Patent Application
20220273711
This application contains a Sequence Listing submitted as an electronic text file named “19-851_PCT_Sequcnce-Listing_ST25.txt”, having a size in bytes of 32 MB, and created on May 14, 2020. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).
Biology is adept at integrating multiple signals to control function; however, natural systems are highly evolved for specific functions that make them difficult to repurpose. Engineering systems that can integrate combinations of binding events and predictively respond remains an outstanding challenge. Such a system would be particularly useful for targeting cells based on recognition of a combination of surface makers: most mammalian cell types differ from other tissues only in the combinations of markers present on their surfaces.
In one aspect, the disclosure provides methods of increasing selectivity of a cell in vitro, ex vivo, or in vivo comprising
(a) contacting 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 colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on or within a cell; and
(b) contacting the cell with a first key polypeptide fused to a second binding domain, wherein upon colocalization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the second binding domain is capable of binding to a second cell moiety present on or within the cell,
wherein the first cell moiety and the second cell moiety are different or the same.
In another aspect, the disclosure provides methods of increasing selectivity of cells that are 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 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 colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on a synapse between the two or more cells; and
(b) contacting the to or more cells with a first key polypeptide fused to a second binding domain, wherein upon colocalization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the second binding domain is capable of binding to a second cell moiety present on the synapse between the two or more cells,
wherein the first cell surface moiety and the second cell surface moiety are the same or different.
In a further aspect, the disclosure provides methods of targeting heterogeneous cells (more than two different cell types) in vitro, ex vivo, or in vivo, wherein a first cell moiety and a second cell moeity are present on the first cell and a first cell moiety and a third cell moiety are present on the second cell, 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, and wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell 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 colocalization, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides and wherein the second binding domain is capable of binding to a second cell moiety present on a cell that also comprises the first cell moiety, and
(c) contacting the two or more cells with a second key polypeptide fused to a third binding domain, wherein upon colocalization, the second key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides and wherein the third binding domain is capable of binding to a third cell moiety present on a cell that comprises the first cell moiety,
wherein the first cell moiety, the second cell moiety, and the third cell moiety are different and the cell that comprises the second cell moiety and the cell that comprises the third cell moiety are different.
In one aspect, the disclosure provides methods 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, and wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on a cell;
(b) contacting the two or more cells with a first key polypeptide fused to a second binding domain, wherein upon colocalization, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides and wherein the second binding domain is capable of binding to a second cell moiety present on a cell that also comprises the first cell moiety, and
(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, which upon colocalization with the key polypeptide and the first cage polypeptide, is capable of preferentially binding to the first key polypeptide and wherein the third binding domain is capable of binding to a third cell moiety present on a cell that comprises the first cell moiety and the second cell moiety.
In another aspect, the disclosure provides protein complexes 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 bioactive peptides, wherein the first key polypeptide binds to the cage structural region, wherein the one or more bioactive peptides are activated, and wherein the first binding domain binds to a first cell moiety present on or within a cell or on a synapse of two interacting cells and the second binding domain binds to a second cell moiety present on or within the cell or on a synapse of the two interacting cells, wherein the first cell moiety and the second cell moiety are different or the same.
In a further aspect, the disclosure provides protein complexes 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, and wherein the first binding domain binds to a first cell moiety present on or within a cell or on a synapse of two interacting cells and the second binding domain binds to a second cell moiety present on or within the cell or on a synapse of the two interacting cells, wherein the first cell moiety and the second cell moiety are different or the same.
In one aspect, the disclosure provides compositions comprising
(a) a first cage polypeptide fused to a first binding domain or a polynucleotide encoding the same, 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 colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on or within a cell; and
(b) a first key polypeptide fused to a second binding domain or a polynucleotide encoding the same, wherein upon colocalization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the second binding domain is capable of binding to a second cell moiety present on or within the cell,
wherein the first cell moiety and the second cell moiety are different or the same.
In another aspect, the disclosure provides compositions comprising
(a) a first cage polypeptide comprising (i) a structural region, (ii) a latch region further comprising one or more bioactive peptides, and (iii) a first binding domain wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides;
(b) a first key polypeptide capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the key polypeptide comprises a second binding domain,
wherein the first binding domain and the second binding domain bind to (i) different moieties on the surface of the same cell, (ii) the same moiety on the surface of the same cell, (iii) different moieties at the synapse between two cells that are in contact, or (iv) the same moiety at the synapse between two cells that are in contact; and
(c) optionally, one or more effector(s) that bind to the one or more bioactive peptides when the one or more bioactive peptides are activated.
In a further aspect, the disclosure provides compositions comprising
(a) one or more expression vectors encoding and/or cells expressing:
wherein the first binding domain and the second binding domain bind to (i) different moieties on the surface of the same cell, (ii) the same moiety on the surface of the same cell, (iii) different moieties at the synapse between two cells that are in contact, or (iv) the same moiety at the synapse between two cells that are in contact; and
(b) optionally, one or more effector(s) that bind to the one or more bioactive peptides when the one or more bioactive peptides are activated, and/or one or more nucleic acids encoding the one or more effectors.
In one aspect, the disclosure provides methods for cell targeting, comprising
(a) contacting a biological sample containing cells with
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, and to promote binding of the key polypeptide to the cage structural region to displace the latch region and activate the one or more bioactive peptides only when the cage polypeptide and the key polypeptide are co-localized to the cell of interest;
(b) contacting the biological sample with one or more effector(s) under conditions to promote binding of the one or more effectors to the one or more activated bioactive peptides to produce an effector-bioactive peptide complex; and
(c) optionally detecting the effector-bioactive peptide complex, wherein the effector-bioactive peptide complex provides a measure of the cell of interest in the biological sample.
In another aspect, the disclosure provides non-naturally occurring polypeptide comprising:
(a) a helical bundle, comprising between 2 and 7 alpha-helices; and
(b) one or more binding domain;
wherein the helical bundle and the one or more binding domain are not both present in a naturally occurring polypeptide.
In a further aspect, the disclosure provides non-naturally occurring polypeptide comprising
(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 cage polypeptide disclosed herein, or selected from the group consisting of SEQ ID NOS: 27359-27392, 1-49, 31-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27278 to 27321 not including optional amino acid residues; or cage polypeptides listed in Table 7, Table 8, or Table 9, wherein the N-terminal and/or C-terminal 60 amino acids of the polypeptides are optional, and
(b) one or more binding domains.
In one aspect, the disclosure provides non-naturally occurring polypeptides comprising
(a) a polypeptide comprising an amino acid sequence at least 40%, 4%, 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, 27,278 to 27,321, not including amino acid residues in the latch region; and
(b) one or more binding domains.
In one aspect, the disclosure provides non-naturally occurring polypeptides, comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 93%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 27359-27392, including optional amino acid residues; or 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 27393-27398, including optional amino acid residues.
la-c. Co-LOCKR ‘AND’ logic distinguishes cancer cell lines based on their combinations of surface antigens. a. Targeting domains directly fused to Bim were used to measure relative expression of Her2, EGFR, and EpCAM based on Bcl2-AF594. b. Co-LOCKR distinguished A431 (Her2low/GFRhigh/EpCAMPlow) and SKBR3 (Her2high/EGFRlow/EpCAMlow) based on their endogenous levels of antigen expression. K562/Her2/EGFR/EpCAMKO cells were used as a specificity control. Co-LOCKR activation was measured by Bcl2-AF594 recruitment. c. Consistent with a stoichiometric mechanism of activation, Co-LOCKR signal is limited by amount of lesser-expressed surface antigen. Furthermore, activation signal is higher when one of the antigens is expressed at high levels compared to when both antigens are expressed at low levels. This suggests that Co-LOCKR can act as a thresholding gate to avoid cells with low antigen expression. Indeed, this may account for the preferential targeting of K562 cells expressing high levels of EpCAM in
As described herein, the polypeptides and compositions described herein can be used to create “protein switches”, 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 so that the cage and key polypeptides are co-localized while bound to their targets.
Targeting specificity has been a long-standing problem in biomedicine. Despite the long-standing goal to target therapeutic agents against specific cell types, general solutions for targeting precise combinations of antigens that unambiguously identify the desired cell type are lacking. Natural systems capable of multiple-input integration are hard-coded to specific biological outputs that are difficult to modularly reassign. The methods, compositions, and polypeptides disclosed herein are modular because they comprised of de novo designed polypeptides that integrate the co-localization of two target antigens so as to conditionally expose a bioactive peptide that can recruit arbitrary effector functions. Before this work, it was not possible to produce a system that can integrate the co-localization of two or more antigens on the surface of a target cell so as to conditionally expose a bioactive peptide that can modularly recruit arbitrary effector functions. Furthermore, it was not previously possible to design such de now proteins that can sequester a bioactive peptide in an inactive confirmation until they are co-localized. Finally, it was not previously possible to tune the sensitivity of a protein actuator to recruit the appropriate amount of effector(s). The methods may comprise use of the polypeptides, nucleic acids, vectors, cells, and/or compositions of any embodiment or combination of embodiments disclosed herein. In various embodiments, the method comprises the use of AND, OR, and/or NOT logic gates, using any embodiment or combination of embodiments as described in detail above and in the examples.
All references cited are herein incorporated by reference in their entirety. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the 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 (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Len; 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).
All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.
The polypeptides are “non-naturally occurring” in that the entire polypeptide is not found in any naturally occurring polypeptide. It will be understood that components of the polypeptide may be naturally occurring, including but not limited to bioactive peptides that may be included in some embodiments.
The cage polypeptides comprise a helical bundle comprising between 2 and 7 alpha-helices. In various embodiments, the helical bundle comprises 3-7, 4-7, 5-7, 6-7, 2-6, 3-6, 4-6, 5-6, 2-5, 3-5, 4-5, 2-4, 3-4, 2-3, 2, 3, 4, 5, 6, or 7 alpha helices.
Design of the helical bundle cage polypeptides of the disclosure may be carried out by any suitable means. In one non-limiting embodiment, a BundleGridSampler™ in the Rosetta™ program may be used to generate backbone geometry based on the Crick expression for a coiled-coil and allows efficient, parallel sampling of a regular grid of coiled-coil expression parameter values, which correspond to a continuum of peptide backbone conformations. This may be supplemented by design for hydrogen bond networks using any suitable means, followed by Rosetta™ sidechain design. In a further non-limiting embodiment, best scoring designs, based on total score, number of unsatisfied hydrogen bonds, and lack of voids in the core of the protein may be selected for helical bundle cage polypeptide design.
Each alpha helix may be of any suitable length and amino acid composition as appropriate for an intended use. In one embodiment, each helix is independently 18 to 60 amino acids in length. In various embodiments, each helix is independently between 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, 35-61, 35-55, 35-50, 35-45, 35-60, 38-55, 38-50, 38-45, 40-60, 40-58, 40-55, 40-50, or 40-45 amino acids in length.
In some aspects, a polypeptide disclosed herein comprises a linker. In some aspects, the linker comprises one or more amino acids, e.g., an amino acid linker or a peptide linker. In some aspects, 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 as appropriate for an intended use. In one non-limiting embodiment, each amino acid linker is independently between 2 and 10 amino acids in length, not including any further functional sequences that may be fused to the linker. In various non-limiting embodiments each amino acid linker is independently 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 2-8, 3-8, 5-8, 6-8, 7-8, 2-7, 3-7, 4-7, 5-7, 6-7, 2-6, 3-6, 4-6, 5-6, 2-5, 3-5, 4-5, 2-4, 3-4, 2-3, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In all embodiments, the linkers may be structured or flexible (e.g. poly-GS). These linkers may encode further functional sequences, including but not limited to protease cleavage sites or one half of a split intein system (see sequences below).
The one or more binding domains may be any polypeptide binding domain suitable for an intended use. In one embodiment, the one or more of the binding domains comprise cell surface protein binding polypeptides. In another embodiment, the helical bundle is linked to the one or more binding domains by any suitable linker polypeptide linker or non-polypeptide linker. In one embodiment, the helical bundle is linked to the one or more binding domains by any suitable polypeptide linker, including but not limited to linkers between helical domains described above.
In some aspects, one or more of the cage polypeptides and the key polypeptides further comprises a linker connecting the cage or key polypeptide and the one or more binding domains. In some aspects, the cage polypeptide comprises a linker connecting the cage polypeptide to the binding domain. In some aspects, the key polypeptide comprises a linker connecting the key polypeptide to the binding domain. Any linker known in the art may be used. In some aspects, the linker comprises one or more amino acids. In some aspects, the linker is cleavable. In some aspect, the linker is any linker disclosed herein. Additional embodiments of the one or more binding domains are described in more detail below.
The polypeptides of this first aspect include a region, termed the “latch region”, which may be used for insertion of a bioactive peptide. The cage polypeptide thus comprises 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 activity of the bioactive peptide. Upon activation by key polypeptide after the cage and key polypeptides are co-localized while the binding domains are bound to their targets (as described below), the latch region dissociates from its interaction with the structural region to expose the bioactive peptide, allowing the peptide to function. As used herein, a “bioactive peptide” is any peptide of any length or amino acid composition that is capable of selectively binding to a defined target (i.e.: capable of binding to an “effector” polypeptide). Such bioactive peptides may comprise peptides of all three types of secondary structure in an inactive conformation; alpha helix beta strand, and loop. The polypeptides of this aspect can be used to control the activity of a wide range of functional peptides. The ability to harness these biological functions with tight, inducible control is useful, for example, in engineering cells (inducible activation of function, engineering complex logic behavior and circuits, etc.), developing sensors, developing inducible protein-based therapeutics, and creating new biomaterials. Additional details of the bioactive peptides are described below.
The latch region may be present near either terminus of the cage polypeptide. In one embodiment, the latch region is placed at the C-terminal helix so as to position the bioactive peptide for maximum burial of the functional residues that need to be sequestered to maintain the bioactive peptide in an inactive state while simultaneously burying hydrophobic residues and promoting solvent exposure/compensatory hydrogen bonds of polar residues. In various embodiments, the latch region may comprise a pan or all of a single alpha helix in the cage polypeptide at the N-terminal or C-terminal portions. In various other embodiments, the latch region may comprise a pan or all of a first, second, third, fourth, fifth, sixth, or seventh alpha helix in the cage polypeptide. In other embodiments, the latch region may comprise all or part of two or more different alpha helices in the cage polypeptide; for example, a C-terminal part of one alpha helix and an N-terminal portion of the next alpha helix, all of two consecutive alpha helices, etc.
As used herein, a “synapse” is a junction between two interacting cells, typically involving protein-protein contacts across the junction. An immunological 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 neuronal synapse is a junction between two nerve cells, consisting of a minute gap across which impulses pass by diffusion of a neurotransmitter. This embodiment is particularly useful, for example, when detecting cells that are in contact with each other, but not cells that are not. For example, one could identify only T cells that are interacting with a specified target cell but avoid all non-interacting T cells.
As used throughout the present application, the term “polypeptide” is used in its broadest sense to refer to a sequence of subunit amino acids. The polypeptides of the invention may comprise L-amino acids+glycine, D-amino acids+glycine (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids+glycine. The polypeptides described herein may be chemically synthesized or recombinantly expressed. The polypeptides may be linked to other compounds to promote an increased half-life in vivo, such as by PEGylation, HESylation, PASylation, glycosylation, or may be produced as an Fc-fusion or in deimmunized variants. Such linkage can be covalent or non-covalent as is understood by those of skill in the art.
An “effector” is any molecule, nucleic acid, protein, nucleoprotein complex, or cell that carries out a biological activity upon interaction with the bioactive peptide. Exemplary biological activities can include binding, recruitment of fluorophores, recruitment of toxins, recruitment of immunomodulators, proteolysis, enzymatic activity, release of signaling proteins (e.g., cytokines, chemokine), induction of cell death, induction of cell differentiation, nuclear import/export, ubiquitination, and fluorophore/chromophore maturation.
The present disclosure is directed to a switch system that can improve a target cell specificity in vitro, in vivo, or ex vivo. In particular, the system can be within a tissue, between cells, in a synapse of cells, or within a cell in which an increased target specificity is needed. In some aspects, the present composition is capable of increasing selectivity of a cell for a therapy. In some aspects, the composition of the present disclosure is capable of increasing selectivity of cells that are interacting with each other for a therapy. In some aspects, the present composition is capable of targeting heterogeneous cells (more than two different cell types) for a therapy, wherein a first cell moiety and a second cell moeity are present on the first cell and a first cell moiety and a third cell moiety are present on the second cell. In some aspects, the composition is also capable of reducing off-target activity for a therapy. Therefore, in some aspects, the present composition can prepare a subject in need of a therapy so that the subject can respond better to the therapy, the efficacy of the therapy is increased, and/or a toxicity due to non-specific binding (or leakiness) is reduced.
Ag1 AND Ag2
In some aspects, the present disclosure is capable of increasing selectivity of a cell that comprises at least two different cell markers (moieties Ag1 AND Ag2). By targeting cells that express two different moieties, cells that comprises only one of the moieties (Ag1 OR Ag2) can be de-selected. In some aspects, the composition comprises:
(a) 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 colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on or within a cell; and
(b) a first key polypeptide fused to a second binding domain, wherein upon colocalization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the second binding domain is capable of binding to a second cell moiety present on or within the cell,
wherein the first cell moiety and the second cell moiety are different or the same.
In some aspects, the present disclosure comprises:
(a) a polynucleotide encoding 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 colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on or within a cell; and
(b) a polynucleotide encoding a first key polypeptide fused to a second binding domain, wherein upon colocalization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the second binding domain is capable of binding to a second cell moiety present on or within the cell,
wherein the first cell moiety and the second cell moiety are different or the same. In some aspects, the polynucleotide encoding the first cage polypeptide and the polynucleotide encoding the second polypeptide are on the same vector. In some aspects, the polynucleotide encoding the first cage polypeptide and the polynucleotide encoding the second polypeptide are on different vectors.
In some aspects, the first cell moiety and the second cell moiety are different. In some aspects, the first cell moiety and the second cell moiety are the same.
For the one or more bioactive peptides are to be activated (e.g., exposed to an effector or capable of transducing its signal downstream), a functional cage polypeptide and a key polypeptide need to be colocalized. The mere expression of the functional cage polypeptide and a key polypeptide is not sufficient. For example, in some aspects, binding of a functional cage polypeptide, e.g., a first cage polypeptide, to a key polypeptide in solution is less efficient to activate the one or more bioactive peptides than binding of the cage and key polypeptides after colocalization. In some aspects, therefore, the colocalization of the first cage polypeptide and the key polypeptide increases selectivity of a cell that highly expresses the cell moiety.
In some aspects, the colocalization of the first cage polypeptide and the first key polypeptide increases the local concentration of the first cage polypeptide and the first key polypeptide and shifts the binding equilibrium in favor of complex formation between the first cage polypeptide and the first key polypeptide.
In order for two cell moieties to be close enough (e.g., in close proximity) to allow colocalization of a cage polypeptide binding the first cell moiety and a key polypeptide binding to the second cell moiety, the two cell moieties may be colocalized as a result of directly or indirectly forming a complex (e.g., two proteins in the same complex such as a Her2-EGFR heterodimer or CD3ξ in complex with LAT or Zap70; two DNA sequences located in close proximity on a chromosome; two RNA sequences located in close proximity on an mRNA). In this case at least one molecule of the first moiety must be colocalized with at least one molecule of the second moiety to result in colocalization. Alternatively, the two cell moieties may be colocalized by virtue of being expressed in sufficient numbers in the same subcellular compartment (e.g., two transmembrane proteins expressed in the cell membrane such as Her2 and EGFR, Her2 and EpCAM, etc.) In some aspects, the cell expresses a first cell moiety and/or the second cell moiety 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 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 copies per cell, or at least about 7000 copies per cell. In some aspects, the first cell moiety and/or the second cell moiety express about 500 to about 10,000 copies per cell, about to about 10,000 copies per cell, about 2000 to about 10,0000 copies per cell, about 3000 to about 10,000 copies per cell, about 4000 to about 10,000 copies per cell, about 5000 to about 10,000 copies per cell, about 1000 to about 9.000 copies per cell, about 2000 to about 90,000 copies per cell, about 3000 to about 9,000 copies per cell, about 4000 to about 9,000 copies per cell, about 5000 to about 9,000 copies per cell, about 1000 to about 8,000 copies per cell, about 2000 to about 8,0000 copies per cell, about 3000 to about 8,000 copies per cell, about 4000 to about 8,000 copies per cell, about 5000 to about 8,000 copies per cell, about 1000 to about 7,000 copies per cell, about 2000 to about 7,0000 copies per cell, about to about 7,000 copies per cell, about 4000 to about 7,000 copies per cell, about 5000 to about 7,000 copies per cell, about 1000 to about 6,000 copies per cell, about 2000 to about 6,0000 copies per cell, about 3000 to about 6,000 copies per cell, about 4000 to about 6,000 copies per cell, about 5000 to about 6,000 copies per cell. In some aspects, the cell expresses a first cell moiety and/or the second cell moiety at least about 5000 copies up to about 6000 copies, up to about 7000 copies or up to about 4000 copies. In some aspects, the first cage polypeptide and the first key polypeptide are colocalized, thereby forming a complex and activating the one or more bioactive peptides.
In some aspects, the first cell moiety and the second cell moiety are present on the surface of the cell. In some aspects, the first cell moiety and the second cell moiety are present within the cytoplasm of the cell. In some aspects, the first cell moiety and the second cell moiety are present within the nucleus of the cell. In some aspects, the first cell moiety and the second cell moiety are present within the secretory pathway of the cell, including the endoplasmic reticulum (ER) and Golgi apparatus.
Ag1 AND (Ag2 OR Ag3)
The present disclosure can also target more than two cells at the same time by utilizing various cell markers. For instance, the disclosure can allow a therapy to target heterogeneous cell types, more than two (Ag1 AND (Ag2 OR Ag3)), more than three (Ag1 AND (Ag2 OR Ag3 OR Ag4)), more than four (Ag1 AND (Ag2 OR Ag3 OR Ag 4 OR Ag5)), more than five (Ag) AND (Ag2 OR Ag3 OR Ag 4 OR Ag5 OR Ag6)), etc. In some embodiments, (Ag1 OR Ag2) AND Ag3 can be accomplished by targeting multiple cage polypeptides to multiple cells at the same time with different binding domains 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 accomplished by targeting multiple cage polypeptides with multiple binding domains and multiple key polypeptides with multiple binding domains.
In some aspects, the composition comprises:
(a) a first cage polypeptide fused to a first binding domain or a polynucleotide encoding de same, 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 colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on or within a first cell (Cell Type t, e.g., cell expressing Ag1 AND Ag2);
(b) a first key polypeptide fused to a second binding domain or a polynucleotide encoding the same, wherein upon colocalization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the second binding domain is capable of binding to a second cell moiety present on or within the first cell; and
(c) a second key polypeptide fused to a third binding domain or a polynucleotide encoding the same, wherein upon colocalization with the first cage polypeptide, the second key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the third binding domain is capable of binding to a third cell moiety present on or within a second cell that also comprises the first cell moiety (Cell type II, e.g., cell expressing Ag1 AND Ag3), wherein the first cell moiety, the second cell moiety, and the third cell moiety are different.
In some aspects, the first key polypeptide comprises a third binding domain, wherein the second binding domain and/or the third binding domain bind to (i) different moieties than the first binding domain on the surface of the same cell, or (ii) different moieties than the first binding domain at the synapse between two cells that are in contact, wherein upon colocalization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the third binding domain is capable of binding to a third cell moiety present on or within the cell that also comprises the first cell moiety, wherein the third cell moiety is different from the first cell moiety or the second cell moiety.
In some aspects, the compositions further comprise:
(d) 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,
wherein the first key and/or the second key polypeptide are capable of binding to the second structural region to activate the one or more bioactive peptides, and
wherein the sixth binding domain and/or the first binding domain bind to (1) different moieties than the second binding domain, third binding domain and/or fourth binding domain on the surface of the same cell, or (ii) different moieties than the second binding domain, third binding domain and/or fourth binding domain at the synapse between two cells that are in contact. Such compositions can be used, for example, to accomplish (Ag1 OR Ag2) AND Ag3 by targeting the 2 cage polypeptides with different binding domains to multiple cells at the same time and targeting one key polypeptide with a single binding domain to those same cells.
In some aspects, the composition can further comprise multiple key polypeptides, a fourth key polypeptide, a fifth key polypeptide, a sixth key polypeptide, or a seventh key polypeptide, to increase selectivity for the first cell and/or the second cell. For example the composition for the first cell can further comprise additional key polypeptides, a fourth key polypeptide, a fifth key polypeptide, a sixth key polypeptide, or a seventh key polypeptide, that can further increase the selectivity of the first cell. In some aspects, the composition for the second cell further comprises additional key polypeptides, a fourth key polypeptide, a fifth key polypeptide, a sixth key polypeptide, or a seventh key polypeptide, that can further increase the selectivity of the second cell. Each of the additional key polypeptides for the present disclosure can be fused to a binding domain, wherein upon colocalization with the first cage polypeptide, the third key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the third binding domain is capable of binding to a cell moiety present on or within the cell that also comprises the first cell moiety. In some aspects, a single key polypeptide can be fused to two or more binding domains such that the same key polypeptide can be targeted to both Cell type I and Cell type II.
(Ag1 AND Ag2) NOT Ag3
The present disclosure can also direct a therapy to avoid normal (healthy) cells, but only target diseased cells, e.g., tumor cells by utilizing various cell markers, thereby reducing off target cell specificity or toxicity. Therefore, the disclosure can allow a therapy to avoid targeting normal cell types that express unique cell markers. For example, if normal cells express Ag3 while the diseased cells don't, the composition for the present disclosure can be constructed to avoid the cells expressing Ag3.
In some aspects, the composition comprises:
(a) a first cage polypeptide fused to a first binding domain or a polynucleotide encoding the same, 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 colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on or within a cell;
(b) a first key polypeptide fused to a second binding domain or a polynucleotide encoding the same, wherein upon colocalization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the second binding domain is capable of binding to a second cell moiety present on or within the cell; and
(c) one or more decoy cage polypeptide fused to one or more binding domain (“decoy binding domain”) or a polynucleotide encoding the same, wherein each decoy cage polypeptide comprises a decoy structural region, which upon colocalization with the first key polypeptide and the first cage polypeptide, is capable of preferentially binding to the first key polypeptide and wherein each decoy binding domain is capable of binding to a cell moiety (“decoy cell moiety”) in the cell that comprises the second cell moiety. In some aspects, the decoy binding domain is capable of binding to a cell moiety (“decoy cell moiety”) in the cell that comprises the first cell moiety and the second cell moiety. In some aspects, each decoy cell moiety is present only on a healthy cell. In some aspects, each decoy cage polypeptide, upon colocalization with the first key polypeptide, binds to the first key polypeptide such that the first key polypeptide does not bind to the first cage polypeptide and wherein the one or more bioactive peptides in the first cage polypeptide are not activated.
Any first cage polypeptide can serve as a decoy polypeptide for any second cage polypeptide, provided that the first cage polypeptide has a higher affinity for the key polypeptide than does the second cage polypeptide.
The compositions and methods of all aspects described herein may comprise use of a single decoy cage polypeptide comprising multiple binding domains, or multiple decoy cage polypeptides each with one (or more) binding domains to avoid cells with different decoy cell moieties (e.g., 1 AND 2 NOT (3 OR 4) logic).
In some aspects, the binding affinity of the decoy cage polypeptide to a key polypeptide (e.g., KD) is stronger (e.g., lower) than the binding affinity of the first cage polypeptide to a key polypeptide (e.g., KD), e.g., by at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 150 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 600 fold, at least about 700 fold, at least about 800 fold, at least about 900 fold, or at least about 1000 fold. In some aspects, 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. In some aspects, the decoy cage polypeptide further comprises a decoy latch region. In some aspects, the decoy latch region is not functional. In some aspects, the decoy latch region does not comprise any bioactive peptide. In some aspects, the decoy latch region is not present. In some aspects, the decoy latch region comprises a non-functional bioactive peptide. In some aspects, the decoy latch region comprises a functional bioactive peptide with a distinct biological function. By way of non-limiting example, the cage polypeptide may comprise a bioactive peptide with immunostimulatory function and the decoy cage polypeptide comprises a bioactive peptide with immunoinhibitory function.
In a fourth aspect, the disclosure provides compositions comprising
(a) a first cage polypeptide comprising (i) a structural region, (ii) a latch region further comprising one or more bioactive peptides, and (iii) a first binding domain wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides;
(b) a first key polypeptide capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the key polypeptide comprises a second binding domain,
wherein the first binding domain and the second binding domain bind to (i) different moieties on the surface of the same cell, (ii) the same moiety on the surface of the same cell, (iii) different moieties at the synapse between two cells that are in contact, or (iv) the same moiety at the synapse between two cells that are in contact; and
optionally, one or more effector(s) that bind to the one or more bioactive peptides when the one or more bioactive peptides are activated.
The compositions disclosed herein, also referred to as “Co-LOCKR system” in the examples that follow, comprise of at least one cage polypeptide and at least one key polypeptide that may be used, for example, as proximity-activated de novo protein switches that perform ‘AND’. ‘OR’, and ‘NOT’ Boolean logic operations and combinations thereof in response to precise combinations of protein-binding events. The switches activate via a conformational change only when all logic conditions are met. The system is demonstrated in the examples to provide for ultraspecific targeting of mammalian cells that are distinguished in a complex cell population only by their precise combination of surface markers. An ‘AND’ gate may be achieved by targeting the cage polypeptide to one antigen and the key polypeptide to a different antigen. A ‘thresholding’ gate may be achieved by targeting the cage polypeptide and key polypeptide to the same antigen (this could be either with binding domains that bind to the same epitope or a different epitope on the same antigen). An ‘OR’ gate may be achieved by targeting the cage polypeptide or the key polypeptide to two different antigens. A ‘NOT’ gate may 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 either antigen 2 OR antigen 3).
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 the synapse between two cells that are in contact. In this embodiment, the composition can be used to establish an AND gate.
In another embodiment, the first binding domain and the second binding domain bind to (ii) the same moiety on the surface of the same cell, or (iv) the same moiety at the synapse between two cells that are in contact. In this embodiment, the composition can be used to establish a thresholding gate.
In one embodiment (c) the first key polypeptide comprises a third binding domain, wherein the second binding domain and/or the third binding domain bind to (i) different moieties than the first binding domain on the surface of the same cell, or (ii) different moieties than the first binding domain at the synapse between two cells that are in contact. In a further embodiment, 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 cither 2 OR 3 logic gate, provided the moiety bound by the first binding domain is present on one of those cells.
In another embodiment, the composition further comprises (d) at least a second key polypeptide capable of binding to the first cage structural region, wherein the key polypeptide comprises a fourth binding domain, wherein the second binding domain and/or the fourth binding domain bind to (i) different moieties than the first binding domain on the surface of the same cell, or (ii) different moieties than the first binding domain at the synapse between two cells that are in contact. In one embodiment, 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 the synapse between two cells that are in contact. 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 either 2 OR 3 logic gate, provided the moiety bound by the first binding domain is present on one of those cells.
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 bind to (i) different moieties than the second binding domain, third binding domain and/or fourth binding domain on the surface of the same cell, or (ii) different moieties than the second binding domain, third binding domain and/or fourth binding domain at the synapse between two cells that are in contact. 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 the synapse between two cells that are in contact. In this embodiment, the composition can be used to establish an OR logic gate, specifically the ((1 OR 5) AND (2 OR 3) logic gate, based on the additional binding domain present on a single cage polypeptide.
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, wherein the first key and/or the second key polypeptide are capable of binding to the second structural region to activate the one or more bioactive peptides, and wherein the sixth binding domain and/or the first binding domain bind to (i) different moieties than the second binding domain, third binding domain and/or fourth binding domain on the surface of the same cell, or (ii) different moieties than the second binding domain, third binding domain and/or fourth binding domain at the synapse between two cells that are in contact in one embodiment, 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 the synapse between two cells that are in contact. In these embodiments, the composition can be used to establish an OR logic gate based on the additional binding domain present on a second cage polypeptide. In one such embodiment, there may be two separate but identical cage polypeptides be each attached to one different binding domain. In another such embodiment, the two cage polypeptides may be different cage polypeptides that both are activated by the same key polypeptide and are each attached to one different binding domain.
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 bioactive 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 the second cage polypeptides, and wherein the seventh binding domain binds to a moiety on the surface of the same cell as the second binding domain, third binding domain, and/or fourth binding domain. In one embodiment, the seventh binding domain binds to a moiety that is present on the cell at an equal or higher level than the moieties to which the second binding domain, the third binding domain, and/or the fourth binding domain bind to. In this embodiment, the composition can be used to establish a NOT logic gate based on the decay cage polypeptide binding to a different target on the same cell as the target of the key polypeptide. In this embodiment, the composition can be used, for example, to establish a 1 AND 2 NOT 7 logic, provided the moieties bound by the first and second binding domains are present the same cell. In one embodiment, the decoy cage polypeptide does not comprise a bioactive peptide. This embodiment can be used, for example, to establish a 1 AND 4 NOT 7 logic (provided that the moieties bound by the first and fourth binding domains are present on the same cell), or a 5 AND 4 NOT 7 logic (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. In one embodiment of all these embodiments of the composition, the first binding domain, the second binding domain, the third binding domain (when present), the fourth binding domain (when present), the fifth binding domain (when present), the sixth binding domain (when present), and/or the seventh binding domain (when present) comprise polypeptides capable of binding moieties present on the cell surface, including proteins, saccharides, and lipids. In one embodiment, the one or more binding proteins comprise cell surface protein binding polypeptides.
All of the compositions above are described as polypeptide compositions. The disclosure also provides compositions comprising expression vectors and/or cells that express the cage polypeptides and key polypeptides as described in the compositions above, and thus can be used for the same purposes (for example, in establishing the same logic gates as for the corresponding polypeptide compositions described above). Thus, in a fifth aspect, the disclosure provides compositions comprising:
The one or more expression vectors may comprise a separate expression vector encoding each separate polypeptide, may comprise an expression vector encoding two or more of the separate polypeptides, or any combination thereof as suitable for an intended use. The expression vector may comprise any suitable expression vector that operatively links a nucleic acid coding region for the cited polypeptide(s) to any control sequences capable of effecting expression of the gene product. Similarly, the cells may be any prokaryotic or eukaryotic cell capable of expressing the recited polypeptide(s); the cells may comprise a single cell capable of expressing all of the recited polypeptides, separate cells capable of expressing each individual polypeptide, or any combination thereof.
In one embodiment the first key polypeptide comprises a third binding domain, wherein the second binding domain and/or the third binding domain bind to (i) different moieties than the first binding domain on the surface of the same cell, or (ii) different moieties than the first binding domain at the synapse between two cells that are in contact. In another embodiment, the second binding domain and the third binding domain bind to different moieties on the surface of different target cells.
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 structural region, wherein the key polypeptide comprises a fourth binding domain, wherein the second binding domain and/or the fourth binding domain bind to (i) different moieties than the first binding domain on the surface of the same cell, or (ii) different moieties than the first binding domain at the synapse between two cells that are in contact. In another embodiment 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 the synapse between two cells that are in contact.
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 to (i) different moieties than the second binding domain, third binding domain, and/or fourth binding domain on the surface of the same cell, or (ii) different moieties than the second binding domain, third binding domain, and/or fourth binding domain at the synapse between two cells that are in contact. 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 the synapse between two cells that are in contact.
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 lath 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, wherein the first key and/or the second key polypeptide are capable of binding to the second structural region to activate the one or more bioactive peptides, and wherein the sixth binding domain and/or the first binding domain bind to (i) different moieties than the second binding domain, third binding domain, and/or fourth binding domain on the surface of the same cell, or (ii) different moieties than the second binding domain, third binding domain, and/or fourth binding domain at the synapse between two cells that are in contact. In one embodiment, 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 the synapse between two cells that are in contact.
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 bioactive 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 the second cage polypeptides, and wherein the seventh binding domain binds to a moiety on the surface of the same cell as the second binding domain, third binding domain, and/or fourth binding domain. In one embodiment, the seventh binding domain and the first binding domain and/or second binding domain bind to (i) different moieties on the surface of the same cell, or (ii) different moieties at the synapse between two cells that are in contact. In another embodiment, the seventh binding domain binds to a moiety that is present on the cell at an equal or higher level than the moieties to which the second binding domain, the third binding domain, and/or the fourth binding domain bind to.
In one embodiment, the first binding domain, the second binding domain, the third binding domain (when present), de fourth binding domain (when present), the fifth binding domain (when present), the sixth binding domain (when present), and/or the seventh binding domain (when present) comprise polypeptides capable of binding moieties present on the cell surface, including proteins, saccharides, and lipids. In one embodiment, the one or more binding proteins comprise cell surface protein binding polypeptides.
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 binding to the cage polypeptide, as described herein), and wherein the binding domain can serve to target the polypeptide to the entity to which the binding domain binds. In one embodiment, the polypeptides are pan of a “protein switch” (together with appropriate key polypeptide(s)), 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 so that the cage and key polypeptides are co-localized while bound to their targets.
In some aspects, the cage polypeptide comprises a helical bundle, comprising between 2 and 7 alpha-helices; wherein the helical bundle is fused to one or more binding domain; wherein the one or more binding domain and the helical bundle are not both present in the same naturally occurring polypeptide.
In each embodiment, the N-terminal and/or C-terminal 60 amino acids of each cage polypeptides may be optional, as the terminal 60 amino acid residues may comprise a latch region that can be modified, such as by replacing all or a portion of a latch with a bioactive 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; 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 the percent sequence identity. In another embodiment, the optional residues may be included in determining percent sequence identity.
In some aspects, the first cage polypeptide comprises no more than 5 alpha helices, no mom than 4 alpha helices, no more than 3 alpha helices, or no more than 2 alpha helices, wherein the structural region comprises at least one alpha helices and the latch region comprises at least one alpha helices. In some aspects, the structural region of the first cage polypeptide comprises one alpha helix. In some aspects, the structural region of the first cage polypeptide comprises two alpha helices. In some aspects, the structural region of the first cage polypeptide comprises three alpha helices.
In some aspects, the first cage polypeptide, the first key polypeptide, the second key polypeptide, and/or the decoy polypeptide are further modified to change (i) hydrophobicity, (ii) a hydrogen bond network, (iii) a binding affinity to each, and/or (iv) any combination thereof. In some aspects, the cage polypeptide and/or the key polypeptide are modified to reduce hydrophobicity. In some aspects, the latch region is mutated to reduce the 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 aspects, one or more hydrophobic amino acids are replaced with a polar amino acid, e.g., serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), glutamine (Gin), and tyrosine (Tyr). In some aspects, an interface between the latch region and the structural region of the first cage polypeptide includes a hydrophobic amino acid to polar amino acid residue ratio of between 1:1 and 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 aspects, an interface between the latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 1:1. In some aspects, an interface between the latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 2:1. In some aspects, an interface between the latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 3:1. In some aspects, an interface between the latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 4:1. In some aspects, an interface between the latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 5:1. In some aspects, an interface between the latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 6:1. In some aspects, an interface between the latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 7:1. In some aspects, an interface between de latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 8:1. In some aspects, an interface between the latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 9:1. In some aspects, an interface between the latch region and the structural region includes a hydrophobic amino acid to polar amino acid residue ratio of 10:1.
In some aspects, 1, 2, 3, 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, e.g., valine (if the starting amino acid is isoleucine or leucine) or alanine. In some aspects, the first cage polypeptide comprises buried amino acid residues at the interface between the latch region and the structural region of the first cage polypeptide, wherein the buried amino acid residues at the interface have side chains comprising nitrogen or oxygen atoms involved in hydrogen bonding.
In some aspects, the disclosure provides non-naturally occurring polypeptides comprising
(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 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 27278-27321 not including optional amino acid residues; or cage polypeptides listed in Table 7, Table 8, or Table 9 wherein the N-terminal and/or C-terminal 60 amino acids of the polypeptides are optional; and
(b) one or more polypeptide binding domains.
In one embodiment, the non-naturally occurring polypeptides comprise
(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 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, 27,278 to 27,321, not including amino acid residues in the latch region; and
(b) one or more polypeptide binding domains.
In another embodiment, the non-naturally occurring polypeptides comprise (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 cage polypeptide disclosed herein, or selected from the group consisting of SEQ ID NOS: 27359-27392 or cage polypeptides listed in Table 7, Table 8, or Table 9, wherein the N-terminal and/or C-terminal 60 amino acids of the polypeptides are optional; and
(b) one or more polypeptide binding domains.
In a further embodiment, the 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% 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, 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27,278 to 27,321, or cage polypeptides listed in Table 7, Table 8, or Table 9, including any optional amino acid residues.
In one embodiment, the non-naturally occurring polypeptide comprises
(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 cage polypeptide disclosed selected from the group consisting of SEQ ID NOS: 27359-27392, not including optional amino acid residues, and
(b) one or more polypeptide binding domains.
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 cage polypeptide disclosed selected from the group consisting of SEQ ID NOS: 2735927392, including optional residues.
indicates data missing or illegible when filed
indicates data missing or illegible when filed
In another embodiment, the disclosure provides 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 the 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 the one or more binding domains, such as those disclosed herein.
As disclosed herein, exemplary polypeptides of the disclosure have been identified and subjected to mutational analysis. Furthermore, different designs starting from the same exemplary polypeptides yield different amino acid sequences while maintaining the same intended function. In various embodiments, a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as lie, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp, or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Polypeptides comprising 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 (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Leu (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 divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro. (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into H is; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; lie into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu. In some aspects, the cage polypeptide comprises an interface between the latch region and the structural region of one or more cage polypeptide of any composition or method disclosed herein. In one embodiment of polypeptides of the first and second aspect of the disclosure, interface residues between the latch and structural regions are primarily (i.e.: 50%, 60%, 70%, 75%, 80%, 85%, 90%, or greater)hydrophobic residues. In one embodiment, interface residues are primarily valine, leucine, isoleucine, and alanine residues. In a further embodiment an interface between a latch region and a structural region of the polypeptide includes a hydrophobic amino acid to polar amino acid residue ratio of between 1:1 and 10:1. The cage polypeptides may be “tuned” to modify strength of the interaction between the latch region and structural region as deemed appropriate for an intended use. In one embodiment 1, 2, 3, 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 including but not limited to valine (if the starting amino acid residue is isoleucine or leucine) or alanine. In this embodiment, the tuning weakens structural region-latch affinity. In some aspects, the cage polypeptide, e.g., the first cage polypeptide, comprises buried amino acid residues at the interface between the latch region and the structural region of the cage polypeptide. In another embodiment, buried amino acid residues at the interface comprise amino acid residues with side chains comprising nitrogen or oxygen atoms involved in hydrogen bonding. Tuning can include increasing or decreasing the number of hydrogen bonds present at the interface. Tuning can include making amino acid changes to increase or decrease the hydrophobicity of the interface. Tuning can include making amino acid changes to decrease the hydrophobic packing of the interface (e.g., by replacing a leucine with an alanine). Tuning can include introducing amino acid changes that create buried unsatisfied hydrogen bonds in the interface (e.g., by replacing a leucine with a serine). Based on the teachings herein, those of skill in the art will understand that such tuning may take any number of forms depending on the desired structural region-latch region affinity.
In certain embodiments, the polypeptides of the first and second aspects of the disclosure comprise one or more bioactive peptides in at least one of the alpha helices, such as in the latch domain, wherein 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 a bioactive peptide in an inactive state (until activated by a key polypeptide binding to the cage polypeptide, as described herein), and wherein the binding domain can serve to target the polypeptide to the entity to which the binding domain binds. In one embodiment, the polypeptides are part of a “protein switch” (together with appropriate key polypeptide(s)), 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 so that the cage and key polypeptides are co-localized while bound to their targets.
Any binding domain may be used as is suitable for an intended use. In non-limiting embodiments, the one or more bioactive peptides may comprise one or more bioactive peptide selected from the group consisting of SEQ ID NOS: 60, 62-64, 66, 27052, 27053, 27059-27093.
In a third aspect, the disclosure provides key polypeptides, comprising a key domain linked to one or more binding domains, wherein the key polypeptide is capable of specifically binding to the cage polypeptide of any embodiment of the first and/or second aspect of the 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 binding to the cage polypeptide, as described herein), and wherein the binding domain can serve to target the polypeptide to the entity to which the binding domain binds. In one embodiment, the polypeptides are part of a “protein switch” (together with appropriate key polypeptide(s)), 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 so 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 comprises (a) a polypeptide comprising an amino acid sequence at east 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95%, 96% 0.97%, 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 27322-27358; and key polypeptides listed in Table 7, Table 8, and/or Table 9; and
(b) one or more binding domains.
In another embodiment, non-naturally occurring polypeptides comprising a polypeptide comprising an amino acid sequence 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 to 27,358, as detailed below.
AHIVMVDAYKPTK
AHIVMVDAYKPTK
indicates data missing or illegible when filed
In a specific embodiment, the key polypeptides comprises an amino acid sequence at least 40%, 50%, 60%, 70%, 75%, 80%, 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 with an odd-numbered SEQ ID NO between SEQ ID NOS: 27127 and 27277), Table 8 and/or Table 9. In another specific embodiment, the key polypeptides 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 the amino acid sequence of a key polypeptide in Table 8. In another specific embodiment, the key polypeptides 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 the amino acid sequence of a key polypeptide in Table 9. In one embodiment of each of the above, the percent identity may be determined without the optional N- and C-terminal 60 amino acids; in another embodiment, the percent identify may be determined with the optional N- and C-terminal 60 amino acids.
indicates data missing or illegible when filed
indicates data missing or illegible when filed
(SEQ ID NO:6)
)
)
indicates data missing or illegible when filed
A. Binding Domains
In various embodiments of the polypeptides of the disclosure, the polypeptide includes one or more (i.e., 1, 2, 3, or more) binding domains. Any suitable binding domain may be used as appropriate for an intended use. In one embodiment, the one or more of the binding domains comprise cell surface protein binding polypeptides. In one such embodiment, the cell surface protein binding polypeptides are on a tumor cell. In another embodiment, the cell surface protein binding polypeptides are oncoproteins. In a further embodiment, the one or more binding domains are selected from the non-limiting group comprising an antigen-binding polypeptide directed against a cell surface moiety to be bound, including but not limited to Fab′, F(ab′)2, Fab, Fv, rIgG, recombinant single chain Fv fragments (scFv). VH single domains, bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies, DARPins; nanobody; affibody; monobody; adnectin; alphabody; Albumin-binding domain; Adhiron; Affilin; Affimer; Affitin/Nanofitin; Anticalin; Armadillo repeat proteins; Atrimer/Tetranectin; Avimer/Maxibody; Centyrin; Fynomer; Kunitz domain; Obody/OB-fold; Pronectin; Repebody; and computationally designed proteins. In another embodiment, the cell surface protein binding domain binds to a cell surface protein on a cell selected from the non-limiting group comprising tumor cells, cancer cells, immune cells, 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, 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, and yeast cells. In yet another embodiment, the cell surface protein binding domain binds to a cell surface protein selected from the non-limiting group comprising 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, 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.
In non-limiting embodiments, 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 the amino acid sequence selected from the group consisting of SEQ ID NOS: 27,399-27,403.
In further non-limiting embodiments, the cage polypeptides with 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 the amino acid sequence selected from the non-limiting group of SEQ ID NOS: 27404-27446.
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 the 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 the amino acid sequence selected from the non-limiting group of SEQ ID NOS: 27404-27446, excluding optional residues.
As disclosed herein, bioactive peptides to be sequestered by the polypeptides of the disclosure are located within the latch region. The latch region is denoted by brackets in the sequence of each cage polypeptide. The bioactive peptide may be added to the latch region without removing any residues of the latch region, or may replace one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid residue in the cage scaffold latch region to produce the final polypeptide. Thus, the latch region may be significantly modified upon inclusion of the bioactive peptide. In one embodiment, the optional residues are not included in determining percent sequence identity. In another embodiment, the latch region residues may be included in determining percent sequence identity. In a further embodiment, each of the optional residues and the latch residues may are not included in determining percent sequence identity.
In one embodiment of this second aspect, the polypeptides are polypeptides according to any embodiment or combination of embodiments of the first aspect and also comprising an amino acid sequence having 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 to the amino acid sequence of the listed reference cage polypeptides disclosed herein. In another embodiment, polypeptides further comprise a bioactive peptide within (or replacing) the latch region of the cage polypeptide.
The cage polypeptide may be a cage decoy polypeptide (i.e.: without a bioactive peptide). For example, see SEQ ID NOS: 1-17, 2034-14317, and certain cage polypeptides listed in Table 7, Table 8, and/or Table 9, or may further include a sequestered bioactive peptide (present as a fusion with the cage scaffold polypeptide) in the latch region of the cage scaffold polypeptide, as described in more detail herein (for example, see SEQ ID NOS: 18-49, 51-52, 54-59, 61, 65, 67-2033, 27094-27117, 27120-27125, and certain cage polypeptides listed in Table 7, 8, and/or 9). Ina specific embodiment, the cage polypeptides comprise an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 73%, 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 in Table 7, Table 8, and/or Table 9.
In another specific embodiment, the cage polypeptides 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% sequence identical to the amino acid sequence of a cage polypeptide in Table 8. In another specific embodiment, the cage polypeptides 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 the amino acid sequence of a cage polypeptide in Table 9. In one embodiment of each of these embodiments, the optional N-terminal and/or C-terminal 60 residues are not included in determining the percent sequence identity. In another embodiment, the optional residues may be included in determining percent sequence identity.
In one embodiment of the key polypeptides disclosed herein, 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% sequence identical to the amino acid sequence selected from the non-limiting group of SEQ ID NOS: 27448-27459, wherein residues in parentheses are optional. In one embodiment, sequence identity determination includes optional residues; in another embodiment, sequence identity determination does not include optional amino acid residues.
In some aspects, the first cage polypeptide, the second cage polypeptide, and/or the decoy cage polypeptide comprise:
(a) a polypeptide comprising an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 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 SEQ IDS NOS: 27359-27392, 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, and 27278-27321 not including optional amino acid residues; or cage polypeptides listed in Table 7, Table 8, or Table 9, wherein the N-terminal and/or C-terminal 60 amino acids of the polypeptides are optional; and (b) one or more first, fifth, sixth, or seventh binding domains.
In some aspects, the first cage polypeptide, the second cage polypeptide, and/or the decoy cage polypeptide comprise:
(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 cage polypeptide disclosed herein, or selected from the group consisting SEQ IDS NOS: 27359-27392, not including optional amino acid residues; and
(b) one or more first, fifth, sixth, or seventh binding domains.
In some aspects, the first cage polypeptide, the second cage polypeptide, and/or the decoy cage polypeptide 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 along its length to the amino acid sequence of a cage polypeptide disclosed herein, or selected from the group consisting SEQ IDS NOS: 27359-27392, including optional amino acid residues in some aspects, the first key polypeptide and/or the second key polypeptide comprise:
(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 selected from SEQ ID NOS: 27393-27398, 14318-26601, 26602-27015, 27016-27050, 27, 322-27,358, and key polypeptides listed in Table 7, Table 8, and/or Table 9; and
(b) one or more second, third, or fourth binding domains.
In some aspects, the first key polypeptide and/or the second key polypeptide comprise:
(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 00% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 27393-27398, or SEQ ID NOS: 27394-27395, not including optional residues, or including optional residues; and
(b) one or more second, third, or fourth binding domains.
In one embodiment of the compositions of any embodiment or combination of embodiments of the disclosure, the one or more bioactive peptides may comprise one or more bioactive peptide selected from the group consisting of SEQ ID NO:60, 62-64, 66, 27052, 27053, 27059-27093.
Nucleic Acids
In one aspect the disclosure provides nucleic acids encoding the polypeptide of any embodiment or combination of embodiments of each aspect disclosed herein. The nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded polypeptide, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the disclosure.
In another aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable “control sequence.” “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences 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 plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may 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 organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.
Cells
In a further aspect, the disclosure provides cells, e.g., host cells, therapeutic cells, or target cells, that comprise the nucleic acids, expression vectors (i.e.: episomal or chromosomally integrated), or polypeptides disclosed herein, wherein the cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. In one embodiment, 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. In another embodiment, the cells comprise:
(a) a first nucleic acid encoding the polypeptide of any embodiment or combination of embodiments of the cage polypeptides of the disclosure, operatively linked to a first promoter; and
(b) a second nucleic acid encoding the polypeptide of any embodiment or combination of embodiments of a key polypeptide of the disclosure, wherein the key polypeptide is capable of binding 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, wherein the second nucleic acid is operatively linked to a second promoter.
In some aspects, the cells can be in vitro cells. In some aspects, the cells are in vivo cells. In some aspects, the cells are ex vivo cells.
The cells may comprise a single cage polypeptide encoding nucleic acid and a single key polypeptide encoding nucleic acid, or may comprise a plurality (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 may encode a key polypeptide capable of binding to a structural region and inducing a conformational change of a different cage polypeptide encoded by the plurality of first nucleic acids. In another embodiment, each second nucleic acid may encode a key polypeptide capable of binding to a structural region and inducing a conformational change of more than one of the cage polypeptides encoded by the plurality of first nucleic acids.
The cells referred to herein can be target cells for a therapy or therapeutic cells. In some aspects, target cells can be tumor cells. In some aspects, target cells can be healthy cells. In some aspects, the first cell moiety, the second cell moiety, or both, are present on or within a healthy cell. In some aspects, the first cell moiety, the second cell moiety, or both, are present on or within a disease cell, in some aspects, the first cell moiety, the second cell moiety, or both, are present on or within a tumor cell or a cancer cell. In some aspects, the first cell moiety, the second cell moiety, or both, are present on or within an immune cell. In some aspects, the first cell moiety, the second cell moiety, or both, are present on or within a cell selected from leukocytes, lymphocytes, T cells, regulatory T cells, effector T cells, CD4+ effector T cells. CD+ 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 aspects, the first cell moiety, the second cell moiety, or both, are present on or within a cell 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.
Any suitable binding domains may be used in the compositions of the disclosure, as appropriate for an intended use. In some aspects, the first, second, third, fourth, fifth, sixth, and/or seventh binding domains are selected from the non-limiting group comprising an antigen-binding polypeptide directed against a cell surface moiety to be bound, including but not limited to Fab′, F(ab′)2, Fab, Fv, rIgG, recombinant single chain Fv fragments (sFv), VH single domains, bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies; DARPins; nanobody; affibody; monobody; adnectin; alphabody; Albumin-binding domain; Adhiron; Affilin; Affimer; Affitin/Nanofitin; Anticalin; Armadillo repeat proteins; Atrimer/Tetranectin; Avimer/Maxibody; Centyrin; Fynomer; Kunitz domain; Obody/OB-fold; Pronectin; Repebody; and computationally designed proteins, and any combination thereof.
In another embodiment, the first, second, third, fourth, fifth, sixth, and/or seventh binding domains bind to a cell surface protein on a cell selected from the non-limiting group comprising tumor cells, cancer cells, immune cells, 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, 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, and yeast cells.
In a further embodiment, the first, second, third, fourth, fifth, sixth, and/or seventh binding domains bind to a cell surface protein selected from the non-limiting group comprising Her2, EGFR, EpCAM, B7-H3, ROR1, GD2, GPC2, αvβ6, Her3, LICAM, 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, LILRB2, LTB4R, TLR2, LILRA2, ITGAX, CR1, EMC10, EMB, DAGLB, P2RY13, L1LRB3, L1LRB4, 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. In a further embodiment, the first, second, third, fourth, fifth, sixth, and/or seventh binding domains comprise 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% identity to the amino acid sequence selected from the group consisting of SEQ ID NOS: 27,399-27,403.
In one embodiment of the compositions of any embodiment or combination of embodiments of the 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 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 cage polypeptide and a key polypeptide, respectively, in the same row or one of 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, and so on), with the proviso that each cage polypeptide and each key polypeptide further comprise one or more binding domain.
In one embodiment, the first cage polypeptide, the second cage polypeptide, and/or the decoy cage polypeptide comprise:
In another embodiment, the first key polypeptide and/or the second key polypeptide comprise:
In another embodiment, the first cage polypeptide, the second cage polypeptide, and/or the decoy cage polypeptide 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 the 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 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 the 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 comprise an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 93%, 96%, 97%, 98%, 99%, or 100% identical to the 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 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 the amino acid sequence selected from the group consisting of SEQ ID NOS: 27448-27439.
Effectors
In some aspects, an effector useful for the present disclosure comprises one or more binding moieties. In some aspects, an effector comprises an antibody or antigen binding fragment thereof, T cell receptor, DARPin, bispecific or bivalent molecule, nanobody, affibody, monobody, adnectin, alphabody, albumin binding domain, adhiron, affilin, affimer, affitin/nanofitin; anticalin; armadillo repeat protein; atrimer/tetranectin; avimer/maxibody; centyrin; fynomer; Kunitz domain; obody/OB-fold; pronectin; repebody; a computationally designed protein; a protease, a ubiquitin ligase, a kinase, a phosphatase, and/or an effector that induces proteolysis; or any combination thereof. In some aspects, the antigen binding portion thereof comprises a Fab′, F(ab′)2, Fab, Fv, rIgG, recombinant single chain Fv fragment (scFv), and/or VH single domain.
In some aspects, the effector is a therapeutic cell. In some aspects, the therapeutic cell comprises an immune cell. In certain aspects, the cell is selected from a T cell, a stem cell, an NK cell, a B cell, or any combination thereof. In some aspects, the stem cell is an induced pluripotent stem cell.
In some aspects, administration of the effector kills the cell that comprises the first binding moiety and the second binding moiety, results in receptor signaling (e.g., cytokine) in the cell that comprises the first binding moiety and the second binding moiety; results in production of signaling molecules (e.g., cytokine, chemokine) nearby the cell that comprises the first binding moiety and the second binding moiety; or results in differentiation of the cell that comprises the first binding moiety and the second binding moiety.
In some aspects, administration of the effector induces receptor signaling (e.g., cytokine) in the cell that comprises the first binding moiety and the second binding moiety. In some aspects, administration of the effector results in production of signaling molecules (e.g., cytokine, chemokine) nearby the cell that comprises the first binding moiety and the second binding moiety, including but not limited to a CD4+ T cell releasing cytokines in the tumor to support CD8+ T cell effector function. In some aspects, administration of the effector induces differentiation in the cell that comprises the first binding moiety and the second binding moiety.
Other aspects of the present disclosure are directed to one or more cells comprising a composition disclosed herein. In some aspects, the cell further comprises an effector disclosed herein. In some aspects, the cell is a tumor cell or a cancer cell. In some aspects, the cell is an immune cell. In some aspects, the cell is selected from leukocytes, lymphocytes, T cells, regulatory T cells, effector T cells, CD4+ effector T cells, CD5+ 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 aspects, the cell is 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.
In some embodiments, the compositions of the fourth and fifth aspects of the disclosure do not include an effector, as the proximity-dependent binding even may be detectable without an effector protein. In one embodiment of the compositions of any embodiment of the fourth and fifth aspects of the disclosure, the effector(s) is/are present. Any effector suitable for an intended use may be used. In certain aspects, the effector binds to the one or more bioactive peptides. In one embodiment, the effector(s) are selected from the non-limiting group comprising Bcl2, GFP1-10, small molecules, antibodies, antibody drug conjugates, immunogenic peptides, proteases, T cell receptors, cytotoxic agents, fluorophores, fluorescent proteins, cell adhesion molecules, endocytic receptors, phagocytic receptors, magnetic beads, and gel filtration resin, and polypeptides 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 the amino acid sequence selected from the group consisting of SEQ ID NOS: 27,460-27,469.
Some aspects of the present disclosure are directed to methods of increasing selectivity of a cell in vitro, ex vivo, or in vivo. Other aspects of the present disclosure are directed to methods of increasing selectivity of cells that are interacting with each other in vitro, ex vivo, or in vivo. Other aspects of the present disclosure are directed to methods of targeting heterogeneous cells (more than two different cell types) in vitro, ex vivo, or in vivo. Other aspects of the present disclosure are directed to methods of reducing off-target activity in vitro, ex vivo, or in vivo.
In some aspects, the present disclosure is directed to a method of increasing selectivity of a cell 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 is directed to a method of increasing selectivity of a cell 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 the cells in vitro, in vivo, or ex vivo together (concurrently) or separately. Some aspects of the present disclosure are directed to a method of increasing selectivity of a cell in vitro, ex vivo, or in vivo comprising (a) contacting cells with (e.g., expressing or adding) a first cage polypeptide fused to a first binding domain, and (b) contacting ((e.g., expressing or adding) the cell with a first key polypeptide fused to a second binding domain. In some aspects, the first cage polypeptide comprises (i) a structural region and (ii) a latch region further comprising one or more bioactive peptides.
Some aspects of the present disclosure are directed to a method of increasing selectivity of cells that are 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 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 colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on 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 colocalization with the first cage polypeptide, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the second binding domain is capable of binding to a second cell moiety present on the synapse between the two or more cells.
In some aspects, 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 a first cell moiety, wherein upon colocalization with the first cage polypeptide, the second key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides, wherein the third binding domain is capable of binding to a third cell moiety present on the synapse of the two or more cells.
In some aspects, the method further comprises contacting the two or more cells with one or more decoy cage polypeptide fused to one or more decoy binding domain with the two or more cells wherein each decoy cage polypeptide comprises a decoy structural region, which upon colocalization with the first key polypeptide and the first cage polypeptide, is capable of preferentially binding to the first key polypeptide and wherein each decoy binding domain is capable of binding to a decoy cell moiety in the synapse of the two or more cells.
Some aspects of the disclosure are directed to a method of targeting heterogeneous cells (i.e., more than two different cell types) in vitro, ex vivo, or in vivo, wherein a first cell moiety and a second cell moeity are present on the first cell and a first cell moiety and a third cell moiety are present on the second cell, comprising, 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, and wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell 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 colocalization, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides and wherein the second binding domain is capable of binding to a second cell moiety present on a cell that also comprises the first cell moiety, and (c) contacting the two or more cells with a second key polypeptide fused to a third binding domain, wherein upon colocalization, the second key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides and wherein the third binding domain is capable of binding to a third cell moiety present on a cell that comprises the first cell moiety.
In some aspects, the method further comprises contacting the two or more cells with a one or more decoy cage polypeptide fused to one or more decoy binding domain, wherein each decoy cage polypeptide comprises a decoy structural region, which upon colocalization with the first key polypeptide, the second key polypeptide, and/or the first cage polypeptide, is capable of preferentially binding to the first key polypeptide or the second key polypeptide, and wherein each decoy binding domain is capable of binding to a decoy cell moiety in a cell that comprises the first cell moiety and the second cell moiety.
Some aspects of the present disclosure are directed to 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, and wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides in the absence of colocalization with a key polypeptide and wherein the first binding domain is capable of binding to a first cell moiety present on a cell; (b) contacting the two or more cells with a first key polypeptide fused to a second binding domain, wherein upon colocalization, the first key polypeptide is capable of binding to the cage structural region to activate the one or more bioactive peptides and wherein the second binding domain is capable of binding to a second cell moiety present on a cell that also comprises the first cell moiety, and (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, which upon colocalization with the key polypeptide and the first cage polypeptide, is capable of preferentially binding to the first key polypeptide and wherein the third binding domain is capable of binding to a third cell moiety in a cell that comprises the first cell moiety and the second cell moiety. In some aspects, the third cell moiety is only present on a healthy cell.
As used herein, “contacting” refers to any means of bring a first element into contact with a second element. In some aspects, contacting includes directly adding a first element, e.g., a polypeptide, to second element, e.g., a cell, such as, for example, by adding a protein into a cell culture. In some aspects, contacting includes expressing the first element, e.g., a protein, by a nucleotide encoding the protein in the target cell or in a cell that is in the same culture as the target cell. In some aspects, the contacting of (a) the cell with a first cage polypeptide fused to a first binding domain, and (b) the contacting of the cell with a first key polypeptide fused to a second binding domain are performed concurrently. In some aspects, the contacting (a) is performed prior to the contacting (b). In some aspects, the contacting (b) is performed prior to the contacting (a). In some aspects, the contacting includes introducing a polynucleotide encoding a polypeptide (e.g., the first cage polypeptide, the first key polypeptide, the second key polypeptide, and the decoy cage polypeptide).
The method disclosed herein increases the selectivity of a cell for a target cell, in some aspects, the colocalization of the first cage polypeptide and the key polypeptide increases the selectivity of a cell that highly expresses the first cell moiety and the second cell moiety. In some aspects, the colocalization of the first cage polypeptide and the key polypeptide increases the selectivity of a cell that highly expresses the first and second cell moiety. In some aspects, the colocalization of the first cage polypeptide and the key polypeptide increases the selectivity of a cell that highly expresses the first and second cell moieties and a call that highly expresses the first and third cell moieties.
In a further aspect, the disclosure provides methods of targeting an effector to a cell comprising contacting a biological sample containing cells with the polypeptides, nucleic acids, vectors, cells, and/or compositions of any embodiment or combination of embodiments of the disclosure.
In another embodiment, the disclosure provides methods for cell targeting, comprising
(a) contacting a biological sample containing cells with
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, and to promote binding of the key polypeptide to the cage structural region to displace the latch region and activate the one or more bioactive peptides only when the cage polypeptide and the key polypeptide are co-localized to the cell of interest;
(b) contacting the biological sample with one or more effector(s) under conditions to promote binding of the one or more effectors to the one or more activated bioactive peptides to produce an effector-bioactive peptide complex; and
(c) optionally detecting the effector-bioactive peptide complex, wherein the effector-bioactive peptide complex provides a measure of the cell of interest in the biological sample.
Other aspects of the disclosure are directed to methods of preparing a subject in need of a therapy comprising administering a composition disclosed herein. Some aspects of the disclosure are directed to methods of preparing a subject in need of a therapy comprising administering a cell disclosed herein.
Some aspects are directed to a method of treating a disease or condition in a subject in need thereof comprising administering an effector to the subject, wherein the subject is also administered a composition disclosed herein. In some aspects, the administering of the effector molecule kills the cell that comprises the first binding moiety and the second binding moiety, results in receptor signaling (e.g., cytokine) in the cell that comprises the first binding moiety and the second binding moiety; results in production of signaling molecules (e.g., cytokine, chemokine) nearby the cell that comprises the first binding moiety and the second binding moiety; or results in differentiation of the cell that comprises the first binding moiety and the second binding moiety. Any effector disclosed herein can be used in the method. In some aspects, the effector binds to the one or mote bioactive peptides. In some aspects, the effector comprises an antibody or antigen binding fragment thereof, T cell receptor, DARPin, bispecific or bivalent molecule, nanobody, affibody, monobody, adnectin, alphabody, albumin binding domain, adhiron, affilin, affimer, affitin nanofitin; anticalin; armadillo repeat protein; atrimer/tetranectin; avimer/maxibody; centyrin; fynomer; Kunitz domain; obody/OB-fold; pronectin; repebody; a computationally designed protein; or any combination thereof. In certain aspects, the effector comprises an antibody or antigen binding fragment thereof. In some aspects, the antigen binding portion thereof comprises a Fab′, F(ab′)2, Fab, Fv, rIgG, recombinant single chain Fv fragment (scFv), and/or VH single domain.
In some aspects, the effector is a therapeutic cell. In some aspects, the therapeutic cell comprises a T cell, a stem cell, an NK cell, a B cell, or any combination thereof. In some aspects, the therapeutic cell comprises an immune cell. In some aspects, therapeutic cell comprises a T cell. In some aspects, therapeutic cell comprises a stem cell. In some aspects, the stem cell is an induced pluripotent stem cell. In some aspects, therapeutic cell comprises an NK cell.
Natural biological systems integrate multiple protein binding inputs through post-translational signaling cascades that are hardcoded to specialized functions; a synthetic system capable of integrating multiple binding inputs through conformational switching could be a general solution for predictively controlling diverse biological functions. We describe the computational design of proximity-activated de novo protein switches that perform ‘AND’, ‘OR’, and ‘NOT’ Boolean logic operations and combinations thereof in response to precise combinations of protein-binding events. The switches activate via a conformational change only when all logic 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 that are distinguished in a complex cell population only by their precise combination of surface markers. Our work shows that de novo designed proteins can perform computations on the surface of cells, integrating multiple distinct binding interactions into a single biological output.
We set out to design a generalizable protein system from scratch that is capable of performing complex logic in response to combinatorial binding events. We aimed for a modular system capable of computing combinations of Boolean logic operations (‘AND’, ‘OR’, and ‘NOT’) when the components are brought into close proximity and actuating a single binding interaction as output (
We set out to design de novo protein switches for which the actuation domain is activated by the proximity of additional designed components. We designed protein switches that activate in solution: Latching Orthogonal Cage-Key pRotein (LOCKR) switches are composed of a structural “Cage” protein that uses a “Latch” domain to sequester a functional peptide in an inactive conformation until binding of a separate “Key” protein induces a conformational change that permits binding to an “Effector” protein. Cage, Key, and Effector bind in a three-way equilibrium, and the sensitivity of the switch can be tuned by adjusting the relative Cage-Latch and Cage-Key affinities. We designed new LOCKR proteins to be inert in solution and strongly activated only when the Cage and Key are colocalized. We designed new LOCKR switches with shorter helices, improved hydrophobic packing, and an additional hydrogen bond network to promote interaction specificity among the helices (
We used the new design as the starting point to develop colocalization-dependent LOCKR (Co-LOCKR) switches (
To evaluate the ability of Co-LOCKR to target cells co-expressing a precise combination of surface antigens, we developed a mixed population flow cytometry assay by combining four K562 cell lines expressing Her2-eGFP, EGFR-iRFP, both, or neither (
We next sought to tune the dynamic range of Co-LOCKR activation to increase colocalization-dependent activation sensitivity and responsiveness. Our initial design was intended to maximize Cage-Latch affinity so as to ensure colocalization-dependence, leading us to ask whether weakening the Cage-Latch affinity could enhance signal intensity without compromising the ability to compute logic. The sensitivity of previous LOCKR switches was timed by shortening the Latch to produce a ‘toehold’, but this also promoted aggregation (
Co-localization dependent activation was also observed at the sub-cellular level by confocal microscopy. CL_CHKE recruited Bcl2-AF680 to the plasma membrane of HEK293T/Her2/EGFR cells but not HEK293T/Her2 or HEK293T/EGFR (
To assess the flexibility of Co-LOCKR, we attempted to specifically target alternative pairwise combinations of three cancer-associated antigens (Her2, EGFR, and EpCAM). Each of these antigens is expressed at differing levels by engineered K562 cell lines or human cancer cell lines (
A truly general technology for targeting any cell type in situ requires more complex logic comprising combinations of ‘AND’, ‘OR’, and ‘NOT’ operations. In principle, the colocalization-dependent activation mechanism of Co-LOCKR should be particularly well suited to accomplish this. ‘OR’ logic can potentially be achieved by adding a second Key fused to a binding domain targeting an alternative surface marker (
Using Her2. EGFR, and EpCAM as model antigens (Ag), we first explored [Ag1 AND either Ag2 OR Ag3] logic on the surface of cells (
We next explored [Ag3 AND Ag2 NOT Ag3] logic using CL_CHKEpDE (D for Decoy) and the same set of model antigens (
The ability to perform complex logic operations using Co-LOCKR affords a level of control and flexibility not reported by previous targeting technologies. Furthermore, the ability to tune responsiveness with rationally designed point mutations enables the rapid optimization of Co-LOCKR for a wide range of applications.
In contrast to current methods. Co-LOCKR computes logic on a single cell expressing precise combinations of antigens in cis, specifically directing cytotoxicity against target cells without harming neighboring off-target cells that only provide a subset of the target antigens. The ability to implement complex logic (e.g., [Ag1 AND either Ag2 OR Ag3] (
Generally, the power of the Co-LOCKR system results from the integration of multiple coherent or competing inputs that determine 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 are no limits on the numbers of each molecule, allowing for arbitrarily complex logic operations. Although our present work has focused on describing the system and demonstrating its ability to improve T cell-based cancer immunotherapies in vitro, the Co-LOCKR system is powerful for engineering biology in any setting that requires proximity-based activation or specific targeting through calculations on the surface of cells.
As a starting point, the backbone of LOCKRa (SEQ ID NO:6) was used as input coordinates to Rosetta protein design software. Latch residues, residues on the Cage making contacts to the Latch (defined by the Interface By Vector Residue Selector in Rosetta™), and existing hydrogen bond networks were held fixed to coordinates of their input rotamers while the remaining residue positions were redesigned as follows: first, additional hydrogen bond networks were designed using HBNet™; second. RosettaDesign™ calculations were performed to optimize hydrophobic packing while the new hydrogen bond networks were maintained using AtomPair restraints on the heavy atoms of each sidechain hydrogen bond. This design procedure produced a new asymmetric Cage scaffold dubbed asymLOCKR. We then created a shorter version of this design by truncating the helical 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 (
We rationally mutated large, hydrophobic residues in the Latch of SEQ ID NO: 27359 (I287A, I287S, I269S) or Cage (L209A) to Alanine or Serine to weaken the Cage-Latch interface and increase Co-LOCKR sensitivity. We deleted several amino acids at the N- or C-terminus of the Key so as to weaken the Cage-Key interface and decrease Co-LOCKR sensitivity/leakiness.
Native Bcl2 was redesigned to improve its solution behavior and stability. As a starting point, the C-terminal 32 residues of the transmembrane domain were deleted, and the long loop between residues 35-91 of Bcl2 was replaced with residues 35-50 of the homolog Bcl-xL, as described previously (30) Additional mutations were made using Rosetta™ and PROSS™ (31) to improve hydrophobic packing and stability. Additional surface mutations were made rationally to improve solubility and remove glycosylation sites.
E. coli Lemo21™ (DE3) cells harboring a pET21 plasmid encoding the gene of interest were grown overnight (10-16 hours) in 3 ml Luria-Bertani (LB) medium supplemented with 50 μg ml−1 carbenicillin with shaking at 225 rpm at 37° C. Starter culture were added to 500 ml Studier TBM-5052 auto induction media supplemented with carbenicillin, grown at 37° C. for 4-7 hours, and then grown at 18° C. for an additional 18-24 hours. Cells were harvested by centrifugation at 5000 g and 4° C. for 15 minutes and resuspended in 20 ml lysis buffer (25 mM Tris pH 8.0 at room temperature, 300 mM NaCl, 20 mM Imidazole, 1 mg ml−1 lysozyme (Sigma L6876, from chicken egg), 0.1 mg ml−1 DNase I (Sigma, DN25, from bovine pancreas). Cells were lysed by microfluidization in the presence of 1 mM phenylmethanesulfonyl fluoride (PMSF). Lysates were clarified by centrifugation at 24,000 g at 4° C. for 30 minutes and passed through 2 ml of nickel-nitrilotriacetic acid agarose (Ni-NTA, Qiagen, 30250) pre-equilibrated in lysis buffer. 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), washed once with 5 CV of high-salt wash buffer (25 mM Tris pH 8.0 at room temperature, 1 M NaCl, 40 mM Imidazole), washed once more 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 proteins were then concentrated (Amicon® Ultra-15 Centrifugal Filter Units. 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 supplemented with glycerol to a final concentration of 10% v/v before being quantitated by absorbance at 280 nm (Nanodrop™), aliquoted, and snap frozen in liquid nitrogen. Protein aliquots were stable at −80° C.
For crystallography screening, the hexahistidine tag was removed via TEV cleavage followed by Ni-NTA affinity chromatography prior to SEC/FPLC. Purified protein samples were concentrated to approximately 12 mg ml−1 and screened using JCSG+ and JCSG Core I-IV screens (Qiagen) on a 5-position deck Mosquito crystallization robot (ttplabtech) with an active humidity chamber. Crystals were obtained after 2 to 14 days by sitting drop vapor diffusion with drop ratios of 1:1, 2:1 and 1:2 protein solution to reservoir solution. The condition that resulted in the crystals that were used for structure determination was 0.2 M di-Sodium tartrate, 20% (w/v) PEG 3350 and no cryoprotectant added.
Protein crystals were looped and flash-frozen in liquid nitrogen. Datasets were collected at the Advanced Light Source at Lawrence Berkeley National Laboratory with beamlines 8.2.1 and 8.2.2. Data sets were indexed and scaled using XDS (34) and phase information was obtained by molecular replacement (MR) using PHASER™ (35) from the Phenix™ software package (36); design models were used for the initial MR searches. Following MR, models were improved using Phenix.autobuild (37); efforts were made to reduce model bias by setting rebuild-in-place to false, and using simulated annealing and prime-and-switch phasing. Iterative rounds of manual building in COOT™ (38) and refinement in Phenix™ were used to produce the final models. Translational non-crystallographic symmetry was present in the data as report by Phenix.Xtriage, which complicated structure refinement and may explain the higher than expected R-values reported, RMSDs of bond lengths, angles and dihedrals from ideal geometries were calculated using Phenix™ (36). The overall quality of the final models was assessed using MOLPROBITY™ (39). Table 16 summarizes diffraction data and refinement statistics.
For BLI experiments, wild-type non-optimized Bcl2 with C-terminal Avi and 6×His-tags was enzymatically biotinylated using BirA according to manufacturer protocols (Avidity), purified by Ni-NTA, eluted into TBS, concentrated, snap frozen in liquid nitrogen, and stored at −80° C. For flow cytometry experiments, Bcl2 with a C-terminal cysteine was purified by Ni-NTA and gel filtration as described above with the addition of 0.5 mM TCEP to the buffers. 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™ 594 C5 Maleimide (Invitrogen A10256) or Alexa Fluor™ 680 C2 Maleimide (Invitrogen A20344). The labeling reaction was then dialyzed overnight into TBS supplemented with 10% glycerol and purified by get filtration as described above. Fractions containing monomeric protein were pooled, concentrated, and supplemented with glycerol to a final concentration of 10% v/v before being quantitated by absorbance at 280 nm, aliquoted, and snap frozen in liquid nitrogen. Protein aliquots were stable at −80° C. After thawing, protein aliquots were stored at 4° C. for up to one week.
BLI measurements were made on an Octet® RED96 System (ForteBio) with streptavidin (SA) coated biosensors and analyzed using ForteBio Data Analysis Software version 9.0.0.10. Assay buffer was HBS-EP+ Buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20, 0.5% non-fat dry milk, pH 7.4 at room temperature). Biotinylated Bcl2 protein was loaded onto the SA tips using a programmed threshold of 0.5 nm. Baseline was obtained by dipping the loaded biosensors into HBS-EP+ buffer; association kinetics were observed by dipping loaded biosensors into wells containing a range of LOCKR Cage and Key concentrations. Dissociation kinetics were observed by dipping tips into the HBS-EP+ Buffer wells that were used to obtain baseline. For
The scFv-targeted Co-LOCKR proteins (anti-Her2_Cage_I269S and Key_anti-EGFR-scFv) were produced using the Daedalus system as previously described (40). Proteins were purified on a HisTrap™ FF Crude protein purification column (GE cat #17528601) followed by size exclusion chromatography (GE Superdex 200 10/300 GL) and eluted in Dulbecco's phosphate-buffered saline supplemented with 5% glycerol.
K562 (CCL-243), Raji (CCL-86), A431 (CRL-1555), and HEK293T (CRL-3216) cells were obtained from 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 RPMI-1640 (Gibco) supplemented with 5% fetal bovine serum (FES), 1 mM L-glutamine, 25 mM HEPES, and 100 U ml−1 penicillin/streptomycin. A431, SKBR3, HEK293T, and LentiX cells were cultured in DMEM high glucose (Gibco) supplemented with 10% FBS, 1 mM L-glutamine, 25 mM HEPES, 100 U ml−1 penicillin/streptomycin, and 1 mM pyruvate. Primary human T cells were cultured in CTL medium consisting of RPMI-1640 supplemented with 10% human serum, 2 mM L-glutamine, 25 mM HEPES. 100 U ml−1 penicillin/streptomycin and 50 μM β-mercaptoethanol. All cells were cultured at 37° C., and 5% CO2, and tested bi-monthly for the absence of Mycoplasma using MycoAlert™ Mycoplasma Detection Kit (Lonza).
HEK293T or LentiX cells were transiently transfected with psPAX2 (Addgene Plasmid #12260), pMD2.G (Addgene Plasmid #12259) packaging plasmids as well as a lentiviral vector encoding either Her2-eGFP, EGFR-iRFP (for K562 cells), or EGFR-mCherry™ (for HEK293T cells) using linear 25-kDa polyethyleneimine (PEI; Polysciences). Two days later, viral supernatant was concentrated by centrifugation at 8000 g for 18 hours and added to K562 cells or REK293T with 4 μg ml−1 Polybrene (Sigma). Flow cytometry indicated that the Her2-eGFP and EGFR-iRFP cell lines were transduced to 98%, and the Her2-eGFP/EGFR-iRFP cell line was transduced to 88%.
Because K562 cells endogenously expressed low levels of EpCAM, EpCAM knockout (KO) cell lines were generated by nucleofection with the Alt-R® CRISPR-Cas9 system (IDT). Pre-designed 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, mixed with tracrRNA at equimolar concentrations, annealed by heating to 95° C. for 5 minutes, followed by slow cooling to room temperature, crRNA-tacrRNA duplexes were combined and complexed with S.p. Cas9 Nuclease V3 and Cas9 Electroporation Enhancer for 15 minutes at room temperature. RNP complexes were added to K562 cell lines and nucleofection was performed using a 4D Nucleofector mCherry™ (Lonza) using SF Cell Line Buffer and FF-120 program according to manufacturer's instructions. Four days later, cells that stained negative for EpCAM were FACS-sorted to greater than 99% purity.
EpCAM high K562 cell lines were generated by transducing Her2-eGFP, Her2-eGFP/EGFR-iRFP, and parental K562 cells with an EpCAM-expressing lentivirus that had been prepared by transiently transfecting LentiX cells with psPAX2, pMD2.G and a lentiviral vector encoding human EpCAM (UniProt P16422, aa1-314) using CalPhos™ Mammalian Transfection Kit (Clontech). Two days after transfection, viral supernatant was filtered using a 0.45 μm PES syringe filter (Millipore) and added to the cell lines with 4 μg ml−1 Polybrene. Five days later, transduced cells that stained high for EpCAM, EGFR, or Her2 were FACS-sorted to greater than 95% purity. Bim-eGFP-expressing K562 cells were generated in an identical manner using a lentivirus encoding a membrane-tethered Bim-eGFP fusion protein (mlgK signal peptide, GS linker, Bim peptide, SGSG linker, eGFP, PDGFR transmembrane domain), and FACS-sorted for eGFP expression five days after transduction.
Cells were stained with a 1:100 dilution of fluorophore-conjugated monoclonal antibodies specific for human EGFR (AY13), EpCAM (9C4), HA 1.1 (16B12), or Her2 (24D2) purchased from ThermoFisher or Biolegend. Cells were also stained with isotype control fluorophore-conjugated antibodies when appropriate. For Bcl2-AF594 binding measurements. K562 cell lines were combined into mixed populations with equal numbers of each cell type. Because EpCAM was not tagged with a fluorescent protein, two distinct populations were evaluated for each logic operation in
Data were acquired on a 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 manufacturer's protocols. All flow cytometry data were analyzed using FlowJo™ (Treestar).
HEK293T cells were grown in ibidi μ-slide 8 well coverslips for 1 day at 37° C. and 5% CO2 (ibidi 80826). Cell staining and incubation were performed in DMEM, high glucose, HEPES, no phenol red (Gibco 21063029). Cell nuclei were stained with Invitrogen Molecular Probes NucBlue™ Live ReadyProbes™ Reagent according to manufacturer's instructions (Invitrogen R37605). Cells were incubated in culture medium containing 1% BSA, 20 nM Her2_Cage-I269S, 20 nM Key_EGFR, and 50 nM Bcl2-AF680 for 1-2 hours at 37° C., and 5% CO2. Images were acquired on a Leica SP8X confocal microscope and analyzed in Fiji. Confocal microscopy heat map analysis
Red, green, and blue (RGB) pseudocolors were assigned to the mCherry™, eGFP, and AF680 channels, respectively, in Fiji. Using a custom python script (see supplement), the ImageIO Python library was used to read the RGB PNG files, the SciPy Python library was used to generate a bidimensional binned statistic from the pseudocolored pixel intensities, and the Matplotlib™ library was used to visualize the results as a heat map.
Statistical analyses were performed using Prism™ (GraphPad). An ordinary one-way ANOVA test followed by Dunnett's post-hoc test was used to compare Co-LOCKR-induced targeting (
favored (%)
allowed (%)
outliers (%)
indicates data missing or illegible when filed
indicates data missing or illegible when filed
heat map analysis
python
coding
47
8 2019
audrey
sky
RGB image pixel intensities
green is on the y-axis
and blue is the heat
import stats
plot as ply
(image)
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_data[0]
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p=′plasma′
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colorbar( )
set_label
AF680 mean pixel intensity
xlabel
CherryTM pixel intensity (EGFR
ylabel
FP pixel intensity (HER2
fig(image
heatmap
(
2016)
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indicates data missing or illegible when filed
Modular Co-LOCKR Cage domains: See Table 1.
Co-LOCKR Cage and Decoy proteins: See Table 11.
Modular Co-LOCKR Key domains: See Table 4.
Co-LOCKR Key proteins: See Table 12.
Effector proteins: See Table 13.
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/848,802 filed May 16, 2019 and 62/964,016 filed Jan. 21, 2020, each incorporated by reference herein in its entirety.
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. R01 CA114536 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/033429 | 5/18/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62964016 | Jan 2020 | US | |
| 62848802 | May 2019 | US |
