGFR DARPins fused to Bim at their N-terminus were pre-complexed with Bcl2-AF594 and serially diluted 3-fold from 300 nM to 0.4 nM. This dilution series was used to label a mixed population of K562 cells expressing Her2-eGFP, EGFR-iRFP, both, or neither in a 50 μl incubation volume at room temperature for 1 h. Cells were then washed with PBS supplemented with 0.1% bovine serum albumin and analyzed on an LSRII flow cytometer. The apparent Kd of the DARPins was approximately 10 nM, consistent with the hypothesis that Co-LOCKR activation is limited by the binding affinity of the DARPins. DETAILED DESCRIPTION OF THE INVENTION

[0020] As described herein, the polypeptides and compositions described herein can be used to create a "protein switch," in which a cage polypeptide and a key polypeptide comprise binding domains that bind to different targets, and the key polypeptide binds to the cage polypeptide and produces a biologically active peptide only when the different targets are closely associated. This induces activation of the CAGE and KEY polypeptides, resulting in colocalization of the CAGE and KEY polypeptides while they are bound to their targets.

[0021] Targeting specificity has been a long-standing problem in biomedicine. Despite the long-standing goal of targeting therapeutics to specific cell types, a general solution for targeting precise combinations of antigens that unambiguously identify the desired cell type remains lacking. Natural systems capable of integrating multiple inputs are hard-coded into specific biological outputs that are difficult to modularly reassign. The methods, compositions, and polypeptides disclosed herein are modular because they involve newly designed polypeptides that integrate the colocalization of two target antigens to conditionally expose bioactive peptides capable of modularly recruiting any effector function. Prior to this work, it had not been possible to create a system capable of integrating the colocalization of two or more antigens on the surface of a target cell to conditionally expose bioactive peptides capable of modularly recruiting any effector function. Furthermore, it was previously impossible to design such novel proteins that could sequester bioactive peptides in an inactive confirmation until they were colocalized. Finally, it was previously impossible to tune the sensitivity of protein actuators to recruit the appropriate amount of effector.

[0022] The method may include the use of polypeptides, nucleic acids, vectors, cells and / or compositions of any embodiment or combination of embodiments disclosed herein. In various embodiments, the method includes the use of AND, OR, and / or NOT logic gates using any embodiment or combination of embodiments as detailed above and in the Examples.

[0023] I. Definition All references cited are incorporated herein by reference in their entirety. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0024] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0025] All embodiments of any aspect of this disclosure can be used in combination unless the context clearly indicates otherwise.

[0026] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the art will recognize.

[0027] A polypeptide is "non-naturally occurring" in that the entire polypeptide is not found in any naturally occurring polypeptide. It will be understood that components of a polypeptide may be naturally occurring, including, but not limited to, biologically active peptides, which may be included in some embodiments.

[0028] A "cage polypeptide" comprises a helix bundle comprising 2 to 7 alpha helices. In various embodiments, the helix bundle comprises 3 to 7, 4 to 7, 5 to 7, 6 to 7, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 2 to 5, 3 to 5, 4 to 5, 2 to 4, 3 to 4, 2 to 3, 2, 3, 4, 5, 6, or 7 alpha helices.

[0029] The design of helix-bundle cage polypeptides of the present disclosure may be carried out by any suitable means. In one non-limiting embodiment, the Rosetta™ program's BundleGridSampler™ is used to generate backbone geometries based on the Crick equation for coiled-coils, allowing efficient, parallel sampling of a regular grid of coiled-coil equation parameter values ​​corresponding to a continuum of peptide backbone conformations. This can be complemented by the design of hydrogen-bond networks using any suitable means, followed by Rosetta™ side-chain design. In a further non-limiting embodiment, the best-scoring design based on total score, number of unsatisfactory hydrogen bonds, and lack of cavities in the protein core can be selected for helix-bundle polypeptide design.

[0030] Each alpha helix can be of any suitable length and amino acid composition appropriate for the intended use, hi one embodiment, each helix is ​​independently 18 to 60 amino acids in length. In various embodiments, each helix is ​​independently 18-60, 18-55, 18-50, 18-45, 22-60, 22-55, 22-50, 22-45, 25-60, 25-55, 25-50, 25-45, 28-60, 28-55, 28-50, 28-45, 32-60, 32-55, 32-50, 32-45, 35-60, 35-55, 35-50, 35-45, 38-60, 38-55, 38-50, 38-45, 40-60, 40-58, 40-55, 40-50, or 40-45 amino acids in length.

[0031] In some embodiments, the polypeptides disclosed herein comprise a linker. In some embodiments, the linker comprises one or more amino acids, e.g., an amino acid linker or a peptide linker. In some embodiments, the linker connects a first alpha helix to a second alpha helix. The amino acid linkers connecting each alpha helix can be of any suitable length or amino acid composition appropriate for the intended use. In one non-limiting embodiment, each amino acid linker is independently 2 to 10 amino acids in length and does not include any additional functional sequence that may be fused to the linker. In various non-limiting embodiments, each amino acid linker is independently 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 2 to 5, 3 to 5, 4 to 5, 2 to 4, 3 to 4, 2-3, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In all embodiments, the linker can be structured or flexible (e.g., polyGS). These linkers may encode additional functional sequences, including but not limited to, protease cleavage sites or half of a split intein system (see sequences below).

[0032] The one or more binding domains may be any polypeptide binding domain suitable for the intended use. In one embodiment, the one or more binding domains comprise a cell surface protein-binding polypeptide. In another embodiment, the helix bundle is linked to the one or more binding domains by any suitable linker, polypeptide linker or non-polypeptide linker. In one embodiment, the helix bundle is linked to the one or more binding domains by any suitable polypeptide linker, including but not limited to the linkers between helix domains described above.

[0033] In some embodiments, one or more of the cage polypeptide and the key polypeptide The cage polypeptide further comprises a linker connecting the cage or key polypeptide to one or more binding domains. In some embodiments, the cage polypeptide comprises a linker connecting the cage polypeptide to the binding domain. In some embodiments, the key polypeptide comprises a linker connecting the key polypeptide to the binding domain. Any linker known in the art can be used. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker is cleavable. In some embodiments, the linker is any linker disclosed herein.

[0034] Additional embodiments of one or more binding domains are described in more detail below.

[0035] The polypeptide of the first embodiment includes a region called the "latch region," which can be used for insertion of a bioactive peptide. Thus, a cage polypeptide includes a latch region and a structural region (i.e., the remainder of the cage polypeptide that is not the latch region). When the latch region is modified to include one or more bioactive peptides, the structural region of the cage polypeptide interacts with the latch region to prevent the activity of the bioactive peptides. When the cage and key polypeptides are colocalized and the binding domains bind to their targets (as described below), upon activation by the key polypeptide, the latch region dissociates from its interaction with the structural region, exposing the bioactive peptides and allowing them to function.

[0036] As used herein, a "bioactive peptide" is any peptide of any length or amino acid composition that can selectively bind to a defined target (i.e., can bind to an "effector" polypeptide). Such bioactive peptides can include peptides with all three types of secondary structure in their inactive conformation: alpha helices, beta strands, and loops. Polypeptides of this nature can be used to control the activity of a wide range of functional peptides. The ability to harness these biological functions with precise, inducible control is useful, for example, for cell engineering (such as inducible activation of function, engineering complex logic operations and circuits), sensor development, the development of inducible protein-based therapeutics, and the creation of novel biological materials. Additional details about bioactive peptides are provided below.

[0037] Latch regions can be located near either end of a cage polypeptide. In one embodiment, the latch region is located in the C-terminal helix, thereby positioning the bioactive peptide for maximum burial of functional residues required to maintain the bioactive peptide in an inactive state, while simultaneously burying hydrophobic residues to facilitate solvent exposure / compensatory hydrogen bonding of polar residues. In various embodiments, the latch region can comprise part or all of a single alpha helix in the cage polypeptide at the N- or C-terminal end. In various other embodiments, the latch region can comprise part or all of the first, second, third, fourth, fifth, sixth, or seventh alpha helix in the cage polypeptide. In other embodiments, the latch region can comprise all or part of two or more different alpha helices in the cage polypeptide, such as the C-terminal portion of one alpha helix and the N-terminal portion of the next alpha helix, all two consecutive alpha helices, etc.

[0038] As used herein, a "synapse" is a junction between two interacting cells, typically involving protein-protein contact across the junction. An immune synapse is the interface between an antigen-presenting cell or target cell and a lymphocyte, such as a T / B cell or natural killer cell. A neural synapse is the junction between two nerve cells, consisting of a small gap through which impulses pass by the diffusion of neurotransmitters. This embodiment is particularly useful, for example, for detecting cells that are in contact with each other, as opposed to cells that are not in contact with each other. For example, it is possible to identify only T cells that are interacting with a designated target cell, while avoiding all T cells that are not interacting.

[0039] As used throughout this application, the term "polypeptide" is used in its broadest sense to refer to a sequence of subunit amino acids. Polypeptides of the invention may contain L-amino acids plus glycine, D-amino acids plus glycine (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids plus glycine. The polypeptides described herein can be chemically synthesized or recombinantly expressed. Polypeptides can be conjugated to other compounds, such as by pegylation, hesyltransferase (HES), persulfation, or glycosylation, to promote increased in vivo half-life, or can be produced as Fc fusions or in deimmunized variants. Such conjugation can be covalent or noncovalent, as understood by those skilled in the art.

[0040] An "effector" is any molecule, nucleic acid, protein, nucleoprotein complex, or cell that performs a biological activity upon interaction with a bioactive peptide. Exemplary biological activities can include binding, fluorophore recruitment, toxin recruitment, immunomodulator recruitment, proteolysis, enzymatic activity, release of signaling proteins (e.g., cytokines, chemokines), induction of cell death, induction of cell differentiation, nuclear import / export, ubiquitination, and fluorophore / chromophore maturation.

[0041] II. Compositions of the Present Disclosure The present disclosure relates to a switch system that can improve target cell specificity in vitro, in vivo, or ex vivo. In particular, the system can be located within a tissue, between cells, within a cellular synapse, or within a cell where increased target specificity is required. In some embodiments, the composition can enhance the selectivity of cells for therapy. In some embodiments, the composition of the present disclosure can enhance the selectivity of cells interacting with each other for therapy. In some embodiments, the composition can target heterogeneous cells (three or more different cell types) for therapy, where a first cell portion and a second cell portion are present on a first cell, and a first cell portion and a third cell portion are present on a second cell. In some embodiments, the composition can also reduce off-target activity for therapy. Thus, in some embodiments, the composition can prepare a subject in need of therapy to respond better to the therapy, increase the efficacy of the therapy, and / or reduce toxicity due to non-specific binding (or leakage).

[0042] Ag1 AND Ag2 In some embodiments, the present disclosure can enhance the selectivity of cells containing at least two different cell markers (portions Ag1 and Ag2). By targeting cells that express the two different portions, cells containing only one of the portions (Ag1 or Ag2) can be deselected. In some embodiments, the composition can: (a) a first cage polypeptide fused to a first binding domain, the first cage polypeptide comprising: (i) a structural region; and (ii) a latch region further comprising one or more bioactive peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of co-localization with the key polypeptide; and the first binding domain is capable of binding to a first cellular moiety present on a cell; (b) a first key polypeptide fused to a second binding domain, wherein when co-localized with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region and activating one or more bioactive peptides, and the second binding domain is capable of binding to a second cellular moiety present on or within a cell; The first cell portion and the second cell portion are different or identical.

[0043] In some aspects, the present disclosure provides: (a) a polynucleotide encoding a first cage polypeptide fused to a first binding domain, the first cage polypeptide comprising: (i) a structural region; and (ii) a latch region further comprising one or more bioactive peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of co-localization with the key polypeptide; and the first binding domain is capable of binding to a first cellular moiety present on a cell; (b) a polynucleotide encoding a first key polypeptide fused to a second binding domain, wherein when co-localized with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region and activating one or more bioactive peptides, and the second binding domain is capable of binding to a second cellular moiety present on or within a cell; The first cell portion and the second cell portion are different or identical. In some embodiments, the polynucleotide encoding the first cage polypeptide and the polynucleotide encoding the second polypeptide are on the same vector. In some embodiments, the polynucleotide encoding the first cage polypeptide and the polynucleotide encoding the second polypeptide are on different vectors.

[0044] In some embodiments, the first cell portion and the second cell portion are different. In some embodiments, the first cell portion and the second cell portion are the same.

[0045] In order for one or more bioactive peptides to be activated (e.g., exposed to an effector or capable of transmitting its signal downstream), the functional cage polypeptide and key polypeptide must be colocalized. Simply expressing the functional cage polypeptide and key polypeptide is not sufficient. For example, in some embodiments, binding of a functional cage polypeptide, e.g., a first cage polypeptide, to a key polypeptide in solution is less efficient at activating one or more bioactive peptides than binding of the cage and key polypeptides after colocalization. Thus, in some embodiments, colocalization of the first cage polypeptide and key polypeptide increases the selectivity of cells that highly express a cellular moiety.

[0046] In some embodiments, colocalization of the first cage polypeptide and the first key polypeptide increases the local concentrations of the first cage polypeptide and the first key polypeptide, shifting the binding equilibrium in favor of complex formation between the first cage polypeptide and the first key polypeptide.

[0047] Two cellular moieties may be colocalized as a result of forming a complex (e.g., two proteins in the same complex, such as a Her2-EGFR heterodimer or CD3ζ complexed with LAT or Zap70, two DNA sequences closely spaced on a chromosome, two RNA sequences closely spaced on an mRNA) directly or indirectly, such that two cellular moieties are sufficiently close (e.g., in close proximity) to allow colocalization of a cage polypeptide associated with a first cellular moiety and a key polypeptide associated with a second cellular moiety. In this case, at least one molecule of the first moiety must colocalize with at least one molecule of the second moiety to result in colocalization. Alternatively, two cellular moieties may be colocalized by being expressed in sufficient numbers in the same intracellular compartment (e.g., two transmembrane proteins expressed in the cell membrane, such as Her2 and EGFR, Her2 and EpCAM. In some embodiments, cells express the first cellular moiety and / or the second cellular moiety at a concentration of at least about 100 copies per cell, at least about 100 copies per cell, or at least about 100 copies per cell. at least about 200 copies per cell, at least about 500 copies per cell, at least about 1000 copies per cell, at least about 1500 copies per cell, at least about 2000 copies per cell, at least about 2500 copies per cell, at least about 3000 copies per cell, at least about 3500 copies per cell, at least about 4000 copies per cell, at least about 4500 copies per cell, at least about 5000 copies per cell, at least about 5500 copies per cell, at least about 6000 copies per cell, at least about 6500 copies per cell, or at least about 7000 copies per cell. In some embodiments, the first cell portion and / or the second cell portion comprises about 500 to about 10,000 copies per cell, about 1,000 to about 10,000 copies per cell, about 2,000 to about 10,000 copies per cell, about 3,000 to about 10,000 copies per cell, about 4,000 to about 10,000 copies per cell, about 5,000 to about 10,000 copies per cell, about 1,000 to about 9,000 copies per cell, about 2,000 to about 9,000 copies per cell, about 3,000 to about 9,000 copies per cell, about 4,000 to about 9,000 copies per cell, about 5,000 to about 9,000 copies per cell, about 1,000 to about 8,000 copies per cell. Expresses about 2,000 to about 8,000 copies per cell, about 3,000 to about 8,000 copies per cell, about 4,000 to about 8,000 copies per cell, about 5,000 to about 8,000 copies per cell, about 1,000 to about 7,000 copies per cell, about 2,000 to about 7,000 copies per cell, about 3,000 to about 7,000 copies per cell, about 4,000 to about 7,000 copies per cell, about 5,000 to about 7,000 copies per cell, about 1,000 to about 6,000 copies per cell, about 2,000 to about 6,000 copies per cell, about 3,000 to about 6,000 copies per cell, about 4,000 to about 6,000 copies per cell, about 5,000 to about 6,000 copies per cell. In some embodiments, the cells express at least about 5000 copies to up to about 6000 copies, up to about 7000 copies, or up to about 8000 copies of the first cell moiety and / or the second cell moiety.In some embodiments, the first cage polypeptide and the first key polypeptide are co-localized, thereby forming a complex and activating the one or more bioactive peptides.

[0048] In some embodiments, the first and second cellular portions are present on the surface of the cell. In some embodiments, the first and second cellular portions are present in the cytoplasm of the cell. In some embodiments, the first and second cellular portions are present in the nucleus of the cell. In some embodiments, the first and second cellular portions are present in the secretory pathway of the cell, including the endoplasmic reticulum (ER) and Golgi apparatus.

[0049] Ag1 AND (Ag2 OR Ag3) The present disclosure also allows for targeting two or more cells simultaneously by utilizing different cell markers. For example, the present disclosure provides for targeting three or more cells simultaneously (Ag1 AND (Ag2 OR Ag3)), four or more cells simultaneously (Ag1 AND (Ag2 OR Ag3)), and the like, in the case of CAR T cell therapy. This allows for targeting of heterogeneous cell types, such as five or more (Ag1 AND (Ag2 OR Ag3 OR Ag4 OR Ag5)), five or more (Ag1 AND (Ag2 OR Ag3 OR Ag4 OR Ag5 OR Ag6)), or six or more (Ag1 AND (Ag2 OR Ag3 OR Ag4 OR Ag5 OR Ag6)). In some embodiments, (Ag1 OR Ag2) AND Ag3 can be achieved by simultaneously targeting multiple cage polypeptides with different binding domains to multiple cells and targeting one key polypeptide with a single binding domain to those same cells. In other embodiments, (Ag1 OR Ag2) AND (Ag3 OR Ag4) can be achieved by targeting multiple cage polypeptides with multiple binding domains and multiple key polypeptides with multiple binding domains.

[0050] In some embodiments, the composition comprises: (a) a first cage polypeptide or a polynucleotide encoding the same fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides in the absence of co-localization with the key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present on or within a first cell (cell type I, e.g., a cell expressing Ag1 and Ag2); (b) a first key polypeptide or a polynucleotide encoding the same fused to a second binding domain, wherein when co-localized with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region and activating one or more biologically active peptides, and the second binding domain is capable of binding to a second cellular moiety present on or within the first cell; (c) a second key polypeptide or a polynucleotide encoding the same fused to a third binding domain, wherein when co-localized with the first cage polypeptide, the second key polypeptide can bind to the cage structural region and activate one or more biologically active peptides, and the third binding domain can bind to a third cellular moiety present on or within a second cell that also comprises a first cellular moiety (cell type II, e.g., a cell expressing Ag1 and Ag3), wherein the first cellular moiety, the second cellular moiety, and the third cellular moiety are different.

[0051] In some embodiments, the first key polypeptide comprises a third binding domain, and the second binding domain and / or the third binding domain bind to (i) a moiety different from the first binding domain on the surface of the same cell, or (ii) a moiety different from the first binding domain at a synapse between two contacting cells, and when co-localized with the first cage polypeptide, the first key polypeptide can bind to the cage structural region and activate one or more biologically active peptides, and the third binding domain can bind to a third cellular moiety present on or within a cell that also contains the first cellular moiety, and the third cellular moiety is different from the first cellular moiety or the second cellular moiety.

[0052] In some embodiments, the composition comprises: (d) at least a second cage polypeptide comprising: (i) a second structural region; (ii) a second latch region further comprising one or more biologically active peptides; and (iii) a sixth binding domain, wherein the second structural region interacts with the second latch region to prevent activity of the one or more biologically active peptides; the first key and / or second key polypeptides are capable of binding to the second structural region to activate one or more biologically active peptides; The sixth binding domain and / or the first binding domain bind (i) to a different portion of the surface of the same cell than the second, third, and / or fourth binding domain, or (ii) to a different portion of the synapse between two contacting cells than the second, third, and / or fourth binding domain. Such compositions can be used, for example, to simultaneously target two cage polypeptides with different binding domains to multiple cells and one key polypeptide with a single binding domain to those same cells, thereby achieving (Ag1 OR Ag2) AND Ag3.

[0053] In some embodiments, the composition comprises multiple key polypeptides, a fourth key polypeptide, a fifth key polypeptide, or both, to enhance selectivity for the first cell and / or the second cell. The composition may further comprise a fourth, sixth, or seventh key polypeptide. For example, the composition for the first cell may further comprise an additional key polypeptide, a fourth, fifth, sixth, or seventh key polypeptide, which can further enhance selectivity for the first cell. In some embodiments, the composition for the second cell further comprises an additional key polypeptide, a fourth, fifth, sixth, or seventh key polypeptide, which can further enhance selectivity for the second cell. Each of the additional key polypeptides for the present disclosure can be fused to a binding domain, and when co-localized with the first cage polypeptide, the third key polypeptide can bind to the cage structural region and activate one or more bioactive peptides, and the third binding domain can bind to a cellular moiety present on or within a cell, including the first cell moiety. In some embodiments, a single key polypeptide can be fused to two or more binding domains so that the same key polypeptide can be targeted to both cell type I and cell type II.

[0054] (Ag1 AND Ag2) NOT Ag3 The present disclosure also allows therapies to avoid normal (healthy) cells, but target only diseased cells, such as tumor cells, by utilizing various cell markers, thereby reducing off-target cell specificity or toxicity. Thus, the present disclosure allows therapies to avoid targeting normal cell types that express unique cell markers. For example, if normal cells express Ag3, but diseased cells do not express Ag3, compositions for the present disclosure can be constructed to avoid cells that express Ag3.

[0055] In some embodiments, the composition comprises: (a) a first cage polypeptide fused to a first binding domain, the first cage polypeptide comprising: (i) a structural region; and (ii) a latch region further comprising one or more bioactive peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of co-localization with the key polypeptide; and the first binding domain is capable of binding to a first cellular moiety present on or within a cell; (b) a first key polypeptide or a polynucleotide encoding the same fused to a second binding domain, wherein, when co-localized with the first cage polypeptide, the first key polypeptide is capable of binding to a cage structural region and activating one or more biologically active peptides, and the second binding domain is capable of binding to a second cellular moiety present on or within a cell; and (c) one or more decoy cage polypeptides or polynucleotides encoding same fused to one or more binding domains ("decoy binding domains"), wherein each decoy cage polypeptide comprises a decoy structural region and, when co-localized with the first key polypeptide and the first cage polypeptide, can preferentially bind to the first key polypeptide, and each decoy binding domain can bind to a cellular portion ("decoy cellular portion") within a cell comprising the second cellular portion. In some embodiments, the decoy binding domain can bind to a cellular portion ("decoy cellular portion") within a cell comprising the first cellular portion and the second cellular portion. In some embodiments, each decoy cellular portion is present only in healthy cells. In some embodiments, when co-localized with the first key polypeptide, each decoy cage polypeptide binds to the first key polypeptide in such a way that the first key polypeptide does not bind to the first cage polypeptide, and one or more biologically active peptides in the first cage polypeptide are not activated.

[0056] Any first cage polypeptide may be a decoy polypeptide for any second cage polypeptide. The first cage polypeptide can function as a key polypeptide, provided that the first cage polypeptide has a higher affinity for the key polypeptide than the second cage polypeptide.

[0057] The compositions and methods of all embodiments described herein may include the use of a single decoy cage polypeptide containing multiple binding domains, or multiple decoy cage polypeptides each having one (or more) binding domains, to avoid cells with different decoy cell moieties (e.g., 1 AND 2 NOT (3 OR 4) logic).

[0058] In some embodiments, the binding affinity (e.g., K) of the decoy cage polypeptide to the key polypeptide is D ) is the binding affinity (e.g., K) of the first cage polypeptide to the key polypeptide. D), at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, or at least about 1000 times stronger (e.g., less) than In some embodiments, the decoy cage polypeptide further comprises a decoy latch region. In some embodiments, the decoy latch region is not functional. In some embodiments, the decoy latch region does not comprise any biologically active peptide. In some embodiments, the decoy latch region is absent. In some embodiments, the decoy latch region comprises a non-functional biologically active peptide. In some embodiments, the decoy latch region comprises a functional biologically active peptide having a distinct biological function. As a non-limiting example, the cage polypeptide may comprise a biologically active peptide having an immunostimulatory function, and the decoy cage polypeptide may comprise a biologically active peptide having an immunoinhibitory function.

[0059] Exemplary Co-LOCK® System In a fourth aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) a first cage polypeptide comprising: (i) a structural region; (ii) a latch region further comprising one or more biologically active peptides; and (iii) a first binding domain, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides; (b) a first key polypeptide capable of binding to the cage structure region and activating one or more biologically active peptides, wherein the key polypeptide comprises a second binding domain; a first key polypeptide, wherein the first binding domain and the second binding domain bind to (i) different portions on the surface of the same cell, (ii) the same portion on the surface of the same cell, (iii) different portions at a synapse between two contacting cells, or (iv) the same portion at a synapse between two contacting cells; Optionally, one or more effectors that bind to the one or more bioactive peptides when the one or more bioactive peptides are activated.

[0060] The compositions disclosed herein, also referred to in the examples below as the "Co-LOCKR System," are proximity-activated proteins that perform Boolean logic operations of "AND," "OR," and "NOT," and combinations thereof, in response to, for example, precise combinations of protein binding events. The system contains at least one cage polypeptide and at least one key polypeptide, which can be used as a novel protein switch. The switch is activated by a conformational change only when all logical conditions are met. This system is demonstrated in an example that provides ultraspecific targeting of mammalian cells, which are distinguished in complex cell populations only by the precise combination of surface markers. An "AND" gate can be achieved by targeting a cage polypeptide to one antigen and a key polypeptide to another antigen. A "threshold" gate can be achieved by targeting a cage polypeptide and a key polypeptide to the same antigen (which may have binding domains that bind to the same epitope or different epitopes on the same antigen). An "OR" gate can be achieved by targeting a cage polypeptide or a key polypeptide to two different antigens. A "NOT" gate can be achieved by supplementing a decoy cage polypeptide that sequesters the key polypeptide and prevents it from interacting with the cage polypeptide. Additional cage polypeptides, key polypeptides, and decoy cage polypeptides can be included to establish the desired logical operation (e.g., antigen 1 AND antigen 2 NOT antigen 3, antigen 1 AND (antigen 2 OR antigen 3)).

[0061] Thus, in one embodiment, the first binding domain and the second binding domain bind to (i) different moieties on the surface of the same cell, or (iii) different moieties at a synapse between two contacting cells. In this embodiment, the composition can be used to establish an AND gate.

[0062] In another embodiment, the first binding domain and the second binding domain bind to (ii) the same portion on the surface of the same cell, or (iv) the same portion at a synapse between two contacting cells. In this embodiment, the composition can be used to establish a thresholding gate.

[0063] In one embodiment, (c) the first key polypeptide comprises a third binding domain, and the second binding domain and / or the third binding domain bind (i) to a different moiety on the surface of the same cell than the first binding domain, or (ii) to a different moiety at a synapse between two contacting cells than the first binding domain. In further embodiments, the second binding domain and the third binding domain bind to different moieties on the surface of different cells. In these embodiments, the composition can be used to establish a 1 AND (2 OR 3) logic gate, where the moiety bound by the first binding domain is present on one of the cells.

[0064] In another embodiment, the composition further comprises (d) at least a second key polypeptide capable of binding to the first cage structure region, wherein the key polypeptide comprises a fourth binding domain, and the second binding domain and / or the fourth binding domain bind (i) to a different moiety on the surface of the same cell than the first binding domain, or (ii) to a different moiety at a synapse between two contacting cells than the first binding domain. In one embodiment, the second binding domain and the fourth binding domain bind (i) to different moieties on the surface of the same cell, or (ii) to different moieties at a synapse between two contacting cells. In a further embodiment, the second binding domain and the fourth binding domain bind to different moieties on the surface of different cells. In these embodiments, the composition can be used to establish a 1 AND (2 OR 3) logic gate, where the moiety bound by the first binding domain is present on one of the cells.

[0065] In a further embodiment, the first cage polypeptide further comprises a fifth binding domain, wherein the fifth binding domain and / or the first binding domain (i) is different from the second binding domain, the third binding domain, and / or the fourth binding domain on the surface of the same cell. In one embodiment, the fifth binding domain and the first binding domain bind to (i) different moieties on the surface of the same cell, or (ii) different moieties at a synapse between two contacting cells than the second binding domain, the third binding domain, and / or the fourth binding domain. In this embodiment, the composition can be used to establish an OR logic gate, specifically a [(1 OR 5) AND (2 OR 3)] logic gate, based on the additional binding domains present in a single cage polypeptide.

[0066] In one embodiment, the composition further comprises (e) at least a second cage polypeptide comprising (i) a second structural region, (ii) a second latch region further comprising one or more bioactive peptides, and (iii) a sixth binding domain, wherein the second structural region interacts with the second latch region to prevent activity of the one or more bioactive peptides, the first key and / or second key polypeptide can bind to the second structural region to activate the one or more bioactive peptides, and the sixth binding domain and / or the first binding domain bind (i) to a different portion of the surface of the same cell from the second, third, and / or fourth binding domain, or (ii) to a different portion of a synapse between two contacting cells from the second, third, and / or fourth binding domain. In one embodiment, the sixth binding domain and the first binding domain bind (i) to different portions of the surface of different cells, or (ii) to different portions of a synapse between two contacting cells. In these embodiments, the compositions can be used to establish an OR logic gate based on an additional binding domain present on a second cage polypeptide. In one such embodiment, two separate but identical cage polypeptides can each bind to a different binding domain. In another such embodiment, the two cage polypeptides can be different cage polypeptides, both activated by the same key polypeptide, each binding to a different binding domain.

[0067] In another embodiment, the composition further comprises (f) a decoy cage polypeptide comprising (i) a decoy structural region, (ii) a decoy latch region optionally further comprising one or more biologically active peptides, and (iii) a seventh binding domain, wherein the decoy structural region interacts with the first key polypeptide and / or the second key polypeptide to prevent them from binding to the first and / or second cage polypeptide, and the seventh binding domain binds to a moiety on the surface of the same cell as the second binding domain, the third binding domain, and / or the fourth binding domain. In one embodiment, the seventh binding domain binds to a moiety present on the cell at a level equal to or greater than that bound by the second binding domain, the third binding domain, and / or the fourth binding domain. In this embodiment, the composition can be used to establish a NOT logic gate based on a decoy cage polypeptide that binds to a different target on the same cell as the target of the key polypeptide. In this embodiment, the composition can be used to establish, for example, 1 AND 2 NOT 7 logic, provided that the portions bound by the first and second binding domains are present on the same cell. In one embodiment, the decoy cage polypeptide does not contain a biologically active peptide. This embodiment can be used to establish, for example, 1 AND 4 NOT 7 logic (provided that the portions bound by the first and fourth binding domains are present on the same cell), or 5 AND 4 NOT 7 logic can be established (provided that the moieties bound by the fifth and fourth binding domains are present on the same cell). Such AND / NOT embodiments require at least one cage polypeptide, at least one key polypeptide, and at least one decoy cage polypeptide.

[0068] In one embodiment of all these embodiments of the composition, the first binding domain, the second binding domain, the third binding domain (if present), the fourth binding domain (if present) The first binding domain (if present), the fifth binding domain (if present), the sixth binding domain (if present), and / or the seventh binding domain (if present) comprise polypeptides capable of binding to moieties present on cell surfaces, including proteins, sugars, and lipids. In one embodiment, one or more binding proteins comprise cell surface protein-binding polypeptides.

[0069] All of the above compositions are described as polypeptide compositions. The present disclosure also provides compositions comprising expression vectors and / or cells expressing the cage polypeptides and key polypeptides described in the above compositions, and thus can be used for the same purposes (e.g., in establishing the same logic gates as for the corresponding polypeptide compositions). Thus, in a fifth aspect, the present disclosure provides: (a) (i) a first cage polypeptide comprising: (i) a structural region; (ii) a latch region further comprising one or more biologically active peptides; and (iii) a first binding domain, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides; and (ii) one or more expression vectors encoding and / or cells expressing a first key polypeptide capable of binding to a cage structure region and activating one or more biologically active peptides, wherein the first key polypeptide comprises a second binding domain; an expression vector and / or a cell, wherein the first binding domain and the second binding domain bind to (i) different portions on the surface of the same cell, (ii) the same portion on the surface of the same cell, (iii) different portions at a synapse between two contacting cells, or (iv) the same portion at a synapse between two contacting cells; (b) optionally, one or more effectors that bind to the one or more biologically active peptides when the one or more biologically active peptides are activated, and / or one or more nucleic acids encoding the one or more effectors.

[0070] The one or more expression vectors may include separate expression vectors encoding each distinct polypeptide, may include expression vectors encoding two or more of the distinct polypeptides, or any combination thereof suitable for the intended use. The expression vector may include any suitable expression vector that operably links the nucleic acid coding region of the referenced polypeptide to any control sequence capable of effecting expression of the gene product. Similarly, the cell may be any prokaryotic or eukaryotic cell capable of expressing one or more of the described polypeptides. The cell may include a single cell capable of expressing all of the described polypeptides, separate cells capable of expressing each individual polypeptide, or any combination thereof.

[0071] In one embodiment, the first key polypeptide comprises a third binding domain, and the second binding domain and / or the third binding domain bind (i) to a different moiety on the surface of the same cell than the first binding domain, or (ii) to a different moiety at a synapse between two contacting cells than the first binding domain. In another embodiment, the second binding domain and the third binding domain bind to different moieties on the surface of different cells.

[0072] In one embodiment, the composition further comprises (c) an expression vector encoding and / or a cell expressing at least a second key polypeptide capable of binding to the first cage structure region, wherein the key polypeptide comprises a fourth binding domain. The second binding domain and / or the fourth binding domain bind (i) to a different portion of the surface of the same cell than the first binding domain, or (ii) to a different portion of the synapse between two contacting cells than the first binding domain. In another embodiment, the second binding domain and the fourth binding domain bind (i) to a different portion of the surface of the same cell than the first binding domain, or (ii) to a different portion of the synapse between two contacting cells than the first binding domain. (ii) They bind to different parts of the synapse between two contacting cells.

[0073] In another embodiment, the first cage polypeptide further comprises a fifth binding domain, wherein the fifth binding domain and / or the first binding domain bind (i) to a different portion of the surface of the same cell than the second, third, and / or fourth binding domain, or (ii) to a different portion of the synapse between two contacting cells than the second, third, and / or fourth binding domain. In one embodiment, the fifth binding domain and the first binding domain bind (i) to a different portion of the surface of the same cell, or (ii) to a different portion of the synapse between two contacting cells.

[0074] In a further embodiment, the composition further comprises (d) an expression vector encoding and / or a cell expressing at least a second cage polypeptide comprising: (i) a second structural region; (ii) a second latch region further comprising one or more bioactive peptides; and (iii) a sixth binding domain, wherein the second structural region interacts with the second latch region to prevent activity of the one or more bioactive peptides. The first key and / or second key polypeptides can bind to the second structural region to activate one or more biologically active peptides. The sixth binding domain and / or the first binding domain bind (i) to a different portion of the surface of the same cell than the second, third, and / or fourth binding domain, or (ii) to a different portion of the synapse between two contacting cells than the second, third, and / or fourth binding domain. In one embodiment, the sixth binding domain and the first binding domain bind (i) to a different portion of the surface of different cells, or (ii) to a different portion of the synapse between two contacting cells.

[0075] In another embodiment, the composition further comprises (e) an expression vector encoding and / or a cell expressing a decoy cage polypeptide comprising: (i) a decoy structural region; (ii) a decoy latch region optionally further comprising one or more biologically active peptides; and (iii) a seventh binding domain, wherein the decoy structural region interacts with the first key polypeptide and / or the second key polypeptide to prevent them from binding to the first and / or second cage polypeptide, and the seventh binding domain binds to the same moiety on the surface of a cell as the second binding domain, the third binding domain, and / or the fourth binding domain. In one embodiment, the seventh binding domain and the first binding domain and / or the second binding domain bind to (i) different moieties on the surface of the same cell, or (ii) different moieties at a synapse between two contacting cells. In another embodiment, the seventh binding domain binds to a moiety present on a cell at a level equal to or greater than that bound by the second binding domain, the third binding domain, and / or the fourth binding domain.

[0076] In one embodiment, the first binding domain, the second binding domain, the third binding domain (if present), the fourth binding domain (if present), the fifth binding domain (if present), the sixth binding domain (if present), and / or the seventh binding domain (if present) comprise a polypeptide capable of binding to a moiety present on a cell surface, including proteins, sugars, and lipids. In one embodiment, one or more binding proteins comprise a cell surface protein-binding polypeptide.

[0077] Cage and key polypeptides The polypeptides disclosed herein can be used as cage polypeptides that sequester a bioactive peptide in an inactive state (until activated by a key polypeptide that binds to the cage polypeptide, as described herein), and the binding domain can be a This may serve to target the polypeptide to a binding entity. In one embodiment, the polypeptide is part of a "protein switch (together with one or more appropriate key polypeptides)," wherein the cage polypeptide and the key polypeptide comprise binding domains that bind to different targets, and the key polypeptide binds to the cage polypeptide and triggers activation of the bioactive peptide only when the different targets are closely associated, such that the cage and key polypeptides are co-localized while bound to their targets.

[0078] In some embodiments, the cage polypeptide comprises a helix bundle comprising 2-7 alpha helices. The helix bundle is fused to one or more binding domains. The one or more binding domains and the helix bundle are not both present in the same naturally occurring polypeptide.

[0079] In each embodiment, the N-terminal and / or C-terminal 60 amino acids of each cage polypeptide may be optional, and the terminal 60 amino acid residues may be modified, such as by substituting all or a portion of the latch with a biologically active peptide. In one embodiment, the N-terminal 60 amino acid residues are optional, in another embodiment, the C-terminal 60 amino acid residues are optional, and in a further embodiment, each of the N-terminal 60 amino acid residues and the C-terminal 60 amino acid residues are optional. In one embodiment, these optional N-terminal and / or C-terminal 60 residues are not included in determining percent sequence identity. In another embodiment, optional residues may be included in determining percent sequence identity.

[0080] In some embodiments, the first cage polypeptide comprises five or fewer alpha helices, four or fewer alpha helices, three or fewer alpha helices, or two or fewer alpha helices, wherein the structural region comprises at least one alpha helix and the latch region comprises at least one alpha helix. In some embodiments, the structural region of the first cage polypeptide comprises one alpha helix. In some embodiments, the structural region of the first cage polypeptide comprises two alpha helices. In some embodiments, the structural region of the first cage polypeptide comprises three alpha helices.

[0081] In some embodiments, the first cage polypeptide, the first key polypeptide, the second key polypeptide, and / or the decoy polypeptide are further modified to alter (i) hydrophobicity, (ii) hydrogen bond network, (iii) binding affinity to each, and / or (iv) any combination thereof. In some embodiments, the cage polypeptide and / or the key polypeptide are modified to reduce hydrophobicity. In some embodiments, the latch region is mutated to reduce hydrophobicity. For example, hydrophobic amino acids are known: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). In some embodiments, one or more hydrophobic amino acids are replaced with polar amino acids, e.g., serine (Ser), threonine (Thr), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), and tyrosine (Tyr). In some embodiments, the interface between the latch region and the structural region of the first cage polypeptide comprises a hydrophobic amino acid to polar amino acid residue ratio of 1:1 to 10:1, e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the interface between the latch region and the structural region comprises a hydrophobic amino acid to polar amino acid residue ratio of 1:1. In some embodiments, the interface between the latch region and the structural region comprises a hydrophobic amino acid to polar amino acid residue ratio of 2:1. In some embodiments, the interface between the latch region and the structural region comprises a hydrophobic amino acid to polar amino acid residue ratio of 3:1. In some In embodiments, the interface between the latch region and the structural region comprises a 4:1 ratio of hydrophobic amino acids to polar amino acid residues. In some embodiments, the interface between the latch region and the structural region comprises a 5:1 ratio of hydrophobic amino acids to polar amino acid residues. In some embodiments, the interface between the latch region and the structural region comprises a 6:1 ratio of hydrophobic amino acids to polar amino acid residues. In some embodiments, the interface between the latch region and the structural region comprises a 7:1 ratio of hydrophobic amino acids to polar amino acid residues. In some embodiments, the interface between the latch region and the structural region comprises an 8:1 ratio of hydrophobic amino acids to polar amino acid residues. In some embodiments, the interface between the latch region and the structural region comprises a 9:1 ratio of hydrophobic amino acids to polar amino acid residues. In some embodiments, the interface between the latch region and the structural region comprises a 10:1 ratio of hydrophobic amino acids to polar amino acid residues.

[0082] In some embodiments, one, two, three, or more large hydrophobic residues (e.g., isoleucine, valine, or leucine) in the latch region are mutated to serine, threonine, or a smaller hydrophobic amino acid residue (e.g., valine (if the starting amino acid is isoleucine or leucine) or alanine).

[0083] In some embodiments, the first cage polypeptide comprises an amino acid residue buried at the interface between the latch region and the structural region of the first cage polypeptide, wherein the buried amino acid residue at the interface has a side chain that includes a nitrogen or oxygen atom that participates in hydrogen bonding.

[0084] In some aspects, the present disclosure provides a non-naturally occurring polypeptide, comprising: (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a caged polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, and 27278-27321, or a caged polypeptide listed in Table 7, Table 8, or Table 9, excluding optional amino acid residues, wherein the N-terminal and / or C-terminal 60 amino acids of the polypeptide are optional; (b) one or more polypeptide binding domains.

[0085] In one embodiment, the non-naturally occurring polypeptide is (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, and 27,278-27,321, excluding amino acid residues within the latch region; (b) one or more polypeptide binding domains.

[0086] In another embodiment, the non-naturally occurring polypeptide is (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a caged polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, or a caged polypeptide listed in Table 7, Table 8, or Table 9, wherein the N-terminal and / or C-terminal 60 amino acids of the polypeptide are optionally, a polypeptide; and (b) one or more polypeptide binding domains.

[0087] In further embodiments, the polypeptide has an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical to the amino acid sequence of a cage polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27,278-27,321, or a cage polypeptide listed in Table 7, Table 8, or Table 9, including optional amino acid residues.

[0088] In one embodiment, the non-naturally occurring polypeptide is (a) a polypeptide comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a disclosed cage polypeptide selected from the group consisting of SEQ ID NOs: 27359-27392, excluding optional amino acid residues; (b) one or more polypeptide binding domains.

[0089] In another embodiment, the polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a disclosed cage polypeptide selected from the group consisting of SEQ ID NOs: 27359-27392, including optional residues. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] Table 2. Other exemplary cage polypeptides (see also SEQ ID NOs: 92-14317, 27094-27117, 27120-27125, 27,728-27321, and the cage polypeptides listed in Tables 7, 8, and 9). Exemplary reference cage polypeptides: Latch regions are indicated in brackets [ ] 6His-MBP-TEV, 6His-TEV, and flexible linker sequences are underlined. The fused functional domains (DARPin, split intein components, and fluorescent protein) are shown in bold. ●Functional peptides are shown in italics and underlined. Exemplary positions that have been mutated to any amino acid to modulate responsiveness are shown in bold and underlined. These positions are exemplary and are not a complete list of residues that can modulate responsiveness. C-terminal sequences that can be deleted to adjust response are included in parentheses. The range from position 1 (1) to all residues included in the parentheses can be removed, starting at the C-terminus and removing consecutive residues there. All sequences within brackets are optional [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] [Table 2-18] [Table 2-19] [Table 2-20] [Table 2-21] [Table 2-22] [Table 2-23]

[0090] In another embodiment, the present disclosure provides a non-naturally occurring polypeptide comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27359-27392, including optional amino acid residues. In one embodiment, the polypeptide further comprises one or more binding domains. In a further embodiment, the polypeptide comprises an amino acid linker connecting the polypeptide and one or more binding domains, such as those disclosed herein.

[0091] As disclosed herein, exemplary polypeptides of the present disclosure have been identified and mutational analysis has been performed. Furthermore, different designs starting from the same exemplary polypeptide result in different amino acid sequences while maintaining the same intended function. In various embodiments, a given amino acid can be substituted with a residue having similar physicochemical properties, for example, substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for each other), or substituting one polar residue for another (such as Lys for Arg, Glu for Asp, or Gln for Asn). Other such conservative substitutions, such as substituting entire regions with similar hydrophobic properties, are known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity is retained. Amino acids can be grouped according to similarities in the properties of their side chains (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)). (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Uncharged Polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); (4) Basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be classified into groups based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral Hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys s, Arg, (5) residues affecting chain orientation: Gly, Pro, (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example, Ala for Gly or Ser, Arg for Lys, Asn for Gln or His, Asp for Glu, Cys for Ser, Gln for Asn, Glu for Asp, Gly for Ala or Pro, His for Asn or Gln, Ile for Leu or Val, Leu for Ile or Val, Lys for Arg, Gln, or Glu, Met for Leu, Tyr, or Ile, Phe for Met, Leu, or Tyr, Ser for Thr, Thr for Ser, Trp for Tyr, Tyr for Trp, and / or Phe for Val, Ile, or Leu.

[0092] In some aspects, a cage polypeptide comprises an interface between the latch region and the structural region of one or more cage polypeptides of any of the compositions or methods disclosed herein. In one embodiment of the polypeptides of the first and second aspects of the present disclosure, the interface residues between the latch region and the structural region are predominantly (i.e., 50%, 60%, 70%, 75%, 80%, 85%, 90%, or more) hydrophobic residues. In one embodiment, the interface residues are predominantly valine, leucine, isoleucine, and alanine residues. In a further embodiment, the interface between the latch region and the structural region of the polypeptide comprises a ratio of hydrophobic amino acids to polar amino acid residues of 1:1 to 10:1. Cage polypeptides can be "tuned" to modify the strength of the interaction between the latch region and the structural region as deemed appropriate for the intended use. In one embodiment, one, two, three, or more large hydrophobic residues in the latch region (including but not limited to, isoleucine, valine, or leucine) are mutated to serine, threonine, or smaller hydrophobic amino acid residues (including but not limited to, valine (if the starting amino acid residue is isoleucine or leucine) or alanine). In this embodiment, the adjustment weakens structural region-latch affinity. In some aspects, the cage polypeptide, e.g., the first cage polypeptide, comprises amino acid residues buried in the interface between the latch region and the structural region of the cage polypeptide. In another embodiment, the buried amino acid residues in the interface comprise amino acid residues having side chains that include nitrogen or oxygen atoms that participate in hydrogen bonding. The adjustment can include increasing or decreasing the number of hydrogen bonds present in the interface. The adjustment can include changing amino acids to increase or decrease the hydrophobicity of the interface. The adjustment can include making amino acid changes to reduce hydrophobic packing at the interface (e.g., by replacing leucine with alanine). The adjustments can include introducing amino acid changes that create insufficient hydrogen bonds buried at the interface (e.g., by replacing leucine with serine). Based on the teachings herein, one of skill in the art will understand that such adjustments can take any number of forms, depending on the desired structural region-latch region affinity.

[0093] In certain embodiments, the polypeptides of the first and second aspects of the present disclosure comprise one or more bioactive peptides in at least one of the alpha helices, such as within the latch domain, where the one or more bioactive peptides are capable of selectively binding to a defined target. As described herein, the non-naturally occurring polypeptides of the first and second aspects disclosed herein can be used as cage polypeptides that sequester the bioactive peptide in an inactive state (until activated by a key polypeptide that binds to the cage polypeptide, as described herein), and the binding domain can serve to target the polypeptide to an entity to which the binding domain binds. In one embodiment, the polypeptide is part of a "protein switch" (together with one or more appropriate key polypeptides) in which the cage polypeptide and the key polypeptide comprise binding domains that bind to different targets, and the key polypeptide binds to the cage polypeptide and triggers activation of the bioactive peptide only when the different targets are closely associated, such that the cage and key polypeptides are co-localized while bound to their targets.

[0094] Any binding domain may be used as appropriate for the intended use. In a non-limiting embodiment, the one or more biologically active peptides may include one or more biologically active peptides selected from the group consisting of SEQ ID NOs: 60, 62-64, 66, 27052, 27053, and 27059-27093. [Table 3-1] [Table 3-2]

[0095] In a third aspect, the present disclosure provides a key polypeptide comprising a key domain linked to one or more binding domains, wherein the key polypeptide is capable of specifically binding to a cage polypeptide of any embodiment of the first and / or second aspects of the present disclosure. As described herein, the non-naturally occurring polypeptides of the first and second aspects disclosed herein can be used as cage polypeptides that sequester a bioactive peptide in an inactive state (until activated by a key polypeptide that binds to the cage polypeptide, as described herein), and the binding domain can serve to target the polypeptide to an entity to which the binding domain binds. In one embodiment, the polypeptide is part of a "protein switch (together with one or more appropriate key polypeptides)," wherein the cage polypeptide and the key polypeptide comprise binding domains that bind to different targets, and the key polypeptide binds to the cage polypeptide and triggers activation of the bioactive peptide only when the different targets are closely associated, such that the cage and key polypeptides are co-localized while bound to their targets. Thus, in one embodiment, the key polypeptide specifically binds to the cage polypeptide and activates one or more bioactive peptides. In various non-limiting embodiments, the key polypeptide is (a) an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a key polypeptide disclosed herein (not including optional amino acid residues), a key polypeptide selected from SEQ ID NOs: 27393-27398, 14318-26601, 26602-27015, 27016-27050, and 27,322-27,358, and a key polypeptide listed in Table 7, Table 8, and / or Table 9; (b) one or more binding domains. [Table 4]

[0096] In another embodiment, a non-naturally occurring polypeptide comprises a polypeptide comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a key polypeptide selected from the group consisting of SEQ ID NOs: 26602-27050 and 27,322-27,358, as described in more detail below. ●Key layout is normal characters 6His-MBP-TEV, 6His-TEV, and flexible linker sequences are underlined. • The sequences in bold and italics are optional residues required for biotinylation of MBP_key. All sequences within brackets are optional - Any number of consecutive amino acids at the N- or C-terminus of the non-optional key sequence can be deleted to adjust responsiveness. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8]

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

Table 5-20

Table 5-21

Table 5-22

Table 5-23

Table 5-24

Table 5-25

[0097] In certain embodiments, the key polypeptide comprises an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a key polypeptide in Table 6, 7 (polypeptides having odd-numbered SEQ ID NOs: 27127 and 27277), Table 8, and / or Table 9. In another embodiment, the key polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a key polypeptide in Table 8. In another embodiment, the key polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a key polypeptide in Table 9. In one embodiment of each of the above, percent specificity may be determined without the optional N- and C-terminal 60 amino acids, and in another embodiment, percent specificity may be determined with the optional N- and C-terminal 60 amino acids. [Table 6-1] [Table 6-2]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 8

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

[0098] A. Binding Domain In various embodiments of the polypeptides of the present disclosure, the polypeptide comprises one or more (i.e., 1, 2, 3, or more) binding domains. Any suitable binding domain may be used as appropriate for the intended use. In one embodiment, the one or more binding domains comprise a cell surface protein-binding polypeptide. In one such embodiment, the cell surface protein-binding polypeptide is on a tumor cell. In another embodiment, the cell surface protein-binding polypeptide is an oncoprotein. In further embodiments, the one or more binding domains comprise an antigen-binding polypeptide (e.g., Fab', F(ab')2, Fab, Fv, rIgG, recombinant single-chain Fv fragment (scFv), VH single domain, bivalent or bispecific molecule, diabody, triabody, and tetrabody) directed against the cell surface moiety to be bound. The protein is selected from the non-limiting group including but not limited to a protein, a DARPin, a nanobody, an affibody, a monobody, an adnectin, an alphabody, an albumin binding domain, an adheren, an affilin, an affimer, an affitin / nanophytin, an anticalin, an armadillo repeat protein, an atrimer / tetranectin, an avimer / maxibody, a centrin, a finomer, a Kunitz domain, an obody / OB-fold, a pronectin, a lipibody, and a computationally designed protein. In another embodiment, the cell surface protein binding domain binds to a cell surface protein on a cell selected from the non-limiting group including: a tumor cell, a cancer cell, an immune cell, a leukocyte, a lymphocyte, a T cell, a regulatory T cell, an effector T cell, a CD4+ effector T cell, a CD8+ effector T cell, a memory T cell, an autoreactive T cell, an exhausted T cell, a natural killer T cell (NKT cell), a B cell, a dendritic cell, a macrophage, an NK cell, a cardiac cell, a lung cell, a muscle cell, an epithelial cell, a pancreatic cell, a skin cell, a CNS cell, a neuron, a muscle cell, a skeletal muscle cell, a smooth muscle cell, a liver cell, a kidney cell, a bacterial cell, and a yeast cell. In yet another embodiment, the cell surface protein binding domain is selected from the group consisting of Her2, EGFR, EpCAM, B7-H3, ROR1, GD2, GPC2, αvβ6, Her3, L1CAM, BCMA, GPCR5d, EGFRvIII, CD20, CD22, CD3, CD4, CD5, CD8, CD19, CD27, CD28, CD30, CD33, CD48, IL3RA, platelet tissue factor, CLEC12A, CD82, TNFRSF1B, ADGRE2, ITGB5, CD96, CCR1, PTPRJ, CD70, and LILRB. 2, LTB4R, TLR2, LILRA2, ITGAX, CR1, EMC10, EMB, DAGLB, P2RY13, LILRB3, LILRB4, SLC30A1, LILRA6, SLC6A6, SEMA4A, TAG72, FRα, PMSA, mesothelin, LIV-1, CEA, MUC1, PD1, BLIMP1, CTLA4, LAG3, TIM3, TIGIT, CD39, nectin-4, cancer markers, healthy tissue markers, and cardiac markers.

[0099] In non-limiting embodiments, one or more binding domains comprise an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399-27403. [Table 10]

[0100] In further non-limiting embodiments, the cage polypeptide having a binding domain comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the non-limiting group of SEQ ID NOs: 27404-27446. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6] [Table 11-7] [Table 11-8] [Table 11-9] [Table 11-10] [Table 11-11]

[0101] In one embodiment, the polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the non-limiting group of SEQ ID NOs: 27404-27446, including optional residues. In another embodiment, the polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the non-limiting group of SEQ ID NOs: 27404-27446, excluding optional residues.

[0102] As disclosed herein, the bioactive peptides sequestered by the polypeptides of the present disclosure are located within a latch region. The latch region is indicated by brackets in the sequence of each cage polypeptide. The bioactive peptide can be added to the latch region without removing any residues from the latch region, or it can replace one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid residues in the cage scaffold latch region to generate the final polypeptide. Thus, the latch region can be significantly modified with the inclusion of a bioactive peptide. In one embodiment, optional residues are not included in determining percent sequence identity. In another embodiment, latch region residues can be included when determining percent sequence identity. In a further embodiment, the optional residues and latch residues can each be excluded when determining percent sequence identity.

[0103] In one embodiment of this second aspect, the polypeptide is a polypeptide according to any embodiment or combination of embodiments of the first aspect and comprises the required 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length to the amino acid sequence of a recited reference cage polypeptide disclosed herein. In another embodiment, the polypeptide further comprises a biologically active peptide within (or substituting for) the latch region of the cage polypeptide.

[0104] The cage polypeptide can be a cage decoy polypeptide (i.e., without a bioactive peptide), see, e.g., SEQ ID NOS: 1-17, 2034-14317, and the specific cage polypeptides listed in Tables 7, 8, and / or 9, or can further comprise a bioactive peptide sequestered within the latch region of the cage scaffold polypeptide (present as a fusion with the cage scaffold polypeptide), as described in more detail herein (see, e.g., SEQ ID NOS: 18-49, 51-52, 54-59, 61, 65, 67-2033, 27094-27117, 27120-27125, and the specific cage polypeptides listed in Tables 7, 8, and / or 9). In certain embodiments, the cage polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide in Tables 7, 8, and / or 9.

[0105] In another embodiment, the cage polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide in Table 8. In another embodiment, the cage polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide in Table 9. In one embodiment of each of these embodiments, these optional N- and / or C-terminal 60 residues are not included in determining percent sequence identity, hi another embodiment, optional residues may be included in determining percent sequence identity.

[0106] In one embodiment of a key polypeptide disclosed herein, the polypeptide comprises an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical to an amino acid sequence selected from the non-limiting group of SEQ ID NOs: 27448-27459, where the parenthesized residues are optional. In one embodiment, the sequence identity determination includes any residue; in another embodiment, the sequence identity determination does not include any amino acid residue. [Table 12-1] [Table 12-2]

[0107] In some embodiments, the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide are (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a caged polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, and 27278-27321, or a caged polypeptide listed in Table 7, Table 8, or Table 9, excluding optional amino acid residues, wherein the N-terminal and / or C-terminal 60 amino acids of the polypeptide are optional; (b) one or more first, fifth, sixth, or seventh binding domains.

[0108] In some embodiments, the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide are (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a caged polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, excluding optional amino acid residues; (b) one or more first, fifth, sixth, or seventh binding domains.

[0109] In some embodiments, the first cage polypeptide, the second polypeptide, and / or the decoy cage polypeptide comprises an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of a cage polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, excluding optional amino acid residues.

[0110] In some embodiments, the first key polypeptide and / or the second key polypeptide is (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 27393-27398, 14318-26601, 26602-27015, 27016-27050, 27,322-27,358, and the cage polypeptides listed in Table 7, Table 8, and / or Table 9; (b) one or more second, third, or fourth binding domains.

[0111] In some embodiments, the first key polypeptide and / or the second key polypeptide is (a) a polypeptide comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27393-27398 or 27394-27395, either excluding optional residues or including optional residues; (b) one or more second, third, or fourth binding domains.

[0112] In one embodiment of the composition of any embodiment or combination of embodiments of the present disclosure, the one or more bioactive peptides may include one or more bioactive peptides selected from the group consisting of SEQ ID NOs: 60, 62-64, 66, 27052, 27053, 27059-27093.

[0113] nucleic acid In one aspect, the present disclosure provides nucleic acids encoding polypeptides of any embodiment or combination of embodiments of each aspect disclosed herein. The nucleic acid sequences may comprise single- or double-stranded RNA or DNA, in genomic or cDNA form, or DNA-RNA hybrids, each of which may contain chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such recombinant nucleic acid sequences may include additional sequences useful for facilitating expression and / or purification of the encoded protein, including, but not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it will be clear to one of skill in the art which nucleic acid sequences encode the fusion proteins of the present disclosure.

[0114] In another aspect, the present disclosure provides an expression vector comprising a nucleic acid of any aspect of the present disclosure operably linked to an appropriate "control sequence." An "expression vector" includes a vector in which a nucleic acid coding region or gene is operably linked to any control sequence capable of effecting expression of the gene product. A "control sequence" operably linked to a nucleic acid sequence of the present disclosure is a nucleic acid sequence capable of effecting expression of a nucleic acid molecule. Control sequences need not be contiguous with the nucleic acid sequence, so long as they function to direct its expression. Thus, for example, intervening non-translated but transcribed sequences can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, enhancers, introns, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including, but not limited to, plasmid- and virus-based expression vectors. The control sequences used to drive expression of the disclosed nucleic acids in mammalian systems can be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF, EF1 alpha, MND, MSCV) or inducible (driven by any of a number of inducible promoters, including but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organism either as an episome or by integration into the host chromosomal DNA. In various embodiments, the expression vector can comprise a plasmid, a virus-based vector, or any other suitable expression vector.

[0115] cell In a further aspect, the present disclosure provides cells, e.g., host cells, therapeutic cells, or target cells, comprising a nucleic acid, expression vector (i.e., episomally or chromosomally integrated), or polypeptide disclosed herein, wherein the cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate an expression vector of the present disclosure using techniques including, but not limited to, bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-, DEAE-dextran-, polycation-, or viral-mediated transfection. In one embodiment, the viral vector comprises an adenoviral vector, a vaccinia virus vector, an AAV vector, a retroviral vector, a lentiviral vector, an alphavirus vector, or any combination thereof. In one embodiment, the cells are (a) a first nucleic acid encoding a polypeptide of any embodiment or combination of embodiments of a cage polypeptide of the present disclosure, operably linked to a first promoter; (b) a second nucleic acid encoding a polypeptide of any embodiment or combination of embodiments of a key polypeptide of the present disclosure, wherein the key polypeptide binds to a structural region of the cage polypeptide to induce a conformational change in the cage polypeptide when the cage and key are co-localized by binding of their respective binding domains to a target, and the second nucleic acid is operably linked to a second promoter.

[0116] In some aspects, the cell can be an in vitro cell. In some aspects, the cell is an in vivo cell. In some aspects, the cell is an ex vivo cell.

[0117] The cells may contain a single cage polypeptide encoding nucleic acid and a single key polypeptide encoding nucleic acid, or may contain multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) first and second nucleic acids, in one such embodiment, each second nucleic acid binds to a structural region and encodes a different cage polypeptide encoded by the multiple first nucleic acids. In another embodiment, each second nucleic acid may encode a key polypeptide that binds to a structural domain and can induce a conformational change in more than one cage polypeptide encoded by multiple first nucleic acids.

[0118] The cells referred to herein may be therapeutic target cells or therapeutic cells. In some embodiments, the target cells may be tumor cells. In some embodiments, the target cells may be healthy cells. In some embodiments, the first cell portion, the second cell portion, or both, are present on or in healthy cells. In some embodiments, the first cell portion, the second cell portion, or both, are present on or in diseased cells. In some embodiments, the first cell portion, the second cell portion, or both, are present on or in tumor cells or cancer cells. In some embodiments, the first cell portion, the second cell portion, or both, are present on or in immune cells. In some embodiments, the first cell portion, the second cell portion, or both, are present on or within a cell selected from a leukocyte, a lymphocyte, a T cell, a regulatory T cell, an effector T cell, a CD4+ effector T cell, a CD8+ effector T cell, a memory T cell, an autoreactive T cell, an exhausted T cell, a natural killer T cell (NKT cell), a B cell, a dendritic cell, a macrophage, an NK cell, and any combination thereof. In some embodiments, the first cell portion, the second cell portion, or both, are present on or within a cell selected from a cardiac cell, a lung cell, a muscle cell, an epithelial cell, a pancreatic cell, a skin cell, a CNS cell, a neuron, a muscle cell, a skeletal muscle cell, a smooth muscle cell, a liver cell, a kidney cell, a bacterial cell, a yeast cell, and any combination thereof.

[0119] Binding domain / cellular part Any suitable binding domain may be used in the compositions of the present disclosure as appropriate for the intended use. In some embodiments, the first, second, third, fourth, fifth, sixth, and / or seventh binding domains are antigen-binding polypeptides (Fab', F(ab')2, Fab, Fv, rIgG, recombinant single-chain Fv fragments (scFv), V) directed against the cell surface moiety to be bound. H and any combination thereof.

[0120] In another embodiment, the first, second, third, fourth, fifth, sixth, and / or seventh binding domain binds to a cell surface protein on a cell selected from the non-limiting group including: a tumor cell, a cancer cell, an immune cell, a leukocyte, a lymphocyte, a T cell, a regulatory T cell, an effector T cell, a CD4+ effector T cell, a CD8+ effector T cell, a memory T cell, an autoreactive T cell, an exhausted T cell, a natural killer T cell (NKT cell), a B cell, a dendritic cell, a macrophage, an NK cell, a cardiac cell, a lung cell, a muscle cell, an epithelial cell, a pancreatic cell, a skin cell, a CNS cell, a neuron, a muscle cell, a skeletal muscle cell, a smooth muscle cell, a liver cell, a kidney cell, a bacterial cell, and a yeast cell.

[0121] In further embodiments, the first, second, third, fourth, fifth, sixth, and / or seventh binding domains are selected from the group consisting of Her2, EGFR, EpCAM, B7-H3, ROR1, GD2, GPC2, αvβ6, Her3, L1CAM, BCMA, GPCR5d, EGFRvIII, CD20, CD22, CD3, CD4, CD5, CD8, CD19, CD27, CD2 8, CD30, CD33, CD48, IL3RA, platelet tissue factor, CLEC12A, CD82, TNFRSF1B, ADGRE2, ITGB5, CD96, CCR1, PTPRJ, CD70, LILRB2, LTB4R, TLR2, LILRA2, ITGAX, CR1, EMC10, EMB, DAGLB, P2RY13, LILRB3, LILRB4, SLC30A1, LILRA6, SLC6A6, SEMA4A, TAG72, FRα, PMSA, mesothelin, LIV-1, CEA, MUC1, PD1, BLIMP1, CTLA4, LAG3, TIM3, TIGIT, CD39, nectin-4, cancer markers, healthy tissue markers, and cardiac markers. In further embodiments, the first, second, third, fourth, fifth, sixth, and / or seventh binding domains comprise an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399-27403.

[0122] In one embodiment of a composition of any embodiment or combination of embodiments of the present disclosure, (i) the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide, and (ii) the first and / or second key polypeptide comprise at least one cage polypeptide and at least one key polypeptide that comprise an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the cage polypeptide and key polypeptide, respectively, in the same row or rows of Tables 7, 8, or 9 (i.e., each cage polypeptide in row 2, column 1 of the Table can be used with each key polypeptide in row 2, column 1 of the Table, etc.), with the proviso that each cage polypeptide and each key polypeptide further comprises one or more binding domains.

[0123] In one embodiment, the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide are (a) an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the non-limiting group consisting of SEQ ID NOs: 27359-27392, with or without optional residues; (b) a binding domain comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399 to 27403.

[0124] In another embodiment, the first key polypeptide and / or the second key polypeptide is (a) an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27393-27398 or 27394-27395, with or without optional residues; (b) a binding domain comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399 to 27403.

[0125] In another embodiment, the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide comprises an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27404-27446. In another embodiment, the first key polypeptide and / or the second key polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27448-27459. In a further embodiment, (i) the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide are at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27404-27446. and (ii) the first key polypeptide and / or the second key polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27448-27459.

[0126] effector In some embodiments, effectors useful in the present disclosure comprise one or more binding moieties. In some embodiments, the effector comprises an antibody or antigen-binding fragment thereof, a T cell receptor, a DARPin, a bispecific or bivalent molecule, a nanobody, an affibody, a monobody, an adnectin, an alfbody, an albumin-binding domain (dmain), an adheren, an affilin, an affimer, an affitin / nanophytin, an anticalin, an armadillo repeat protein, an atrimer / tetranectin, an avimer / maxibody, a centilin, a finomer, a Kunitz domain, an obody / OB-fold, a pronectin, a lipibody, a computationally designed protein, a protease, a ubiquitin ligase, a kinase, a phosphatase, and / or a proteolysis-inducing effector, or any combination thereof. In some embodiments, the antigen-binding moiety is a Fab', F(ab')2, Fab, Fv, rIgG, a recombinant single-chain Fv fragment (scFv), and / or a V H Contains a single domain.

[0127] In some embodiments, the effector is a therapeutic cell. In some embodiments, the therapeutic cell comprises an immune cell. In certain embodiments, the cell is selected from a T cell, a stem cell, a NK cell, a B cell, or any combination thereof. In some embodiments, the stem cell is an induced pluripotent stem cell.

[0128] In some embodiments, the effector kills a cell comprising the first binding moiety and the second binding moiety, resulting in receptor signaling (e.g., cytokines) in the cell comprising the first binding moiety and the second binding moiety, producing signaling molecules (e.g., cytokines, chemokines) in the vicinity of the cell comprising the first binding moiety and the second binding moiety, or causing differentiation of the cell comprising the first binding moiety and the second binding moiety.

[0129] In some embodiments, administration of the effector induces receptor signaling (e.g., cytokines) in cells comprising the first binding moiety and the second binding moiety. In some embodiments, administration of the effector induces signaling molecules (e.g., cytokines) in the vicinity of cells comprising the first binding moiety and the second binding moiety, including, but not limited to, CD4+ T cells that release cytokines within tumors to support CD8+ T cell effector function. In some embodiments, administration of the effector induces differentiation in cells comprising the first binding moiety and the second binding moiety.

[0130] Other embodiments of the present disclosure are directed to one or more cells comprising the compositions disclosed herein. In some embodiments, the cells further comprise an effector as disclosed herein. In some embodiments, the cells are tumor cells or cancer cells. In some embodiments, the therapeutic cells comprise immune cells. In some embodiments, the cells are selected from leukocytes, lymphocytes, T cells, regulatory T cells, effector T cells, CD4+ effector T cells, CD8+ effector T cells, memory T cells, autoreactive T cells, exhausted T cells, natural killer T cells (NKT cells), B cells, dendritic cells, macrophages, NK cells, and any combination thereof. In some embodiments, the cells are selected from cardiac cells, lung cells, muscle cells, epithelial cells, pancreatic cells, skin cells, CNS cells, neurons, myocytes, skeletal muscle cells, smooth muscle cells, liver cells, kidney cells, bacterial cells, yeast cells, and any combination thereof.

[0131] In some embodiments, proximity-dependent binding can be detected without an effector protein, so the compositions of the fourth and fifth aspects of the present disclosure do not contain an effector. In one embodiment of the compositions of any embodiment of the fourth and fifth aspects of the present disclosure, an effector is present. Any effector suitable for the intended use can be used. In certain embodiments, the effector binds to one or more biologically active peptides. In one embodiment, the effector is selected from the non-limiting group including Bcl2, GFP1-10, a small molecule, an antibody, an antibody drug conjugate, an immunogenic peptide, a protease, a T cell receptor, a cytotoxic agent, a fluorophore, a fluorescent protein, a cell adhesion molecule, an endocytic receptor, a phagocyte receptor, a magnetic bead, and a gel filtration resin, and a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27460-27469. [Table 13-1] [Table 13-2]

[0132] III. Methods of the Disclosure Some aspects of the present disclosure relate to methods of increasing cell selectivity in vitro, ex vivo, or in vivo. Other aspects of the present disclosure relate to methods of increasing selectivity of cells interacting with each other in vitro, ex vivo, or in vivo. Other aspects of the present disclosure relate to methods of targeting heterologous cells (three or more different cell types) in vitro, ex vivo, or in vivo. Other aspects of the present disclosure relate to methods of reducing off-target activity in vitro, ex vivo, or in vivo.

[0133] In some aspects, the present disclosure relates to methods for enhancing the selectivity of cells, comprising expressing a first cage polypeptide disclosed herein and a first key polypeptide disclosed herein in vitro, in vivo, or ex vivo. In some aspects, the present disclosure relates to methods for enhancing the selectivity of cells, comprising adding a first cage polypeptide disclosed herein and a first key polypeptide disclosed herein in vitro, in vivo, or ex vivo. The first cage polypeptide and one or more key polypeptides can be added to cells together (simultaneously) or separately, in vitro, in vivo, or ex vivo. Some embodiments of the present disclosure relate to methods of enhancing cell selectivity in vitro, ex vivo, or in vivo, comprising: (a) contacting a cell with (e.g., expressing or adding) a first cage polypeptide fused to a first binding domain; and (b) contacting a cell with (e.g., expressing or adding) a first key polypeptide fused to a second binding domain. In some embodiments, the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides.

[0134] Some embodiments of the present disclosure provide methods for increasing selectivity of cells interacting with each other in vitro, ex vivo, or in vivo, comprising: (a) contacting two or more cells with a first cage polypeptide fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides in the absence of co-localization with the key polypeptide, and wherein the first binding domain is capable of binding to a first cellular moiety present at a synapse between the two or more cells; and (b) contacting the two or more cells with a first key polypeptide fused to a second binding domain, wherein, upon co-localization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region and activating the one or more biologically active peptides, and wherein the second binding domain is capable of binding to a second cellular moiety present at a synapse between the two or more cells.

[0135] In some embodiments, the method further comprises contacting a second key polypeptide fused to a third binding domain with a synapse of two or more cells that also express the first cellular moiety, wherein upon co-localization with the first cage polypeptide, the second key polypeptide can bind to the cage structural region and activate one or more biologically active peptides, and the third binding domain can bind to a third cellular moiety present on the synapse of the two or more cells.

[0136] In some embodiments, the method further comprises contacting two or more cells with one or more decoy cage polypeptides fused to one or more decoy binding domains comprising the two or more cells, wherein each decoy cage polypeptide comprises a decoy structural region and, when co-localized with the first key polypeptide and the first cage polypeptide, can preferentially bind to the first key polypeptide, and each decoy binding domain can bind to a decoy cell portion within a synapse of the two or more cells.

[0137] Some embodiments of the present disclosure include a method of targeting heterologous cells (i.e., two or more different cell types) in vitro, ex vivo, or in vivo, wherein a first cell moiety and a second cell moiety are present on a first cell, and a first cell moiety and a third cell moiety are present on a second cell, the method comprising: (a) contacting the two or more cells with a first cage polypeptide fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more bioactive peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of co-localization with the key polypeptide, and wherein the first binding domain is capable of binding to the first cell moiety present on or within the two or more cells. (b) contacting two or more cells with a first key polypeptide fused to a second binding domain, wherein upon co-localization, the first key polypeptide is capable of binding to the cage structure region and activating one or more biologically active peptides, and the second binding domain is capable of binding to a second cellular moiety present on a cell that also comprises the first cellular moiety; and (c) contacting two or more cells with a second key polypeptide fused to a third binding domain, wherein upon co-localization, the second key polypeptide is capable of binding to the cage structure region and activating one or more biologically active peptides, and the third binding domain is capable of binding to a third cellular moiety present on a cell that also comprises the first cellular moiety.

[0138] In some embodiments, the method further comprises contacting the two or more cells with one or more decoy cage polypeptides fused to one or more decoy binding domains, each decoy cage polypeptide comprising a decoy structural region and capable of preferentially binding to the first key polypeptide or the second key polypeptide when co-localized with the first key polypeptide, the second key polypeptide, and / or the first cage polypeptide, and each decoy binding domain capable of binding to a decoy cell portion in a cell comprising the first cell portion and the second cell portion.

[0139] Some embodiments of the present disclosure include a method for reducing off-target activity in vitro, ex vivo, or in vivo, comprising: (a) contacting two or more cells with a first cage polypeptide fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more bioactive peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of co-localization with the key polypeptide, and wherein the first binding domain is capable of binding to a first cellular moiety present on the cells; and (b) contacting two or more cells with the first cage polypeptide fused to a second binding domain and the second key polypeptide. (c) contacting the two or more cells with a decoy cage polypeptide fused to a third binding domain, wherein the decoy cage polypeptide comprises a decoy structural region and, when co-localized with the key polypeptide and the first cage polypeptide, is capable of preferentially binding to the first key polypeptide and activating one or more biologically active peptides, and the second binding domain is capable of binding to a second cellular portion present on the cell that also includes the first cellular portion; and and contacting said third cellular moiety, said third cellular moiety being capable of binding to said third cellular moiety. In some embodiments, said third cellular moiety is present only on healthy cells.

[0140] As used herein, "contacting" refers to any means of contacting a first element with a second element. In some embodiments, contacting includes directly adding a first element, such as a polypeptide, to a second element, such as a cell, for example, by adding the protein to a cell culture. In some embodiments, contacting includes expressing a first element, such as a protein, in a target cell or in a cell in the same culture as the target cell with a nucleotide encoding the protein. In some embodiments, (a) contacting a cell with a first cage polypeptide fused to a first binding domain and (b) contacting a cell with a first key polypeptide fused to a second binding domain are performed simultaneously. In some embodiments, contacting (a) is performed before contacting (b). In some embodiments, contacting (b) is performed before contacting (a). In some embodiments, contacting includes introducing a polynucleotide encoding a polypeptide (e.g., a first cage polypeptide, a first key polypeptide, a second key polypeptide, and a decoy cage polypeptide).

[0141] The methods disclosed herein enhance the selectivity of cells for target cells. In some embodiments, co-localization of the first cage polypeptide and the key polypeptide enhances the selectivity of cells that express a high amount of the first cell moiety and the second cell moiety. In some embodiments, co-localization of the first cage polypeptide and the key polypeptide enhances the selectivity of cells that highly express the first and second cell moieties. In some embodiments, co-localization of the first cage polypeptide and the key polypeptide enhances the selectivity of cells that highly express the first and second cell moieties and cells that highly express the first and third cell moieties.

[0142] In a further aspect, the present disclosure provides a method of targeting an effector to a cell, comprising contacting a biological sample containing the cell with a polypeptide, nucleic acid, vector, cell, and / or composition of any embodiment or combination of embodiments of the present disclosure.

[0143] In another embodiment, the present disclosure provides a method for cell targeting, comprising: (a) a biological sample containing cells, (i) a cage polypeptide comprising: (i) a structural region; (ii) a latch region further comprising one or more bioactive peptides; and (iii) a first binding domain that targets a cell of interest, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides; and (ii) contacting the cell of interest with a key polypeptide comprising a second binding domain that targets the cell of interest, wherein the first binding domain and the second binding domain bind to (i) different portions on the surface of the same cell, (ii) the same portion on the surface of the same cell, (iii) different portions at a synapse between two contacting cells, or (iv) the same portion at a synapse between two contacting cells; contacting, wherein the contacting occurs for a time and under conditions to promote binding of the cage polypeptide and the key polypeptide to the cell of interest only if the cage polypeptide and the key polypeptide are co-localized in the cell of interest, promote binding of the key polypeptide to the cage structural region to displace the latch region and activate one or more biologically active peptides; (b) contacting the biological sample with one or more effectors under conditions that promote binding of the one or more effectors to the one or more activated biologically active peptides to form effector-bioactive peptide complexes; (c) optionally detecting the effector-bioactive peptide complex, wherein the effector-bioactive peptide complex is a measure of a cell of interest in the biological sample; and detecting.

[0144] Other aspects of the present disclosure relate to methods of preparing a subject in need of therapy, comprising administering a composition disclosed herein.Some aspects of the present disclosure relate to methods of preparing a subject in need of therapy, comprising administering a cell disclosed herein.

[0145] Some embodiments relate to methods of treating a disease or condition in a subject in need thereof, comprising administering an effector to the subject, wherein the subject is further administered a composition disclosed herein. In some embodiments, administration of the effector molecule kills cells comprising the first and second binding moieties, resulting in receptor signaling (e.g., cytokines) in the cells comprising the first and second binding moieties, producing signaling molecules (e.g., cytokines, chemokines) near the cells comprising the first and second binding moieties, or differentiating the cells comprising the first and second binding moieties. Any effector disclosed herein can be used in this method. In some embodiments, the effector binds to one or more bioactive peptides. In some embodiments, the effector comprises an antibody or antigen-binding fragment thereof, a T cell receptor, a DARPin, a bispecific or bivalent molecule, a nanobody, an affibody, a monobody, an adnectin, an alfbody, an albumin-binding domain (dmain), an adheren, an affilin, an affimer, an affitin / nanophytin, an anticalin, an armadillo repeat protein, an atrimer / tetranectin, an avimer / maxibody, a centilin, a finomer, a Kunitz domain, an obody / OB-fold, a pronectin, a lipibody, a computationally designed protein, or any combination thereof. In certain embodiments, the effector comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding portion thereof is selected from the group consisting of Fab', F(ab'), Fab, Fv, rIgG, recombinant single-chain Fv fragment (scFv), and / or V. H Contains a single domain.

[0146] In some embodiments, the effector is a therapeutic cell. In some embodiments, the therapeutic cell comprises a T cell, a stem cell, a NK cell, a B cell, or any combination thereof. In some embodiments, the therapeutic cell comprises an immune cell. In some embodiments, the therapeutic cell comprises a T cell. In some embodiments, the therapeutic cell comprises a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the therapeutic cell comprises a NK cell. [Example]

[0147] overview Natural biological systems integrate multiple protein-binding inputs through post-translational signaling cascades hard-coded for specialized functions. Synthetic systems capable of integrating multiple binding inputs through conformational switching may offer a general solution for predictively controlling diverse biological functions. We describe the computational design of a novel proximity-activated protein switch that performs "AND," "OR," and "NOT" Boolean logic operations and their combinations in response to precise combinations of protein-binding events. The switch activates via a conformational change only when all logical conditions are met, and a high-resolution X-ray crystal structure confirms the design model. We demonstrate the utility of this system for ultraspecific targeting of mammalian cells, which are distinguished in complex cell populations only by precise combinations of surface markers. Our study demonstrates that a de novo designed protein can perform computations at the surface of cells and integrate multiple distinct binding interactions into a single biological output.

[0148] A generalizable task that can perform complex logic in response to combinatorial binding events. We set out to design a protein system from scratch. We aimed for a modular system that could compute combinations of Boolean logic operations ("AND," "OR," and "NOT") when components are in close proximity and actuate a single binding interaction as the output (Figure 1a). Such a system would be broadly useful for regulating a wide range of cellular transactions in the nucleus, cytoplasm, and cell surface. Here, we develop such a system and apply it to cell targeting applications. We sought to integrate multiple protein binding inputs into a single output biological function by using Boolean logic to distinguish between cell subpopulations and exploiting the property that antigen binding at the cell surface increases the local concentration of bound proteins. For this system to be generally useful, actuation must be modular and independent of the identity of the target antigen.

[0149] We set out to design a novel protein switch in which the actuation domain is activated by the proximity of an additional engineered component. We designed a protein switch that is activated in solution. The pRotein (LOCKR) switch consists of a structural "cage" protein that uses a "latch" domain to sequester a functional peptide in an inactive conformation until the binding of another "key" protein triggers a conformational change that allows binding to an "effector" protein. The cage, key, and effector bind in a three-way equilibrium state, and the sensitivity of the switch can be tuned by adjusting the relative cage-latch and cage-key affinities. A new LOCKR protein was designed to be inactive in solution and potently activated only when the cage and key colocalize. We designed a new LOCKR switch with shorter helices, improved hydrophobic packing, and an additional hydrogen-bonding network to promote interaction specificity between the helices (Figure 4a-c and the "Computational Protein Design" section of the Methods chapter). The new design was nearly 100% monomeric and exhibited significantly reduced aggregation compared to other exemplary LOCKR switches (Figure 5a). The improved solution behavior of the new design allowed us to solve a 2.1 Å X-ray crystal structure. This closely matched the designed model (Fig. 1b, Table 16) with a root mean square deviation (RMSD) of 1.1 Å across all backbone atoms and an RMSD of 0.5 Å across all side-chain heavy atoms within the newly designed hydrogen-bonding network (Fig. 1b).

[0150] Using this novel design as a starting point, we developed a colocalization-dependent LOCKR (Co-LOCKR) switch (Figure 1c). To install an output function into Co-LOCKR, we chose the Bim-Bcl2 pair as a well-studied model system for peptide-protein binding (12). Bim was encoded into the latch as a sequestered peptide, and Bcl2 was used as the effector. Next, we added a targeting domain, which recruits the Co-LOCKR Cage and Key, to cells expressing the target antigen. While the targeting domain needs to bind to any cells expressing the target antigen, only cells bearing both antigens are required to recruit both the Cage and Key proteins and achieve colocalization-dependent activation (Figure 1d-e). Co-LOCKR operates via a thermodynamic mechanism based on reversible protein-protein interactions. Thus, complex formation can occur in solution (Figure 6a) or on a surface (Figure 6b). Cage-Key colocalization increases the local concentration, shifting the binding equilibrium to favor complex formation (Figure 6c). Below, we demonstrate the use of a Co-LOCKR switch to regulate the recruitment of a fluorophore-containing effector protein.

[0151] To assess the ability of Co-LOCKR to target cells co-expressing precise combinations of surface antigens, we performed mixed population flow cytometry by combining four K562 cell lines expressing Her2-eGFP, EGFR-iRFP, both, or neither. We developed a cytometry assay (Figure 1d). Using engineered ankyrin repeat protein (DARPin) domains (13, 14), we targeted the Cage and Key to Her2 and EGFR, respectively. If the system works as designed, only cells co-expressing both Her2 and EGFR should activate Co-LOCKR and bind Bcl2. The Cage contains a sequestered Bim peptide, the exposure of which requires the Key. This Co-LOCKR construct is called CL_C. H K E In this nomenclature, "CL" refers to Co-LOCKR, and C Hindicates that the cage is targeted to Her2, and K E indicates that the key is targeted to EGFR (Table 17). When mixed cell populations were co-incubated with equimolar dilutions of cage and key (3 μM to 1.4 nM), washed, and then AlexaFluor™ 594-labeled Bcl2 (Bcl2-AF594) was added, the expected sigmoidal binding curve was observed for Her2 / EGFR cells, but not for cells expressing either protein alone (Figure 1f). When cells were co-incubated with cage, key, and Bcl2-AF594 without washing, binding was similarly observed for Her2 / EGFR cells, although the Bcl2-AF594 signal was significantly higher at 111 nM CL_C. H K Eで The free cage and key may compete with cage-key-Bcl2 formed in solution for binding to a limited number of surface Her2 and EGFR proteins.

[0152] Next, we sought to tune the dynamic range of Co-LOCKR activation to enhance the sensitivity and responsiveness of colocalization-dependent activation. Our initial design aimed to maximize cage-latch affinity to ensure colocalization-dependence. We then investigated whether weakening cage-latch affinity could enhance signal strength without compromising logic computational power. Previous LOCKR switch sensitivity was tuned by shortening the latch to create a "toehold," but this also promoted aggregation (Figure 5b). Therefore, we focused on rationally designed mutations to tune the relative interaction affinity of the Co-LOCKR system to make it colocalization-dependent (Figure 7a-c). We mutated large hydrophobic residues in the latch region of the polypeptide at sequence number 27359 (I287A, I287S, I269S) or the cage (L209A) to weaken cage-latch affinity (Figure 2a). Biolayer interferometry showed that increasingly disruptive mutations improved responsiveness (Figure 8b), and flow cytometry demonstrated that tuning the cage-latch interface enhanced colocalization-dependent activation.H K E The regulated mutants of CL_C showed greater Bcl2-AF594 fluorescence in the same K562 / Her2 / EGFR cells (Fig. 2b, Fig. 8c). Colocalization-dependent activation of CL_C H K Eの This occurred even at low nanomolar concentrations and may be limited by the number of LOCKR proteins available in small incubation volumes (Figure 8d-e). Little effector binding was observed in cells expressing only Her2 or EGFR, suggesting that Co-LOCKR avoids targeting nearby cells in trans. Of the switches tested, I269S exhibited the greatest activation (Figure 9a), while the parent Co-LOCKR design exhibited the least off-target activation (Figure 9b), and I287A exhibited the highest fold specificity (Figure 9c).

[0153] Confocal microscopy revealed colocalization-dependent activation at the subcellular level. H K E recruited Bcl2-AF680 to the plasma membrane of HEK293T / Her2 / EGFR cells, but not to HEK293T / Her2 or HEK293T / EGFR (Fig. 2c). There was a close correspondence between regions of the plasma membrane showing colocalized Her2-eGFP and EGFR-iRFP signals with Co-LOCKR activation (Fig. 2c, column 6, quantified in Fig. 2d).

[0154] To assess the flexibility of Co-LOCKR, we specifically targeted alternative pairwise combinations of three cancer-associated antigens (Her2, EGFR, and EpCAM). We attempted to target these antigens at different levels by engineered K562 cell lines or human cancer cell lines (Figures 10a and 11a). Using the I269S mutant to maximize detection of low-level antigens, we found that (1) Co-LOCKR was able to distinguish the correct antigen pair in all cases, and (2) the magnitude of Bcl2 binding corresponded to the expression level of the lower-expressing of the two target antigens (Figures 3a and 11b-c). This is consistent with a stoichiometric binding mechanism for colocalization-dependent activation. Collectively, these results demonstrate the modularity of Co-LOCKR, which allows it to target several antigens produced at a wide range of different expression levels. While we chose DARPins as the targeting domain to allow for easy expression of Co-LOCKR mutants, any binding domain, including single-chain variable fragments, could alternatively be used (Figure 12).

[0155] A truly general technique for targeting any cell type in situ would require more complex logic, involving a combination of "AND," "OR," and "NOT" operations. In principle, Co-LOCKR's colocalization-dependent activation mechanism should be particularly well-suited to achieving this. "OR" logic could potentially be realized by adding a second key fused to the binding domain that targets an alternative surface marker (Figure 3b). "NOT" logic could potentially be realized by adding a decoy protein fused to the binding domain that targets a surface marker to be avoided. The decoy would act as a sponge to sequester the key, thereby preventing cage activation (Figure 3d).

[0156] Using Her2, EGFR, and EpCAM as model antigens (Ags), we first investigated the [Ag1 AND (Ag2 OR Ag3)] logic on the surface of cells (Figure 3b). To assess the configurability of Co-LOCKR targeting, we tested all three combinations: [Her2 AND (EGFR OR EpCAM)], [EGFR AND (Her2 OR EpCAM)], and [EpCAM AND (Her2 OR EGFR)]. In all cases, the correct cell subpopulations were targeted at levels consistent with the restrictive target antigen (Figure 3c). For example, CL_C E K H K Ep EGFR / EpCAM 低 10x background, Her2 / EGFR / EpCAM 低 59-fold above background, Her2 / EGFR / EpCAM 高 Target cells expressing 56-fold above background but lacking at least one antigen showed minimal off-target activation ( Fig. 3c , middle panel).

[0157] Next, CL_C H K Ep D E (D is decoy) and the same model antigen set was used to explore the [Ag1 AND Ag2 NOT Ag3] logic (Figure 3d). Consistent with the predicted stoichiometric activation mechanism, Ag3 needed to be expressed at higher levels than Ag2 so that excess decoy could sequester all of the Key molecules. Targeting the decoy to highly expressed EGFR completely abolished activation by Key targeted to low, but not high, levels of EpCAM. Cage-latch affinity (Figure 3d, Figure 13a) and decoy-key affinity (Figure 13b, Figure 14a-d) can be easily tuned to minimize leakiness or maximize activation.

[0158] The ability to perform complex logical operations with Co-LOCKR provides a level of control and flexibility not previously reported in targeting technologies. Furthermore, the ability to tune responsiveness with rationally designed point mutations allows for the rapid optimization of Co-LOCKR for a wide range of applications.

[0159] In contrast to current methods, Co-LOCKR computes the logic of single cells expressing precise combinations of antigens in cis, specifically inducing cytotoxicity against target cells without harming neighboring off-target cells that deliver only a subset of the target antigens. This complex logic (e.g., [Ag1 AND (Ag2 OR Ag3)] (Figure 3c) and [Ag The ability to perform [Ag1 AND Ag2 NOT Ag3] (Figure 3d) is unique to Co-LOCKR and cannot be achieved with existing technologies.

[0160] In general, the power of the Co-LOCKR system derives from the integration of multiple coherent or competing inputs, which determines the magnitude of a single response. The output signal exposure of the functional peptide on the latch is increased by key binding and countered by decoy competition. In principle, there is no limit to the number of individual molecules, allowing for arbitrarily complex logical operations. While the current work focuses on describing the system and demonstrating its ability to improve T cell-based cancer immunotherapy in vitro, the Co-LOCKR system is powerful for manipulating biology in any setting requiring computational specific targeting or proximity-based activation on the surface of cells.

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[0162] Method Computational protein design New LOCKR switch design As a starting point, the backbone of LOCKRa (SEQ ID NO: 6) was used as input coordinates into Rosetta protein design software. The latch residues, residues on the cage that contact the latch (defined by Rosetta™'s InterfaceByVector ResidueSelector), and the existing hydrogen bond network were held fixed in the coordinates of the input rotamer, while the positions of the remaining residues were redesigned as follows: First, an additional hydrogen bond network was designed using HBNet™. Next, a RosettaDesign™ calculation was performed to optimize hydrophobic packing and maintain the new hydrogen bond network using AtomPair constraints on the heavy atoms of each side chain hydrogen bond. This design procedure resulted in a novel asymmetric cage scaffold called asymLOCKR. Shorter versions of this design were then created by shortening the helix bundle by 12 residues based on visual inspection, reconnecting the helices with SGSGS linkers, and mutating several surface-exposed Arg and Lys residues to Glu to reduce the pI (Figure 1b). Finally, these scaffolds were converted to LOCKR by encoding the Bim sequence into a latch. The shorter version (sequence number 27359) was used as the parent Co-LOCKR. The RosettaScripts™ XML file used to perform these design calculations is shown below.

[0163] Design to tune relative cage-latch-key affinities to achieve colocalization-dependent activation Large hydrophobic residues in the latch of sequence number 27359 (I287A, I287S, I269S) or the cage (L209A) were rationally mutated to alanine or serine to weaken the cage-latch interface and increase Co-LOCKR sensitivity. Several amino acids at the N- or C-termini of the key were deleted to weaken the cage-key interface and decrease Co-LOCKR sensitivity / leakyness.

[0164] Designed to optimize Bcl2 Native Bcl2 was redesigned to improve solution behavior and stability. As a starting point, the C-terminal 32 residues of the transmembrane domain were deleted, and the long loop from residues 35–91 of Bcl2 was replaced with residues 35–50 of the homolog Bcl-xL, as previously described (30). Additional mutations were made using Rosetta™ and PROSS™ (31) to improve hydrophobic packing and stability. Additional surface mutations were rationally made to improve solubility and remove glycosylation sites.

[0165] Experimental Method Bacterial protein expression and purification E. coli Lemo21™ (DE3) cells harboring the pET21 plasmid encoding the gene of interest were incubated at 50 μg ml- with shaking at 225 rpm and 37°C. 1 The starter culture was grown overnight (10-16 hours) in 3 ml Luria-Bertani (LB) medium supplemented with carbenicillin. The starter culture was added to 500 ml of Studier TBM-5052 autoinduction medium supplemented with carbenicillin and grown at 37°C for 4-7 hours, followed by an additional 18-24 hours at 18°C. Cells were harvested by centrifugation at 5000 g for 15 minutes at 4°C and lysed in 20 ml of lysis buffer (25 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 mg ml) at room temperature. -1 Lysozyme (Sigma L6876, from chicken eggs), 0.1 mg ml -1DNase I (Sigma, DN25, from bovine pancreas). Cells were lysed by microfluidization in the presence of 1 mM phenylmethanesulfonyl fluoride (PMSF). The lysate was clarified by centrifugation at 24,000 g for 30 min at 4°C and passed through 2 ml of nickel-nitrilotriacetic acid agarose (Ni-NTA, Qiagen, 30250) pre-equilibrated with lysis buffer. The immobilized protein was washed twice with 15 column volumes (CV) of wash buffer (25 mM Tris pH 8.0 at room temperature, 300 mM NaCl, 40 mM imidazole), once with 5 CV of high-salt wash buffer (25 mM Tris pH 8.0 at room temperature, 1 M NaCl, 40 mM imidazole), once with 15 CV of wash buffer, and then eluted with 10 ml of elution buffer (25 mM Tris pH 8.0 at room temperature, 300 mM NaCl, 250 mM imidazole). The eluted protein was then concentrated (Amicon® Ultra-15 centrifugal filter unit, 10 kDa NMWL) and further purified by FPLC gel filtration using a Superdex™ 75 Increase 10 / 300 GL (GE) size exclusion column in Tris-buffered saline (TBS, 25 mM Tris pH 8.0 at room temperature, 150 mM NaCl). Fractions containing non-aggregated protein were pooled, concentrated, and mobilized with glycerol to a final concentration of 10% v / v, then quantified by absorbance at 280 nm (Nanodrop™), aliquoted, and flash-frozen in liquid nitrogen. Protein aliquots were stable at -80°C.

[0166] X-ray crystallography For crystallography screening, the hexahistidine tag was removed by TEV cleavage followed by Ni-NTA affinity chromatography prior to SEC / FPLC. Purified protein samples were collected at approximately 12 mg ml -1The crystals were concentrated to 100°C and screened using the JCSG+ and JCSG Core I-IV Screen (Qiagen) on a five-position deck Mosquito crystallization robot (ttplabtech) equipped with an active humidity chamber. Crystals were obtained after 2 to 14 days by droplet vapor diffusion at droplet ratios of 1:1, 2:1, and 1:2 protein solution to reservoir solution. The conditions under which crystals used for structure determination were obtained were 0.2 M disodium tartrate, 20% (w / v) PEG 3350, and no added cryoprotectant.

[0167] X-ray data collection and structure determination Protein crystals were looped and flash-frozen in liquid nitrogen. X-ray data sets were acquired at Lawrence Berkeley National Laboratory's Advanced Light Source via beamlines 8.2.1 and 8.2.2. The data sets were indexed and scaled using XDS (34), and phase information was obtained by molecular replacement (MR) using PHASER™ (35) in the Phenix™ software package (36). The design model was used for the initial MR search. Following MR, the model was refined using Phenix.autobuild (37). Efforts were made to reduce model bias by setting in-place rebuild to false and using simulated annealing and prime-and-switch phasing. The final model was created using an iterative process of manual building in COOT™ (38) and refinement in Phenix™. As reported by Phenix.Xtriage, translational non-crystallographic symmetry was present in the data. This complicated structure refinement and may explain the higher-than-expected R values ​​reported. The RMSDs of bond lengths, angles, and dihedral angles for idealized geometries were calculated using Phenix™. (36) The overall quality of the final model was assessed using MOLPROBITY™. (39) Table 16 summarizes the diffraction data and refinement statistics.

[0168] Bcl2 labeling For BLI experiments, wild-type, unoptimized Bcl2 with C-terminal Avi and 6xHis tags was enzymatically biotinylated using BirA according to the manufacturer's protocol (Avidity), purified with Ni-NTA, eluted in TBS, concentrated, flash-frozen in liquid nitrogen, and stored at -80°C. For flow cytometry experiments, Bcl2 containing a C-terminal cysteine ​​was purified by Ni-NTA and gel filtration as described above, with 0.5 mM TCEP added to the buffer. All fractions containing monomeric Bcl2 were combined, concentrated to 100 μM in TBS supplemented with 2% glycerol and 1 mM TCEP, and labeled overnight at 4°C with a 5-fold molar excess of Alexa Fluor™ 594C5 maleimide (Invitrogen A10256) or Alexa Fluor™ 680C2 maleimide (Invitrogen A20344). The labeling reaction was then dialyzed overnight against TBS supplemented with 10% glycerol and purified by gel filtration as described above. Fractions containing monomeric protein were pooled, concentrated, and mobilized with glycerol to a final concentration of 10% v / v. The fractions were then quantified by absorbance at 280 nm, aliquoted, and flash-frozen in liquid nitrogen. Protein aliquots were stable at -80°C. After thawing, protein aliquots were stored at 4°C for up to 1 week.

[0169] Biolayer Interferometry (BLI) BLI measurements were performed on an Octet® RED96 system (ForteBio) equipped with streptavidin (SA)-coated biosensors and analyzed with ForteBio data analysis software version 9.0.0.10. The assay buffer was HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM The buffer was 0.05% v / v EDTA, 0.05% v / v surfactant P20, and 0.5% nonfat dry milk, pH 7.4 at room temperature. Biotinylated Bcl2 protein was loaded onto the SA chip using a programmed threshold of 0.5 nm. A baseline was acquired by immersing the loaded biosensor in HBS-EP+ buffer. Association rates were observed by immersing the loaded biosensor in wells containing a range of LOCKR cage and key concentrations. Dissociation rates were observed by immersing the chip in the HBS-EP+ buffer wells used to acquire the baseline. For Figure 2 and Figure 8b-e, cage and key were diluted simultaneously to maintain a 1:1 stoichiometry.

[0170] Mammalian protein expression and purification The scFv-targeted Co-LOCKR proteins (anti-Her2_Cage_I269S and Key_anti-EGFR-scFv) were generated using the Daedalus system as previously described (40). The proteins were purified on a HisTrap™ FF crude protein purification column (GE catalog no. 17528601) followed by size-exclusion chromatography (GE Superdex 200 10 / 300 GL) and eluted with Dulbecco's phosphate-buffered saline supplemented with 5% glycerol.

[0171] cell culture K562 (CCL-243), Raji (CCL-86), A431 (CRL-1555), and HEK293T (CRL-3216) cells were obtained from the American Type Culture Collection (ATCC). 293T LentiX cells were purchased from Clontech. SKBR3 cells were a gift from David Hockenbery (Fred Hutchinson Cancer Research Center). K562 and Raji cells were cultured in 5% fetal bovine serum (FBS), 1 mM L-glutamine, 25 mM HEPES, and 100 μg / ml -1A431, SKBR3, HEK293T, and LentiX cells were cultured in RPMI-1640 (Gibco) supplemented with penicillin / streptomycin. Cells were cultured in 10% FBS, 1 mM L-glutamine, 25 mM HEPES, 100 μg / ml -1 Primary human T cells were cultured in DMEM high glucose (Gibco) supplemented with penicillin / streptomycin and 1 mM pyruvate. Primary human T cells were cultured in 10% human serum, 2 mM L-glutamine, 25 mM HEPES, 100 μg / ml -1 Cells were cultured in CTL medium consisting of RPMI-1640 supplemented with penicillin / streptomycin and 50 μM β-mercaptoethanol. All cells were cultured at 37°C and 5% CO2 and tested bimonthly for the presence of mycoplasma using the MycoAlert™ Mycoplasma Detection Kit (Lonza).

[0172] Generation of K562 and HEK293T cell lines HEK293T or LentiX cells were transiently transfected with lentiviral vectors encoding psPAX2 (Addgene plasmid #12260), pMD2.G (Addgene plasmid #12259) packaging plasmids, and either Her2-eGFP, EGFR-iRFP (for K562 cells), or EGFR-mCherry™ (for HEK293T cells) using linear 25 kDa polyethyleneimine (PEI; Polysciences). After 2 days, viral supernatants were concentrated by centrifugation at 8000 g for 18 h and diluted to 4 μg ml. -1 Polybrene (Sigma) was added to K562 cells or HEK293T cells. Flow cytometry was performed using the Herbicide showed that the 2-eGFP and EGFR-iRFP cell lines were transduced to 98% and the Her2-eGFP / EGFR-iRFP cell line was transduced to 88%.

[0173] Because K562 cells endogenously express low levels of EpCAM, an EpCAM knockout (KO) cell line was generated by nucleofection using the Alt-R® CRISPR-Cas9 System (IDT). Predesigned crRNAs specific for the human EpCAM gene (Hs.Cas9.EPCAM.1.AA and Hs.Cas9.EPCAM.1.AB, IDT) were reconstituted in nuclease-free duplex buffer and mixed with an equimolar concentration of tracrRNA. Annealing was performed by heating at 95°C for 5 minutes, followed by slow cooling to room temperature. The crRNA-tracrRNA duplex was complexed with SpCas9 nuclease V3 and Cas9 electroporation enhancer for 15 minutes at room temperature. Cas9 nuclease V3 and Cas9 electroporation enhancer were then added for 15 minutes at room temperature. The RNP complex was added to K562 cell line, and nucleofection was performed using a 4D Nucleofector mCherry™ (Lonza) with SF cell line buffer and the FF-120 program according to the manufacturer's instructions. After 4 days, cells that stained negative for EpCAM were FACS-sorted to greater than 99% purity.

[0174] EpCAM-high K562 cell lines were generated by transfecting Her2-eGFP, Her2-eGFP / EGFR-iRFP, and parental K562 cells with EpCAM-expressing lentiviruses prepared by transiently transfecting LentiX cells with psPAX2, pMD2.G, and a lentiviral vector encoding human EpCAM (UniProt:P16422, aa 1-314) using the CalPhos™ mammalian transfection kit (Clontech). Two days after transfection, the viral supernatant was filtered using a 0.45 μm PES syringe filter (Millipore) and diluted to 4 μg ml. -1The cell lines were transduced with polybrene. Five days later, transduced cells that stained highly for EpCAM, EGFR, or Her2 were FACS-sorted to greater than 95% purity. K562 cells expressing Bim-eGFP were generated in an identical manner using a lentivirus encoding a membrane-tethered Bim-eGFP fusion protein (mIgK signal peptide, GS linker, Bim peptide, SGSG linker, eGFP, PDGFR transmembrane domain) and FACS-sorted for eGFP expression five days after transduction.

[0175] Flow cytometry and cell phenotyping Cells were stained with a 1:100 dilution of fluorophore-conjugated monoclonal antibodies specific for human EGFR (AY13), EpCAM (9C4), HA1.1 (16B12), or Her2 (24D2) purchased from ThermoFisher or Biolegend. Where appropriate, cells were stained with an isotype control fluorophore-conjugated antibody. For Bcl2-AF594 binding measurements, K562 cell lines were combined to form a mixed population containing equal numbers of each cell type. Because EpCAM is not tagged with a fluorescent protein, two separate populations were evaluated for each logical operation in Figure 3. The "low EpCAM" population contained K562 / EpCAM. 低 , K562 / Her2-eGFP / EpCAM 低 , K562 / EGFR-iRFP / EpCAM 低 , and K562 / EGFR-iRFP / EpCAM 低 The "high EpCAM" population includes K562 / EpCAM 低 , K562 / Her2-eGFP / EpCAM 高 , K562 / EGFR-iRFP / EpCAM 低 , and K562 / EGFR-iRFP / EpCAM 高The cell mixture was washed with flow buffer (20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 1% BSA) and dispensed into V-bottom plates at 200,000 cells / well. Unless otherwise stated, samples were incubated for 1 hour at room temperature with 50 nM Bcl2-AF594 and a final concentration of 20 nM cage, key, and / or decoy. Samples were then transferred to 150 μl of PBS. They were washed once with flow buffer and resuspended in 150 μl of flow buffer 15–30 min before analysis.

[0176] Data were acquired using an LSRII or FACSCelesta™ (BD Biosciences). K562 cells were FACS-purified using a FACSAria II™ (BD Biosciences). The absolute number of EGFR, EpCAM, and Her2 molecules on the surface of K562 cells was determined using Quantibrite™ beads (BD Biosciences) according to the manufacturer's protocol. All flow cytometry data were analyzed using FlowJo™ (Treestar).

[0177] Confocal microscopy HEK293T cells were cultured on ibidi μ-slide 8-well coverslips at 37°C and 5% CO2 (ibidi 80826) for 1 day. Cell staining and incubation were performed in DMEM, high glucose, HEPES, phenol red-free (Gibco 21063029). Cell nuclei were stained with Invitrogen Molecular Probes NucBlue™ Live ReadyProbes™ reagent according to the manufacturer's instructions (Invitrogen R37605). Cells were incubated for 1-2 hours at 37°C and 5% CO2 in medium containing 1% BSA, 20 nM Her2_Cage-I269S, 20 nM Key_EGFR, and 50 nM Bcl2-AF680. Images were acquired with a Leica SP8X confocal microscope and analyzed in Fiji.

[0178] Confocal microscopy heat map analysis In Fiji, red, green, and blue (RGB) pseudocolors were assigned to the mCherry™, eGFP, and AF680 channels, respectively. Custom python scripts (see Supplementary Table) were used to read the RGB PNG files using the ImageIO Python library, generate two-dimensional binned statistics from the pseudocolor pixel intensities using the SciPy Python library, and visualize the results as heatmaps using the Matplotlib™ library.

[0179] statistical analysis Statistical analysis was performed using Prism™ (GraphPad). Conventional one-way ANOVA tests followed by Dunnett's post-hoc test were used to compare Co-LOCKR-induced targeting (Figure 3a, c, e) and CAR T cell cytokine production (Figure 4). For "AND" targeting, the control group was set as a double-negative cell line. For "OR" and "NOT" targeting, the control group was set as a triple-negative cell line. Only p-values ​​meeting the statistical significance cutoff of alpha = 0.05 are shown on the graphs. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. [Table 14] [Table 15] #Custom Python script for confocal microscopy heatmap analysis: #! / usr / bin / env python3 # -*- coding: utf-8 -*- """ Created on Thursday, June 6, 2019 at 13:47:38 @Author:audreyolshefsky Heatmap of pixel intensities of co-LOCKR confocal RGB images. Red is the x-axis, green is the y-axis, and blue is heat. """ import imageio from scipy import stats import matplotlib.pyplot as plt def heatmap(image): im=imageio.imread(image) #Fast access to image data copies imcopy=im #Print(imcopy.shape)#RGB shape is (1024, 1024, 3) pixel_list=[] counter = 0 for y in range(imcopy.shape[0]): for x in range(imcopy.shape[1]): counter+=1 #Print (Counter) color=tuple(imcopy[y][x]) #r,g,b=color pixel_list.append(color) r=[x[0] for x in pixel_list] g=[x[1] for x in pixel_list] b=[x[2] for x in pixel_list] binned_data=stats.binned_statistic_2d(r,g,b) im=plt.imshow(binned_data[0].T,cmap='plasma',origin='lower',extent=[0,255,0,255]) cb=plt.colorbar() cb.set_label('AF680 mean pixel intensity(Bcl2)') plt.xlabel('mCherryTM pixel intensity (EGFR)') plt.ylabel('eGFP pixel intensity (HER2)') plt.savefig(image[:-4]+'_heatmap.png') RosettaScripts XML used in the design of #Co-LOCKR #Rosetta XML script to redesign LOCKR to be more asymmetric #The original scaffold was constructed from a symmetric homotrimer (Boyken 2016). #Scott Boyken and Marc Lajoie <rosettascripts> <scorefxns> <scorefunction name=""hard”" weights=""beta” / "> < / scorefunction> < / scorefxns> <RESIDUE_SELECTORS> <index name=""latch”" resnums=""302-350” / "> <index name=""cage”" resnums=""1-301” / "> #Maintain the original hydrogen bond network <index name=""HBNet"" resnums=""9,12,24,27,30,45,48,75,93,111,130,133,148,151,166,169,196,214,232,251,254,269,272,287,2" 90,335,339” / > <interfacebyvector name=""switch_interface”" grp1_selector=""latch”" grp2_selector=""cage” / "> <and name=""main_scaffold”"> <not selector=""switch_interface” / "> <not selector=""HBNet” / "> <secondarystructure ss=""H” / "> < / secondarystructure> < / not> < / not> < / and> <not name=""no_design”" selector=""main_scaffold” / "> < / RESIDUE_SELECTORS> <taskoperations> <operateonresiduesubset name=""repack_new”" selector=""no_design”"> <preventrepackingrlt / > < / operateonresiduesubset> < / taskoperations> <movers> <switchchainorder name=""rechain”" chain_order=""12” / "> #Search for a new hydrogen bond network while preserving the original network <HBNetStapleInterface scorefxn="hard” name="hbnet” design_residues="HYNQST” task_operations="repack_new” hb_threshold="-0.5” minimize="true” show_task="true” verbose="true” all_helical_interfaces="true” min_connectivity="0.6” min_helices_contacted_by_network="2” min_networks_per_pose="1” max_networks_per_pose="3” min_network_size="3” min_core_res="2” max_unsat="3” max_replicates_before_branch="3” use_aa_dependent_weights="true” write_network_pdbs="true” write_cst_files="false” / > <multipleposemover name=""switch_peptide_design_MPM”" max_input_poses=""100”"> <rosettascripts> <scorefxns> <scorefunction name=""hard”" wei ghts=""beta” / "> <scorefunction name=""hard_cart”" weights=""beta_cart” / "> <scorefunction name=""soft_cst”" weights="" / home / sboyken / weights / beta_soft_rep_cst.wts” / "> <scorefunction name=""hard_cst”" weights=""beta_cst” / "> <scorefunction name=""up_ele”" weights=""beta”"> <reweight scoretype=""fa_elec”" weight=""1.4” / "> <reweight scoretype=""hbond_sc”" weight=""2.0” / "> < / reweight> < / reweight> < / scorefunction> < / scorefunction> < / scorefunction> < / scorefunction> < / scorefunction> < / scorefxns> <RESIDUE_SELECTORS> <layer name=""hbnet_core”" select_core=""true”" core_cutoff=""3.6” / "> <and name=""terminal_loop”"> <secondarystructure ss=""L”" include_terminal_loops=""true”" use_dssp=""true” / "> <index resnums=""1-15” / "> <chain chains=""A” / "> < / chain> < / index> < / secondarystructure> < / and> <secondarystructure name=""loops”" use_dssp=""true”" ss=""L” / "> <not name=""not_redesign”" selector=""loops” / "> <layer name=""pick_core_and_boundary”" select_core=""true”" select_boundary=""true”" core_cutoff=""5.2” / "> <layer name=""pick_core_and_surface”" select_core=""true”" select_surface=""true”" core_cutoff=""5.2” / "> <layer name=""pick_surface_and_boundary”" select_surface=""true”" select_boundary=""true”core_cutoff="5.2” / "> <chain name=""chain_a”" chains=""A” / "> <layer name=""core”" select_core=""true”" core_cutoff=""5.2” / "> <residuename name=""ala_and_met”" residue_name3=""ALA,MET” / "> <not name=""not_ala_or_met”" selector=""ala_and_met” / "> <!-- <residuepdbinfohaslabel n ame=""hbnet_residues”" property=""HBNet” / "> --> <index name=""latch”" resnums=""302-350” / "> <index name=""cage”" resnums=""1-301” / "> <index name=""HBNet”" resnums=""9,12,24,27,30,45,48,75,93,111,130,133,148,151,166,169,196,214,232,251,254,269,272,287,290,335,339” / "> <interfacebyvector name=""switch_interface”" grp1_selector=""latch”" grp2_selector=""cage” / "> #Select all residues to avoid touching during design <and name=""main_scaffold”"> <not selector=""switch_interface” / "> #Comment out this line to repack HBNet res with AtomPair csts during design. Use "hbnet_task" #HBNet Leave it alone to fix the rotamer <not selector=""HBNet” / "> <secondarystructure ss=""H” / "> < / secondarystructure> < / not> < / not> < / and> <not name=""no_design”" selector=""main_scaffold” / "> < / RESIDUE_SELECTORS> <taskoperations> <consensusloopdesign name=""disallow_non_abego_aas” / "> <layerdesign name=""layer_all”" layer=""core_boundary_surface_Nterm_Cterm”" make_pymol_script=""0”" use_sidechain_neighbors=""True”" core=""4.2”"> <core> Helix append="M” / > <helix exclude=""WY” / "> < / helix> < / core> <boundary> <helix exclude=""WMY” / "> < / helix> < / boundary> <surface> <helix append=""A” / "> < / helix> < / surface> < / layerdesign> <operateonresiduesubset name=""loop_design”" selector=""not_redesign”"> <preventrepackingrlt / > < / operateonresiduesubset> <operateonresiduesubset name=""repack_new”" selector=""no_design”"> <preventrepackingrlt / > < / operateonresiduesubset> <operateonresiduesubset name=""design_core”" selector=""pick_surface_and_boundary”"> <preventrepackingrlt / > < / operateonresiduesubset> <operateonresiduesubset name=""design_boundary”" selector=""pick_core_and_surface”"> <preventrepackingrlt / > < / operateonresiduesubset> <operateonresiduesubset name=""design_surface”" selector=""pick_core_and_boundary”"> <preventrepackingrlt / > < / operateonresiduesubset> <operateonresiduesubset name=""repack_not_ala_or_met”" selector=""not_ala_or_met”"> <restricttorepackingrlt / > < / operateonresiduesubset> <operateonresiduesubset name=""redesign_ala_met”" selector=""ala_and_met”"> <restrictabsentcanonicalaasrlt aas=""AMILVF” / "> < / restrictabsentcanonicalaasrlt> < / operateonresiduesubset> <initializefromcommandline name=""init” / "> <constrainhbondnetwork name=""hbnet_task” / "> <includecurrent name=""current”" / > <limitaromachi2 name=""arochi” / "> <extrarotamersgeneric name=""ex1_ex2”" ex1=""1”" ex2=""1” / "> <extrarotamersgeneric name=""ex1”" ex1=""1” / "> <restrictabsentcanonicalaas name=""ala_only”" resnum=""0”" keep_aas=""A” / "> <restricttorepacking name=""repack_only” / "> BundleReporter name=bundle_filter scorefxn=hard / > < / restricttorepacking> < / restrictabsentcanonicalaas> < / extrarotamersgeneric> < / extrarotamersgeneric> < / limitaromachi2> < / constrainhbondnetwork> < / initializefromcommandline> < / consensusloopdesign> < / taskoperations> <movers> <packrotamersmover name=""softpack_core”" scorefxn=""soft_cst”" task_operations=""layer_all,design_core,current,arochi,disallow_non_abego_aas,repack_new,hbnet_task” / "> <packrotamersmover name=""softpack_boundary”" scorefxn=""soft_cst”" task_operations=""layer_all,design_boundary,current,arochi,disallow_non_abego_aas,repack_new,hbnet_task” / "> <packrotamersmover name=""softpack_surface”" scorefxn=""soft_cst”" task_operations=""layer_all,design_surface,current,arochi,disallow_non_abego_aas,repack_new,hbnet_task” / "> <packrotamersmover name=""hardpack_core”" scorefxn=""hard_cst”" task_operations=""layer_all,design_core,current,arochi,ex1_ex2,disallow_non_abego_aas,repack_new,hbnet_task” / "> <packrotamersmover name=""hardpack_boundary”" scorefxn=""hard_cst”" task_operations=""layer_all,design_boundary,current,arochi,ex1_ex2,disallow_non_abego_aas,repack_new,hbnet_task” / "> <packrotamersmover name=""hardpack_surface”" scorefxn=""up_ele”" task_operations=""layer_all,design_surface,current,arochi,ex1,disallow_non_abego_aas,repack_new,hbnet_task” / "> <packrotamersmover name=""design_loops”" scorefxn=""hard_cst”" task_operations=""current,arochi,ex1_ex2,loop_design,layer_all,disallow_non_abego_aas,hbnet_task” / "> <dumppdb name=""dump1”" fname=""dump1.pdb”" scorefxn=""hard” / "> ConnectChainsMover name=closer chain_connections="[A+B],[B+A]” / > InterfaceAnalyzerMover name=interface_analyzer scorefxn=hard packstat=1 pack_separated=0 / > <minmover name=""hardmin_sconly”" scorefxn=""hard_cst”" chi=""1”" bb=""0”" bondangle=""0”" bondlength=""0” / "> < / minmover> < / dumppdb> < / packrotamersmover> < / packrotamersmover> < / packrotamersmover> < / packrotamersmover> < / packrotamersmover> < / packrotamersmover> < / packrotamersmover> < / movers> <protocols> <add mover=""softpack_core” / "> <add mover=""softpack_boundary” / "> <add mover=""softpack_surface” / "> <add mover=""hardmin_sconly” / "> <add mover=""hardpack_core” / "> <add mover=""hardpack_boundary” / "> <add mover=""hardpack_surface” / "> < / add> < / add> < / add> < / add> < / add> < / add> < / add> < / protocols> < / not> < / interfacebyvector> < / index> < / index> < / index> < / residuepdbinfohaslabel> < / not> < / residuename> < / layer> < / chain> < / layer> < / layer> < / layer> < / not> < / secondarystructure> < / layer> < / rosettascripts> < / multipleposemover> <multipleposemover name=""MPM_filters”"> <rosettascripts> <scorefxns> <scorefunction name=""hard_cst”" weights=""beta_cst” / "> < / scorefunction> < / scorefxns> <RESIDUE_SELECTORS> <chain name=""peptide”" chains=""B” / "> <chain name=""SB76_trunc”" chains=""A” / "> <interfacebyvector name=""scaffold_interface”"> <chain chains=""A” / "> <not selector=""SB76_trunc” / "> < / not> < / chain> < / interfacebyvector> < / RESIDUE_SELECTORS> <taskoperations> <layerdesign name=""layer_all”" layer=""core_boundary_surface_Nterm_Cterm”" make_pymol_script=""0”" use_sidechain_neighbors=""True”" core=""5.2”"> <core> Helix append="M” / > <helix exclude=""WY” / "> < / helix> < / core> <boundary> <helix exclude=""DWMY” / "> < / helix> < / boundary> <surface> <helix append=""A” / "> < / helix> < / surface> < / layerdesign> < / taskoperations> <filters> <preproline name=""prepro”" use_statistical_potential=""0” / "> <scoretype name=""scorefilter”" scorefxn=""hard_cst”" score_type=""total_score”" threshold=""0.0”" confidence=""0” / "> <enzscore name=""cst_score”" score_type=""cstE”" scorefxn=""hard_cst”" whole_pose=""1”" energy_cutoff=""5”" confidence=""0” / "> <buriedunsathbonds name=""buns3”" scorefxn=""beta”" cutoff=""10”" print_out_info_to_pdb=""true”" use_hbnet_behavior=""true”" confidence=""0” / "> <residuecount name=""ala_count”" max_residue_count=""15”" residue_types=""ALA”" residue_selector=""scaffold_interface”" confidence=""0” / "> < / residuecount> < / buriedunsathbonds> < / enzscore> < / scoretype> < / preproline> < / filters> <protocols> <add filter=""scorefilter” / "> <add filter=""cst_score” / "> <add filter=""ala_count” / "> <add filter=""buns3” / "> < / add> < / add> < / add> < / add> < / protocols> < / chain> < / chain> < / rosettascripts> < / multipleposemover> < / switchchainorder> < / movers> <protocols> <add mover=""hbnet” / "> <add mover=""switch_peptide_design_MPM” / "> <add mover=""MPM_filters” / "> < / add> < / add> < / add> < / protocols> < / not> < / interfacebyvector> < / index> < / index> < / rosettascripts>

[0180] All amino acid sequences Modular Arrays: 1. Co-LOCKR is composed of one or more cage polypeptides, one or more key polypeptides, and optionally one or more decoy polypeptides. a. Cage polypeptides are composed of one or more modular targeting moieties, one or more modular Co-LOCKR cage domains, and, optionally, one or more modular Co-LOCKR linkers. b. The Key Polypeptide is composed of one or more modular targeting moieties, one or more modular Co-LOCKR Key Domains, and, optionally, one or more modular Co-LOCKR Linkers. c. The decoy polypeptide is composed of one or more modular targeting moieties, one or more modular Co-LOCKR decoy domains, and optionally, one or more modular Co-LOCKR linkers. Modular targeting moiety: See Table 10 [Table 16] Modular Co-LOCKR cage domain: See Table 1. Co-LOCKR cage and decoy proteins: See Table 11. Modular Co-LOCKR key domain: see Table 4. Co-LOCKR key proteins: see Table 12. Effector proteins: see Table 13. [Table 17] [Table 18-1] [Table 18-2] [Table 18-3]

Claims

1. 1. A method for enhancing cell selectivity in vitro, ex vivo, or in vivo, comprising: (a) contacting a cell with a first cage polypeptide fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides in the absence of co-localization with a key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present on or within the cell; (b) contacting the cell with a first key polypeptide fused to a second binding domain, wherein, upon co-localization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region and activating the one or more biologically active peptides, and the second binding domain is capable of binding to a second cellular moiety present on or within the cell; The method, wherein said first cell portion and said second cell portion are different or identical.

2. The method of claim 1 , wherein the first cell portion and the second cell portion are different.

3. The method of claim 1 , wherein the first cell portion and the second cell portion are the same.

4. 4. The method of claim 3, wherein the co-localization of the first cage polypeptide and the first key polypeptide enhances selectivity of the effector for a cell comprising the first cell portion and the second cell portion.

5. The method of any one of claims 1 to 4, wherein the contacting (a) and the contacting (b) are carried out simultaneously or sequentially.

6. the first cell portion and the second cell portion are in proximity to each other, and optionally, (a) the first cellular portion and the second cellular portion co-localize as a result of forming, directly or indirectly, a complex; and / or 6. The method of any one of claims 1 to 5, wherein (b) the first cellular portion and the second cellular portion are co-localized as a result of being expressed in sufficient numbers in the same subcellular compartment.

7. 7. The method of any one of claims 1 to 6, wherein the first cell portion and / or the second cell portion are present at at least about 100 copies per cell, at least about 200 copies per cell, at least about 500 copies per cell, at least about 1000 copies per cell, at least about 1500 copies per cell, at least about 2000 copies per cell, at least about 2500 copies per cell, at least about 3000 copies per cell, at least about 3500 copies per cell, at least about 4000 copies per cell, at least about 4500 copies per cell, at least about 5000 copies per cell, at least about 5500 copies per cell, at least about 6000 copies per cell, at least about 6500 copies per cell, or at least about 7000 copies per cell.

8. 8. The method of any one of claims 1 to 7, further comprising co-localizing the first cage polypeptide and the first key polypeptide, thereby forming a complex and activating the one or more biologically active peptides.

9. The method of any one of claims 1 to 8, wherein the first cell portion and the second cell portion are present on the surface of the cell.

10. The method of any one of claims 1 to 8, wherein the first cell portion and the second cell portion are present within the cytoplasm of the cell.

11. The method of any one of claims 1 to 8, wherein the first cell portion and the second cell portion are present in the nucleus of the cell.

12. 12. The method of any one of claims 1 to 11, further comprising contacting the cell with a second key polypeptide fused to a third binding domain, wherein when co-localized with the first cage polypeptide, the second key polypeptide is capable of binding to the cage structure region and activating the one or more biologically active peptides, and the third binding domain is capable of binding to a third cellular moiety present on or within the cell that also comprises the first cellular moiety, the third cellular moiety being different from the first cellular moiety or the second cellular moiety and optionally further comprising a third key polypeptide, a fourth key polypeptide, a fifth key polypeptide, a sixth key polypeptide, or a seventh key polypeptide, wherein one or more of the third, fourth, fifth, sixth, or seventh key polypeptides are fused to a binding domain, and the binding domain is capable of binding to a cellular moiety present on or within the cell that also comprises the first cellular moiety.

13. (i) the first key polypeptide comprises a third binding domain, and the second binding domain and / or the third binding domain bind to (i) a moiety different from the first binding domain on the surface of the same cell, or (ii) a moiety different from the first binding domain at a synapse between two contacting cells, such that when co-localized with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region and activating the one or more biologically active peptides; the third binding domain is capable of binding to a third cellular moiety present on or within the cell that also contains the first cellular moiety, the third cellular moiety being different from the first cellular moiety or the second cellular moiety; and / or (ii) further comprising contacting the cell with at least a second cage polypeptide comprising: (A) a second structural region; (B) a second latch region further comprising one or more biologically active peptides; and (C) a sixth binding domain, wherein the second structural region interacts with the second latch region to prevent activity of the one or more biologically active peptides; the first key and / or the second key polypeptide can bind to the second structural region to activate the one or more biologically active peptides; and the sixth binding domain and / or the first binding domain can bind to the same. The method of any one of claims 1 to 11, wherein the first key polypeptide binds to a portion on the surface of a cell that is different from the second binding domain, the third binding domain, and / or the fourth binding domain, or (II) a portion in a synapse between two contacting cells that is different from the second binding domain, the third binding domain, and / or the fourth binding domain, and when co-localized with the first cage or the second cage polypeptide, can bind to the first cage or the second cage structural region and activate the one or more biologically active peptides.

14. 12. The method of any one of claims 1 to 11, further comprising contacting a second key polypeptide fused to a third binding domain with the cell, the second cell also comprising a first cellular moiety, wherein upon co-localization with the first cage polypeptide, the second key polypeptide is capable of binding to the cage structural region and activating the one or more biologically active peptides, and the third binding domain is capable of binding to a third cellular moiety present on or within the second cell.

15. contacting the cells with a third key polypeptide fused to a fourth binding domain.

15. The method of any one of claims 1 to 11 or 14, further comprising: when co-localized with the first cage polypeptide, the third key polypeptide is capable of binding to the cage structure region and activating the one or more biologically active peptides; and the third binding domain is capable of binding to a third cellular moiety present on or within the cell that also contains the first cellular moiety, the third cellular moiety being different from the first cellular moiety or the second cellular moiety.

16. The method of claim 15, further comprising contacting the cell with a fourth key polypeptide, a fifth key polypeptide, a sixth key polypeptide, or a seventh key polypeptide, wherein one or more of the fourth, fifth, sixth, or seventh key polypeptides are fused to a binding domain, and the binding domain is capable of binding to a cellular moiety present on or within the cell.

17. The method of any one of claims 1 to 16, further comprising contacting the cell with one or more decoy cage polypeptides fused to one or more binding domains ("decoy binding domains"), each decoy cage polypeptide comprising a decoy structural region and capable of preferentially binding to the first key polypeptide when co-localized with the first key polypeptide and the first cage polypeptide, and each decoy binding domain capable of binding to a cellular portion ("decoy cellular portion") within the cell, including the first cellular portion and / or the second cellular portion.

18. The method of claim 17, wherein each decoy cell portion is present only on healthy cells.

19. The method of claim 17 or 18, wherein when co-localized with the first key polypeptide, the decoy cage polypeptide binds to the first key polypeptide and the one or more biologically active peptides in the first cage polypeptide are not activated.

20. 1. A method for enhancing the selectivity of cells interacting with each other in vitro, ex vivo, or in vivo, comprising: (a) contacting two or more cells with a first cage polypeptide fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides in the absence of co-localization with a key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present within a synapse between the two or more cells; (b) contacting the two or more cells with a first key polypeptide fused to a second binding domain, wherein, when co-localized with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region and activating the one or more biologically active peptides, and the second binding domain is capable of binding to a second cellular moiety present within a synapse between the two or more cells; The method, wherein said first cell surface moiety and said second cell surface moiety are the same or different.

21. 21. The method of claim 20, wherein the first cell portion and the second cell portion are in close proximity to each other.

22. 22. The method of claim 20 or 21, further comprising co-localizing the first cage polypeptide and the first key polypeptide, thereby forming a complex, and activating the one or more biologically active peptides.

23. The method of any one of claims 20 to 22, wherein the first cell portion and the second cell portion are different or identical.

24. The method of any one of claims 20 to 23, wherein the contacting (a) and the contacting (b) are carried out simultaneously or sequentially.

25. 25. The method of any one of claims 20 to 24, further comprising contacting a second key polypeptide fused to a third binding domain with a synapse of two or more cells that also includes a first cell portion, wherein, upon co-localization with the first cage polypeptide, the second key polypeptide can bind to the cage structural region and activate the one or more biologically active peptides, and the third binding domain can bind to a third cell portion present within the synapse of the two or more cells.

26. The method of any one of claims 20 to 25, further comprising contacting the two or more cells with one or more decoy cage polypeptides fused to one or more decoy binding domains comprising the two or more cells, wherein each decoy cage polypeptide comprises a decoy structural region and, when co-localized with the first key polypeptide and the first cage polypeptide, can preferentially bind to the first key polypeptide, and each decoy binding domain can bind to a decoy cell portion present within the synapse of the two or more cells.

27. 1. A method of targeting heterologous cells (three or more different cell types) in vitro, ex vivo, or in vivo, wherein a first cell portion and a second cell portion are present on the first cell, and a first cell portion and a third cell portion are present on the second cell, the method comprising: (a) contacting two or more cells with a first cage polypeptide fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides in the absence of co-localization with a key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present on or within the two or more cells; (b) contacting the two or more cells with a first key polypeptide fused to a second binding domain, wherein upon co-localization, the first key polypeptide is capable of binding to the cage structure region and activating the one or more biologically active peptides, and the second binding domain is capable of binding to a second cellular moiety present on a cell that also contains the first cellular moiety; (c) contacting the two or more cells with a second key polypeptide fused to a third binding domain, wherein upon co-localization, the second key polypeptide is capable of binding to the cage structure region and activating the one or more biologically active peptides, and the third binding domain is capable of binding to a third cellular moiety present on a cell comprising the first cellular moiety; The method, wherein the first cell portion, the second cell portion, and the third cell portion are different, and the cells comprising the second cell portion and the cells comprising the third cell portion are different.

28. 28. The method of claim 27, wherein the first key polypeptide and the second key polypeptide are the same.

29. 28. The method of claim 27, wherein the first key polypeptide and the second key polypeptide are not identical.

30. The method of any one of claims 27 to 29, further comprising contacting the two or more cells with one or more decoy cage polypeptides fused to one or more decoy binding domains, wherein each decoy cage polypeptide comprises a decoy structural region, and when co-localized with the first key polypeptide, the second key polypeptide and / or the first cage polypeptide can preferentially bind to the first key polypeptide or the second key polypeptide, and each decoy binding domain can bind to a decoy cell portion in a cell comprising the first cell portion and the second cell portion.

31. 1. A method of reducing off-target activity in vitro, ex vivo, or in vivo, comprising: (a) contacting two or more cells with a first cage polypeptide fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides in the absence of co-localization with a key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present on the cells; (b) contacting the two or more cells with a first key polypeptide fused to a second binding domain, wherein, upon co-localization, the first key polypeptide is capable of binding to the cage structure region and activating the one or more biologically active peptides, and the second binding domain is capable of binding to a second cellular moiety present on a cell that also contains the first cellular moiety; (c) contacting the two or more cells with a decoy cage polypeptide fused to a third binding domain, wherein the decoy cage polypeptide comprises a decoy structural region and, when co-localized with the key polypeptide and the first cage polypeptide, is capable of preferentially binding to the first key polypeptide, and the third binding domain is capable of binding to a third cell portion present on a cell comprising the first cell portion and the second cell portion.

32. 32. The method of claim 31, wherein the third cellular portion is present only on healthy cells.

33. 33. The method of any one of claims 1-32, wherein the first cage polypeptide comprises no more than seven alpha helices, no more than six alpha helices, no more than five alpha helices, no more than four alpha helices, no more than three alpha helices, or no more than two alpha helices, the structural region comprises at least one alpha helix, and the latch region comprises at least one alpha helix.

34. 34. The method of any one of claims 1-33, wherein the structural region of the first cage polypeptide comprises 1 alpha helix, 2 alpha helices, 3 alpha helices, 4 alpha helices, 5 alpha helices, or 6 alpha helices, and the latch region of the first key polypeptide comprises no more than 1 alpha helix.

35. The method of claims 17 to 19 and 26 to 34, wherein each decoy cage polypeptide comprises at least one alpha helix, at least two alpha helices, at least three alpha helices, at least four alpha helices, at least five alpha helices, at least six alpha helices, or at least seven alpha helices.

36. The binding affinity (e.g., K) of the decoy cage polypeptide to the key polypeptide Ｄ ) determines the binding affinity (e.g., K) of the first cage polypeptide to the key polypeptide. Ｄ 36. The method of any one of claims 17-19 and 26-35, wherein the cleavage is at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, or at least about 1000 times stronger (e.g., less strong) than

37. 37. The method of any one of claims 1-36, wherein binding of the first cage polypeptide and the first key polypeptide in solution is less efficient than binding of the first cage polypeptide and the first key polypeptide when co-localized on or within the cell.

38. 38. The method of any one of claims 1-37, wherein the co-localization of the first cage polypeptide and the first key polypeptide increases the local concentrations of the first cage polypeptide and the first key polypeptide, shifting the binding equilibrium in favor of complex formation between the first cage polypeptide and the first key polypeptide.

39. 39. The method of any one of claims 1-38, wherein the contacting comprises introducing polynucleotides encoding polypeptides (e.g., the first cage polypeptide, the first key polypeptide, the second key polypeptide, and the decoy cage polypeptide).

40. 40. The method of any one of claims 1 to 39, wherein the first cage polypeptide, the first key polypeptide, the second key polypeptide, and / or the decoy polypeptide are further modified to alter (i) their hydrophobicity, (ii) their hydrogen bond network, (iii) their binding affinity to each other, and / or (iv) any combination thereof.

41. 41. The method of any one of claims 1 to 40, wherein the interface between the latch region and the structural region of the first cage polypeptide comprises a ratio of hydrophobic to polar amino acid residues of 1:1 to 10:1, e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:

1.

42. 42. The method of any one of claims 1 to 41, wherein the latch region is mutated to reduce the hydrophobicity.

43. 43. The method of claim 42, wherein one, two, three, or more large hydrophobic residues in the latch region, e.g., isoleucine, valine, or leucine, are mutated to serine, threonine, or a small hydrophobic amino acid residue, e.g., valine or alanine.

44. 44. The method of any one of claims 1-43, wherein the first cage polypeptide comprises an amino acid residue buried in the interface between the latch region and the structural region of the first cage polypeptide, the amino acid residue buried in the interface having a side chain comprising a nitrogen or oxygen atom that participates in hydrogen bonding.

45. 45. The method of any one of claims 1 to 44, wherein the cells of the first cell portion and / or the second cell portion are present on or within a tumor cell, a cancer cell, an immune cell, a leukocyte, a lymphocyte, a T cell, a regulatory T cell, an effector T cell, a CD4+ effector T cell, a CD8+ effector T cell, a memory T cell, an autoreactive T cell, an exhausted T cell, a natural killer T cell (NKT cell), a B cell, a dendritic cell, a macrophage, an NK cell, a cardiac cell, a lung cell, a muscle cell, an epithelial cell, a pancreatic cell, a skin cell, a CNS cell, a neuron, a muscle cell, a skeletal muscle cell, a smooth muscle cell, a liver cell, a kidney cell, a bacterial cell, a yeast cell, or any combination thereof.

46. One or more of the first, second, third, fourth, fifth, sixth, seventh, and / or decoy binding domains may be an antibody or antigen-binding portion thereof, Fab', F(ab') ２ , Fab, Fv, rIgG, recombinant single chain Fv fragment (scFv), V Ｈ 46. ​​The method of any one of claims 1 to 45, comprising a single domain, bivalent or bispecific molecule, diabody, triabody, and tetrabody, DARPin, nanobody, affibody, monobody, adnectin, alphabody, albumin binding domain, adheren, affilin, affimer, affitin / nanophytin, anticalin, armadillo repeat protein, atrimer / tetranectin, avimer / maxibody, centirin, finomer, Kunitz domain, obody / OB-fold, pronectin, lipibody, computationally designed protein, or any combination thereof.

47. and / or one or more of the first, second, third, fourth, fifth, sixth, seventh, and / or decoy binding domains are selected from the group consisting of Her2, EGFR, EpCAM, B7-H3, ROR1, GD2, GPC2, αvβ6, Her3, L1CAM, BCMA, GPCR5d, EGFRvIII, CD20, CD22, CD3, CD4, CD5, CD8, CD19, CD27, CD28, CD30, CD33, CD48, IL3RA, platelet tissue factor, CLEC12A, CD82, TNFRSF1B, ADGRE2, ITGB5, CD96, CCR1, PTPRJ, CD70, LILRB 2, LTB4R, TLR2, LILRA2, ITGAX, CR1, EMC10, EMB, DAGLB, P2RY13, LILRB3, LILRB4, SLC30A1, LILRA6, SLC6A6, SEMA4A, TAG72, FRα, PMSA, mesothelin, LIV-1, CEA, MUC1, PD1, BLIMP1, CTLA4, LAG3, TIM3, TIGIT, CD39, Nectin-4, a cancer marker, a healthy tissue marker, a cardiac marker, or any combination thereof.

48. 48. The method of any one of claims 1-47, wherein one or more of the cage polypeptide and the key polypeptide further comprises a linker connecting the cage or key polypeptide and the one or more binding domains.

49. 50. The method of any one of claims 1 to 49, further comprising administering an effector to said cells.

50. 50. The method of any one of claims 1 to 49, wherein the cell is in vivo.

51. 50. The method of any one of claims 1 to 49, wherein the cell is in vitro or ex vivo.

52. 52. The method of any one of claims 49 to 51, wherein the effector binds to one or more biologically active peptides.

53. The effector may be an antibody or an antigen-binding fragment thereof, a T cell receptor, a DARPin, 53. The method of claim 52, comprising a bispecific or bivalent molecule, nanobody, affibody, monobody, adnectin, alfbody, albumin binding domain, adheren, affilin, affimer, affitin / nanophytin, anticalin, armadillo repeat protein, atrimer / tetranectin, avimer / maxibody, centirin, finomer, Kunitz domain, obody / OB-fold, pronectin, lipibody, computationally designed protein, protease, ubiquitin ligase, kinase, phosphatase, and / or effector that induces proteolysis.

54. The antigen-binding portion thereof may be Fab', F(ab') ２ , Fab, Fv, rIgG, recombinant single chain Fv fragment (scFv), and / or V Ｈ 54. The method of claim 53, comprising a single domain.

55. The method of any one of claims 49 to 54, wherein the effector is a therapeutic cell.

56. 56. The method of claim 55, wherein the therapeutic cells comprise immune cells.

57. 57. The method of claim 56, wherein the therapeutic cells comprise T cells, stem cells, NK cells, B cells, or any combination thereof.

58. (a) said administering kills said cells containing said first binding moiety and said second binding moiety; or (b) said administering results in receptor signaling (e.g., cytokine) in said cell comprising said first binding moiety and said second binding moiety; (c) said administration produces a signaling molecule (e.g., a cytokine, a chemokine) in the vicinity of said cells comprising said first binding moiety and said second binding moiety; or (d) said administering results in differentiation of said cells comprising said first binding moiety and said second binding moiety.

59. A protein complex formed by any one of methods 1-58.

60. A polynucleotide encoding the protein complex of claim 59.

61. 1. A protein complex comprising: (i) a first cage polypeptide fused to a first binding domain; and (ii) a first key polypeptide fused to a second binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides; wherein the first key polypeptide binds to the cage structural region, activating the one or more biologically active peptides; wherein the first binding domain binds to a first cellular moiety present on or within a cell, or at a synapse of two interacting cells; and wherein the second binding domain binds to a second cellular moiety present on or within the cell, or at a synapse of the two interacting cells; and wherein the first cellular moiety and the second cellular moiety are different or identical.

62. A protein complex comprising: (i) a first key polypeptide fused to a first binding domain; and (ii) a decoy cage polypeptide fused to a second binding domain, wherein the first key polypeptide binds to the decoy cage polypeptide, the first binding domain binds to a first cellular moiety present on or within a cell or at a synapse of two interacting cells, and the second binding domain binds to a second cellular moiety present on or within the cell or at a synapse of the two interacting cells. a protein complex that binds to a first cellular portion and a second cellular portion, wherein the first cellular portion and the second cellular portion are different or identical.

63. (a) a first cage polypeptide or a polynucleotide encoding the same fused to a first binding domain, wherein the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more biologically active peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides in the absence of co-localization with a key polypeptide, and the first binding domain is capable of binding to a first cellular moiety present on or within a cell; (b) a first key polypeptide or a polynucleotide encoding the same fused to a second binding domain, wherein when co-localized with the first cage polypeptide, the first key polypeptide can bind to the cage structural region and activate the one or more biologically active peptides, and the second binding domain can bind to a second cellular moiety present on or within the cell, A composition wherein said first cell portion and said second cell portion are different or identical.

64. 64. The composition of claim 63, wherein the first cell portion and the second cell portion are different.

65. 64. The composition of claim 63, wherein the first cell portion and the second cell portion are the same.

66. 66. The composition of claim 65, wherein the co-localization of the first cage polypeptide and the first key polypeptide enhances selectivity of the effector for a cell comprising the first cell portion and the second cell portion.

67. 67. The composition of any one of claims 63 to 66, wherein the first cage polynucleotide and the first key polynucleotide are encoded on the same or different nucleic acid sequences.

68. the first cell portion and the second cell portion are in proximity to each other, and optionally, (a) the first cellular portion and the second cellular portion co-localize as a result of forming, directly or indirectly, a complex; or 68. The composition of any one of claims 63-67, wherein (b) the first cellular portion and the second cellular portion are present in sufficient numbers in the same subcellular compartment such that they co-localize.

69. 69. The composition of any one of claims 63-68, wherein the first cell portion and / or the second cell portion are present at at least about 100 copies per cell, at least about 200 copies per cell, at least about 500 copies per cell, at least about 1000 copies per cell, at least about 1500 copies per cell, at least about 2000 copies per cell, at least about 2500 copies per cell, at least about 3000 copies per cell, at least about 3500 copies per cell, at least about 4000 copies per cell, at least about 4500 copies per cell, at least about 5000 copies per cell, at least about 5500 copies per cell, at least about 6000 copies per cell, at least about 6500 copies per cell, or at least about 7000 copies per cell.

70. The first cage polypeptide and the first key polypeptide are co-localized, The composition of any one of claims 63 to 69, thereby forming a complex and activating the one or more biologically active peptides.

71. 71. The composition of any one of claims 63 to 70, wherein the first cell portion and the second cell portion are present on the surface of the cell.

72. 71. The composition of any one of claims 63 to 70, wherein the first cell portion and the second cell portion are present within the cytoplasm of the cell.

73. 71. The composition of any one of claims 63 to 70, wherein the first cell portion and the second cell portion are present in the nucleus of the cell.

74. 74. The composition of any one of claims 63 to 73, further comprising a second key polypeptide or a polynucleotide encoding the same fused to a third binding domain, wherein when co-localized with the first cage polypeptide, the second key polypeptide is capable of binding to the cage structural region and activating the one or more biologically active peptides, and the third binding domain is capable of binding to a third cellular moiety present on or within the cell that also contains the first cellular moiety, wherein the third cellular moiety is different from the first cellular moiety or the second cellular moiety.

75. The composition of claim 74, further comprising a third key polypeptide, a fourth key polypeptide, a fifth key polypeptide, a sixth key polypeptide, or a seventh key polypeptide, or a polynucleotide encoding the same, wherein one or more of the third, fourth, fifth, sixth, or seventh key polypeptides are fused to a binding domain, and the binding domain is capable of binding to a cellular moiety present on or within the cell, including the first cellular moiety.

76. 74. The composition of any one of claims 63 to 73, further comprising a second key polypeptide or a polynucleotide encoding the same fused to a third binding domain, wherein when co-localized with the first cage polypeptide, the second key polypeptide is capable of binding to the cage structural region and activating the one or more biologically active peptides, and the third binding domain is capable of binding to a third cellular moiety present on or within a second cell that also comprises the first cellular moiety.

77. 77. The composition of any one of claims 63-73 or 76, further comprising a third key polypeptide or a polynucleotide encoding the same fused to a fourth binding domain, wherein when co-localized with the first cage polypeptide, the third key polypeptide is capable of binding to the cage structural region and activating the one or more biologically active peptides, and the third binding domain is capable of binding to a third cellular moiety present on or within the cell that also contains the first cellular moiety, wherein the third cellular moiety is different from the first cellular moiety or the second cellular moiety.

78. The composition of claim 77, further comprising a fourth key polypeptide, a fifth key polypeptide, a sixth key polypeptide, or a seventh key polypeptide, or a polynucleotide encoding the same, wherein one or more of the fourth, fifth, sixth, or seventh key polypeptides are fused to a binding domain, and the binding domain is capable of binding to a cellular moiety present on or within the cell.

79. Each decoy cage further comprises one or more decoy cage polypeptides or polynucleotides encoding same fused to one or more binding domains ("decoy binding domains"). The composition of any one of claims 63 to 78, wherein the polypeptide comprises a decoy structural region and, when co-localized with the first key polypeptide and the first cage polypeptide, is capable of preferentially binding to the first key polypeptide, and each decoy binding domain is capable of binding to a cellular portion (a "decoy cellular portion") within the cell, including the first cellular portion and / or the second cellular portion.

80. 80. The composition of claim 79, wherein each decoy cell portion is present only on healthy cells.

81. The composition of claim 79 or 80, wherein when co-localized with the first key polypeptide, the decoy cage polypeptide binds to the first key polypeptide and the one or more biologically active peptides in the first cage polypeptide are not activated.

82. 82. The composition of any one of claims 63-81, wherein the first cage polypeptide comprises no more than seven alpha helices, no more than six alpha helices, no more than five alpha helices, no more than four alpha helices, no more than three alpha helices, or no more than two alpha helices, the structural region comprises at least one alpha helix, and the latch region comprises at least one alpha helix.

83. 83. The composition of any one of claims 63-82, wherein the structural region of the first cage polypeptide comprises one alpha helix, two alpha helices, three alpha helices, four alpha helices, five alpha helices, or six alpha helices, and the latch region of the first key polypeptide comprises no more than one alpha helix.

84. The composition of claims 79 to 83, wherein the decoy cage polypeptide comprises at least one alpha helix, at least two alpha helices, at least three alpha helices, at least four alpha helices, or at least five alpha helices.

85. The binding affinity (e.g., K) of the decoy cage polypeptide to the key polypeptide Ｄ ) determines the binding affinity (e.g., K) of the first cage polypeptide to the key polypeptide. Ｄ 85. The composition of any one of claims 79-84, wherein the composition is at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, or at least about 1000 times stronger (e.g., less potent) than the composition of any one of claims 79-84.

86. 86. The composition of any one of claims 63-85, wherein binding of the first cage polypeptide and the first key polypeptide in solution is less efficient than binding of the first cage polypeptide and the first key polypeptide when co-localized on or within the cell.

87. 87. The method of claim 63, wherein the co-localization of the first cage polypeptide and the first key polypeptide increases the local concentrations of the first cage polypeptide and the first key polypeptide, shifting the binding equilibrium in favor of complex formation between the first cage polypeptide and the first key polypeptide. Finished product.

88. The composition of any one of claims 63 to 87, wherein the first cage polypeptide, the first key polypeptide, the second key polypeptide, and / or the decoy polypeptide are further modified to change (i) their hydrophobicity, (ii) their hydrogen bond network, (iii) their binding affinity to each other, and / or (iv) any combination thereof.

89. 89. The composition of any one of claims 63-88, wherein the interface between the latch region and the structural region of the first cage polypeptide comprises a ratio of hydrophobic to polar amino acid residues of 1:1 to 10:1, e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:

1.

90. 90. The composition of any one of claims 63 to 89, wherein the latch region is mutated to reduce the hydrophobicity.

91. 91. The composition of claim 90, wherein one, two, three, or more large hydrophobic residues in the latch region, e.g., isoleucine, valine, or leucine, are mutated to serine, threonine, or a smaller hydrophobic amino acid residue or serine.

92. 92. The composition of any one of claims 63-91, wherein the first cage polypeptide comprises an amino acid residue buried at the interface between the latch region and the structural region of the first cage polypeptide, the amino acid residue buried at the interface having a side chain comprising a nitrogen or oxygen atom that participates in hydrogen bonding.

93. 93. The composition of any one of claims 63-92, wherein the cells of the first cell portion and / or the second cell portion are present on or within a tumor cell, a cancer cell, an immune cell, a leukocyte, a lymphocyte, a T cell, a regulatory T cell, an effector T cell, a CD4+ effector T cell, a CD8+ effector T cell, a memory T cell, an autoreactive T cell, an exhausted T cell, a natural killer T cell (NKT cell), a B cell, a dendritic cell, a macrophage, an NK cell, a cardiac cell, a lung cell, a muscle cell, an epithelial cell, a pancreatic cell, a skin cell, a CNS cell, a neuron, a muscle cell, a skeletal muscle cell, a smooth muscle cell, a liver cell, a kidney cell, a bacterial cell, a yeast cell, or any combination thereof.

94. One or more of the first, second, third, fourth, fifth, sixth, seventh, and / or decoy binding domains may be an antibody or antigen-binding portion thereof, Fab', F(ab') ２ , Fab, Fv, rIgG, recombinant single chain Fv fragment (scFv), V Ｈ 94. The composition of any one of claims 63-93, comprising a single domain, bivalent or bispecific molecule, diabody, triabody, and tetrabody, DARPin, nanobody, affibody, monobody, adnectin, alphabody, albumin binding domain, adheren, affilin, affimer, affitin / nanophytin, anticalin, armadillo repeat protein, atrimer / tetranectin, avimer / maxibody, centirin, finomer, Kunitz domain, obody / OB-fold, pronectin, lipibody, computationally designed protein, or any combination thereof.

95. One or more of the first, second, third, fourth, fifth, sixth, seventh, and / or decoy binding domains are selected from the group consisting of Her2, EGFR, EpCAM, B7-H3, ROR1, GD2, GPC2, αvβ6, Her3, L1CAM, BCMA, GPCR5d, EGFRvIII, CD20, CD22, CD3, CD4, CD5, CD8, CD19, CD27, CD28, CD30, CD33, CD48, IL3RA, platelet tissue factor, CLEC12 95. The composition of any one of claims 63-94, wherein the composition binds to a cell surface protein comprising: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49 ...0, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-4

96. 96. The composition of any one of claims 63-95, wherein one or more of the cage polypeptide and the key polypeptide further comprises a linker connecting the cage or key polypeptide and the one or more binding domains.

97. The composition of any one of claims 63 to 96, further comprising an effector.

98. A cell comprising the composition of any one of claims 63 to 96.

99. 99. The cell of claim 98, further comprising an effector.

100. 97. A method of preparing a subject in need thereof, comprising administering to said subject a composition according to any one of claims 63 to 96.

101. 101. The method of claim 100, wherein one or more cells of the subject exhibit one or more activated bioactive peptides.

102. 97. A method of treating a disease or condition in a subject in need thereof, comprising administering an effector to the subject, wherein the subject has also been administered a composition of any one of claims 63 and 96.

103. 103. The method of any one of claims 99 or 102, wherein the effector binds to one or more biologically active peptides.

104. 104. The method of claim 103, wherein the effector comprises an antibody or antigen-binding fragment thereof, a T-cell receptor, a DARPin, a bispecific or bivalent molecule, a nanobody, an affibody, a monobody, an adnectin, an alfbody, an albumin-binding domain (dmain), an adheren, an affilin, an affimer, an affitin / nanophytin, an anticalin, an armadillo repeat protein, an atrimer / tetranectin, an avimer / maxibody, a centrin, a finomer, a Kunitz domain, an obody / OB-fold, a pronectin, a lipibody, a computationally designed protein, a protease, a ubiquitin ligase, a kinase, a phosphatase, an effector that induces proteolysis, or any combination thereof.

105. The antigen-binding portion thereof may be Fab', F(ab') ２ , Fab, Fv, rIgG, recombinant single chain Fv fragment (scFv), and / or V Ｈ 105. The method of claim 104, comprising a single domain.

106. 104. The method of any one of claims 99, 102, and 103, wherein the effector is a therapeutic cell.

107. 107. The method of claim 106, wherein the therapeutic cells comprise immune cells.

108. 108. The method of claim 107, wherein the therapeutic cells comprise T cells, stem cells, NK cells, B cells, or any combination thereof.

109. (a) said administering kills said cells containing said first binding moiety and said second binding moiety; or (b) said administering results in receptor signaling (e.g., cytokine) in said cell comprising said first binding moiety and said second binding moiety; (c) said administration produces a signaling molecule (e.g., a cytokine, a chemokine) in the vicinity of said cells comprising said first binding moiety and said second binding moiety; or (d) said administering results in differentiation of said cells comprising said first binding moiety and said second binding moiety.

110. (a) a first cage polypeptide comprising: (i) a structural region; (ii) a latch region further comprising one or more biologically active peptides; and (iii) a first binding domain, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides; (b) a first key polypeptide capable of binding to the cage structure region and activating the one or more biologically active peptides, the key polypeptide comprising a second binding domain; a first key polypeptide, wherein the first binding domain and the second binding domain bind to (i) different portions on the surface of the same cell, (ii) the same portion on the surface of the same cell, (iii) different portions at a synapse between two contacting cells, or (iv) the same portion at a synapse between two contacting cells; (c) optionally, one or more effectors that bind to said one or more biologically active peptides when said one or more biologically active peptides are activated.

111. The composition of claim 110, wherein the first key polypeptide comprises a third binding domain, and the second binding domain and / or the third binding domain bind to (i) a different portion from the first binding domain on the surface of the same cell, or (ii) a different portion from the first binding domain at a synapse between two contacting cells.

112. 112. The composition of claim 111, wherein the second binding domain and the third binding domain bind to different moieties on the surface of different cells.

113. (d) at least a second key polypeptide capable of binding to the first cage structure region, wherein the key polypeptide further comprises a fourth binding domain; 113. The composition of any one of claims 110 to 112, wherein the second binding domain and / or the fourth binding domain bind to (i) a different moiety from the first binding domain on the surface of the same cell, or (ii) a different moiety from the first binding domain at a synapse between two contacting cells.

114. The composition of claim 113, wherein the second binding domain and the fourth binding domain bind to (i) different moieties on the surface of the same cell, or (ii) different moieties at a synapse between two contacting cells, or the second binding domain and the fourth binding domain bind to different moieties on the surface of different cells.

115. The first cage polypeptide further comprises a fifth binding domain, and the fifth binding domain and / or the first binding domain are capable of binding to (i) the first cage polypeptide on the surface of the same cell.

115. The composition of any one of claims 110 to 114, wherein the composition binds to (i) a moiety distinct from the second binding domain, the third binding domain, and / or the fourth binding domain, or (ii) a moiety distinct from the second binding domain, the third binding domain, and / or the fourth binding domain at a synapse between two contacting cells.

116. The composition of claim 115, wherein the fifth binding domain and the first binding domain bind to (i) different portions on the surface of the same cell, or (ii) different portions at a synapse between two contacting cells.

117. (e) at least a second cage polypeptide comprising: (i) a second structural region; (ii) a second latch region further comprising one or more biologically active peptides; and (iii) a sixth binding domain, wherein the second structural region interacts with the second latch region to prevent activity of the one or more biologically active peptides; wherein the polypeptide of said first key and / or said second key is capable of binding to said second structural region to activate said one or more biologically active peptides; 117. The composition of any one of claims 110 to 116, wherein the sixth binding domain and / or the first binding domain binds to (i) a moiety on the surface of the same cell that is distinct from the second, third, and / or fourth binding domain, or (ii) a moiety at a synapse between two contacting cells that is distinct from the second, third, and / or fourth binding domain.

118. The composition of claim 117, wherein the sixth binding domain and the first binding domain bind to (i) different moieties on the surface of different cells, or (ii) different moieties at a synapse between two contacting cells.

119. (f) the composition of any one of claims 110-118, further comprising one or more decoy cage polypeptides each comprising (i) a decoy structural region, (ii) a decoy latch region optionally further comprising one or more biologically active peptides, and (iii) a seventh binding domain, wherein the decoy structural region interacts with the first key polypeptide and / or the second key polypeptide to prevent them from binding to the first and / or second cage polypeptides, and the seventh binding domain binds to the same moiety on the surface of a cell as the second binding domain, the third binding domain, and / or the fourth binding domain.

120. The composition of claim 119, wherein the seventh binding domain and the first binding domain, and / or the second binding domain bind to (i) different portions on the surface of the same cell, or (ii) different portions at a synapse between two contacting cells.

121. 121. The composition of claim 119 or 120, wherein the seventh binding domain binds to a moiety present on the cell at a level equal to or greater than the moiety bound by the second binding domain, the third binding domain, and / or the fourth binding domain.

122. 122. The composition of any one of claims 110 to 121, wherein the first binding domain, the second binding domain, the third binding domain (if present), the fourth binding domain (if present), the fifth binding domain (if present), the sixth binding domain (if present), and / or the seventh binding domain (if present) comprise a polypeptide capable of binding to a moiety present on the cell surface, including proteins, sugars, and lipids, or comprise a cell surface protein-binding polypeptide.

123. (a) (i) a first cage polypeptide comprising: (i) a structural region; (ii) a latch region further comprising one or more biologically active peptides; and (iii) a first binding domain, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides; (ii) one or more expression vectors encoding and / or cells expressing a first key polypeptide capable of binding to the cage structure region and activating the one or more biologically active peptides, wherein the first key polypeptide comprises a second binding domain; an expression vector and / or a cell, wherein the first binding domain and the second binding domain bind to (i) different portions on the surface of the same cell, (ii) the same portion on the surface of the same cell, (iii) different portions at a synapse between two contacting cells, or (iv) the same portion at a synapse between two contacting cells; (b) optionally, one or more effectors that bind to the one or more biologically active peptides when the one or more biologically active peptides are activated, and / or one or more nucleic acids encoding the one or more effectors.

124. The composition of claim 123, wherein the first key polypeptide comprises a third binding domain, and the second binding domain and / or the third binding domain bind to (i) a different portion from the first binding domain on the surface of the same cell, or (ii) a different portion from the first binding domain at a synapse between two contacting cells.

125. 125. The composition of claim 124, wherein the second binding domain and the third binding domain bind to different moieties on the surface of different target cells.

126. (c) an expression vector encoding and / or a cell expressing at least a second key polypeptide capable of binding to the first cage structure region, wherein the key polypeptide comprises a fourth binding domain; 126. The composition of any one of claims 123 to 125, wherein the second binding domain and / or the fourth binding domain bind to (i) a different moiety from the first binding domain on the surface of the same cell, or (ii) a different moiety from the first binding domain at a synapse between two contacting cells.

127. 127. The composition of claim 126, wherein the second binding domain and the fourth binding domain bind to (i) different moieties on the surface of the same cell, or (ii) different moieties at a synapse between two contacting cells, or wherein the second binding domain and the fourth binding domain bind to different moieties on the surface of different cells.

128. 128. The composition of any one of claims 123-127, wherein the first cage polypeptide further comprises a fifth binding domain, and the fifth binding domain and / or the first binding domain binds to (i) a moiety on the surface of the same cell that is distinct from the second, third, and / or fourth binding domain, or (ii) a moiety at a synapse between two contacting cells that is distinct from the second, third, and / or fourth binding domain.

129. 129. The composition of claim 128, wherein the fifth binding domain and the first binding domain bind to (i) different portions on the surface of the same cell, or (ii) different portions at a synapse between two contacting cells.

130. (d) a second structural domain further comprising: (i) a second structural domain; and (ii) one or more biologically active peptides. and (iii) a sixth binding domain, wherein the second structural domain interacts with the second latch domain to prevent activity of the one or more biologically active peptides. the first key and / or the second key polypeptide can bind to the second structural region to activate the one or more biologically active peptides; 130. The composition of any one of claims 123 to 129, wherein the sixth binding domain and / or the first binding domain binds to (i) a moiety on the surface of the same cell that is distinct from the second, third, and / or fourth binding domain, or (ii) a moiety at a synapse between two contacting cells that is distinct from the second, third, and / or fourth binding domain.

131. 131. The composition of claim 130, wherein the sixth binding domain and the first binding domain bind to (i) different moieties on the surface of different cells, or (ii) different moieties at a synapse between two contacting cells.

132. (e) The composition of any one of claims 123 to 131, further comprising an expression vector encoding and / or a cell expressing a decoy cage polypeptide comprising: (i) a decoy structural region; (ii) a decoy latch region optionally further comprising one or more biologically active peptides; and (iii) a seventh binding domain, wherein the decoy structural region interacts with the first key polypeptide and / or the second key polypeptide to prevent them from binding to the first and / or second cage polypeptides, and the seventh binding domain binds to the same moiety on the surface of a cell as the second binding domain, the third binding domain, and / or the fourth binding domain.

133. The composition of claim 132, wherein the seventh binding domain and the first binding domain, and / or the second binding domain bind to (i) different portions on the surface of the same cell, or (ii) different portions at a synapse between two contacting cells.

134. 134. The composition of claim 132 or 133, wherein the seventh binding domain binds to a moiety present on the cell at a level equal to or greater than the moiety bound by the second binding domain, the third binding domain, and / or the fourth binding domain.

135. 135. The composition of any one of claims 123 to 134, wherein the first binding domain, the second binding domain, the third binding domain (if present), the fourth binding domain (if present), the fifth binding domain (if present), the sixth binding domain (if present), and / or the seventh binding domain (if present) comprise a polypeptide capable of binding to a moiety present on the cell surface, including proteins, sugars, and lipids, or comprise a cell surface protein-binding polypeptide.

136. The composition of any one of claims 110 to 134, wherein the effector is present.

137. 137. The composition of claim 136, wherein the effector is selected from the non-limiting group including Bcl2, GFP1-10, a small molecule, an antibody, an antibody drug conjugate, an immunogenic peptide, a protease, a T cell receptor, a cytotoxic agent, a fluorophore, a fluorescent protein, a cell adhesion molecule, an endocytic receptor, a phagocyte receptor, a magnetic bead, and a gel filtration resin, and a polypeptide comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27460-27469.

138. the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide are (a) an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, and 27278-27321, or a cage polypeptide listed in Table 7, Table 8, or Table 9, excluding optional amino acid residues, wherein the N-terminal and / or C-terminal 60 amino acids of the polypeptide are optional; (b) one or more of the first, fifth, sixth, or seventh binding domains.

139. the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide are (a) an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27359-27392, excluding optional amino acid residues; (b) one or more of the first, fifth, sixth, or seventh binding domains.

140. the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide are (a) an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27359-27392, including optional amino acid residues; (b) one or more of the first, fifth, sixth, or seventh binding domains.

141. the first key polypeptide and / or the second key polypeptide are (a) a polypeptide comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 14318-26601, 26602-27015, 27016-27050, 27322-27358, and the key polypeptides listed in Table 7, Table 8, and / or Table 9, and SEQ ID NOs: 27393-27398; (b) one or more second, third, or fourth binding domains.

142. the first key polypeptide and / or the second key polypeptide are (a) a polypeptide comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27393-27398, excluding optional residues; (b) one or more second, third, or fourth binding domains.

140. The composition of any one of claims 140.

143. the first key polypeptide and / or the second key polypeptide are (a) a polypeptide comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27393-27398, including optional residues; (b) one or more second, third, or fourth binding domains.

144. the first key polypeptide and / or the second key polypeptide are (a) a polypeptide comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27394-27395; (b) one or more second, third, or fourth binding domains.

145. 145. The composition of any one of claims 110-144, wherein the one or more biologically active peptides comprise one or more biologically active peptides selected from the group consisting of SEQ ID NOs: 60, 62-64, 66, 27052, 27053, and 27059-27093.

146. The first, second, third, fourth, fifth, sixth, and / or seventh binding domains are antigen-binding polypeptides (Fab', F(ab')) directed against the cell surface moiety to be bound. ２ , Fab, Fv, rIgG, recombinant single chain Fv fragment (scFv), V Ｈ 146. The composition of any one of claims 110-145, wherein the protein is selected from the non-limiting group comprising: a single domain, bivalent or bispecific molecule, diabody, triabody, and tetrabody), DARPin, nanobody, affibody, monobody, adnectin, alphabody, albumin binding domain, adheren, affilin, affimer, affitin / nanophytin, anticalin, armadillo repeat protein, atrimer / tetranectin, avimer / maxibody, centirin, finomer, Kunitz domain, obody / OB-fold, pronectin, lipibody, and computationally designed protein.

147. 147. The composition of any one of claims 110-146, wherein the first, second, third, fourth, fifth, sixth, and / or seventh binding domain binds to a cell surface protein on a cell selected from the non-limiting group including: a tumor cell, a cancer cell, an immune cell, a leukocyte, a lymphocyte, a T cell, a regulatory T cell, an effector T cell, a CD4+ effector T cell, a CD8+ effector T cell, a memory T cell, an autoreactive T cell, an exhausted T cell, a natural killer T cell (NKT cell), a B cell, a dendritic cell, a macrophage, an NK cell, a cardiac cell, a lung cell, a muscle cell, an epithelial cell, a pancreatic cell, a skin cell, a CNS cell, a neuron, a muscle cell, a skeletal muscle cell, a smooth muscle cell, a liver cell, a kidney cell, a bacterial cell, and a yeast cell.

148. The first, second, third, fourth, fifth, sixth, and / or seventh binding domains are selected from the group consisting of Her2, EGFR, EpCAM, B7-H3, ROR1, GD2, GPC2, αvβ6, Her3, L1CAM, BCMA, GPCR5d, EGFRvIII, CD20, CD22, CD3, CD4, CD5, CD8, CD19, CD27, CD28, CD30, CD33, CD48, IL3RA, platelet tissue factor, CLEC12A, CD82, TNFRSF1B, ADGRE2, ITGB5, CD96, CCR1, PTPRJ, CD70, LI 148. The composition of any one of claims 110-147, which binds to a cell surface protein selected from the non-limiting group including: LRB2, LTB4R, TLR2, LILRA2, ITGAX, CR1, EMC10, EMB, DAGLB, P2RY13, LILRB3, LILRB4, SLC30A1, LILRA6, SLC6A6, SEMA4A, TAG72, FRα, PMSA, mesothelin, LIV-1, CEA, MUC1, PD1, BLIMP1, CTLA4, LAG3, TIM3, TIGIT, CD39, Nectin-4, a cancer marker, a healthy tissue marker, and a cardiac marker.

149. 149. The composition of any one of claims 110-148, wherein the first, second, third, fourth, fifth, sixth, and / or seventh binding domain comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399-27403.

150. 150. The composition of any one of claims 110-149, wherein (i) the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide, and (ii) the first and / or second key polypeptide comprise at least one cage polypeptide and at least one key polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide and key polypeptide, respectively, in the same row of Table 7, 8, or 9 (i.e., each cage polypeptide in row 2, column 1 of the Table can be used with each key polypeptide in row 2, column 1 of the Table, etc.), with the proviso that each cage polypeptide and each key polypeptide comprises a binding domain.

151. the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide are (a) an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the non-limiting group consisting of SEQ ID NOs: 27359-27392; (b) a binding domain comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399-27403.

152. the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide are (a) an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the non-limiting group consisting of SEQ ID NOs: 27359-27392, including optional amino acid residues; (b) a binding domain comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399-27403. The composition described in

153. the first key polypeptide and / or the second key polypeptide are (a) an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27393-27398; (b) a binding domain comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399-27403.

154. the first key polypeptide and / or the second key polypeptide are (a) an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27393-27398, including optional amino acid residues; (b) a binding domain comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399-27403.

155. the first key polypeptide and / or the second key polypeptide are (a) an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27394-27395; (b) a binding domain comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399-27403.

156. 156. The composition of any one of claims 110-155, wherein the first cage polypeptide, the second cage polypeptide, and / or the decoy cage polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27404-27446.

157. 157. The composition of any one of claims 110-156, wherein the first key polypeptide and / or the second key polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27448-27459.

158. 158. A method of targeting an effector to a cell, comprising contacting a biological sample containing the cell with a composition of any one of claims 110 to 157.

159. 159. The method of claim 158, further comprising contacting said cell with said effector.

160. 1. A method for cell targeting, comprising: (a) a biological sample containing cells, (i) a cage polypeptide comprising: (i) a structural region; (ii) a latch region further comprising one or more biologically active peptides; and (iii) a first binding domain that targets a cell of interest, wherein the structural region interacts with the latch region to prevent activity of the one or more biologically active peptides; and (ii) contacting the cell of interest with a key polypeptide comprising a second binding domain that targets the cell of interest, wherein the first binding domain and the second binding domain bind to (i) different portions on the surface of the same cell, (ii) the same portion on the surface of the same cell, (iii) different portions at a synapse between two contacting cells, or (iv) the same portion at a synapse between two contacting cells; contacting, wherein the contacting occurs for a time and under conditions to promote binding of the cage polypeptide and the key polypeptide to the cell of interest, promote binding of the key polypeptide to the cage structural region to displace the latch region, and activate the one or more biologically active peptides only if the cage polypeptide and the key polypeptide are co-localized in the cell of interest; (b) contacting the biological sample with the one or more effectors under conditions that promote binding of the one or more effectors to the one or more activated biologically active peptides to form effector-bioactive peptide complexes; (c) optionally detecting said effector-bioactive peptide complex, wherein said effector-bioactive peptide complex provides a measure of said cells of interest in said biological sample.

161. 161. The method of claim 160, wherein the detecting step is performed.

162. 162. The method of claim 160 or 161, wherein the method comprises the use of a composition according to any one of claims 110 to 157.

163. 163. The method of any one of claims 158 to 162, wherein the method comprises the use of AND, OR and / or NOT logic using any embodiment or combination of embodiments disclosed herein.

164. A method according to any one of claims 158 to 163, wherein the method comprises the use of AND logic.

165. 165. The method of claim 164, wherein the method comprises the use of a composition according to any one of claims 110 to 112 or 123 to 125 or any claim dependent thereon.

166. A method according to any one of claims 158 to 165, wherein the method comprises the use of OR logic.

167. 167. The method of claim 166, wherein the method comprises the use of a composition according to any one of claims 113 to 118 or 126 to 131 or any claim dependent thereon.

168. A method according to any one of claims 158 to 167, wherein the method comprises the use of NOT logic.

169. 169. The method of claim 168, wherein the method comprises the use of a composition according to any one of claims 119 to 121 and 132 to 144, or any claim dependent thereon.

170. A non-naturally occurring polypeptide, (a) a helical bundle comprising 2 to 7 alpha-helices; (b) one or more binding domains; A non-naturally occurring polypeptide, wherein said helix bundle and said one or more binding domains are both absent from naturally occurring polypeptides.

171. (c) a polypeptide according to claim 170, further comprising an amino acid linker connecting adjacent alpha helices.

172. 172. The polypeptide of claim 170 or 171, wherein the one or more binding domains comprise a cell surface protein-binding polypeptide.

173. 173. The polypeptide of any one of claims 170-172, wherein each helix is ​​independently 18-60, 18-55, 18-50, 18-45, 22-60, 22-55, 22-50, 22-45, 25-60, 25-55, 25-50, 25-45, 28-60, 28-55, 28-50, 28-45, 32-60, 32-55, 32-50, 32-45, 35-60, 35-55, 35-50, 35-45, 38-60, 38-55, 38-50, 38-45, 40-60, 40-58, 40-55, 40-50, or 40-45 amino acids in length.

174. 174. The polypeptide of any one of claims 170-173, wherein each amino acid linker is independently 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 2 to 5, 3 to 5, 4 to 5, 2 to 4, 3 to 4, 2 to 3, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length and does not include any additional functional sequences that may be fused to the linker.

175. 175. The polypeptide of any one of claims 170 to 174, wherein the helix bundle is connected to the one or more binding domains by a linker.

176. 176. The polypeptide of claim 175, wherein the linker comprises a polypeptide linker or a non-polypeptide linker.

177. A non-naturally occurring polypeptide, (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs: 27359-27392, SEQ ID NOs: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27278-27321, or a cage polypeptide listed in Table 7, Table 8, or Table 9, excluding optional amino acid residues, wherein the N-terminal and / or C-terminal 60 amino acids of said polypeptide are optional; (b) one or more binding domains.

178. A non-naturally occurring polypeptide, (a) a polypeptide comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs:27359-27392, SEQ ID NOs:1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27278-27321, excluding amino acid residues within the latch region; (b) one or more binding domains.

179. 179. The polypeptide of claim 177 or 178, wherein the polypeptide has an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a cage polypeptide disclosed herein or selected from the group consisting of SEQ ID NOs:27359-27392, SEQ ID NOs:1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27278-27321, or a cage polypeptide listed in Table 7, Table 8, or Table 9, including optional amino acid residues.

180. (a) a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length to the amino acid sequence of a disclosed cage polypeptide selected from the group consisting of SEQ ID NOs: 27359-27392, excluding optional amino acid residues; (b) one or more binding domains.

181. 181. The polypeptide of claim 180, wherein the polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along its length to the amino acid sequence of a disclosed cage polypeptide selected from the group consisting of SEQ ID NOs: 27359-27392, including optional residues.

182. 182. The polypeptide of any one of claims 170 to 181, wherein the interface between the latch region and the structural region of the polypeptide comprises a ratio of hydrophobic amino acid residues to polar amino acid residues of 1:1 to 10:

1.

183. 183. The polypeptide of any one of claims 170-182, wherein one, two, three, or more large hydrophobic residues in the latch region, including but not limited to, isoleucine, valine, or leucine, are mutated to serine, threonine, or a smaller hydrophobic amino acid residue.

184. 184. The polypeptide of any one of claims 170-183, wherein one, two, three or more large hydrophobic residues within the structural region, including but not limited to isoleucine, valine, or leucine, are mutated to serine, threonine, or smaller hydrophobic amino acid residues.

185. 185. The polypeptide of any one of claims 170 to 184, comprising an amino acid residue buried in the interface having a side chain that includes a nitrogen or oxygen atom that participates in hydrogen bonding.

186. A non-naturally occurring polypeptide comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27359-27392, including optional amino acid residues.

187. 187. The non-naturally occurring polypeptide of claim 186, further comprising one or more binding domains.

188. 188. The polypeptide of claim 187, further comprising an amino acid linker connecting the polypeptide and the one or more binding domains.

189. 189. The polypeptide of any one of claims 186 to 188, wherein the interface between the latch region and the structural region of the polypeptide comprises a ratio of hydrophobic amino acid residues to polar amino acid residues of 1:1 to 10:

1.

190. 190. The polypeptide of any one of claims 186-189, wherein one, two, three, or more large hydrophobic residues in the latch region, including but not limited to, isoleucine, valine, or leucine, are mutated to serine, threonine, or a smaller hydrophobic amino acid residue.

191. 191. The polypeptide of any one of claims 186-190, wherein one, two, three, or more large hydrophobic residues within the structural region, including but not limited to isoleucine, valine, or leucine, are mutated to serine, threonine, or smaller hydrophobic amino acid residues.

192. 192. The polypeptide of any one of claims 186 to 191, comprising an amino acid residue buried in the interface having a side chain that includes a nitrogen or oxygen atom that participates in hydrogen bonding.

193. 193. The polypeptide of any one of claims 170 to 192, wherein the one or more binding domains comprise a cell surface protein-binding polypeptide.

194. The polypeptide of claim 193, wherein the cell surface protein-binding polypeptide is on a tumor cell.

195. 195. The polypeptide of claim 194, wherein the cell surface protein-binding polypeptide is an oncoprotein.

196. 196. The polypeptide of any one of claims 170 to 195, wherein the polypeptide comprises one or more biologically active peptides in at least one of the alpha helices, and the one or more biologically active peptides are capable of selectively binding to a defined target.

197. 200. The polypeptide of claim 196, wherein the one or more biologically active peptides can comprise one or more biologically active peptides selected from the group consisting of SEQ ID NOs: 60, 62-64, 66, 27052, 27053, and 27059-27093.

198. 202. A non-naturally occurring key polypeptide comprising a key domain and one or more binding domains, said key polypeptide capable of specifically binding to a polypeptide of any one of claims 179-197.

199. 200. The polypeptide of claim 198, wherein the key specifically binds to the cage polypeptide and activates one or more biologically active peptides.

200. (a) the key polypeptide has an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a key polypeptide disclosed herein (not including optional amino acid residues) or the amino acid sequence of a key polypeptide listed in SEQ ID NOs: 27393-27398, 14318-26601, 26602-27015, 27016-27050, 27322-27358, and Table 7, Table 8, and / or Table 9; and 200. The polypeptide of claim 198 or 199, comprising (b) one or more binding domains.

201. (a) the key polypeptide has an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27393-27398; and (b) one or more binding domains.

202. (a) the key polypeptide has an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27394-27395; and (b) one or more binding domains.

203. 203. The polypeptide of any one of claims 198 to 202, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acid residues are deleted at the N-terminus and / or C-terminus of the polypeptide.

204. A non-naturally occurring polypeptide comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27393-27398, including optional amino acid residues.

205. 205. The non-naturally occurring polypeptide of claim 204, comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27394-27395.

206. 206. The non-naturally occurring polypeptide of claim 204 or 205, further comprising one or more binding domains.

207. 207. The polypeptide of claim 206, further comprising an amino acid linker connecting the polypeptide and the one or more binding domains.

208. 208. The polypeptide of any one of claims 205 to 207, wherein one, two, three or more residues are deleted at the N-terminus and / or C-terminus of the polypeptide.

209. The polypeptide of claims 170-208, wherein the one or more binding domains comprise a cell surface protein-binding polypeptide.

210. The one or more binding domains are antigen-binding polypeptides (Fab', F(ab')) directed against the cell surface moiety to be bound. ２ , Fab, Fv, rIgG, recombinant single chain Fv fragment (scFv), V Ｈ 210. The polypeptide of any one of claims 170-209, selected from the non-limiting group comprising: single domain, bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies), DARPins, nanobodies, affibodies, monobodies, adnectins, alphabodies, albumin binding domains, adherens, affilins, affimers, affitins / nanophytins, anticalins, armadillo repeat proteins, atrimers / tetranectins, avimers / maxibodies, centrins, finomers, Kunitz domains, obodies / OB-folds, pronectins, lipibodies, and computationally designed proteins.

211. 211. The polypeptide of any one of claims 170-210, wherein said cell surface protein binding domain binds to a cell surface protein on a cell selected from the non-limiting group including: a tumor cell, a cancer cell, an immune cell, a leukocyte, a lymphocyte, a T cell, a regulatory T cell, an effector T cell, a CD4+ effector T cell, a CD8+ effector T cell, a memory T cell, an autoreactive T cell, an exhausted T cell, a natural killer T cell (NKT cell), a B cell, a dendritic cell, a macrophage, an NK cell, a cardiac cell, a lung cell, a muscle cell, an epithelial cell, a pancreatic cell, a skin cell, a CNS cell, a neuron, a muscle cell, a skeletal muscle cell, a smooth muscle cell, a liver cell, a kidney cell, a bacterial cell, and a yeast cell.

212. The cell surface protein binding domain is Her2, EGFR, EpCAM, B7-H3, ROR1, GD2, GPC2, αvβ6, Her3, L1CAM, BCMA, GPCR5d, EGFRvIII, CD20, CD22, CD3, CD4, CD5, CD8, CD1 9, CD27, CD28, CD30, CD33, CD48, IL3RA, platelet tissue factor, CLEC12A, CD82, TNFRSF 1B, ADGRE2, ITGB5, CD96, CCR1, PTPRJ, CD70, LILRB2, LTB4R, TLR2, LILRA2 , ITGAX, CR1, EMC10, EMB, DAGLB, P2RY13, LILRB3, LILRB4, SLC30A1, LILRA6, SLC6A6, SEMA4A, TAG72, FRα, PMSA, mesothelin, LIV-1, CEA, MUC1, PD1, BLIMP1, CTLA4, LAG3, TIM3, TIGIT, CD39, Nectin-4, a cancer marker, a healthy tissue marker, and a cardiac marker.

213. 213. The polypeptide of any one of claims 170-212, wherein the one or more binding domains comprise an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27399-27403.

214. 214. The polypeptide of any one of claims 170-197 and 209-213, wherein the polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the non-limiting group of SEQ ID NOs: 27404-27446.

215. The polypeptide is a non-SEQ ID NO: 27404-27446 polypeptide, including optional residues.

213. The polypeptide of any one of claims 170-197 and 209-212, comprising an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from a non-limiting group.

216. 209. The polypeptide of any one of claims 198-208, wherein the polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the non-limiting group of SEQ ID NOs: 27448-27459.

217. 209. The polypeptide of any one of claims 198-208, wherein the polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the non-limiting group of SEQ ID NOs: 27448-27459, including optional residues.

218. A nucleic acid encoding the polypeptide of any one of claims 170 to 217.

219. 219. A vector, including but not limited to an expression vector, comprising the nucleic acid of claim 218 operably linked to a promoter.

220. 220. The vector of claim 219, wherein the vector is a viral vector.

221. 221. The vector of claim 220, wherein the viral vector comprises an adenoviral vector, a vaccinia viral vector, an AAV vector, a retroviral vector, a lentiviral vector, an alphaviral vector, or any combination thereof.

222. 222. A cell comprising a polypeptide according to any one of claims 170 to 217, a nucleic acid according to claims 218, and / or a vector according to claims 219 to 221, optionally wherein the nucleic acid and / or the expression vector is integrated into a cellular chromosome, or optionally wherein the nucleic acid and / or the expression vector is episomal.

223. Use of the polypeptide, nucleic acid, expression vector, cell and / or composition of any one of claims 110 to 222 for any suitable purpose, including but not limited to those disclosed herein.

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