Human carbonic anhydrase 2 compositions and methods for tunable modulation

Abstract

The present disclosure provides tunable biocircuit systems. Such systems provide a modular and tunable protein expression system that supports the discovery and development of therapeutic modalities. In particular, the present application relates to fusion proteins comprising a human carbonic anhydrase 2 fragment and a chimeric antigen receptor (CAR). The activity of the destabilizing domain can be tuned by an externally administered agent.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 815,399, filed March 8, 2019; U.S. Provisional Application No. 62 / 815,402, filed March 8, 2019; U.S. Provisional Application No. 62 / 826,487, filed March 29, 2019; U.S. Provisional Application No. 62 / 826,443, filed March 29, 2019; U.S. Provisional Application No. 62 / 835,548, filed April 18, 2019; U.S. Provisional Application No. 62 / 835,552, filed April 18, 2019; and U.S. Provisional Application No. 62 / 860,388, filed June 12, 2019. The entire contents of the foregoing applications are incorporated herein by reference.

[0003] References to sequence lists

[0004] This application includes a “long” sequence list of CD-Rs that have been submitted in lieu of printed paper copies and are incorporated herein by reference in their entirety. The CD-Rs, created and documented on March 6, 2020, are labeled “CRF,” “Copy 1,” “Copy 2,” “Copy 3,” and “Copy 4,” each containing only a single, identical 699,270,904-byte (measured in MS-WINDOWS) file named 268052-462540_SL.txt. Each CD-R is machine-readable in IBM-PC format, and each disc runs on MS-Windows. Technical Field

[0005] This disclosure relates to a destabilizing domain (DD) derived from human carbonic anhydrase 2 (CA2) capable of modulating protein stability for at least one payload, as well as compositions thereof and methods of use thereof. Provided in this disclosure are peptides, CA2 effector submodules, stimulus-response elements (SREs), polynucleotides encoding them, vectors comprising said peptides and / or polynucleotides, and cells for use in cancer immunotherapy. Background Technology

[0006] Gene and cell therapies are revolutionizing medicine, offering new hope for treating previously intractable diseases. However, most current technologies do not allow for the titration of target protein induction time or levels. This makes the safe and effective deployment of many potential gene and cell therapies difficult or impossible.

[0007] Inadequate regulation of exogenous and / or endogenous genes is a key issue in many gene and cell therapy settings. This lack of tunability also makes it difficult to safely express proteins with narrow or indeterminate therapeutic windows or proteins that require further titration or transient expression.

[0008] One approach to regulating protein expression or function is the use of destabilizing domains (DDs). A destabilizing domain is a small protein domain that can be attached to a target protein of interest. In the absence of a DD-binding ligand, the attached protein of interest becomes unstable and is rapidly degraded by the cell's ubiquitin-proteasome system. However, when a specific small molecule DD-binding ligand binds to the DD, the attached protein of interest is stabilized, and its function is restored.

[0009] DD technology forms the basis for a new class of cell and gene therapies that allow for tunable temporal control of gene expression and function, expanding the range of protein therapeutics that can be safely and effectively incorporated into cell and gene therapy modalities. Summary of the Invention

[0010] This disclosure provides novel protein domains derived from human carbonic anhydrase 2 (CA2) exhibiting small molecule-dependent stability. Such protein domains are referred to as destabilizing domains (DDs). In the absence of their binding ligands, DDs are destabilized and cause degradation of the payload fused with DDs (e.g., the protein of interest (POI)). In the presence of their binding ligands, the fused DDs and payloads can be stabilized, and their stability is dose-dependent.

[0011] In a first aspect, this disclosure provides compositions comprising an effector submodule. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises an H122Y mutation at amino acid position 122 (H122) of SEQ ID NO. 11717.

[0012] In a second aspect, this disclosure provides compositions comprising an effector submodule. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises an E106D mutation at amino acid position 106 (E106) of SEQ ID NO. 11717.

[0013] In a third aspect of this disclosure, a composition comprising an effector submodule is provided. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises a W208S mutation at amino acid position 208 (W208) of SEQ ID NO. 11717.

[0014] In a fourth aspect of this disclosure, a composition comprising an effector submodule is provided. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises an I59N mutation at amino acid position 59 (159) of SEQ ID NO. 11717.

[0015] In a fifth aspect of this disclosure, a composition comprising an effector submodule is provided. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises an L156H mutation at amino acid position 156 (L156) of SEQ ID NO. 11717. In an embodiment related to the fifth aspect, DD further comprises: (i) a W4Y mutation at amino acid position 4 (W4) of SEQ ID NO. 11717; (ii) a F225L mutation at amino acid position 225 (F225) of SEQ ID NO. 11717; (iii) a deletion of amino acids 257-260 of SEQ ID NO. 11717; (iv) a deletion of amino acids 1-5 of SEQ ID NO. 11717; or (v) a deletion of amino acids G234, E235 and P236 of SEQ ID NO. 11717; or DD comprises four mutations relative to SEQ ID NO. 11717, including mutations corresponding to: (i) L156H, S172C, F178Y and E186D; or (ii) D70N, D74N, D100N and L156H.

[0016] In a sixth aspect, this disclosure provides compositions comprising an effector submodule. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD) comprising a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises a first mutation and a second mutation relative to SEQ ID NO. 11717, wherein: (i) the first mutation is an S73N mutation at amino acid position 73 (S73) of SEQ ID NO. 11717; and (ii) the second mutation is a substitution of F or Y at amino acid position 89 (R89) of SEQ ID NO. 11717.

[0017] In a seventh aspect, a composition comprising an effector submodule is provided. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD) comprising a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises a substitution of N or F for amino acid position 56 (S56) of SEQ ID NO. 11717.

[0018] In an eighth aspect, a composition comprising an effector submodule is provided. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively connected to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD) comprising a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises one or more substitutions relative to SEQ ID NO. 11717, wherein at least one substitution is a substitution of D or N at amino acid position 63 (G63) of SEQ ID NO. 11717, and wherein the one or more substitutions correspond to: G63D; G63D and M240L; G63D, E69V and N23II; or T55K, G63N and Q248N.

[0019] In a ninth aspect, a composition comprising an effector submodule is provided. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD) comprising a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises two or more substitutions relative to SEQ ID NO. 11717, one of which is an L or K substitution at amino acid position 71 (D71) of SEQ ID NO. 11717. In various embodiments, the two or more substitutions correspond to: D71L and T87N; D71L and L250R; D71L, T87N, and L250R; or D71K and T192F.

[0020] In a tenth aspect, a composition comprising an effector submodule is provided. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively connected to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD) comprising a region or integral of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises two or more substitutions relative to SEQ ID NO. 11717. At least one of the two or more substitutions is: (i) a substitution of F in amino acid position 241 (V241) of SEQ ID NO. 11717; or (ii) a substitution of F or L in amino acid position 249 (P249) of SEQ ID NO. 11717; and wherein the two or more substitutions correspond to: D72F and V241F; D72F and P249L; D72F and P249F; D72F, V241F and P249L; A77I and P249F; or V241F and P249L.

[0021] In an eleventh aspect, this disclosure provides compositions comprising an effector submodule. The effector submodule comprises a stimulus-response element (SRE) and at least one payload operatively connected to the SRE. The SRE comprises a destabilizing domain (DD) comprising a region or entirely of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises one or more substitutions relative to SEQ ID NO. 11717, said substitutions being selected from combinations of Y51T, L183S, Y193I, L197P, and V134F and L228F.

[0022] In all the exemplary embodiments provided above, the SRE responds to one or more stimuli. In various embodiments, the stimuli are small molecules selected from acetazolamide, celecoxib, vardecoxib, rofecoxib, acetazolamide, dazolamide, brinzolamide, diclofenac, esoxazolamide, zonisamide, dansylamide, or dichlorobenzenesulfonamide. Furthermore, the compositions described and illustrated herein comprise a DD having at least one mutation or substitution in the DD, said mutation or substitution destabilizing the DD and at least one payload in the absence of a stimuli, and wherein the DD and payload are stabilized in the presence of a stimuli.

[0023] In a twelfth aspect, this disclosure provides: a biological circuit system comprising any one or more compositions described in aspects 1-10; a pharmaceutical composition comprising the compositions described in aspects 1-10 and a pharmaceutically acceptable excipient; a polynucleotide encoding the compositions described in aspects 1-10; a carrier comprising a polynucleotide encoding the compositions described in aspects 1-10; a cell comprising a polynucleotide encoding the compositions described in aspects 1-10; and a pharmaceutical composition comprising a cell and a pharmaceutically acceptable excipient, wherein the cell comprises a polynucleotide encoding the compositions described in aspects 1-10.

[0024] In a thirteenth aspect, this disclosure provides a method for treating a disease in a subject in need. The method includes: (a) administering to the subject a therapeutically effective amount of cells of aspect 11, wherein the cells contain a therapeutically effective payload; and (b) administering to the subject a therapeutically effective amount of a stimulus, wherein the SRE responds to the stimulus, and wherein the expression of the payload is modulated in response to the stimulus, thereby treating the disease.

[0025] In some embodiments, this disclosure provides a stimulus-response element (SRE) that may comprise a destabilizing domain (DD) derived wholly or partially from human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717). In one embodiment, the DD may comprise the entire CA2 (SEQ ID NO. 11717).

[0026] In some embodiments, this disclosure provides a region or the entirety comprising human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717) and further comprising a DD with a mutation relative to SEQ ID NO. 11717, said mutation being selected from A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54 Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C205R, C205V, C205W, C205Y , D101G, D101M, D110I, D129I, D138G, D138M, D138N, D161*, D161M, D161V, D164G, D164 I, D174*, D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34T, D41T, D52I, D52L, D71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D72T, D72X, D 75T, D75V, D85M, E106D, E106G, E106S, E117*, E117N, E14N, E186*, E186N, E204A, E204 D. E204G, E204N, E213*, E213G, E213N, E220K, E220R, E220S, E233D, E233G, E233R, E23 5*, E235G, E235N, E237K, E237R, E238*, E238N, E238R, E26S, E69D, E69K, E69S, F130L, F146V, F175I, F175L, F17 5S, F178L, F178S, F20L, F20S, F225I, F225L, F225S, F225Y, F230I, F230L, F230S, F259L, F259S, F66S, F70I, F70 L, F95Y, G102D, G104R, G104V, G128R, G12D, G12E, G131E, G131R, G131W, G139D, G144D, G144V, G150A, G150S, G15 0W, G155A, G155C, G155D, G155S, G170A, G170D, G182A, G182W, G195A, G195R, G232R, G232W, G234L, G234V, G25E,G63D、G63V、G81E、G81V、G82D、G86A、G86D、G98V、H107I、H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I215S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153*、K153N、K158E、K158N、K167*、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227*、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143*、L147*、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197*、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211*、L211A、L211S、L223*、L223I、L223V、L228F、L228H、L228T、L239*、L239F、L239T、L250*、L250P、L250T、L44*、L44M、L47C、L47V、L57*、L57X、L60S、L79F、L79S、L84W、L90*、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177*、N177T、N229*、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、P13A、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、R181S、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206*、V206C、V206M、V210C、V217L、V217R、V217S、V222A、V222C、V222G、V241G、V241W、V241X、V31L、V49F、V68L、V68W、V78C、W123G、W123R、W16G、W191*、W191G、W191L、W208G、W208L、W208S、W244*、W244G、W244L、W97C、W97G、Y114H, Y114M, Y127M, Y190*, Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, and S29A.

[0027] In some embodiments, this disclosure provides a region or the entirety comprising human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717) and further comprising a DD with a mutation relative to SEQ ID NO. 11717, said mutation being selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0028] In some embodiments, this disclosure provides a region or the whole comprising human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717) and also comprising DD with two or more mutations relative to SEQ ID NO. 11717. In some embodiments, DD may comprise CA2 (WT amino acids 2-260, R27L, H122Y), CA2 (WT amino acids 2-260, T87I, H122Y), CA2 (WT amino acids 2-260, H122Y, N252D), CA2 (WT amino acids 2-260, D72F, V241F), CA2 (WT amino acids 2-260, V241F, P249L), CA2 (WT amino acids 2-260, D72F, P249L), CA2 (WT amino acids 2-260, D71L, L250R), CA2 (WT amino acids 2-260, D72F, P249F), CA2 (WT amino acids 2-260, T55K, G63N, Q248N), C A2 (WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT amino acids 2-260, W4Y, L156H), CA2 (WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2 (WT amino acids 2-260, L156H, F225L), CA2 (WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2 (WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 ( NO.210505), CA2(WT amino acid 2-260, A77I, P249F) (SEQ ID NO. 210514), CA2(WT amino acid 2-260, D71K, P249H) (SEQ ID NO. 210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 2105 ... A77I, P249F) (SEQ ID NO. 210514), CA2(WT amino acid 2-260, D71K, P249H) (SEQ ID NO. 210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2 (WT amino acids 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2 (WT amino acids 2-260, Y193L, K260L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71F, V241F, P249L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, L147F, Q248F) (SEQ ID NO. 210548), CA2 (WT amino acids 2-260, D52I, S258P) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D72S, T192N) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210532), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210534), CA2 (WT amino acids 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2 (WT amino acids 2-260, Y193L, K260L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210534), CA2 (WT amino acids 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2 (WT amino SEQ ID NO. 210554), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210560), CA2 (WT amino acids 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acids 2-260, D72F, P249I) (SEQ ID NO. 210568), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210560), CA2 (WT amino acids 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D NO.210572), CA2 (WT amino acids 2-260, A54X, S56X, L57X, T192X) (SEQ ID NO.).210574), CA2(WT amino acid 2-260, Y193V, K260F) (SEQ ID NO. 210576), CA2(WT amino acid 2-260, G63D, M240L) (SEQ ID NO. 210578), CA2(WT amino acid 2-260, V134F, L228F) (SEQ ID NO. 210580), CA2(WT amino acid 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acid 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acid 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 21057 ...6), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210577), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210577), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210577), CA2(WT amino acid 2-260, G63D, M240L) (SEQ ID NO. 210578), CA2(WT amino acid 2-260, S (SEQ ID NO. 210588), CA2 (WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210588), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 2105992), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 2105998 ... (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, KI HE, E220K, F225I) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, KI HE, E220K, F225I) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210708 ...). NO.210712), CA2 (WT amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO.210714), CA2 (WT amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO.210716), CA2 (WT amino acids 2-260, L79F, P180S) (SEQ ID NO. 210718), CA2 (WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO. 210724), CA2 (WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2 (WT amino acids 2-260, T199N, L202P, L228F) (SEQ ID NO. 210728), CA2 (WT amino acids 2-260, K9N, H122Y, T168K) (SEQ ID NO. 210730), CA2 (WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210718), CA2 (WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO. 210724), CA2 (WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2 (WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210728 ... (SEQ ID NO. 210732), CA2 (WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, D75V, K169N, F259L) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210732), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210734 ... SEQ ID NO. 210750), CA2 (amino acids 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L of WT) (SEQ ID NO. 210752), CA2 (amino acids 2-260, G12R, H15Y, D19V of WT) (SEQ ID NO. 210754), CA2 (amino acids 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I of WT) (SEQ ID NO. 210758), and / or CA2 (amino acids 2-260, G63D, E69V, N231I of WT) (SEQ ID NO. 210851 or 210847).

[0029] In some embodiments, DD may comprise CA2 (WT amino acids 2-260, R27L, H122Y), CA2 (WT amino acids 2-260, T87I, H122Y), CA2 (WT amino acids 2-260, H122Y, N252D), CA2 (WT amino acids 2-260, D72F, V241F), CA2 (WT amino acids 2-260, V241F, P249L), CA2 (WT amino acids 2-260, D72F, P249L), CA2 (WT amino acids 2-260, D71L, L250R), CA2 (WT amino acids 2-260, D72F, P249F), CA2 (WT amino acids 2-260, T55K, G63N, Q248N), C A2 (WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT amino acids 2-260, W4Y, L156H), CA2 (WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2 (WT amino acids 2-260, L156H, F225L), CA2 (WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2 (WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 21074 ... R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. (SEQ ID NO. 210514), CA2 (amino acids 2-260, E106D, C205S of WT) (SEQ ID NO. 210523), CA2 (amino acids 2-260, C205S, W208S of WT) (SEQ ID NO. 210523).CA2(210525), CA2(2-260, S73N, R89Y of WT amino acids) (SEQ ID NO. 210532), CA2(2-260, D71K, T192F of WT amino acids) (SEQ ID NO. 210534), CA2(2-260, S73N, R89F of WT amino acids) (SEQ ID NO. 210562), CA2(2-260, G63D, M240L of WT amino acids) (SEQ ID NO. 210578), CA2(2-260, V134F, L228F of WT amino acids) (SEQ ID NO. 210580), and / or CA2(2-260, S56F, D71S of WT amino acids) (SEQ ID NO. 210584).

[0030] The SRE described herein can respond to one or more stimuli. Such stimuli can be small molecules, such as, but not limited to, acetazolamide, celecoxib, vardicoxib, rofecoxib, acetazolamide, dazolamide, brinzolamide, diclofenac, itazolamide, zonisamide, dansylamide, or dichlorobenzylsulfonamide. In embodiments, the small molecule can be acetazolamide. In some aspects, the stimuli can be celecoxib.

[0031] This disclosure provides a CA2 biological circuit system comprising at least one effector submodule. Such effector submodules may include stimulus-response elements (SREs). This document provides biological circuits having an SRE comprising a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717).

[0032] The payload included in the CA2 biological loop system can be a therapeutic agent, a natural protein, a fusion peptide, an antibody, or a variant or fragment thereof.

[0033] In some embodiments, the payload may be a therapeutic agent. In some embodiments, the therapeutic agent may be a cytokine, a chimeric antigen receptor, a cytokine, or a cytokine-cytokine receptor fusion protein.

[0034] The CA2 biological circuit system can respond to one or more stimuli. In one aspect, the stimulus can be a small molecule, such as, but not limited to, acetazolamide, celecoxib, vardicoxib, rofecoxib, acetazolamide, dazolamide, brinzolamide, diclofenac, itazolamide, zonisamide, dansylamide, or dichlorobenzylsulfonamide. In one embodiment, the small molecule can be acetazolamide. In another embodiment, the small molecule can be celecoxib.

[0035] This document also provides polynucleotides encoding the SREs, biological circuit systems, and / or compositions described herein, as well as vectors comprising said polynucleotides. This disclosure also describes pharmaceutical compositions comprising the CA2 biological circuits and / or compositions described herein, along with pharmaceutically acceptable excipients.

[0036] Brief description of the attached figures

[0037] As illustrated in the accompanying drawings, the foregoing and other objects, features, and advantages will become apparent from the following description of specific embodiments of this disclosure. The components in these drawings are not necessarily to scale; rather, the focus is on explaining the principles of the various embodiments of this disclosure.

[0038] Figure 1 This demonstrates the ligand-dependent regulation of the CA2 chimeric antigen receptor.

[0039] Figure 2 The response of the CA2 biological circuit to different doses of acetazolamide was shown.

[0040] Figure 3 The acetazolamide response to OT-002347 (labeled CA2-070) and OT-001978 (labeled CA2-026) is shown. Detailed Implementation

[0041] Details of one or more embodiments of this disclosure are set forth in the following accompanying description. While any materials and methods similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred materials and methods are now described. Other features, objects, and advantages of this disclosure will be apparent from the description. In this specification, the singular form also includes the plural meaning unless the context clearly specifies otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the event of any conflict, this specification shall prevail.

[0042] 1. Composition

[0043] Biological circuits or biological circuit systems

[0044] According to this disclosure, a biological circuit system comprising at least one effector submodule at its core is provided. Such an effector submodule is independently associated with or integrated with one or more stimulus-response elements (SREs). Generally, a stimulus-response element (SRE) can be operatively connected to a payload, which can be any protein of interest (POI) (e.g., an immunotherapeutic agent), thereby forming an effector submodule. When activated by a specific stimulus (e.g., a small molecule), the SRE can generate a signal or result that upregulates or downregulates the transcriptional and / or protein levels of the connected payload by persistent signal stabilization or destabilization or any other type of regulation. Very detailed descriptions of biological circuit systems are taught in co-owned U.S. Provisional Patent Application No. 62 / 320,864, filed April 11, 2016; No. 62 / 466,596, filed March 3, 2017; and International Publication WO2017 / 180587 (the contents of which are incorporated herein by reference in their entirety). According to this disclosure, biological circuit systems, effector modules, SREs, and components are provided for regulating the expression level and activity of any agent used in immunotherapy.

[0045] As used herein, a “biological circuit” or “biological circuit system” is defined as a circuit within or useful within a biological system, comprising a stimulus and at least one effector submodule that responds to the stimulus, wherein the response to the stimulus produces at least one signal or result within, between, as an indicator of, or on the biological system. A biological system is generally understood to be any cell, tissue, organ, organ system, or organism, whether animal, plant, fungus, bacteria, or virus. It should also be understood that a biological circuit can be an artificial circuit that employs the stimulus or effector submodules taught in this disclosure and, for example, utilizes diagnostic, reporting systems, devices, assays, or kits to produce a signal or result in a cell-free environment. Artificial circuits can be associated with one or more electronic, magnetic, or radioactive components or parts.

[0046] According to this disclosure, the biological circuit system can be a destabilizing domain (DD) biological circuit system, a chimeric antigen receptor (CAR) biological circuit system (e.g., an I / O biological circuit system), a dimerization biological circuit system, a receptor biological circuit system, and a cell biological circuit system. Any of these systems can be used as a signal for any other system within these biological circuit systems.

[0047] Effect submodule

[0048] The biological circuits disclosed herein include at least one effector submodule. As used herein, an "effector submodule" is a single-component or multi-component construct or complex that includes at least (a) one or more stimulus-response elements (SREs) and (b) one or more payloads (e.g., a protein of interest (POI)).

[0049] The effect submodule can be designed to include one or more payloads, one or more SREs, one or more cut sites, one or more signal sequences, and one or more additional features (including the presence or absence of one or more connectors). (See international publication number WO2017 / 180587). Figure 2 Figure 6 illustrates a representative implementation of the effector module of this disclosure, the contents of which are incorporated herein by reference in their entirety. Figures 7-12 of International Publication No. WO2017 / 180587 illustrate biological circuits and components utilizing such effector modules, the contents of which are incorporated herein by reference in their entirety.

[0050] As in international publication number WO2017 / 180587 Figure 2 The diagram illustrates a representative implementation of an effector submodule comprising a single payload (i.e., an immunotherapeutic agent). Each component of the effector submodule can be positioned or arranged in various configurations, with or without (A to F) cleavage sites (G to Z, and AA to DD). Optional connectors can be inserted between each component of the effector submodule.

[0051] International Publication No. WO2017 / 180587 Figure 3 Figures 6 to 6 illustrate representative implementations of an effector submodule comprising two payloads, i.e., two immunotherapeutic agents. In some aspects, an effector submodule may include more than two immunotherapeutic agents (payloads) regulated by the same SRE (e.g., the same DD). The two or more agents may be directly interconnected or separate. The SRE may be located at the N-terminus of the construct, or at the C-terminus of the construct, or internally.

[0052] In some embodiments, the biological circuits of this disclosure may be modified to reduce their immunogenicity. Immunogenicity is the result of a complex series of responses to what is considered a foreign substance, including the production of neutralizing and non-neutralizing antibodies, the formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity, and allergic responses. Several factors, including but not limited to protein sequence, route and frequency of administration, and patient population, can contribute to protein immunogenicity. In a preferred embodiment, protein engineering may be employed to reduce the immunogenicity of the compositions of this disclosure. In some embodiments, modifications to reduce immunogenicity may include modifications that reduce the binding of processed peptides derived from parental sequences to MHC proteins. For example, amino acid modifications may be engineered such that no or only a minimal number of immunoepitaxes are predicted to bind with high affinity to any common MHC allele. Several methods for identifying MHC-binding epitopes of known protein sequences are known in the art and can be used to score epitopes in the compositions of this disclosure. Such methods are disclosed in US Patent Publications US20020119492, US20040230380 and US20060148009; the contents of each patent are incorporated herein by reference in their entirety.

[0053] Effector modules (including their SREs and payloads) can be nucleic acid-based, protein-based, or a combination thereof. They can be in the form of DNA, RNA, mRNA, protein, fusion protein, or any combination thereof.

[0054] Effector submodules (including their SREs and payloads) may contain peptides, polypeptides, or proteins individually, collectively, or independently. At the protein level, such payloads can be any natural or artificial peptide or polypeptide or fragment thereof. The natural peptide or polypeptide component of the payload can be derived from any known protein of any species.

[0055] Effector submodules can be designed to function within a group of one, two, three, four, or more modules. When more than one effector submodule is used in a biological circuit, it is referred to as an effector submodule system for that biological circuit.

[0056] Stimulus-response element (SRE)

[0057] As used herein, a “stimulus-response element” (SRE) is a component of an effector submodule that is connected, attached, linked, or associated with one or more payloads, and in some cases, is responsible for the responsiveness of the effector submodule to one or more stimuli. As used herein, the “response” nature of an SRE to a stimulus can be characterized by covalent or non-covalent interactions with the stimulus, direct or indirect associations, or structural or chemical responses. Furthermore, the response of any SRE to a stimulus can be a matter of degree or kind. The response can be partial. The response can be reversible. The response can ultimately lead to modulation of a signal or output. Such an output signal can be an output signal of a relative nature of the stimulus, for example, producing a modulation effect between 1% and 100%, or multiplying or decreasing it, for example, by 2, 3, 4, 5, 10, or more times. In some embodiments, the SRE is a peptide fused to a peptide payload.

[0058] In some embodiments, this disclosure provides methods for regulating protein expression, function, or level. In some aspects, regulation of protein expression, function, or level means that the expression, function, or level is regulated by at least about 20%, such as at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-10 ... 00%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.

[0059] Destabilized structural domains

[0060] Destabilizing domains (DDs) are small protein domains that can be attached to target proteins of interest. The term destabilizing domain (DD) is interchangeable with the term drug-response domain (DRD). In the absence of a DD-binding ligand, the attached protein of interest is destabilized, leading to its rapid degradation by the cell's ubiquitin-proteasome system (Stankunas, K. et al., Mol. Cell, 2003, 12: 1615-1624; Banazzynski et al., Cell; 2006, 126(5): 995-1004; reviews in Banazzynski, LA, and Wandless, TJ Chem. Biol:, 2006, 13: 11-21 and Rakhit R et al., Chem Biol. 2014; 21(9): 1238-1252). However, when a specific small molecule ligand binds to its intended DD as a ligand-binding partner, the instability is reversed and protein function is restored. The conditional nature of DD stability allows for rapid, undisturbed switching from stable proteins to unstable substrates to facilitate degradation. Furthermore, its dependence on ligand concentration provides tunable control over the degradation rate.

[0061] In one implementation, the SRE is a destabilizing domain (DD). The presence, absence, or amount of small molecule ligands that bind to or interact with the DD can modulate the stability of the payload upon such binding or interaction, thereby modulating the function of the payload. Depending on the degree of binding and / or interaction, the function of the payload can vary, thus providing "modulation" of the payload function.

[0062] In some embodiments, desired characteristics of DD may include, but are not limited to, low protein levels in the absence of a DD ligand (e.g., low basal stability), a large dynamic range, robust and predictable dose-response behavior, and rapid degradation kinetics. DD that binds to the desired ligand but not to endogenous molecules may be preferred.

[0063] In some embodiments, the DD of this disclosure can be developed from a known protein referred to herein as a parent protein. In some embodiments, the CA2 destabilizing domain described herein or known in the art can be used as a SRE in the biological circuit system of this disclosure, said SRE being associated with any payload taught herein (e.g., a protein of interest or an immunotherapeutic agent).

[0064] Regions, portions, or domains of wild-type proteins (e.g., CA2) can be used, in whole or in part, as SREs / DDs. They can be combined or rearranged to produce new peptides, proteins, regions, or domains, any of which can be used as SREs / DDs or as a starting point for designing further SREs and / or DDs.

[0065] In one implementation, the SRE originates from a region of the parent protein (e.g., CA2) or from a mutant protein. The length of the parental protein region can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 14 9, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450 or more amino acids. The length of the parental protein region can be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 amino acids. As a non-limiting example, the length of the parental protein region can be 250-270 amino acids. As a non-limiting example, the length of the parental protein region can be 225-250 amino acids. As a non-limiting example, the length of the parental protein region can be 225-260 amino acids.

[0066] In one embodiment, the SRE is derived from a parental protein (e.g., CA2) or from a mutant protein, and includes a region of the parental protein. The SRE may include a region of the parental protein, said region being 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the parental protein or mutant protein, 5-10%, 10-15%, 15-20%, 20-25%, 25- 30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70 -80%, 80-90%, 90-100%, 10-30%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 10-40%, 20-50%, 30-60%, 40-70%, 50-80%, 60-90%, 70-100%, 10-50%, 20-60%, 30-70% 40-80%, 50-90%, 60-100%, 10-60%, 20-70%, 30-80%, 40-90%, 50-100%, 10-70%, 20-80%, 30-90%, 40-100%, 10-80%, 20-90%, 30-100%, 10-90%, 20-100%, 25-50%, 50-75% or 75-100%.

[0067] In one embodiment, the SRE is derived from a parental protein (e.g., CA2) or from a mutant protein, and the content of the parental or mutant protein can be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, 5-10%, 10-15%, or 15%. -20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 10-30%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 10-40%, 20-50%, 30-60%, 40-70%, 50-80%, 60-90%, 70-100%, 10-50%, 20-60%, 30- The sameness of 70%, 40-80%, 50-90%, 60-100%, 10-60%, 20-70%, 30-80%, 40-90%, 50-100%, 10-70%, 20-80%, 30-90%, 40-100%, 10-80%, 20-90%, 30-100%, 10-90%, 20-100%, 25-50%, 50-75%, or 75-100%.

[0068] Candidate destabilizing domain sequences identified from the protein domains of a parent protein (as templates) can be mutated to generate a library of mutants based on the template candidate domain sequences. Mutagenesis strategies for generating DD libraries can include site-directed mutagenesis, for example, using structural guidance information; or random mutagenesis, for example, using error-prone PCR; or a combination of both. In some embodiments, destabilizing domains identified using random mutagenesis can be used to identify structural characteristics of candidate DDs that may be needed for destabilization, and then can be used to further generate mutant libraries using site-directed mutagenesis.

[0069] In some implementations, a DD mutant library can be screened for mutations that exhibit altered, preferably higher, binding affinity to the ligand compared to the wild-type protein. Two or more ligands can also be used to screen the DD library, with preferential selection of DD mutations stabilized by some ligands but not others. DD mutations that preferentially bind to the ligand compared to naturally occurring proteins can also be selected. Such methods can be used to optimize ligand selection and ligand binding affinity for DD. Furthermore, such methods can be used to minimize the detrimental effects caused by off-target ligand binding.

[0070] In some implementations, suitable DD mutants can be identified by screening mutant libraries using barcodes. Such methods can be used to detect, identify, and quantify individual mutant clones within heterogeneous mutant libraries. Each DD mutant in the library may have a different barcode sequence (with respect to each other). In other cases, the polynucleotide may also have different barcode sequences for 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid bases. Each DD mutant in the library may also contain multiple barcode sequences. When multiple are used, they can be used such that each barcode is unique to any other barcode. Alternatively, each barcode used may not be unique, but combinations of barcodes used can create unique sequences that can be individually traced. Barcode sequences can be placed upstream of the SRE, downstream of the SRE, or, in some cases, within the SRE. DD mutants can be identified using barcodes obtained through sequencing methods such as Sanger sequencing and next-generation sequencing, but can also be identified using polymerase chain reaction (PCR) and quantitative polymerase chain reaction (QPCR). In some implementations, polymerase chain reaction (PCR) primers that amplify products of different sizes for each barcode can be used to identify each barcode on an agarose gel. In other cases, each barcode may have a unique quantitative PCR probe sequence, enabling targeted amplification of each barcode.

[0071] In one embodiment, the effect submodule and / or SRE of this disclosure may include at least one destabilizing domain (DD). The effect submodule and / or SRE may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more DDs. When there is more than one DD, each DD may originate from the same parent protein, from different parent proteins, be a fusion of two different parent proteins, or be artificial.

[0072] In one embodiment, the effect submodule and / or SRE of this disclosure may include 2 DDs. In one embodiment, the effect submodule and / or SRE of this disclosure may include 3 DDs. In one embodiment, the effect submodule and / or SRE of this disclosure may include 4 DDs. In one embodiment, the effect submodule and / or SRE of this disclosure may include 5 DDs. In one embodiment, the effect submodule and / or SRE of this disclosure may include 6 DDs. In one embodiment, the effect submodule and / or SRE of this disclosure may include 7 DDs. In one embodiment, the effect submodule and / or SRE of this disclosure may include 8 DDs. In one embodiment, the effect submodule and / or SRE of this disclosure may include 9 DDs. In one embodiment, the effect submodule and / or SRE of this disclosure may include 10 DDs. DDs may be derived from any parental protein known in the art and / or described herein. In some embodiments, DDs are derived from the same parental protein. In some embodiments, DDs are derived from different regions of the same parental protein. In some embodiments, DDs are derived from different parental proteins.

[0073] CA2 destabilized structural domain

[0074] In some embodiments, the DD of this disclosure may be derived from human carbonic anhydrase 2CA2, a member of carbonic anhydrases (CA, EC 4.2.1.1), a superfamily of metalloenzymes present in all biological kingdoms. CAs balance reactions among three chemical species: CO2, bicarbonate, and protons. CAs are evolutionarily convergent, with seven genetically distinct CA families that have evolved independently in bacteria, archaea, and eukaryotes: α-, β-, γ-, δ-, ζ-, η-, and θ-CAs. In some embodiments, the DD described herein may be derived from at least one parental protein selected from, but not limited to, carbonic anhydrase 2 (CA2), carbonic anhydrase 1 (CA1), carbonic anhydrase 3 (CA3), carbonic anhydrase 4 (CA4), carbonic anhydrase 5A (CA5A), carbonic anhydrase 5B (CA5B), carbonic anhydrase 6 (CA6), carbonic anhydrase 7 (CA7), carbonic anhydrase 8 (CA8), carbonic anhydrase 9 (CA9), carbonic anhydrase 10 (CA10), carbonic anhydrase 11 (CA11), carbonic anhydrase 12 (CA12), carbonic anhydrase 13 (CA13), and carbonic anhydrase 14 (CA14).

[0075] In one embodiment, DD may be derived from cytosolic CA, such as, but not limited to, carbonic anhydrase 2 (CA2), carbonic anhydrase 1 (CA1), carbonic anhydrase 3 (CA3), carbonic anhydrase 7 (CA7), and carbonic anhydrase 13 (CA13). In one embodiment, DD may be derived from mitochondrial CA, such as, but not limited to, carbonic anhydrase 5A (CA5A) and carbonic anhydrase 5B (CA5B). In one embodiment, DD may be derived from secretory CA, such as, but not limited to, carbonic anhydrase 6 (CA6). In one embodiment, DD may be derived from membrane-bound CA, such as, but not limited to, carbonic anhydrase 4 (CA4), carbonic anhydrase 9 (CA9), carbonic anhydrase 12 (CA12), and carbonic anhydrase 14 (CA14). In one embodiment, DD is derived from CA2. In another embodiment, DD may be derived from CA9.

[0076] In some embodiments, the DD disclosed herein may be derived from CA2 (SEQ ID NO.11717; Uniprot ID: P00918), which may be stabilized by a small molecule inhibitor of ligands such as CA2. As used herein, the term "CA2 WT" refers to the human wild-type CA2 protein sequence, defined as SEQ ID NO. 11717, GenBank accession number P00918, having the following amino acid sequence: MSHHWGYGKHNGPEHWHKDFPIAKGERQS PVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLG IFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK. In some respects, DD may be derived from SEQ ID NO. 11717. SEQ ID NO. 11718 (having the amino acid sequence: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDT HTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDS QDKAAS LGSLEHQIWGFWESCAAT) or SEQ ID NO. 11719 (having the amino acid sequence: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYD PSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAEKGISMLRK KDVKNIHSPDNACEE) contains CA2. In some embodiments, the DDs of this disclosure can be identified by utilizing a mixture of CA2 inhibitors. In other cases, suitable DDs can be identified by first screening with one CA2 inhibitor and then screening with a second CA2 inhibitor.

[0077] The amino acid sequences with destabilized domains covered in this disclosure share at least about 40%, 50%, or 60% identity with the amino acid sequences listed therein, more preferably at least about 70% identity, preferably at least about 75% or 80% identity, more preferably at least about 85%, 86%, 87%, 88%, 89%, or 90% identity, and even more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. For example, the percentage of identity can be determined by comparing sequence information using an advanced BLAST computer program (including Magic-BLAST version 1.2.0 available from the National Institutes of Health). The BLAST program is based on the alignment method discussed in Karl and Altschul (1990) Proc. Natl. Acad. Sci. USA, 87:2264-68 (the contents of which are incorporated herein by reference in their entirety).

[0078] In some embodiments, the DD derived from CA2 may contain amino acids 2-260 of the parental CA2 sequence. This is referred to herein as the M1del mutation. In one embodiment, the DD derived from CA2 may contain amino acids 2-237 of the parental CA2 sequence.

[0079] Tables 1, 2, 3, 4, and 6 of this document provide CA2 mutants identified by mutagenesis, such as random mutagenesis screening using a combination of nucleotide analog mutagenesis and error-prone PCR to generate a mutant library; or saturation mutagenesis. CA2 destabilizing mutants can also be identified by structure-guided mutagenesis, which is provided in Table 1. The positions of the mutant amino acids listed in Tables 1, 2, 3, 4, 5, and 6 are relative to the full-length CA2 of SEQ ID NO. 11717.

[0080] Table 1: CA2 DD

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] Table 2 provides additional CA2 destabilizing structural domains.

[0097] Table 2: CA2DD

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] In some embodiments, the CA2 DD described herein may include any sequence provided in Table 3. In Table 3, "*" represents the translation of a stop codon. When the amino acid sequence in Table 3 contains one or more stop codons, the "AA SEQ ID" column provides the SEQ ID NO. of the individual components before and after the stop codons, in the order they appear in the amino acid sequence.

[0109] Table 3: CA2DD

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Table 4 provides additional CA2 destabilizing domains. Table 3 provides examples of CA2 destabilizing mutants identified as described above (e.g., through structure-guided mutagenesis or by combining single mutants).

[0121] Table 4: CA2DD

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] In some embodiments, a region or portion of the CA2 WT can be used as a template for generating CA2DD. In some embodiments, CA2 DD can exclude lysine at position 260 of SEQ ID NO. 11717. In some aspects, the CA2 region can include, but is not limited to, those described in Table 5.

[0133] Table 5: CA2 Region

[0134]

[0135]

[0136]

[0137] CA2 DDs can be generated using any CA2 region described in this paper. Table 6 provides CA2 DDs derived from CA2 regions.

[0138] Table 6: CA2 DDs originating from the CA2 region

[0139]

[0140]

[0141]

[0142]

[0143] In some implementations, the DD derived from CA2 may include one, two, three, four, five or more mutations as described in the table above.

[0144] In some embodiments, the mutation can be conserved (having similar physicochemical properties to the amino acid at the mutation site), semi-conserved (e.g., an amino acid that is negatively to positively charged), or non-conserved (an amino acid with different physicochemical properties than the amino acid at the mutation site). In some embodiments, the amino acid lysine can be mutated to glutamic acid or arginine; the amino acid phenylalanine can be mutated to leucine; the amino acid leucine can be mutated to phenylalanine; or the amino acid asparagine can be mutated to serine. Regions or portions or domains of wild-type proteins can be used wholly or partially as SRE / DD. They can be combined or rearranged to produce new peptides, proteins, regions, or domains, any of which can be used as SRE / DD or as a starting point for designing further SREs and / or DDs.

[0145] The destabilizing domains described herein may also include amino acid and nucleotide substitutions that do not affect stability, including conserved, non-conserved substitutions and / or polymorphisms. In some embodiments, the CA2 DD described herein may also be fragments of the aforementioned destabilizing domains, including fragments containing variant amino acid sequences. Preferred fragments are unstable in the absence of stimulation but become stable upon the addition of stimulation. Preferred fragments retain the ability to interact with stimulation, exhibiting similar efficiency to the DD described herein.

[0146] In one implementation, the SRE includes a region of the CA2 protein. The length of the CA2 protein region can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 8 3, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 1 46, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260 or more amino acids.The length of the parental protein region can be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, or 225-260 amino acids.

[0147] In some implementations, the CA2 DD described herein may include one or more mutations relative to Uniprot ID: P00918 (SEQ ID NO. 11717). These mutations may include, but are not limited to, A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, and D138. G, D138M, D138N, D161*, D161M, D161V, D164G, D164I, D174*, D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34 T, D41T, D52I, D52L, D71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D 72T, D72X, D75T, D75V, D85M, E106D, E106G, E106S, E117*, E117N, E14N, E186*, E186N, E204A, E204D, E204G, E204N, E213*, E213G, E213N, E220K, E220R, E220S, E233D, E233G, E233R, E235*, E235G, E235N, E237K, E2 37R, E238*, E238N, E238R, E26S, E69D, E69K, E69S, F130L, F146V, F175 I, F175L, F175S, F178L, F178S, F20L, F20S, F225I, F225L, F225S, F225Y , F230I, F230L, F230S, F259L, F259S, F66S, F70I, F70L, F95Y, G102D, G 104R, G104V, G128R, G12D, G12E, G131E, G131R, G131W, G139D, G144D, G 144V, G150A, G150S, G150W, G155A, G155C, G155D, G155S, G170A, G170D , G182A, G182W, G195A, G195R, G232R, G232W, G234L, G234V, G25E, G63D,G63V、G81E、G81V、G82D、G86A、G86D、G98V、H107I、H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I215S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153*、K153N、K158E、K158N、K167*、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227*、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143*、L147*、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197*、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211*、L211A、L211S、L223*、L223I、L223V、L228F、L228H、L228T、L239*、L239F、L239T、L250*、L250P、L250T、L44*、L44M、L47C、L47V、L57*、L57X、L60S、L79F、L79S、L84W、L90*、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177*、N177T、N229*、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、P13A、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、R181S、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206*、V206C、V206M、V210C、V217L、V217R、V217S、V222A、V222C、V222G、V241G、V241W、V241X、V31L、V49F、V68L、V68W、V78C、W123G、W123R、W16G、W191*、W191G、W191L、W208G、W208L、W208S、W244*、W244G、W244L、W97C、W97G、Y114H、Y114M, Y127M, Y190*, Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, S29A. As used herein, “*” indicates the translation of a stop codon, and X indicates any amino acid.

[0148] In one embodiment, the CA2 DD described herein includes a mutation relative to Uniprot ID: P00918 (SEQ ID NO. 11717), the mutation being selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0149] In some implementations, the CA2 DD described herein may include a mutation relative to UniprotID:P00918 (SEQ ID NO.11717). These mutations may include, but are not limited to, CA2(WT amino acid 2-260, R27L, H122Y), CA2(WT amino acid 2-260, T87I, H122Y), CA2(WT amino acid 2-260, H122Y, N252D), CA2(WT amino acid 2-260, D72F, V241F), CA2(WT amino acid 2-260, V241F, P249L), CA2(WT amino acid 2-260, D72F, P249L), CA2(WT amino acid 2-260, D71L, L250R), CA2(WT amino acid 2-260, D72F, P249F), CA2(WT amino acid 2-260, T55K, G63N, Q248N), CA 2(WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2(WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2(WT amino acids 2-260, W4Y, L156H), CA2(WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2(WT amino acids 2-260, L156H, F225L), CA2(WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2(WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 ( ID NO. 210505), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO.).210514), CA2(WT amino acids 2-260, D71K, P249H) (SEQ ID NO. 210516), CA2(WT amino acids 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acids 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acids 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acids 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acids 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acids 2-260, D71K, T192F) (SEQ ID NO. 210514), CA2(WT amino acids 2-260, D71K, T192F) (SEQ ID NO. 210516), CA2(WT amino acids 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acids 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acids 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acids 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acids 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acids 2-260, D71K, T192 (SEQ ID NO. 210534), CA2 (WT amino acids 2-260, Y193L, K260L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71F, V241F, P249L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, L147F, Q248F) (SEQ ID NO. 210548), CA2 (WT amino acids 2-260, D52I, S258P) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D72S, T192N) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210534), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210540 ... D71F, V241F, P249L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210554), CA2 (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210560), CA2 (WT amino acids 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acids 2-260, D72F, P249I) (SEQ ID NO. 210568), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210572), CA2 (WT amino acids 2-260, A54X, S56X, L57X, T192X) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210560), CA2 (WT amino acids 2-260, A54X, S56X, L57X, T192X ... NO.210574), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO).210576), CA2(WT amino acid 2-260, G63D, M240L) (SEQ ID NO. 210578), CA2(WT amino acid 2-260, V134F, L228F) (SEQ ID NO. 210580), CA2(WT amino acid 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acid 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acid 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210576), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210578), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210578), CA2(WT amino acid 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acid 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acid 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210578 ... (SEQ ID NO. 210588), CA2 (WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210588), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 2105992), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 2105998 ... (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, PBS, P83A, D101G, K111N, F230I) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E ...5), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210705), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210705), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210705), CA2 (WT amino acids 2-260, G6 (SEQ ID NO. 210712), CA2 (amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO. 210714), CA2 (amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210716), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210712), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210712), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210714), CA2 (amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210716), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210712), CA2 (amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO. 210714), CA2 (amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210715), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210712 ...210718), CA2(WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, T199N, L202P, L228F) (SEQ ID NO. 210728), CA2(WT amino acids 2-260, K9N, H122Y, T168K) (SEQ ID NO. 210730), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210718), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210728 ... (SEQ ID NO. 210732), CA2 (WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, D75V, K169N, F259L) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210732), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210734 ... SEQ ID NO. 210750), CA2 (amino acids 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L of WT) (SEQ ID NO. 210752), CA2 (amino acids 2-260, G12R, H15Y, D19V of WT) (SEQ ID NO. 210754), CA2 (amino acids 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I of WT) (SEQ ID NO. 210758), and / or CA2 (amino acids 2-260, G63D, E69V, N231I of WT) (SEQ ID NO. 210851 or 210847).

[0150] In one implementation, the CA2 DD described herein includes a value relative to Uniprot ID: P00918 (SEQ ID NO: 1). Multiple mutations in IDNO.11717, selected from CA2 (amino acids 2-260, R27L, H122Y in WT), CA2 (amino acids 2-260, T87I, H122Y in WT), CA2 (amino acids 2-260, H122Y, N252D in WT), CA2 (amino acids 2-260, D72F, V241F in WT), CA2 (amino acids 2-260, V241F, P249L in WT), CA2 (amino acids 2-260, D72F, P249L in WT), CA2 (amino acids 2-260, D71L, L250R in WT), CA2 (amino acids 2-260, D72F, P249F in WT), CA2 (amino acids 2-260, T55K, G63N, Q24 in WT). 8N), CA2(WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2(WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2(WT amino acids 2-260, W4Y, L156H), CA2(WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2(WT amino acids 2-260, L156H, F225L), CA2(WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2(WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 21074 ... R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. (SEQ ID NO. 210514), CA2 (WT amino acids 2-260, E106D, C205S) (SEQ ID NO.).CA2 (210523), CA2 (2-260 amino acids of WT, C205S, W208S) (SEQ ID NO. 210525), CA2 (2-260 amino acids of WT, S73N, R89Y) (SEQ ID NO. 210532), CA2 (2-260 amino acids of WT, D71K, T192F) (SEQ ID NO. 210534), CA2 (2-260 amino acids of WT, S73N, R89F) (SEQ ID NO. 210562), CA2 (2-260 amino acids of WT, G63D, M240L) (SEQ ID NO. 210578), CA2 (2-260 amino acids of WT, V134F, L228F) (SEQ ID NO. 210580) and / or CA2 (2-260 amino acids of WT, S56F, D71S) (SEQ ID NO. 210523), CA2 (2-260 amino acids of WT, C205S, W208S) (SEQ ID NO. 210525), CA2 (2-260 amino acids of WT, S73N, R89Y) (SEQ ID NO. 210532), CA2 (2-260 amino acids of WT, D71K, T192F) (SEQ ID NO. 210534), CA2 (2-260 amino acids of WT, S73N, R89F) (SEQ ID NO. 210562), CA2 (2-260 amino acids of WT, G63D, M240L) (SEQ ID NO. 210578), CA2 (2-260 amino acids of WT, V134F, L228F) (SEQ ID NO. 210580) and / or CA2 NO.210584).

[0151] In some embodiments, CA2 may be derived from Homo sapiens' carbonic anhydrase. In some embodiments, the CA2 DD described herein may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity with a specific reference polynucleotide or polypeptide, as determined by sequence alignment procedures and parameters described herein and known to those skilled in the art. In some embodiments, the reference polypeptide may be SEQ ID NO. 11717. Tools used for alignment may include those from BLAST suites (Stephen F. Altschul et al., (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402).

[0152] In some implementations, CA2 DD may be derived from carbonic anhydrases from species other than Homo sapiens. In some implementations, CA2 DD may be derived from carbonic anhydrases of the following species, including, but not limited to, cheetah (Acinonyx jubatus), giant panda (Ailuropoda melanoleuca), North Pacific minke whale (Balaenoptera acutorostratascammoni), common marmoset (Callithrix jacchus), northern fur seal (Callorhinus ursinus), Bactrian camel (Camelus bactrianus), dromedary camel (Camelus dromedarius), wild Bactrian camel (Camelus ferus), dingo (Canis lupus dingo), domestic dog (Canis lupus familiaris), Philippine tarsier (Carlitosyrichta), American beaver (Castor canadensis), Panamanian capuchin monkey (Cebus capucinusimitator), white rhinoceros (Ceratotherium simum), white-naped mangabey (Cercocebus atys), and chinchilla (Chinchilla). Lanigera, Green Monkey (Chlorocebus sabaeus), Angolan Colobus (Colobus angolensis palliatus), Beluga Whale (Delphinapterus leucas), Oligocephal Rat (Dipodomys ordii), Northern Sea Otter (Enhydra lutris kenyoni), Domestic Ass (Equus asinus), Domestic Horse (Equus caballus), Przewalski's Horse (Equus przewalskii), Western European Hedgehog (Erinaceus europaeus), Northern Sea Lion (Eumetopias jubatus), Domestic Cat (Felis catus), Malayan Flying Squirrel (Galeopterus variegatus), Gorilla, Homo sapiens, Thirteen-striped Ground Squirrel (Ictidomys tridecemlineatus), African Jerboa (Jaculus), Pacific White Dolphin (Lagenorhynchus obliquidens), Ring-tailed Lemur (Lemuria) catta, Weddell seal (Leptonychotes weddellii), Baiji dolphin (Lipotes vexillifer), African savanna elephant (Loxodonta)African macaques (Macaca fascicularis), common macaques (Macaca mulatta), pig-tailed macaques (Macaca nemestrina), mandarin ducks (Mandrillus leucophaeus), Malayan pangolins (Manisjavanica), yellow-bellied marmots (Marmota flaviventris), marmots (Marmota), dwarf lemurs (Microcebusmurinus), field mice (Mus caroli), house mice (Mus musculus), Sikkimese mice (Mus pahari), ferrets (Mustela putorius furo), Israeli moles (Nannospalax galili), Hawaiian monk seals (Neomonachus schauinslandi), Yangtze finless porpoises (Neophocaena asiaeorientalis), white-cheeked gibbons (Nomascus leucogenys), Pacific walruses (Odobenus rosmarus divergens), killer whales (Orcinusorca), and European rabbits (Oryctolagus). cuniculus, small-eared baby monkey (Otolemur garnettii), bonobo (Panpaniscus), chimpanzee (Pan troglodytes), leopard (Panthera pardus), Siberian tiger (Pantheratigris altaica), East African baboon (Papio anubis), sperm whale (Physeter catodon), Ugandan red colobus monkey (Piliocolobus tephrosceles), Sumatran orangutan (Pongo abelii), Kremlin's crowned lemur (Propithecus coquereli), cougar (Puma concolor), Yunnan snub-nosed monkey (Rhinopithecus bieti), Sichuan snub-nosed monkey (Rhinopithecus roxellana), Amazon squirrel monkey (Saimiri boliviensis), wild boar (Sus scrofa), lion-tailed baboon (Theropithecus gelada), Florida manatee (Trichechus manatus latirostris), Chinese tree shrew (Tupaia) chinensis, bottlenose dolphin (Tursiops truncatus), Arctic ground squirrel (Urocitellus parryii), grizzly bear (Ursus arctos horribilis), polar bear (Ursusmaritimus, fox (Vulpes) and / or California sea lion (Zalophus californianus).

[0153] The SREs described in this article may include, but are not limited to, CA2 mutations with one, two, three or more mutations. DD, the mutations being, but not limited to, A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, D138G, and D138M. , D138N, D161*, D161M, D161V, D164G, D164I, D174*, D174T, D179E, D179I , D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34T, D41T, D52I , D52L, D71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D72T, D72X, D75T , D75V, D85M, E106D, E106G, E106S, E117*, E117N, E14N, E186*, E186N, E20 4A, E204D, E204G, E204N, E213*, E213G, E213N, E220K, E220R, E220S, E23 3D, E233G, E233R, E235*, E235G, E235N, E237K, E237R, E238*, E238N, E23 8R, E26S, E69D, E69K, E69S, F130L, F146V, F175I, F175L, F175S, F178L, F 178S, F20L, F20S, F225I, F225L, F225S, F225Y, F230I, F230L, F230S, F25 9L, F259S, F66S, F70I, F70L, F95Y, G102D, G104R, G104V, G128R, G12D, G1 2E, G131E, G131R, G131W, G139D, G144D, G144V, G150A, G150S, G150W, G15 5A, G155C, G155D, G155S, G170A, G170D, G182A, G182W, G195A, G195R, G23 2R, G232W, G234L, G234V, G25E, G63D, G63V, G81E, G81V, G82D, G86A, G86D,G98V、H107I、H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I215S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153*、K153N、K158E、K158N、K167*、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227*、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143*、L147*、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197*、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211*、L211A、L211S、L223*、L223I、L223V、L228F、L228H、L228T、L239*、L239F、L239T、L250*、L250P、L250T、L44*、L44M、L47C、L47V、L57*、L57X、L60S、L79F、L79S、L84W、L90*、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177*、N177T、N229*、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、P13A、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、R181S、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206*、V206C、V206M、V210C、V217L、V217R、V217S、V222A、V222C、V222G、V241G、V241W、V241X、V31L、V49F、V68L、V68W、V78C、W123G、W123R、W16G、W191*、W191G、W191L、W208G、W208L、W208S、W244*、W244G、W244L、W97C、W97G、Y114H、Y114M、Y127M、Y190*、Y190L、Y190T、Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, S29A. ,

[0154] In one embodiment, the SRE described herein may include a CA2DD containing a mutation selected from: E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0155] The SRE described herein may include CA2 DD containing mutations, such as, but not limited to, CA2(WT amino acid 2-260, R27L, H122Y), CA2(WT amino acid 2-260, T87I, H122Y), CA2(WT amino acid 2-260, H122Y, N252D), CA2(WT amino acid 2-260, D72F, V241F), CA2(WT amino acid 2-260, V241F, P249L), CA2(WT amino acid 2-260, D72F, P249L), CA2(WT amino acid 2-260, D71L, L250R), CA2(WT amino acid 2-260, D72F, P249F), CA2(WT amino acid 2-260, T55K, G63N, Q248N), C A2 (WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT amino acids 2-260, W4Y, L156H), CA2 (WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2 (WT amino acids 2-260, L156H, F225L), CA2 (WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2 (WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 ( NO.210505), CA2 (amino acids 2-260, A77I, P249F of WT) (SEQ ID NO.210514), CA2 (amino acids 2-260, D71K, P249H of WT) (SEQ ID NO.SEQ ID NO. 210516), CA2 (WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2 (WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2 (WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2 (WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2 (WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2 (WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2 (WT amino acid 2-260, Y193L, K260L) (SEQ ID NO. 210516), CA2 (WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2 (WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2 (WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2 (WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2 (WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2 (WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2 (WT amino acid 2-260, Y19 (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71F, V241F, P249L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, L147F, Q248F) (SEQ ID NO. 210548), CA2 (WT amino acids 2-260, D52I, S258P) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D72S, T192N) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210548 ... (SEQ ID NO. 210560), CA2 (WT amino acid 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acid 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acid 2-260, D72F, P249I) (SEQ ID NO. 210568), CA2 (WT amino acid 2-260, D72X, V241X, P249X) (SEQ ID NO. 210572), CA2 (WT amino acid 2-260, A54X, S56X, L57X, T192X) (SEQ ID NO. 210574), CA2 (WT amino acid 2-260, Y193V, K260F) (SEQ ID NO. 210560), CA2 (WT amino acid 2-260, Y193V, K260F) (SEQ ID NO. 210562), CA2 (WT amino acid 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564 ... NO.210576), CA2 (WT amino acids 2-260, G63D, M240L) (SEQ ID NO.).210578), CA2 (WT amino acids 2-260, V134F, L228F) (SEQ ID NO. 210580), CA2 (WT amino acids 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2 (WT amino acids 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2 (WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2 (WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2 (WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 21057 ... S56F, D71S) (SEQ ID NO. 210584), CA2 (WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2 (WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2 (WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 210578), CA2 (WT amino acids (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 2105994), CA2 (WT amino acids 2-260, K45N, V68L, (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, PBS, P83A, D101G, K111N, F230I) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210712), CA2 (WT amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO. 210714), CA2 (WT amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, I59N, G102R, A173T ... (SEQ ID NO.210716), CA2 (WT amino acids 2-260, L79F, Pl 80S) (SEQ ID NO.210718), CA2 (WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO.210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, T199N, L202P, L228F) (SEQ ID NO. 210728), CA2(WT amino acids 2-260, K9N, H122Y, T168K) (SEQ ID NO. 210730), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210732), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210726 ... (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, D75V, K169N, F259L) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738 ...F, V206M, G23 SEQ ID NO. 210750), CA2 (amino acids 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L of WT) (SEQ ID NO. 210752), CA2 (amino acids 2-260, G12R, H15Y, D19V of WT) (SEQ ID NO. 210754), CA2 (amino acids 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I of WT) (SEQ ID NO. 210758), and / or CA2 (amino acids 2-260, G63D, E69V, N231I of WT) (SEQ ID NO. 210851 or 210847).

[0156] In one embodiment, the SRE described herein may include a CA2DD containing a mutation selected from CA2(WT amino acid 2-260, R27L, H122Y), CA2(WT amino acid 2-260, T87I, H122Y), CA2(WT amino acid 2-260, H122Y, N252D), CA2(WT amino acid 2-260, D72F, V241F), CA2(WT amino acid 2-260, V241F, P249L), CA2(WT amino acid 2-260, D72F, P249L), CA2(WT amino acid 2-260, D71L, L250R), CA2(WT amino acid 2-260, D72F, P249F), CA2(WT amino acid 2-260, T55K, G 63N, Q248N), CA2(WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2(WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2(WT amino acids 2-260, W4Y, L156H), CA2(WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2(WT amino acids 2-260, L156H, F225L), CA2(WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2(WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 21074 ... R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. NO.210514), CA2 (amino acids 2-260, E106D, C205S of WT) (SEQ ID NO.210523), CA2 (amino acids 2-260, C205S, W208S of WT) (SEQ ID NO.

[0157] CA2(210525), CA2(2-260, S73N, R89Y of WT) (SEQ ID NO.210532), CA2(2-260, D71K, T192F of WT) (SEQ ID NO.210534), CA2(2-260, S73N, R89F of WT) (SEQ ID NO.210562), CA2(2-260, G63D, M240L of WT) (SEQ ID NO.210578), CA2(2-260, V134F, L228F of WT) (SEQ ID NO.210580) and / or CA2(2-260, S56F, D71S of WT) (SEQ ID NO.210584).

[0158] This document also provides a biological circuit system comprising at least one effector submodule. The effector submodule of the biological circuit may include a stimulus-response element (SRE) comprising, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717). The biological circuit may also include at least one payload that can be attached, supplemented, or associated with the SRE.

[0159] SREs of biological circuit systems that include, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717) may include one, two, three or more mutations, such as, but not limited to, A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, D138G, D138M, D138N, D161*, D161M, D161V, D164G, D 164I, D174*, D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242 G, D242T, D32T, D34T, D41T, D52I, D52L, D71F, D71G, D71K, D71M, D71S, D71 Y, D72I, D72S, D72T, D72X, D75T, D75V, D85M, E106D, E106G, E106S, E117*, E117N, E14N, E186*, E186N, E204A, E204D, E204G, E204N, E213*, E213G, E 213N, E220K, E220R, E220S, E233D, E233G, E233R, E235*, E235G, E235N, E2 37K, E237R, E238*, E238N, E238R, E26S, E69D, E69K, E69S, F130L, F146V, F 175I, F175L, F175S, F178L, F178S, F20L, F20S, F225I, F225L, F225S, F225 Y, F230I, F230L, F230S, F259L, F259S, F66S, F70I, F70L, F95Y, G102D, G10 4R, G104V, G128R, G12D, G12E, G131E, G131R, G131W, G139D, G144D, G144V, G150A, G150S, G150W, G155A, G155C, G155D, G155S, G170A, G170D, G182A, G 182W, G195A, G195R, G232R, G232W, G234L, G234V, G25E, G63D, G63V, G81E,G81V、G82D、G86A、G86D、G98V、H107I、H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I2I5S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153*、K153N、K158E、K158N、K167*、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227*、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143*、L147*、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197*、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211*、L211A、L211S、L223*、L223I、L223V、L228F、L228H、L228T、L239*、L239F、L239T、L250*、L250P、L250T、L44*、L44M、L47C、L47V、L57*、L57X、L60S、L79F、L79S、L84W、L90*、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177*、N177T、N229*、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、P13A、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、R181S、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206*、V206C、V206M、V210C、V217L、V217R、V217S、V222A、V222C、V222G、V241G、V241W、V241X、V31L、V49F、V68L、V68W、V78C、W123G、W123R、W16G、W191*、W191G、W191L、W208G、W208L、W208S、W244*、W244G、W244L、W97C、W97G、Y114H、Y114M、Y127M, Y190*, Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, S29A. ,

[0160] In one embodiment, the SRE of a biological circuit system comprising, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717) may include mutations selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0161] SREs of biological circuit systems that include, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717) may include multiple mutations, such as, but not limited to, CA2 (WT amino acid 2-260, R27L, H122Y), CA2 (WT amino acid 2-260, T87I, H122Y), CA2 (WT amino acid 2-260, H122Y, N252D), CA2 (WT amino acid 2-260, D72F, V241F), CA2 (WT amino acid 2-260, V241F, P249L), CA2 (WT amino acid 2-260, D72F, P249L), CA2 (WT amino acid 2-260, D71L, L250R), CA2 (WT amino acid 2-260, D72F, P249F), and CA2 (WT amino acid 2-260, T55K). G63N, Q248N), CA2(WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2(WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2(WT amino acids 2-260, W4Y, L156H), CA2(WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2(WT amino acids 2-260, L156H, F225L), CA2(WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2(WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 ( ID NO. 210505), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO.).210514), CA2(WT amino acids 2-260, D71K, P249H) (SEQ ID NO. 210516), CA2(WT amino acids 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acids 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acids 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acids 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acids 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acids 2-260, D71K, T192F) (SEQ ID NO. 210514), CA2(WT amino acids 2-260, D71K, T192F) (SEQ ID NO. 210516), CA2(WT amino acids 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acids 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acids 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acids 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acids 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acids 2-260, D71K, T192 (SEQ ID NO. 210534), CA2 (WT amino acids 2-260, Y193L, K260L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71F, V241F, P249L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, L147F, Q248F) (SEQ ID NO. 210548), CA2 (WT amino acids 2-260, D52I, S258P) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D72S, T192N) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210534), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210540 ... D71F, V241F, P249L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210554), CA2 (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210560), CA2 (WT amino acids 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acids 2-260, D72F, P249I) (SEQ ID NO. 210568), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210572), CA2 (WT amino acids 2-260, A54X, S56X, L57X, T192X) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210560), CA2 (WT amino acids 2-260, A54X, S56X, L57X, T192X ... NO.210574), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO).210576), CA2(WT amino acid 2-260, G63D, M240L) (SEQ ID NO. 210578), CA2(WT amino acid 2-260, V134F, L228F) (SEQ ID NO. 210580), CA2(WT amino acid 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acid 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acid 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210576), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210578), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210578), CA2(WT amino acid 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acid 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acid 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acid 2-260, S73X, R89X) (SEQ ID NO. 210578 ... (SEQ ID NO. 210588), CA2 (WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210588), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 2105992), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 2105998 ... (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210705), CA2 (WT amino acids 2-260, G63D, W123R, E220K ... (SEQ ID NO. 210712), CA2 (amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO. 210714), CA2 (amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210716), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210712), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210712), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210714), CA2 (amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210716), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210712), CA2 (amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO. 210714), CA2 (amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210715), CA2 (amino acids 2-260, L79F, P180S) (SEQ ID NO. 210712 ...210718), CA2(WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, T199N, L202P, L228F) (SEQ ID NO. 210728), CA2(WT amino acids 2-260, K9N, H122Y, T168K) (SEQ ID NO. 210730), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210718), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210728 ... (SEQ ID NO. 210732), CA2 (WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, D75V, K169N, F259L) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210732), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210734 ... SEQ ID NO. 210750), CA2 (amino acids 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L of WT) (SEQ ID NO. 210752), CA2 (amino acids 2-260, G12R, H15Y, D19V of WT) (SEQ ID NO. 210754), CA2 (amino acids 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I of WT) (SEQ ID NO. 210758), and / or CA2 (amino acids 2-260, G63D, E69V, N231I of WT) (SEQ ID NO. 210851 or 210847).

[0162] In one embodiment, the SRE of a biological circuit system comprising, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717) may include multiple mutations selected from CA2 (amino acid 2-260, R27L, H122Y of WT), CA2 (amino acid 2-260, T87I, H122Y of WT), CA2 (amino acid 2-260, H122Y, N252D of WT), CA2 (amino acid 2-260, D72F, V241F of WT), CA2 (amino acid 2-260, V241F, P249L of WT), CA2 (amino acid 2-260, D72F, P249L of WT), CA2 (amino acid 2-260, D71L, L250R of WT), CA2 (amino acid 2-260, D72F, P249F of WT), and CA2 (amino acid 2-260, T55K, G63 of WT). N, Q248N), CA2 (WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT amino acids 2-260, W4Y, L156H), CA2 (WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2 (WT amino acids 2-260, L156H, F225L), CA2 (WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2 (WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 21074 ... R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. (SEQ ID NO. 210514), CA2 (WT amino acids 2-260, E106D, C205S) (SEQ ID NO.).CA2 (210523), CA2 (2-260 amino acids of WT, C205S, W208S) (SEQ ID NO. 210525), CA2 (2-260 amino acids of WT, S73N, R89Y) (SEQ ID NO. 210532), CA2 (2-260 amino acids of WT, D71K, T192F) (SEQ ID NO. 210534), CA2 (2-260 amino acids of WT, S73N, R89F) (SEQ ID NO. 210562), CA2 (2-260 amino acids of WT, G63D, M240L) (SEQ ID NO. 210578), CA2 (2-260 amino acids of WT, V134F, L228F) (SEQ ID NO. 210580) and / or CA2 (2-260 amino acids of WT, S56F, D71S) (SEQ ID NO. 210523), CA2 (2-260 amino acids of WT, C205S, W208S) (SEQ ID NO. 210525), CA2 (2-260 amino acids of WT, S73N, R89Y) (SEQ ID NO. 210532), CA2 (2-260 amino acids of WT, D71K, T192F) (SEQ ID NO. 210534), CA2 (2-260 amino acids of WT, S73N, R89F) (SEQ ID NO. 210562), CA2 (2-260 amino acids of WT, G63D, M240L) (SEQ ID NO. 210578), CA2 (2-260 amino acids of WT, V134F, L228F) (SEQ ID NO. 210580) and / or CA2 NO.210584).

[0163] Stability to Destabilization Ratio of SRE

[0164] In some embodiments, this disclosure provides methods for regulating protein expression, function, or level by measuring a stability ratio and a destabilization ratio. As used herein, a stability ratio can be defined as the ratio of the expression, function, or level of the protein of interest in response to a stimulus to the expression, function, or level of the protein of interest in the absence of a stimulus specific to SRE. In some aspects, the stability ratio is at least 1, for example at least 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-95, 20-100, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-95, 30-100, 40-50, 40-60, 40-70, 40-80, 40-90, 40-95, 40-100, 50-60, 50-70, 50-80, 50-90, 50-95, 50-100, 60-70, 60-80, 60-90, 60-95, 60-100, 70-80, 70-90, 70-95, 70-100, 80-90, 80-95, 80-100, 90-95, 90-100, or 95-100. As used herein, the destabilization ratio can be defined as the ratio of the expression, function, or level of the protein of interest in the absence of an effector module-specific stimulus to the expression, function, or level of the protein of interest constitutively expressed in the absence of an SRE-specific stimulus. As used in this article, “constitutive” refers to the expression, function, or level of a protein of interest that is not linked to an SRE and is therefore expressed in the presence or absence of a stimulus.In some respects, the destabilization ratio is at least 0, for example at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or at least 0-0.1, 0-0.2, 0-0.3, 0-0.4, 0-0.5, 0-0.6, 0-0.7, 0-0.8, 0-0.9, 0.1-0.2, 0.1-0.3, 0.1-0.4, 0.1-0.5, 0.1-0.6, 0.1-0.7, 0.1-0.8, 0.1-0.9, 0.2-0.3, 0.2-0. 4. 0.2-0.5, 0.2-0.6, 0.2-0.7, 0.2-0.8, 0.2-0.9, 0.3-0.4, 0.3-0.5, 0.3-0.6, 0.3-0.7, 0.3-0.8, 0.3-0.9, 0.4-0.5, 0.4-0.6, 0.4-0.7, 0.4-0.8, 0.4-0.9, 0.5-0.6, 0.5-0.7, 0.5-0.8, 0.5-0.9, 0.6-0.7, 0.6-0.8, 0.6-0.9, 0.7-0.8, 0.7-0.9 or 0.8-0.9.

[0165] In some implementations, the SRE of the effect submodule can stabilize the payload of interest with a stabilization ratio of 1 or greater, wherein the stabilization ratio can include the ratio of the expression, function, or level of the payload of interest in the presence of a stimulus to the expression, function, or level of the payload of interest in the absence of a stimulus.

[0166] In some implementations, the SRE can destabilize the payload of interest with a destabilization ratio between 0 and 0.09, wherein the destabilization ratio can include the ratio of the expression, function, or level of the payload of interest in the absence of an SRE-specific stimulus to the expression, function, or level of the payload of interest constitutively expressed in the absence of an SRE-specific stimulus.

[0167] Protein-protein interactions in SRE

[0168] In some embodiments, this disclosure provides a stimulus-response element (SRE) that may comprise, wholly or partially, a destabilizing domain (DD) derived from human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717). In one embodiment, the DD may comprise the entire CA2 (SEQ ID NO. 11717). In some embodiments, the DD may comprise a portion or region of human carbonic anhydrase.A portion or region of CA2 may be selected from, but is not limited to, amino acids 2-260 of CA2 (SEQ ID NO. 11717), for example, but not limited to, SEQ ID NO. (210492); amino acids 1-142 of CA2 (SEQ ID NO. 11717), for example, but not limited to, SEQ ID NO. (210820); amino acids 2-142 of CA2 (SEQ ID NO. 11717), for example, but not limited to, SEQ ID NO. (210821); amino acids 1-190 of CA2 (SEQ ID NO. 11717), for example, but not limited to, SEQ ID NO. (210822); amino acids 2-190 of CA2 (SEQ ID NO. 11717), for example, but not limited to, SEQ ID NO. (210823); amino acids 1-89 of CA2 (SEQ ID NO. 11717), for example, but not limited to, SEQ ID NO. (210824); CA2 (SEQ ID NO. 11717) Amino acids 2-89 of CA2 (SEQ ID NO. 11717), such as but not limited to, SEQ ID NO. (210825); amino acids 1-243 of CA2 (SEQ ID NO. 11717), such as but not limited to, SEQ ID NO. (210826); amino acids 2-243 of CA2 (SEQ ID NO. 11717), such as but not limited to, SEQ ID NO. (210827); amino acids 1-166 of CA2 (SEQ ID NO. 11717), such as but not limited to, SEQ ID NO. (210828); amino acids 2-166 of CA2 (SEQ ID NO. 11717), such as but not limited to, SEQ ID NO. (210782); amino acids 1-116 of CA2 (SEQ ID NO. 11717), such as but not limited to, SEQ ID NO. (210830); CA2 (SEQ ID NO. 11717) Amino acids 2-116 of CA2 (SEQ ID NO. 11717), for example but not limited to, SEQ ID NO. (210831); amino acids 1-152 of CA2 (SEQ ID NO. 11717), for example but not limited to, SEQ ID NO. (210832); amino acids 2-152 of CA2 (SEQ ID NO. 11717), for example but not limited to, SEQ ID NO. (210833); amino acids 1-43 of CA2 (SEQ ID NO. 11717), for example but not limited to, SEQ ID NO. (210834); or amino acids 2-43 of CA2 (SEQ ID NO. 11717), for example but not limited to, SEQ ID NO. (210835).In one embodiment, DD may include amino acids 2 to 260 of CA2 (SEQ ID NO. 11717). In another embodiment, DD may include amino acids 2 to 260 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. 210492.

[0169] In one embodiment, DD may include amino acids 2 to 260 of CA2 (SEQ ID NO. 11717).

[0170] In some implementations, DD may include one, two, three, or more mutations, such as, but not limited to, A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D12 9I, D138G, D138M, D138N, D161*, D161M, D161V, D164G, D164I, D174*, D174T , D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34T , D41T, D52I, D52L, D71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D72T, D 72X, D75T, D75V, D85M, E106D, E106G, E106S, E117*, E117N, E14N, E186*, E18 6N, E204A, E204D, E204G, E204N, E213*, E213G, E213N, E220K, E220R, E220S , E233D, E233G, E233R, E235*, E235G, E235N, E237K, E237R, E238*, E238N, E 238R, E26S, E69D, E69K, E69S, F130L, F146V, F175I, F175L, F175S, F178L, F 178S, F20L, F20S, F225I, F225L, F225S, F225Y, F230I, F230L, F230S, F259L, F259S, F66S, F70I, F70L, F95Y, G102D, G104R, G104V, G128R, G12D, G12E, G1 31E, G131R, G131W, G139D, G144D, G144V, G150A, G150S, G150W, G155A, G155 C. G155D, G155S, G170A, G170D, G182A, G182W, G195A, G195R, G232R, G232W, G234L, G234V, G25E, G63D, G63V, G81E, G81V, G82D, G86A, G86D, G98V, H107I,H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I215S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153*、K153N、K158E、K158N、K167*、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227*、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143*、L147*、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197*、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211*、L211A、L211S、L223*、L223I、L223V、L228F、L228H、L228T、L239*、L239F、L239T、L250*、L250P、L250T、L44*、L44M、L47C、L47V、L57*、L57X、L60S、L79F、L79S、L84W、L90*、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177*、N177T、N229*、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、P13A、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、R181S、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206*、V206C、V206M、V210C、V217L、V217R、V217S、V222A、V222C、V222G、V241G、V241W、V241X、V31L、V49F、V68L、V68W、V78C、W123G、W123R、W16G、W191*、W191G、W191L、W208G、W208L、W208S、W244*、W244G、W244L、W97C、W97G、Y114H、Y114M、Y127M、Y190*、Y190L、Y190T、Y193C、Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, S29A. As used herein, "*" indicates the translation of a stop codon, and X indicates any amino acid.

[0171] In one implementation, DD may include E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, or Y51T mutations.

[0172] In some implementations, DD may include multiple mutations, such as, but not limited to, CA2(WT amino acid 2-260, R27L, H122Y), CA2(WT amino acid 2-260, T87I, H122Y), CA2(WT amino acid 2-260, H122Y, N252D), CA2(WT amino acid 2-260, D72F, V241F), CA2(WT amino acid 2-260, V241F, P249L), CA2(WT amino acid 2-260, D72F, P249L), CA2(WT amino acid 2-260, D71L, L250R), CA2(WT amino acid 2-260, D72F, P249F), CA2(WT amino acid 2-260, T55K, G63N, Q2). 48N), CA2(WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2(WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2(WT amino acids 2-260, W4Y, L156H), CA2(WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2(WT amino acids 2-260, L156H, F225L), CA2(WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2(WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 ( NO.210505), CA2 (amino acids 2-260, A77I, P249F of WT) (SEQ ID NO.210514), CA2 (amino acids 2-260, D71K, P249H of WT) (SEQ ID NO.210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K260L) (SEQ ID NO. 210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K2 (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71F, V241F, P249L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, L147F, Q248F) (SEQ ID NO. 210548), CA2 (WT amino acids 2-260, D52I, S258P) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D72S, T192N) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210548 ... SEQ ID NO. 210560), CA2 (WT amino acids 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acids 2-260, D72F, P249I) (SEQ ID NO. 210568), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210572), CA2 (WT amino acids 2-260, A54X, S56X, L57X, T192X) (SEQ ID NO. 210574), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO. 210560), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO. 210565 ... NO.210576), CA2 (WT amino acids 2-260, G63D, M240L) (SEQ ID NO).210578), CA2(WT amino acids 2-260, V134F, L228F) (SEQ ID NO. 210580), CA2(WT amino acids 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acids 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2(WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 21057 ... S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2(WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 210578), CA2(WT amino acids (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 2105994), CA2 (WT amino acids 2-260, K45N, V68L, (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, PBS, P83A, D101G, K111N, F230I) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210712), CA2 (WT amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO. 210714), CA2 (WT amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, I59N, G102R, A173T ... (SEQ ID NO.210716), CA2 (WT amino acids 2-260, L79F, Pl 80S) (SEQ ID NO.210718), CA2 (WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO.210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, T199N, L202P, L228F) (SEQ ID NO. 210728), CA2(WT amino acids 2-260, K9N, H122Y, T168K) (SEQ ID NO. 210730), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210732), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210726 ... (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, D75V, K169N, F259L) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59F, R232R) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59F, R232R) (SEQ SEQ ID NO. 210750), CA2 (amino acids 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L of WT) (SEQ ID NO. 210752), CA2 (amino acids 2-260, G12R, H15Y, D19V of WT) (SEQ ID NO. 210754), CA2 (amino acids 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I of WT) (SEQ ID NO. 210758), and / or CA2 (amino acids 2-260, G63D, E69V, N231I of WT) (SEQ ID NO. 210851 or 210847).

[0173] In one embodiment, DD may include CA2 (amino acids 2-260 of WT, R27L, H122Y), CA2 (amino acids 2-260 of WT, T87I, H122Y), CA2 (amino acids 2-260 of WT, H122Y, N252D), CA2 (amino acids 2-260 of WT, D72F, V241F), CA2 (amino acids 2-260 of WT, V241F, P249L), CA2 (amino acids 2-260 of WT, D72F, P249L), CA2 (amino acids 2-260 of WT, D71L, L250R), CA2 (amino acids 2-260 of WT, D72F, P249F), CA2 (amino acids 2-260 of WT, T55K, G63N, Q248N), C A2 (WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT amino acids 2-260, W4Y, L156H), CA2 (WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2 (WT amino acids 2-260, L156H, F225L), CA2 (WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2 (WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, A77I, P249F) (SEQ ID NO. 21074 ... R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. (SEQ ID NO. 210514), CA2 (amino acids 2-260, E106D, C205S of WT) (SEQ ID NO. 210523), CA2 (amino acids 2-260, C205S, W208S of WT) (SEQ ID NO. 210523).CA2(210525), CA2(2-260, S73N, R89Y of WT amino acids) (SEQ ID NO. 210532), CA2(2-260, D71K, T192F of WT amino acids) (SEQ ID NO. 210534), CA2(2-260, S73N, R89F of WT amino acids) (SEQ ID NO. 210562), CA2(2-260, G63D, M240L of WT amino acids) (SEQ ID NO. 210578), CA2(2-260, V134F, L228F of WT amino acids) (SEQ ID NO. 210580), and / or CA2(2-260, S56F, D71S of WT amino acids) (SEQ ID NO. 210584).

[0174] In one embodiment, DD may include amino acids 2 to 260 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210492).

[0175] In some implementations, the SRE can be, but is not limited to, CA2(WT amino acid 2-260, R27L, H122Y), CA2(WT amino acid 2-260, T87I, H122Y), CA2(WT amino acid 2-260, H122Y, N252D), CA2(WT amino acid 2-260, D72F, V241F), CA2(WT amino acid 2-260, V241F, P249L), CA2(WT amino acid 2-260, D72F, P249L), CA2(WT amino acid 2-260, D71L, L250R), CA2(WT amino acid 2-260, D72F, P249F), CA2(WT amino acid 2-260, T55K, G63N, Q248N). CA2(WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2(WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2(WT amino acids 2-260, W4Y, L156H), CA2(WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2(WT amino acids 2-260, L156H, F225L), CA2(WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2(WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 ( NO.210505), CA2 (amino acids 2-260, A77I, P249F of WT) (SEQ ID NO.210514), CA2 (amino acids 2-260, D71K, P249H of WT) (SEQ ID NO.210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K260L) (SEQ ID NO. 210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K2 (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71F, V241F, P249L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, L147F, Q248F) (SEQ ID NO. 210548), CA2 (WT amino acids 2-260, D52I, S258P) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D72S, T192N) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210548 ... SEQ ID NO. 210560), CA2 (WT amino acids 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acids 2-260, D72F, P249I) (SEQ ID NO. 210568), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210572), CA2 (WT amino acids 2-260, A54X, S56X, L57X, T192X) (SEQ ID NO. 210574), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO. 210560), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO. 210565 ... NO.210576), CA2 (WT amino acids 2-260, G63D, M240L) (SEQ ID NO).210578), CA2(WT amino acids 2-260, V134F, L228F) (SEQ ID NO. 210580), CA2(WT amino acids 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acids 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2(WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 21057 ... S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2(WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 210578), CA2(WT amino acids (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, KI) HE, E220K, F225I)(SEQ ID NO.210706), CA2 (WT amino acids 2-260, P13S, P83A, D101G, K111N, F230I)(SEQ ID NO.210708), CA2 (WT amino acids 2-260, G63D, W123R, E220K)(SEQ ID NO.210712), CA2 (WT amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S)(SEQ ID NO.210714), CA2 (WT amino acids 2-260, I59N, G102R, A173T)(SEQ ID NO.210706), CA2 (WT amino acids 2-260, I59N, G102R, A173T)(SEQ ID NO.210706), CA2 (WT amino acids 2-260, I59N, G102R, A173T))(SEQ ID NO.210712), CA2 (WT amino acids 2-260, I59N, G102R, A173T))(SEQ ID NO.210712), CA2 (WT amino acids 2-260, I13S, P83A, D101G, K111N, F230I ... NO.210716), CA2 (amino acids 2-260, L79F, P180S of WT) (SEQ ID NO.210718), CA2 (amino acids 2-260, A77P, G102R, D138N of WT) (SEQ ID NO.210716), CA2 (amino acids 2-260, A77P, G102R, D138N of WT) (SEQ ID NO.210718).210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, T199N, L202P, L228F) (SEQ ID NO. 210728), CA2(WT amino acids 2-260, K9N, H122Y, T168K) (SEQ ID NO. 210730), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210732), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210726 ... (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, D75V, K169N, F259L) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738 ...F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A1 SEQ ID NO. 210750), CA2 (amino acids 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L of WT) (SEQ ID NO. 210752), CA2 (amino acids 2-260, G12R, H15Y, D19V of WT) (SEQ ID NO. 210754), CA2 (amino acids 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I of WT) (SEQ ID NO. 210758), and / or CA2 (amino acids 2-260, G63D, E69V, N231I of WT) (SEQ ID NO. 210851 or 210847).

[0176] In one embodiment, DD can be CA2 (WT amino acid 2-260, R27L, H122Y), CA2 (WT amino acid 2-260, T87I, H122Y), CA2 (WT amino acid 2-260, H122Y, N252D), CA2 (WT amino acid 2-260, D72F, V241F), CA2 (WT amino acid 2-260, V241F, P249L), CA2 (WT amino acid 2-260, D72F, P249L), CA2 (WT amino acid 2-260, D71L, L250R), CA2 (WT amino acid 2-260, D72F, P249F), CA2 (WT amino acid 2-260, T55K, G63N, Q248N), C A2 (WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT amino acids 2-260, W4Y, L156H), CA2 (WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2 (WT amino acids 2-260, L156H, F225L), CA2 (WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2 (WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 ( (SEQ ID NO. 210505), CA2 (amino acids 2-260, A77I, P249F of WT) (SEQ ID NO. 210514), CA2 (amino acids 2-260, D71K, P249H of WT) (SEQ ID NO. 210514).210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K260L) (SEQ ID NO. 210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K2 SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71F, V241F, P249L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, L147F, Q248F) (SEQ ID NO. 210548), CA2 (WT amino acids 2-260, D52I, S258P) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D72S, T192N) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210558) (SEQ ID NO. 210559) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D71Y, Q248L ... SEQ ID NO. 210560), CA2 (WT amino acid 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acid 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acid 2-260, D72F, P249I) (SEQ ID NO. 210568), CA2 (WT amino acid 2-260, D72X, V241X, P249X) (SEQ ID NO. 210572), CA2 (WT amino acid 2-260, A54X, S56X, L57X, T192X) (SEQ ID NO. 210574), CA2 (WT amino acid 2-260, Y193V, K260F) (SEQ ID NO. 210560), CA2 (WT amino acid 2-260, Y193V, K260F) (SEQ ID NO. 210562), CA2 (WT amino acid 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564 ... (SEQ ID NO. 210576), CA2 (WT amino acids 2-260, G63D, M240L) (SEQ ID NO. 210576).210578), CA2(WT amino acids 2-260, V134F, L228F) (SEQ ID NO. 210580), CA2(WT amino acids 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acids 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2(WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 21057 ... D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acids 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, KI) HE, E220K, F225I)(SEQ ID NO.210706), CA2(WT amino acids 2-260, PBS, P83A, D101G, K111N, F230I)(SEQ ID NO.210708), CA2(WT amino acids 2-260, G63D, W123R, E220K)(SEQ ID NO.210712), CA2(WT amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S)(SEQ ID NO.210714), CA2(WT amino acids 2-260, I59N, G102R, A173T)(SEQ ID NO.210716), CA2(WT amino acids 2-260, L79F, P180S)(SEQ ID NO.210706), NO.210718), CA2 (WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO.).210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, T199N, L202P, L228F) (SEQ ID NO. 210728), CA2(WT amino acids 2-260, K9N, H122Y, T168K) (SEQ ID NO. 210730), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210732), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210726 ... (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, D75V, K169N, F259L) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59F, R232R) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59F, R232R) (SEQ SEQ ID NO. 210750), CA2 (amino acids 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L of WT) (SEQ ID NO. 210752), CA2 (amino acids 2-260, G12R, H15Y, D19V of WT) (SEQ ID NO. 210754), CA2 (amino acids 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I of WT) (SEQ ID NO. 210758), and / or CA2 (amino acids 2-260, G63D, E69V, N231I of WT) (SEQ ID NO. 210851 or 210847).

[0177] This document also provides isolated peptide variants comprising at least one mutation relative to SEQ ID NO. 11717. (Relative to SEQ ID) Non-restrictive examples of mutations in NO.11717 include A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, D 138G, D138M, D138N, D161*, D161M, D161V, D164G, D164I, D174*, D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D3 4T, D41T, D52I, D52L, D71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D 72T, D72X, D75T, D75V, D85M, E106D, E106G, E106S, E117*, E117N, E14N, E 186*, E186N, E204A, E204D, E204G, E204N, E213*, E213G, E213N, E220K, E220R, E220S, E233D, E233G, E233R, E235*, E235G, E235N, E237K, E237R , E238*, E238N, E238R, E26S, E69D, E69K, E69S, F130L, F146V, F175I, F1 75L, F175S, F178L, F178S, F20L, F20S, F225I, F225L, F225S, F225Y, F230 I, F230L, F230S, F259L, F259S, F66S, F70I, F70L, F95Y, G102D, G104R, G 104V, G128R, G12D, G12E, G131E, G131R, G131W, G139D, G144D, G144V, G15 0A, G150S, G150W, G155A, G155C, G155D, G155S, G170A, G170D, G182A, G1 82W, G195A, G195R, G232R, G232W, G234L, G234V, G25E, G63D, G63V, G81E,G81V、G82D、G86A、G86D、G98V、H107I、H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I215S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153*、K153N、K158E、K158N、K167*、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227*、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143*、L147*、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197*、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211*、L211A、L211S、L223*、L223I、L223V、L228F、L228H、L228T、L239*、L239F、L239T、L250*、L250P、L250T、L44*、L44M、L47C、L47V、L57*、L57X、L60S、L79F、L79S、L84W、L90*、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177*、N177T、N229*、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、P13A、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、R181S、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206*、V206C、V206M、V210C、V217L、V217R、V217S、V222A、V222C、V222G、V241G、V241W、V241X、V31L、V49F、V68L、V68W、V78C、W123G、W123R、W16G、W191*、W191G、W191L、W208G、W208L、W208S、W244*、W244G、W244L、W97C、W97G、Y114H、Y114M、Y127M、Y190*、Y190L、Y190T、Y193C、Y193F、Y193I、Y193L、Y193T、Y193V、Y193X、Y40M、Y51F、Y51M、Y51T、Y51X、Y88T、K9N、S29A。、

[0178] In some embodiments, the isolated peptide variants may be CA2(WT amino acid 2-260, R27L, H122Y), CA2(WT amino acid 2-260, T87I, H122Y), CA2(WT amino acid 2-260, H122Y, N252D), CA2(WT amino acid 2-260, D72F, V241F), CA2(WT amino acid 2-260, V241F, P249L), CA2(WT amino acid 2-260, D72F, P249L), CA2(WT amino acid 2-260, D71L, L250R), CA2(WT amino acid 2-260, D72F, P249F), CA2(WT amino acid 2-260, T55K, G63N, Q248N). CA2(WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2(WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2(WT amino acids 2-260, W4Y, L156H), CA2(WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2(WT amino acids 2-260, L156H, F225L), CA2(WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2(WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 ( NO.210505), CA2 (amino acids 2-260, A77I, P249F of WT) (SEQ ID NO.210514), CA2 (amino acids 2-260, D71K, P249H of WT) (SEQ ID NO.210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K260L) (SEQ ID NO. 210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K2 (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71F, V241F, P249L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, L147F, Q248F) (SEQ ID NO. 210548), CA2 (WT amino acids 2-260, D52I, S258P) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D72S, T192N) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210548 ... SEQ ID NO. 210560), CA2 (WT amino acids 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acids 2-260, D72F, P249I) (SEQ ID NO. 210568), CA2 (WT amino acids 2-260, D72X, V241X, P249X) (SEQ ID NO. 210572), CA2 (WT amino acids 2-260, A54X, S56X, L57X, T192X) (SEQ ID NO. 210574), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO. 210560), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO. 210562), CA2 (WT amino acids 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acids 2-260, Y193V, K260F) (SEQ ID NO. 210565 ... NO.210576), CA2 (WT amino acids 2-260, G63D, M240L) (SEQ ID NO).210578), CA2(WT amino acids 2-260, V134F, L228F) (SEQ ID NO. 210580), CA2(WT amino acids 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acids 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2(WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 21057 ... S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2(WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 210578), CA2(WT amino acids (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, KI) HE, E220K, F225I)(SEQ ID NO.210706), CA2 (WT amino acids 2-260, P13S, P83A, D101G, K111N, F230I)(SEQ ID NO.210708), CA2 (WT amino acids 2-260, G63D, W123R, E220K)(SEQ ID NO.210712), CA2 (WT amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S)(SEQ ID NO.210714), CA2 (WT amino acids 2-260, I59N, G102R, A173T)(SEQ ID NO.210706), CA2 (WT amino acids 2-260, I59N, G102R, A173T)(SEQ ID NO.210706), CA2 (WT amino acids 2-260, I59N, G102R, A173T))(SEQ ID NO.210712), CA2 (WT amino acids 2-260, I59N, G102R, A173T))(SEQ ID NO.210712), CA2 (WT amino acids 2-260, I13S, P83A, D101G, K111N, F230I ... NO.210716), CA2 (amino acids 2-260, L79F, P180S of WT) (SEQ ID NO.210718), CA2 (amino acids 2-260, A77P, G102R, D138N of WT) (SEQ ID NO.210716), CA2 (amino acids 2-260, A77P, G102R, D138N of WT) (SEQ ID NO.210718).210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, T199N, L202P, L228F) (SEQ ID NO. 210728), CA2(WT amino acids 2-260, K9N, H122Y, T168K) (SEQ ID NO. 210730), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210732), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210726 ... (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, D75V, K169N, F259L) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738 ...F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A1 SEQ ID NO. 210750), CA2 (amino acids 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L of WT) (SEQ ID NO. 210752), CA2 (amino acids 2-260, G12R, H15Y, D19V of WT) (SEQ ID NO. 210754), CA2 (amino acids 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I of WT) (SEQ ID NO. 210758), and / or CA2 (amino acids 2-260, G63D, E69V, N231I of WT) (SEQ ID NO. 210851 or 210847).

[0179] This document also provides a biological circuit system comprising at least one effector submodule. The effector submodule of the biological circuit may include a stimulus-response element (SRE), and the SRE may comprise, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717). The biological circuit may also include at least one payload, which may be attached, supplemented, or associated with the SRE.

[0180] SREs of biological circuit systems that include, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717) may include one, two, three or more mutations, such as, but not limited to, A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, D138G, D138M, D138N, D161*, D161M, D161V, D164G, D 164I, D174*, D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242 G, D242T, D32T, D34T, D41T, D52I, D52L, D71F, D71G, D71K, D71M, D71S, D71 Y, D72I, D72S, D72T, D72X, D75T, D75V, D85M, E106D, E106G, E106S, E117*, E117N, E14N, E186*, E186N, E204A, E204D, E204G, E204N, E213*, E213G, E 213N, E220K, E220R, E220S, E233D, E233G, E233R, E235*, E235G, E235N, E2 37K, E237R, E238*, E238N, E238R, E26S, E69D, E69K, E69S, F130L, F146V, F 175I, F175L, F175S, F178L, F178S, F20L, F20S, F225I, F225L, F225S, F225 Y, F230I, F230L, F230S, F259L, F259S, F66S, F70I, F70L, F95Y, G102D, G10 4R, G104V, G128R, G12D, G12E, G131E, G131R, G131W, G139D, G144D, G144V, G150A, G150S, G150W, G155A, G155C, G155D, G155S, G170A, G170D, G182A, G 182W, G195A, G195R, G232R, G232W, G234L, G234V, G25E, G63D, G63V, G81E,G81V、G82D、G86A、G86D、G98V、H107I、H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I215S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153*、K153N、K158E、K158N、K167*、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227*、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143*、L147*、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197*、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211*、L211A、L211S、L223*、L223I、L223V、L228F、L228H、L228T、L239*、L239F、L239T、L250*、L250P、L250T、L44*、L44M、L47C、L47V、L57*、L57X、L60S、L79F、L79S、L84W、L90*、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177*、N177T、N229*、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、P13A、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、R181S、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206*、V206C、V206M、V210C、V217L、V217R、V217S、V222A、V222C、V222G、V241G、V241W、V241X、V31L、V49F、V68L、V68W、V78C、W123G、W123R、W16G、W191*、W191G、W191L、W208G、W208L、W208S、W244*、W244G、W244L、W97C、W97G、Y114H、Y114M、Y127M、Y190*、Y190L、Y190T、Y193C、Y193F、Y193I、Y193L、Y193T、Y193V、Y193X、Y40M、Y51F、Y51M、Y51T、Y51X、Y88T、K9N、S29A。、

[0181] In some respects, SRE may be selected from, but is not limited to, CA2(WT amino acid 2-260, R27L, H122Y), CA2(WT amino acid 2-260, T87I, H122Y), CA2(WT amino acid 2-260, H122Y, N252D), CA2(WT amino acid 2-260, D72F, V241F), CA2(WT amino acid 2-260, V241F, P249L), CA2(WT amino acid 2-260, D72F, P249L), CA2(WT amino acid 2-260, D71L, L250R), CA2(WT amino acid 2-260, D72F, P249F), and CA2(WT amino acid 2-260, T55K, G63N, Q248N). CA2(WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2(WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2(WT amino acids 2-260, W4Y, L156H), CA2(WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2(WT amino acids 2-260, L156H, F225L), CA2(WT amino acids 2-260, D70N, D74N, D100N, L156H), CA2(WT amino acids 2-260, I59N, G102R) (SEQ) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, G63D, E69V, N231I) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, R27L, T87I, H122Y, N252D) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, L156H, S172C, F178Y, E186D) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210598), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210748), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210702), CA2 (WT amino acids 2-260, D72F, V241F, P249L) (SEQ ID NO. 210503), CA2 (WT amino acids 2-260, D71L, T87N, L250R) (SEQ ID NO. 210510), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 (WT amino acids 2-260, D71F, N231F) (SEQ ID NO. 210756), CA2 ( (SEQ ID NO. 210505), CA2 (amino acids 2-260, A77I, P249F of WT) (SEQ ID NO. 210514), CA2 (amino acids 2-260, D71K, P249H of WT) (SEQ ID NO. 210514).210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K260L) (SEQ ID NO. 210516), CA2(WT amino acid 2-260, D72F, P249H) (SEQ ID NO. 210518), CA2(WT amino acid 2-260, Q53N, N61Y) (SEQ ID NO. 210521), CA2(WT amino acid 2-260, E106D, C205S) (SEQ ID NO. 210523), CA2(WT amino acid 2-260, C205S, W208S) (SEQ ID NO. 210525), CA2(WT amino acid 2-260, S73N, R89Y) (SEQ ID NO. 210532), CA2(WT amino acid 2-260, D71K, T192F) (SEQ ID NO. 210534), CA2(WT amino acid 2-260, Y193L, K2 SEQ ID NO. 210540), CA2 (WT amino acids 2-260, D71F, V241F, P249L) (SEQ ID NO. 210544), CA2 (WT amino acids 2-260, L147F, Q248F) (SEQ ID NO. 210548), CA2 (WT amino acids 2-260, D52I, S258P) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D72S, T192N) (SEQ ID NO. 210552), CA2 (WT amino acids 2-260, D179E, T192I) (SEQ ID NO. 210554), CA2 (WT amino acids 2-260, S56N, Q103K) (SEQ ID NO. 210558), CA2 (WT amino acids 2-260, D71Y, Q248L) (SEQ ID NO. 210558) (SEQ ID NO. 210559) (SEQ ID NO. 210550), CA2 (WT amino acids 2-260, D71Y, Q248L ... SEQ ID NO. 210560), CA2 (WT amino acid 2-260, S73N, R89F) (SEQ ID NO. 210562), CA2 (WT amino acid 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564), CA2 (WT amino acid 2-260, D72F, P249I) (SEQ ID NO. 210568), CA2 (WT amino acid 2-260, D72X, V241X, P249X) (SEQ ID NO. 210572), CA2 (WT amino acid 2-260, A54X, S56X, L57X, T192X) (SEQ ID NO. 210574), CA2 (WT amino acid 2-260, Y193V, K260F) (SEQ ID NO. 210560), CA2 (WT amino acid 2-260, Y193V, K260F) (SEQ ID NO. 210562), CA2 (WT amino acid 2-260, D71K, N231L, E235G, L239F) (SEQ ID NO. 210564 ... (SEQ ID NO. 210576), CA2 (WT amino acids 2-260, G63D, M240L) (SEQ ID NO. 210576).210578), CA2(WT amino acids 2-260, V134F, L228F) (SEQ ID NO. 210580), CA2(WT amino acids 2-260, D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acids 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino acids 2-260, S73X, R89X) (SEQ ID NO. 210588), CA2(WT amino acids 2-260, Y51X, D72X, V241X, P249X) (SEQ ID NO. 21057 ... D71G, N231K) (SEQ ID NO. 210582), CA2(WT amino acids 2-260, S56F, D71S) (SEQ ID NO. 210584), CA2(WT amino acids 2-260, D52L, G128R, Q248F) (SEQ ID NO. 210586), CA2(WT amino (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, D72I, W97C) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210592), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 210594), CA2 (WT amino acids 2-260, D71K, T192F, N231F) (SEQ ID NO. 210596), CA2 (WT amino acids 2-260, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO. 210698), CA2 (WT amino acids 2-260, F70I, F146V) (SEQ ID NO. 210700), CA2 (WT amino acids 2-260, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO. 2105994), CA2 (WT amino acids 2-260, K45N, V68L, (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO. 210708), CA2 (WT amino acids 2-260, G63D, W123R, E220K) (SEQ ID NO. 210712), CA2 (WT amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO. 210704), CA2 (WT amino acids 2-260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO. 210706), CA2 (WT amino acids 2-260, P13S, A54V, K111E, E220K, F225I ... (SEQ ID NO. 210714), CA2 (WT amino acids 2-260, I59N, G102R, A173T) (SEQ ID NO. 210716), CA2 (WT amino acids 2-260, L79F, P180S) (SEQ ID NO. 210718), CA2 (WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO. 210714), CA2 (WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO. 210716 ... L79F, P180S) (SEQ ID NO. 210718), CA2 (WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO. 210714), CA2 (WT amino acids 2-260, L79F, P180S) (SEQ ID NO. 210718), CA2 (WT amino acids 2-260, A77P, G102R, D138N) (SEQ ID NO. 210714), CA2 (WT amino acids 2-260, L79F, P180S) (SEQ ID NO. 210718), CA2 (WT amino acids 2-260, L79F, P180S) (SEQ ID NO. 210714), CA2 (WT amino acids 2-260210724), CA2(WT amino acids 2-260, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO. 210726), CA2(WT amino acids 2-260, T199N, L202P, L228F) (SEQ ID NO. 210728), CA2(WT amino acids 2-260, K9N, H122Y, T168K) (SEQ ID NO. 210730), CA2(WT amino acids 2-260, Q53H, L90V, Q92H, G131E) (SEQ ID NO. 210732), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210724), CA2(WT amino acids 2-260, L44M, L47V, N62K, E69D) (SEQ ID NO. 210726 ... (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, D75V, K169N, F259L) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210734), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, T207S, V222A, N231D) (SEQ ID NO. 210740), CA2 (WT amino acids 2-260, I59F, V206M, G232R) (SEQ ID NO. 210742), CA2 (WT amino acids 2-260, P13A, A133T) (SEQ ID NO. 210744), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59N, R89I) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59F, R232R) (SEQ ID NO. 210738), CA2 (WT amino acids 2-260, I59F, R232R) (SEQ SEQ ID NO. 210750), CA2 (amino acids 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L of WT) (SEQ ID NO. 210752), CA2 (amino acids 2-260, G12R, H15Y, D19V of WT) (SEQ ID NO. 210754), CA2 (amino acids 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I of WT) (SEQ ID NO. 210758), and / or CA2 (amino acids 2-260, G63D, E69V, N231I of WT) (SEQ ID NO. 210851 or 210847).

[0182] The biological circuit systems described herein may include SREs that respond to one or more stimuli.

[0183] In some embodiments, the stimulant may be a small molecule, such as celecoxib, vardecoxib, rofecoxib, acetazolamide, metronidazole, dzodamine, brinzolamide, diclofenac, itazolamide, zonisamide, dansylsulfonamide, or dichlorobenzenesulfonamide. In some embodiments, the small molecule may be acetazolamide.

[0184] This article also describes carriers that encode biological circuit systems, and pharmaceutical compositions comprising said biological circuit systems and pharmaceutically acceptable excipients.

[0185] Payload

[0186] As used herein, “payload”, “target payload”, or “payload of interest (POI)” is defined as any protein or nucleic acid whose function is to be altered.

[0187] The payload may include any coding or non-coding gene or any protein or fragment thereof.

[0188] The payload is typically associated with one or more SREs in the polynucleotides of this disclosure and can be encoded individually or in combination with one or more SREs. The payload itself can be modified (at the protein or nucleic acid level) to provide an additional, sustainable layer of effector submodule. For example, the payload can be engineered or designed to contain one or more mutations that affect the stability of the payload or its sensitivity to degradation, cleavage, or transport. Combining an SRE that may have a stimulus-response profile with a payload modified to display various responses or levels (e.g., expression levels) of output signal creates biological circuits superior to those in the art. For example, mutations or substitution designs, such as those created for IL12 in WO2016048903 (particularly in Example 1 therein), the contents of which are incorporated herein by reference in their entirety, can be combined with the SREs of this disclosure for any protein payload to create a dually tunable biological circuit. The ability to independently modulate both the SRE and the payload greatly expands the applicability of the effector submodules of this disclosure.

[0189] Artificial peptides or loaded polypeptide components can be derived from any known polypeptide that does not exist naturally.

[0190] As used herein, when referring to an effector module, SRE, or payload, the phrase “derived from” means that the effector module, SRE, or payload is at least partially derived from the stated parent molecule or sequence. For example, in designing an SRE, such an SRE may be derived from an epitope or region of a naturally occurring protein but subsequently modified in any manner taught herein to optimize SRE function.

[0191] In one implementation, the payload is derived from a region of the parent protein or from a mutant protein. The length of the parental protein region can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 21 0, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 31 6, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 3 62, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450 or more amino acids. The length of the parental protein region can be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 amino acids.

[0192] In one embodiment, the payload is derived from a region of the parental protein or from a mutant protein and includes a region of the parental protein. The payload may include a region of the parental protein, which is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the parental protein or mutant protein, 5-10%, 10-15%, 15-20%, 20-25%, 25- 30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70 -80%, 80-90%, 90-100%, 10-30%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 10-40%, 20-50%, 30-60%, 40-70%, 50-80%, 60-90%, 70-100%, 10-50%, 20-60%, 30-70% 40-80%, 50-90%, 60-100%, 10-60%, 20-70%, 30-80%, 40-90%, 50-100%, 10-70%, 20-80%, 30-90%, 40-100%, 10-80%, 20-90%, 30-100%, 10-90%, 20-100%, 25-50%, 50-75% or 75-100%.

[0193] In one embodiment, the payload is derived from the parental protein or from a mutant protein, and may contain 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, 5-10%, 10-15%, or 15-20% of the parental or mutant protein. 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, 70-80%, 80-90%, 90-100%, 10-30%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 10-40%, 20-50%, 30-60%, 40-70%, 50-80%, 60-90%, 70-100%, 10-50%, 20-60%, 30-70% The sameness of %, 40-80%, 50-90%, 60-100%, 10-60%, 20-70%, 30-80%, 40-90%, 50-100%, 10-70%, 20-80%, 30-90%, 40-100%, 10-80%, 20-90%, 30-100%, 10-90%, 20-100%, 25-50%, 50-75%, or 75-100%.

[0194] In one implementation, the transmembrane domain region of the first payload may be replaced by a transmembrane domain, variant, or fragment from the second parent protein.

[0195] Peptides and peptides as payload

[0196] The stimuli, biological circuit components, and effector submodules (including their SREs and payloads) disclosed herein may exist as whole peptides, multiple peptides, or peptide fragments, which may be independently encoded by one or more nucleic acids, multiple nucleic acids, nucleic acid fragments, or variants of any of the foregoing.

[0197] As used herein, the term "peptide" refers to a polymer of amino acid residues (natural or non-natural) most commonly linked together by peptide bonds. The term as used herein refers to proteins, polypeptides, and peptides of any size, structure, or function. In some cases, the encoded polypeptide is less than about 50 amino acids, and thus the polypeptide is referred to as a peptide. If a polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Therefore, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the aforementioned substances. Polypeptides can be single molecules or multi-molecule complexes, such as dimers, trimers, or tetramers. They may also comprise single-chain or multi-chain polypeptides and can associate or link together. The term polypeptide can also be applied to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0198] As used herein, the term "peptide variant" refers to a molecule whose amino acid sequence differs from that of the native or reference sequence. Compared to the native or reference sequence, amino acid sequence variants may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence. Typically, variants have at least about 50% identity (homology) with the native or reference sequence, preferably at least about 80%, more preferably at least about 90%, with the native or reference sequence.

[0199] In some embodiments, "variant mimics" are provided. As used herein, the term "variant mimic" refers to a variant containing one or more amino acids that will mimic the activating sequence. For example, glutamic acid can be used as a mimic of phosphorylated threonine and / or phosphorylated serine. Alternatively, variant mimics can lead to inactivation or produce inactivating products containing the mimic, such as phenylalanine as an inactivating substitute for tyrosine; or alanine as an inactivating substitute for serine. The amino acid sequences of the pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) of this disclosure may contain naturally occurring amino acids and can therefore be considered as proteins, peptides, polypeptides, or fragments thereof. Alternatively, pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) may contain both naturally occurring and non-naturally occurring amino acids.

[0200] As used herein, the term "amino acid sequence variant" refers to a molecule whose amino acid sequence differs from the native or originating sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence. As used herein, the terms "native" or "originating," when referring to a sequence, are relative terms and refer to the original molecule to which it can be compared. Native or originating sequences should not be confused with wild-type sequences. A native sequence or molecule may represent the wild-type (a sequence found in nature), but need not be identical to the wild-type sequence.

[0201] Typically, the variants share at least about 70% homology with the natural sequence, preferably at least about 80%, more preferably at least about 90%.

[0202] As used herein, the term "homology," when applied to amino acid sequences, is defined as the percentage of residues in a candidate amino acid sequence that are safely identical to residues in the amino acid sequence of a second sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage of homology. Methods and computer programs used for alignment are well known in the art. It should be understood that homology depends on the calculation of the percentage of identity, but its value may differ due to vacancies and penalties introduced in the calculation.

[0203] As used herein, the term "homology," when applied to amino acid sequences, refers to the corresponding sequence of another species that is substantially identical to the second sequence of the second species.

[0204] As used herein, the term "analyte" is intended to include peptide variants that differ due to one or more amino acid alterations, such as substitution, addition, or deletion of amino acid residues, but still retain the characteristics of the parent peptide.

[0205] As used herein, the term “derivative” and the term “variant” are used synonymously and refer to a molecule that has been modified or altered in any way relative to a reference molecule or a starting molecule.

[0206] This disclosure contemplates several types of pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their amino acid-based SREs or payloads), including variants and derivatives. These include substitution, insertion, deletion, and covalent variants and derivatives. Therefore, pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) are included within the scope of this disclosure, including substitution, insertion, addition, deletion, and / or covalent modifications. Sequence tags or amino acids, such as one or more lysine residues, can be added to the peptide sequence of this disclosure (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide purification or localization. Lysine residues can be used to increase peptide solubility or allow biotinylation. Alternatively, amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to provide a truncated sequence. Alternatively, certain amino acids (e.g., C-terminal or N-terminal residues) can be deleted, depending on the intended use of the sequence, for example, expressing the sequence as part of a larger sequence that is soluble or linked to a solid support.

[0207] When referring to proteins, a "substitution variant" is a protein in which at least one amino acid residue is removed from the native or starting sequence and a different amino acid is inserted at the same position as said residue. Substitution can be single, where only one amino acid in the molecule is substituted, or multiple, where two or more amino acids in the same molecule are substituted.

[0208] As used herein, the term "conservative amino acid substitution" refers to the substitution of a normally present amino acid in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitution include the substitution of another nonpolar residue with a nonpolar (hydrophobic) residue such as isoleucine, valine, or leucine. Similarly, examples of conservative substitution include the substitution of one polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of another basic residue with a basic residue such as lysine, arginine, or histidine, or the substitution of another acidic residue with an acidic residue such as aspartic acid or glutamic acid, are further examples of conservative substitution. Examples of nonconservative substitution include the substitution of a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine with a nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine, and / or the substitution of a nonpolar residue with a polar residue.

[0209] As used herein, when referring to proteins, the term "insertion variant" refers to a protein in which one or more amino acids are inserted at a specific position immediately adjacent to the native or starting sequence. As used herein, the term "immediately adjacent" refers to the adjacent amino acid linked to the α-carboxyl or α-amino functional group of the starting or reference amino acid.

[0210] As used herein, when referring to proteins, the term "deletion variant" refers to a protein in which one or more amino acids are removed from the native or starting amino acid sequence. Typically, deletion variants will have one or more amino acids missing from a specific region of the molecule.

[0211] As used herein, the term "derivative" includes variants of native or starting proteins that contain one or more modifications, including post-translational modifications, made with organic protein or non-protein derivatizing agents. Traditionally, covalent modifications are introduced by reacting a target amino acid residue of a protein with an organic derivatizing agent capable of reacting with selected side-chain or terminal residues, or by utilizing post-translational modification mechanisms that function in selected recombinant host cells. The resulting covalent derivative can be used in procedures aimed at identifying residues important for the bioactivity, immunoassay, or preparation of antiprotein antibodies for immunoaffinity purification of recombinant glycoproteins. Such modifications are within the general scope of the art and are performed without excessive experimentation.

[0212] As used herein, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" when it pertains to an amino acid-based embodiment. A site represents a location within a peptide or polypeptide, within the polypeptide-based molecule of this disclosure, which may be modified, manipulated, altered, derived, or changed.

[0213] As used herein, the term "terminus," when referring to a protein, means the end of a peptide or polypeptide. Such a terminus is not limited to the first or final site of the peptide or polypeptide, but may include additional amino acids in the terminal region. The polypeptide-based molecules of this disclosure are characterized by having an N-terminus (terminated by an amino acid having a free amino group (NH2)) and a C-terminus (terminated by an amino acid having a free carboxyl group (COOH)).

[0214] The polypeptides or proteins disclosed herein are, in some cases, composed of multiple polypeptide chains (polymers, oligomers) linked together by disulfide bonds or by non-covalent forces. These types of proteins will have multiple N-termini and C-termini. Alternatively, depending on the circumstances, the ends of the polypeptides may be modified such that they begin or end with a non-peptide-based portion (e.g., an organic conjugate).

[0215] Once any feature is identified or defined as a component, stimulus, or effector submodule (including SREs or payloads) of the biological circuit system of this disclosure, any of these features can be manipulated and / or modified by moving, exchanging, inverting, deleting, randomizing, or replicating. Furthermore, it should be understood that manipulation of a feature can produce the same results as modification of the compositions of this disclosure. For example, manipulation involving the deletion of a domain will result in a change in molecular length, just as modifying a nucleic acid to encode a molecule shorter than its full length.

[0216] Modification and manipulation can be accomplished using methods known in the art, such as site-directed mutagenesis. The activity of the resulting modified molecules can then be tested using in vitro or in vivo assays as described herein or any other suitable screening assays known in the art.

[0217] In some embodiments, the compositions of this disclosure may comprise one or more isotopic atoms. As used herein, the term "isotope" refers to a chemical element having one or more additional neutrons. In some embodiments, the compounds of this disclosure may be deuterated. As used herein, the term "deuteration" refers to the process of replacing one or more hydrogen atoms in a substance with a deuterium isotope. Deuterium isotopes are isotopes of hydrogen. A hydrogen nucleus contains one proton, while a deuterium nucleus contains one proton and one neutron. The pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) of this disclosure may be deuterated to alter one or more physical properties, such as stability, or to allow the pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) to be used for diagnostic and / or experimental applications.

[0218] At the protein level, any biological circuit component may contain one or more post-translational modifications (PTMs). Such PTMs can occur intracellularly after the administration of a protein-based biological circuit component, or at or after the translation of a biological circuit component administered as a nucleic acid encoding the biological circuit component.

[0219] The post-translational modifications (PTMs) disclosed herein include, but are not limited to, acetylation, phosphorylation, ubiquitination, carboxylation, deamidation, deamination, deacetylation, dihydroxylation, dephosphorylation, formylation, γ-carboxyglutamate, glutathioneization, glycosylation, hydroxylation, methylation, nitration, sumoylation, N- or O-transglutamination, glycosylation, and farnesylation.

[0220] Effect submodules (including their SREs and payloads) can independently have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more identical or different PTMs.

[0221] Effector submodules can be designed to include one or more structural or functional domains, repetitive sequences, or motifs belonging to a protein family. Such domains, repetitive sequences, and motifs are classified according to protein families; and located in... http: / / www.ebi.ac.uk / The EMBL-EBI database provides representative families.

[0222] In some embodiments, protein modifications (such as glycosylation and polyethylene glycolation) engineered into the structure of the compositions disclosed herein may also be used in this disclosure to interfere with antigen processing and peptide loading. The compositions of this disclosure may also be designed to include non-classical amino acid side chains in order to design compositions with lower immunogenicity. Any methods for reducing immunogenicity discussed in International Patent Publication No. WO2005051975 may be used in this disclosure (the contents of which are incorporated herein by reference in their entirety).

[0223] SREs can be, but are not limited to, peptides, peptide complexes, peptide-protein complexes, proteins, fusion proteins, protein complexes, and protein-protein complexes. SREs may include one or more regions derived from any natural or mutant protein or antibody. In this respect, SREs are elements capable of modulating the intracellular localization, intramolecular activation, and / or degradation of the payload when responding to a stimulus.

[0224] In some embodiments, the effector submodule of this disclosure may include additional features that promote the expression and regulation of the effector submodule, such as one or more signaling sequences (SS), one or more cleavage and / or processing sites, one or more targeting and / or penetrating peptides, one or more tags and / or one or more adapters. Additionally, the effector submodule of this disclosure may also include other regulatory components, such as inducible promoters, enhancer sequences, microRNA sites, and / or microRNA targeting sites. Each aspect or mode of regulation can impart different regulatory features to the effector submodule or biological circuit. For example, SRE can represent a destabilizing domain, while mutations in the protein payload can alter its cleavage site, dimerization properties, or half-life, and including one or more microRNAs or microRNA binding sites can confer cellular detargeting or transport characteristics. Thus, this disclosure includes biological circuits that are multifactorial in terms of their maintainability. Such biological circuits can be engineered to contain one, two, three, four, or more regulatory features.

[0225] In some embodiments, the effector submodules of this disclosure may include one or more degradation determinants that regulate expression. As used herein, a “degradation determinant” refers to the smallest sequence within a protein that is sufficient to be recognized and degraded by a proteolytic system. An important characteristic of degradation determinants is that they are transferable, meaning that attaching a degradation determinant to a sequence confers degradation on that sequence. In some embodiments, degradation determinants may be attached to destabilizing domains, payloads, or both. The incorporation of degradation determinants into the effector submodules of this disclosure confers additional protein instability to the effector submodule and can be used to minimize basal expression. In some embodiments, degradation determinants may be N-degradation determinants, phosphorylation degradation determinants, heat-inducible degradation determinants, photosensitized degradation determinants, or oxygen-dependent degradation determinants. As a non-limiting example, a degradation determinant may be an ornithine decarboxylase degradation determinant, which is described by Takeuchi et al. (Takeuchi J et al., (2008). Biochem J. 2008 Mar 1; 410(2):401-7; the contents of which are incorporated herein by reference in their entirety). Other examples of degradation determinants useful in this disclosure include those described in International Patent Publications WO2017004022, WO2016210343 and WO2011062962, the contents of which are incorporated herein by reference in their entirety.

[0226] Immunotherapy agents

[0227] The biological circuits described herein may include immunotherapeutic agents. In some embodiments, the payload of this disclosure may be an immunotherapeutic agent that induces an immune response in an organism. Immunotherapeutic agents may be, but are not limited to, antibodies and their fragments and variants, chimeric antigen receptors (CARs), chimeric switch receptors, cytokines, chemokines, cytokine receptors, chemokine receptors, cytokine-cytokine receptor fusion peptides, or any agent that induces an immune response. In one embodiment, the immunotherapeutic agent induces an anticancer immune response in cells or a subject.

[0228] Cytokines, chemokines and other factors

[0229] According to this disclosure, the payload can be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells, and other cell types, which act as chemical communication substances between cells and tissues in the body. These proteins mediate a wide range of physiological functions, from influencing cell growth, differentiation, migration, and survival to the activity of many effectors. For example, activated T cells produce a variety of cytokines with cytotoxic functions to eliminate tumor cells.

[0230] In some embodiments, the payload of this disclosure may be cytokines and their fragments, variants, analogs, and derivatives, including but not limited to interleukins, tumor necrosis factor (TNF), interferon (IFN), TGFβ, and chemokines. It should be understood in the art that the nomenclature of a gene or protein may include or exclude punctuation marks “-”, such as dashes, or symbols such as Greek letters. Whether these are included or excluded herein, their meaning is not altered, as understood by those skilled in the art. For example, IL2, IL2, and IL-2 refer to the same interleukin. Similarly, TNFα, TNFα, TNF-α, TNF-a, TNFalpha, and TNFα all refer to the same protein. Likewise, CD40L, CD40L, and CD40LG refer to the same protein.

[0231] In some embodiments, the payload of this disclosure may be a cytokine that stimulates an immune response. In other embodiments, the payload of this disclosure may be an antagonist of a cytokine that negatively affects an anticancer immune response.

[0232] In some embodiments, the payload of this disclosure may be a cytokine receptor, a recombinant receptor, its variants, analogs and derivatives; or a signaling component of a cytokine.

[0233] In some embodiments, the cytokines of this disclosure can be used to enhance the expansion, survival, persistence, and potency of immune cells used in immunotherapy, such as CD8+ TEMs, natural killer cells, and tumor-infiltrating lymphocytes (TILs). In other embodiments, T cells are utilized to provide kinetic control of T cell activation and tumor microenvironment remodeling, said T cells being engineered with cytokines regulated by two or more DDs. In one aspect, this disclosure provides biological circuits and compositions for minimizing cytokine therapy-related toxicities. While systemic cytokine therapy has been successful in reducing tumor burden, it often results in severe dose-limiting side effects. Two factors contribute to the observed toxicities: (a) pleiotropic effects, where cytokines affect different cell types, sometimes producing opposite effects on the same cells depending on the situation; and (b) the short serum half-life of cytokines, thus requiring high doses to achieve therapeutic effects, which exacerbates pleiotropic effects. In one aspect, the cytokines of this disclosure can be used to modulate cytokine expression in the presence of side effects. In some embodiments, the cytokines of this disclosure can be engineered to have prolonged lifespan or enhanced specificity, thereby minimizing toxicity.

[0234] In some embodiments, the payload of this disclosure may be interleukin (IL) cytokines. Interleukins (ILs) are a class of glycoproteins produced by leukocytes that regulate immune responses. As used herein, the term "interleukin (IL)" refers to an interleukin polypeptide from any species or source and includes full-length proteins as well as fragments or portions of said proteins. In some respects, the effective load of interleukins is selected from IL1, IL1α (also known as hematopoietic 1), IL1β (catabolism), IL1δ, IL1ε, IL1η, IL1ζ, interleukin-1 family members 1 to 11 (IL1F1 to IL1F11), interleukin-1 homologs 1 to 4 (IL1H1 to IL1H4), IL1-associated proteins 1 to 3 (IL1RP1 to IL1RP3), IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL10C, IL10D, IL11, IL11a, IL11b, IL12, IL13, IL14, IL15, and I. L16, IL17, IL17A, I117B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL20-like (IL20L), I121, IL22, IL23, IL23A, IL23-p19, IL23-p40, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36α, IL36β, IL36γ, IL36RN, IL37, IL37a, IL37b, IL37c, IL37d, IL37e, and IL38. In other respects, the payload of this disclosure may be an interleukin receptor selected from CD121a, CDw121b, IL2Rα / CD25, IL2Rβ / CD122, IL2Rγ / CD132, CDw131, CD124, CD131, CDw125, CD126, CD130, CD127, CDw210, IL8RA, IL11Rα, CD212, CD213α1, CD213α2, IL14R, IL15Rα, CDw217, IL18Rα, IL18Rβ, IL20Rα, and IL20Rβ. In other respects, the payload of this disclosure may be a member of the TNF superfamily, including but not limited to TNFα, CD40L, lymphotoxin (LTA)α, LTAβ, and OX40L.

[0235] Antibodies, as well as antibody fragments and variants

[0236] The biological circuits described herein may include one or more antibodies described herein. In some embodiments, the one or more antibodies described herein may be a payload.

[0237] In some implementations, antibody fragments and variants may include an antigen-binding region derived from the intact antibody. Examples of antibody fragments and variants include, but are not limited to, Fab, Fab-1999, and Fab-19 ... 1 F(ab')2 and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules such as single-chain variable fragments (scFv); and multispecific antibodies formed from antibody fragments. Pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) disclosed herein may contain one or more of these fragments.

[0238] For the purposes of this article, an "antibody" may include a heavy chain variable domain, a light chain variable domain, and an Fc region. As used herein, the term "natural antibody" generally refers to a heterotetraglycosaccharide protein of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains linked together by disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domains of the light chain are aligned with the first constant domain of the heavy chain, and the variable domains of the light chain are aligned with the variable domains of the heavy chain.

[0239] As used herein, the term "variable domain" refers to a specific antibody domain found on the heavy and light chains of an antibody. These domains vary significantly in sequence between antibodies and are responsible for the binding and specificity of each particular antibody to its specific antigen. Variable domains contain hypervariable regions. As used herein, the term "hypervariable region" refers to a region within a variable domain that contains the amino acid residues responsible for antigen binding. The amino acids present within the hypervariable region determine the structure of the complementarity-determining region (CDR), which becomes part of the antibody's antigen-binding site. As used herein, the term "CDR" refers to an antibody region containing a structure complementary to its target antigen or epitope. The remaining portions of the variable domain that do not interact with the antigen are referred to as frame (FW) regions. The antigen-binding site (also called the antigen-binding site or complementation site) contains the amino acid residues necessary for interaction with a specific antigen.

[0240] The VH and VL domains each have three CDRs. As the peptide moves along the variable domain from the N-terminus to the C-terminus, the VL CDRs are referred to in this paper as CDR-L1, CDR-L2, and CDR-L3, in order of appearance. Similarly, as the peptide moves along the variable domain from the N-terminus to the C-terminus, the VH CDRs are referred to in this paper as CDR-H1, CDR-H2, and CDR-H3, in order of appearance.

[0241] As used herein, the term "Fv" refers to an antibody fragment containing the smallest segment on the antibody required to form a complete antigen-binding site. These regions consist of a dimer of a tightly non-covalently associated heavy chain variable region domain and a light chain variable region domain. Fv fragments can be generated by proteolytic cleavage but are largely unstable. Recombinant methods for generating stable Fv fragments are known in the art and are typically employed by inserting a flexible linker between the light chain variable region and the heavy chain variable region (to form a single-chain Fv (scFv)) or by introducing a disulfide bridge between the heavy chain and the light chain variable region (Strohl, WR Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Chapter 3, pp. 46-47, the contents of which are incorporated herein by reference in their entirety).

[0242] As used herein, the term "light chain" refers to an antibody component from any vertebrate species, designated as one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. Antibodies can be designated as different types based on the amino acid sequence of their heavy chain's constant domain. There are five main types of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0243] As used herein, the terms "single-chain Fv" and "scFv" refer to a fusion protein of VH and VL antibody domains, wherein these domains are linked together by a flexible peptide linker to form a single polypeptide chain. In some embodiments, the Fv peptide linker enables the scFv to form a desired structure for antigen binding. In some embodiments, scFvs are used in conjunction with phage display, yeast display, or other display methods, wherein they can be expressed in conjunction with surface members (e.g., phage coat proteins) and used to identify high-affinity peptides against a given antigen.

[0244] Using molecular genetics, two single-chain antibodies can be tandemly engineered into a single polypeptide separated by a linker domain, called a “tandem scFv” (tascFv). Constructing a tascFv with genes for two different scFvs produces a “bispecific single-chain variable fragment” (bis-scFv). Commercial companies have developed only two tascFvs clinically; both are bispecific agents being actively developed by Micromet for oncology indications and are described as “bispecific T-cell binders (BiTEs).” Blinatumomab is an anti-CD19 / anti-CD3 bispecific tascFv that enhances the T-cell response in stage 2 B-cell non-Hodgkin lymphoma. MT110 is an anti-EP-CAM / anti-CD3 bispecific tascFv that enhances the T-cell response in stage 1 solid tumors. Affimed (Nelson, AL, MAbs., 2010, Jan-Feb; 2(1):77–83) is also investigating a bispecific tetravalent “TandAb”. It may also include large antibodies against IgG (bivalent scFv fused to the amino terminus of IgG Fc (CH2-CH3 domain)).

[0245] As used herein, the term “bispecific antibody” refers to an antibody capable of binding to two different antigens. Such antibodies typically contain regions derived from at least two different antibodies. Bispecific antibodies may include any of those described in the following references: Riethmuller, G. Cancer Immunity. 2012, 12:12-18; Marvin et al., 2005. Acta Pharmacologica Sinica. 2005, 26(6):649-658; and Schaefer et al., PNAS. 2011, 108(27):11187-11192, the contents of which are incorporated herein by reference in their entirety.

[0246] As used herein, the term "biantibody" refers to a small antibody fragment having two antigen-binding sites. A biantibody is a functional bispecific single-chain antibody (bscAb). A biantibody contains a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) on the same polypeptide chain. By using a linker that is too short to allow pairing of two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, resulting in two antigen-binding sites.

[0247] The term "intracellular antibody" refers to a form of antibody that is not secreted from the cell that produces it but targets one or more intracellular proteins. Intracellular antibodies can be used to influence many cellular processes, including but not limited to intracellular transport, transcription, translation, metabolic processes, proliferation signaling, and cell division. In some embodiments, the methods of this disclosure may include intracellular antibody-based therapies. In some such embodiments, the variable domain sequences and / or CDR sequences disclosed herein may be incorporated into one or more constructs for use in intracellular antibody-based therapies.

[0248] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous cells (or clones), meaning that the individual antibodies comprising that population are identical and / or bind to the same epitope, except for possible variants that may arise during the monoclonal antibody production process, which are typically present in small quantities. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody targets a single determinant on the antigen.

[0249] The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the antibody to be produced by any particular method. Monoclonal antibodies as used herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; fragments of such antibodies are also included.

[0250] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising a minimal portion derived from one or more non-human (e.g., mouse) antibody sources, with the remainder derived from one or more human immunoglobulin sources. In most cases, humanized antibodies are human immunoglobulin (receptor antibodies) in which residues of the hypervariable region of an antibody derived from a receptor are replaced by residues of the hypervariable region of an antibody (donor antibody) from a non-human species such as a mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and / or ability. In one embodiment, the antibody may be a humanized full-length antibody. As a non-limiting example, antibodies can be humanized using the methods taught in U.S. Patent Publication No. US20130303399, the contents of which are incorporated herein by reference in their entirety.

[0251] As used herein, the term "antibody variant" refers to a modified antibody (as opposed to a natural or initiating antibody) or a biomolecule (e.g., an antibody mimic) that is structurally and / or functionally similar to a natural or initiating antibody. Antibody variants may differ from natural antibodies in their amino acid sequence, composition, or structure. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.

[0252] In some embodiments, the pharmaceutical compositions, biological circuits, components of biological circuits, and effector submodules (including their SREs or payloads) of this disclosure may be antibody mimics. As used herein, the term "antibody mimic" refers to any molecule that mimics the function or action of an antibody and specifically and with high affinity binds to its molecular target. In some embodiments, the antibody mimic may be a monoclonal antibody designed to incorporate a fibronectin type III domain (Fn3) as a protein scaffold (US 6,673,901; US ​​6,348,584). In some embodiments, the antibody mimic may be those known in the art, including but not limited to affibody molecules, affilin, affitin, anticarrier proteins, avimers, centyrins, DARPINS™, fenomeres, and Kunitz and domain peptides. In other embodiments, the antibody mimic may include one or more non-peptide regions.

[0253] In one embodiment, the antibody may comprise a modified Fc region. As a non-limiting example, the modified Fc region may be prepared by the method described in U.S. Patent Publication No. US20150065690 or may be any region described in that patent, the entire contents of which are incorporated herein by reference.

[0254] In some embodiments, the payload of this disclosure may encode a multispecific antibody that binds to more than one epitope. As used herein, the term "multibody" or "multispecific antibody" refers to an antibody in which two or more variable regions bind to different epitopes. The epitopes may be on the same or different targets. In one embodiment, multispecific antibodies can be generated and optimized by the methods described in International Patent Publication No. WO2011109726 and U.S. Patent Publication No. US20150252119, the contents of which are incorporated herein by reference in their entirety. These antibodies are capable of binding to a variety of antigens with high specificity and high affinity.

[0255] In some implementations, a multispecific antibody is a “bispecific antibody” that recognizes two different epitopes on the same or different antigens. In one respect, a bispecific antibody is capable of binding to two different antigens. Such antibodies typically contain antigen-binding regions from at least two different antibodies. For example, a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein composed of fragments from two different monoclonal antibodies, thus allowing BsAb to bind to two different types of antigens. A bispecific antibody framework may include any of those frameworks described in the following literature: Riethmuller, G., 2012. Cancer Immunity, 2012, 12:12-18; Marvin et al., Acta Pharmacologica Sinica. 2005, 26(6):649-658; and Schaefer et al., PNAS. 2011, 108(27):11187-11192, the contents of which are incorporated herein by reference in their entirety. A new generation of BsMAbs known as “trifunctional bispecific” antibodies has been developed. These consist of two heavy chains and two light chains, each derived from two different antibodies. The two Fab regions (arms) target two antigens, and the Fc region (foot) contains the two heavy chains and forms a third binding site.

[0256] In some implementations, the payload may encode an antibody containing a single antigen-binding domain. These molecules are extremely small, with a molecular weight approximately one-tenth that of a full-size mAb. Further antibodies may include “nanobodies” derived from the antigen-binding variable heavy chain region (VHH) of heavy chain antibodies found in the genus *Laminaria* that lack light chains (Nelson, AL, MAbs. 2010. Jan-Feb; 2(1):77–83).

[0257] In some implementations, the antibody can be "miniaturized." A prime example of mAb miniaturization is Trubion Pharmaceuticals' Small Modular Immunotherapy Pulses (SMIPs). These molecules, which can be monovalent or bivalent, are recombinant single-chain molecules containing a VL and a VH antigen-binding domain and one or two constant "effector" domains, all linked by a linker domain. It is presumed that such molecules may offer the advantages of increased tissue or tumor penetration claimed by the fragment, while retaining the effector immune function conferred by the constant domains. At least three "miniaturized" SMIPs are in clinical development.

[0258] One example of a miniaturized antibody is called a "unibody," in which the hinge region has been removed from the IgG4 molecule. Although IgG4 molecules are unstable and can exchange light-heavy chain heterodimers, the absence of the hinge region completely prevents heavy chain-heavy chain pairing, leaving highly specific monovalent light / heavy heterodimers, while retaining the Fc region to ensure stability and in vivo half-life.

[0259] In some embodiments, the payload of this disclosure may encode a single-domain antibody (sdAb or nanobody), which is an antibody fragment composed of a single monomeric variable antibody domain. Like a whole antibody, it is capable of selectively binding to a specific antigen. In one aspect, the sdAb may be “camel Ig” or “camel family VHH”. As used herein, the term “camel Ig” refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolte et al., FASEB). J., 2007, 21:3490-3498). "Heavy chain antibody" or "cameloid antibody" refers to an antibody containing two VH domains and lacking a light chain (Riechmann L. et al., J. Immunol. Methods, 1999, 231:25-38; International Patent Publications WO1994 / 04678 and WO1994 / 025591; and US Patent No. 6,005,079). On the other hand, sdAb can be an "immunoglobulin neoantigen receptor" (IgNAR). As used herein, the term "immunoglobulin neoantigen receptor" refers to a class of antibodies derived from a shark immune repertoire that consists of a homodimer of a variable neoantigen receptor (VNAR) domain and five constant neoantigen receptor (CNAR) domains.

[0260] In some embodiments, the payload of this disclosure may encode an intracellular antibody. An intracellular antibody is a form of antibody that is not secreted from the cell that produces it but targets one or more intracellular proteins. Intracellular antibodies are expressed and function within cells and can be used to influence many cellular processes, including but not limited to intracellular transport, transcription, translation, metabolic processes, proliferation signaling, and cell division. In some embodiments, the methods described herein include intracellular antibody-based therapies. In some such embodiments, the variable domain sequences and / or CDR sequences disclosed herein are incorporated into one or more constructs for use in intracellular antibody-based therapies. For example, an intracellular antibody may target one or more glycosylated intracellular proteins, or may modulate the interaction between one or more glycosylated intracellular proteins and alternative proteins.

[0261] In some aspects, the payload of this disclosure may encode the biosynthetic antibody described in U.S. Patent No. 5,091,513, the contents of which are incorporated herein by reference in their entirety. Such an antibody may comprise one or more amino acid sequences that constitute a region serving as a biosynthetic antibody binding site (BABS). These sites comprise 1) a non-covalently associated or disulfide-bonded synthetic VH and VL dimer, 2) a VH-VL or VL-VH single chain, wherein the VH and VL are attached via a polypeptide linker, or 3) a separate VH or VL domain. The binding domain includes linked CDR and FR regions, which may be derived from individual immunoglobulins. Biosynthetic antibodies may also include other polypeptide sequences that function, for example, as an enzyme, toxin, binding site, or attachment site for immobilization media or radioactive atoms. Methods for generating biosynthetic antibodies, for designing BABS having any specificity that can be initiated by in vivo antibody production, and for generating analogues thereof are disclosed.

[0262] In some embodiments, the payload may encode an antibody having the antibody receptor framework taught in U.S. Patent No. 8,399,625. Such an antibody receptor framework may be particularly well-suited to accept a CDR from an antibody of interest.

[0263] In one embodiment, the antibody may be a conditionally active biological protein. The antibody can be used to generate conditionally active biological proteins that are reversibly or irreversibly inactivated under normal physiological conditions of wild type, as well as the use of such conditionally active biological proteins. Such methods and conditionally active proteins are taught in, for example, International Publications WO2015175375 and WO2016036916 and U.S. Patent Publication US20140378660, the contents of which are incorporated herein by reference in their entirety.

[0264] Antibodies used in immunotherapy

[0265] In some embodiments, the payload of this disclosure may be antibodies, fragments thereof, and variants specific to tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). The antibodies circulate systemically until they find and attach to a TSA / TAA. Once attached, they recruit other parts of the immune system, thereby enhancing ADCC (antibody-dependent cell-mediated cytotoxicity) and ADCP (antibody-dependent cell-mediated phagocytosis) to destroy tumor cells. As used herein, the term "tumor-specific antigen (TSA)" refers to an antigenic substance produced in tumor cells that can trigger an anti-tumor immune response in the host organism. In one embodiment, the TSA may be a neotumor antigen. Tumor antigen-specific antibodies mediate a complement-dependent cytotoxic response against tumor cells expressing the same antigen.

[0266] In some embodiments, tumor-specific antigens (TSAs), tumor-associated antigens (TAAs), pathogen-associated antigens, or fragments thereof may be expressed as peptides or complete proteins or portions thereof. Complete proteins or portions thereof may be natural or mutagenic. Antigens associated with cancer or virus-induced cancer as described herein are well known in the art. Such TSAs or TAAs may have been previously associated with cancer or may be identified by any method known in the art.

[0267] In one implementation, the antigen is CD19, a B cell surface protein expressed throughout B cell development. CD19 is a well-known B cell surface molecule that enhances B cell antigen receptor-induced signaling and amplification in the B cell population upon activation of B cell receptors. CD19 is widely expressed in both normal and neoplastic B cells. Malignant tumors derived from B cells, such as chronic lymphocytic leukemia, acute lymphoblastic leukemia, and many non-Hodgkin's lymphomas, frequently retain CD19 expression. This near-universal expression and specificity to single-cell lineages make CD19 an attractive target for immunotherapy. Human CD19 has 14 exons, with exons 1-4 encoding the extracellular portion of CD19, exon 5 encoding the transmembrane portion, and exons 6-14 encoding the cytoplasmic tail.

[0268] In one embodiment, the payload of this disclosure may be an antibody specific to the CD19 antigen, a fragment thereof, or a variant thereof.

[0269] In some implementations, the immunotherapeutic agent may be an antibody that has a specific immune response to an antigen selected from tumor-specific antigens (TSA), tumor-associated antigens (TAA), or antigenic epitopes.

[0270] On one hand, the antigen can be an antigenic epitope. In some implementations, the antigenic epitope can be CD19.

[0271] Tumor-specific antigens (TSAs) can be neoantigens. Neoantigens are mutated antigens expressed only by tumor cells, resulting from gene mutations or transcriptional alterations that change the protein-coding sequence, thus creating new foreign antigens. Genetic changes are caused by genetic substitution, insertion, deletion, or any other genetic alteration of the native homologous protein (i.e., the molecule expressed in normal cells).

[0272] Chimeric antigen receptor (CAR)

[0273] The biological circuits described herein may include chimeric antigen receptors. In some embodiments, the payload of this disclosure may be a chimeric antigen receptor (CAR) that, when transduced into immune cells (e.g., T cells and NK cells), can redirect immune cells to targets (e.g., tumor cells) expressing molecules recognized by the extracellular target portion of the CAR.

[0274] As used herein, the term "chimeric antigen receptor (CAR)" refers to a synthetic receptor that mimics the TCR on the surface of T cells. Generally, a CAR consists of an extracellular targeting domain, a transmembrane domain / region, and an intracellular signaling / activation domain. In a standard CAR receptor, the extracellular targeting domain, transmembrane domain, and intracellular signaling / activation domain are constructed linearly as a single fusion protein. The extracellular region contains the targeting domain / part (e.g., scFv), which recognizes specific tumor antigens or other tumor cell surface molecules. The intracellular region may contain signaling domains of the TCR complex (e.g., the CD3ζ signaling region and / or one or more co-stimulatory signaling domains, such as signaling domains from CD28, 4-1BB (CD137), and OX-40 (CD134)). For example, "first-generation CARs" contain only the CD3ζ signaling domain. To enhance T cell persistence and proliferation, co-stimulatory intracellular domains have been added, resulting in second-generation CARs with a CD3ζ signaling domain plus one co-stimulatory signaling domain, and third-generation CARs with a CD3ζ signaling domain plus two or more co-stimulatory signaling domains. When expressed by T cells, the CAR confers antigen specificity to the T cells determined by the extracellular targeting portion of the CAR. Recently, there has also been a desire to add one or more elements, such as homing and suicide genes, to develop more active and safer CAR architectures, the so-called fourth-generation CARs.

[0275] In some implementations, the immunotherapeutic agent having an effector submodule is a chimeric antigen receptor (CAR). The chimeric antigen may include an extracellular target portion; a transmembrane domain; an intracellular signal transduction domain; and optionally, one or more co-stimulatory domains.

[0276] In some implementations, the extracellular targeting domain is connected to the intracellular signaling domain via hinges (also called spatial domains or spacers) and transmembrane regions. The hinges connect the extracellular targeting domain to the transmembrane domain, which traverses the cell membrane laterally and connects to the intracellular signaling domain. Due to the size of the target protein bound to the targeting moiety, as well as the size and affinity of the targeting domain itself, the hinges may need to be modified to optimize the potency of CAR-expressing cells against cancer cells. After the targeting moiety is recognized and bound to the target cell, the intracellular signaling domain generates an activation signal for CAR T cells, which is further amplified by a “second signal” from one or more intracellular co-stimulatory domains. Once activated, the CAR T cell can destroy the target cell.

[0277] In some embodiments, the CAR of this disclosure can be divided into two parts, each connected to a dimerization domain, such that inputs triggering dimerization promote the assembly of a fully functional receptor. Wu and Lim recently reported a splitting CAR in which an extracellular CD19-binding domain and an intracellular signaling element are separated and connected to an FKBP domain and an FRB* (FKBP-rapamycin-binding T2089L mutant) domain (which heterodimerize in the presence of the rapamycin analog AP21967). The splitting receptor assembles in the presence of AP21967, simultaneously binding to a specific antigen and activating T cells (Wu et al., Science, 2015, 625(6258):aab4077).

[0278] In some embodiments, the CAR of this disclosure can be designed as an inducible CAR. Sakemura et al. recently reported the combination of the Tet-On induction system with a CD19 CAR construct. The CD19 CAR is activated only in the presence of doxycycline (Dox). Sakemura reported that Tet-CD19CAR T cells in the presence of Dox exhibited equivalent cytotoxicity to CD19+ cell lines and equivalent cytokine production and proliferation upon CD19 stimulation compared to conventional CD19CAR T cells (Sakemura et al., Cancer Immuno. Res., June 21, 2016, pre-print electronic publication). In one instance, such a Tet-CAR can be a payload of an effector submodule controlled by the SRE (e.g., DD) of this disclosure. The dual system provides greater flexibility in turning CAR expression on and off in transduced T cells.

[0279] According to this disclosure, the payload may be a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR. In some embodiments, the payload may be a full CAR construct consisting of an extracellular domain, a hinge and transmembrane domain, and an intracellular signal transduction region. In other embodiments, the payload may be a component of a full CAR construct, including an extracellular targeting portion, a hinge region, a transmembrane domain, an intracellular signal transduction region, one or more co-stimulatory domains, and other additional elements that improve the CAR architecture and functionality, including but not limited to leader sequences, homing elements, and safety switches, or combinations of these components.

[0280] The CAR regulated by the biological circuits and compositions of this disclosure is tunable, thus providing several advantages. A reversible on-off switch mechanism allows for the management of acute toxicity caused by excessive CAR-T cell expansion. Pulsating CAR expression using the SRE of this disclosure can be achieved through circulating ligand levels. Ligand-conferred regulation of the CAR effectively counteracts tumor escape induced by antigen loss, thereby avoiding functional exhaustion due to enhanced signaling caused by chronic antigen exposure and improving the persistence of CAR-expressing cells in vivo.

[0281] In some embodiments, the biological circuits and compositions of this disclosure can be used to downregulate CAR expression to limit intermediate-target tissue toxicity caused by tumor lysis syndrome. Downregulating the CAR expression of this disclosure after antitumor efficacy can prevent (1) off-target tumor toxicity caused by antigen expression in normal tissues, and (2) antigen-independent activation in vivo.

[0282] Extracellular targeting domain / part

[0283] According to this disclosure, the extracellular target portion of a CAR can be any agent that recognizes and binds to a given target molecule (e.g., a neoantigen on tumor cells) with high specificity and affinity. The target portion can be an antibody or its variant that specifically binds to a target molecule on tumor cells, or a peptide aptamer selected from a random sequence pool based on its ability to bind to a target molecule on tumor cells, or a variant or fragment thereof that can bind to a target molecule on tumor cells, or an antigen recognition domain from a natural T-cell receptor (TCR) (e.g., the CD4 extracellular domain that recognizes HIV-infected cells), or an exogenous recognition component, such as a linker cytokine that causes recognition by a target cell carrying a cytokine receptor, or a natural ligand of the receptor.

[0284] In some embodiments, the targeting domain of the CAR can be an Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-stranded variable fragment (scFv), bis-scFv, (scFv)2, microantibody, biantibody, triantibody, tetraantibody, disulfide-stabilized Fv protein (dsFv), monoantibody, nanobody, or an antigen-binding region derived from an antibody that specifically recognizes a target molecule, such as a tumor-specific antigen (TSA). In some embodiments, the targeting portion is scFv. When the scFv domain is expressed on the surface of CAR T cells and subsequently binds to target proteins on cancer cells, it is able to keep the CAR T cells near the cancer cells and trigger T cell activation. scFv can be generated using conventional recombinant DNA techniques, as discussed in this disclosure.

[0285] On the one hand, the extracellular target portion can be an antibody-derived scFv. On the other hand, scFv can specifically bind to the CD19 antigen.

[0286] Intracellular signal transduction domains

[0287] The intracellular domain of a CAR fusion peptide, upon binding to its target molecule, signals to effector immune cells, activating at least one normal effector function of the effector immune cells, including cytolytic activity (e.g., cytokine secretion) or helper cell activity. Therefore, the intracellular domain includes the "intracellular signal transduction domain" of the T cell receptor (TCR).

[0288] In some respects, the entire intracellular signal transduction domain can be used. In other respects, a truncated portion of the intracellular signal transduction domain can be used instead of the complete chain, as long as its transduction effector function is utilized.

[0289] In some embodiments, the intracellular signal transduction domain of this disclosure may contain a signal transduction motif known as an immune receptor tyrosine activation motif (ITAM). Examples of primary cytoplasmic signal transduction sequences containing ITAMs include those derived from TCR CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In one example, the intracellular signal transduction domain is the CD3ζ (CD3ζ) signal transduction domain.

[0290] In some embodiments, the intracellular region of this disclosure further includes one or more co-stimulatory signaling domains that provide additional signals to effector immune cells. These co-stimulatory signaling domains, combined with signaling domains, can further enhance the expansion, activation, memory, persistence, and tumor eradication efficiency of CAR-engineered immune cells (e.g., CAR T cells). In some cases, the co-stimulatory signaling region contains one, two, three, or four cytoplasmic domains of one or more intracellular signaling and / or co-stimulatory molecules. The co-stimulatory signaling domain can be an intracellular / cytoplasmic domain of a co-stimulatory molecule, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), GITR (glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function-associated antigen-1), LIGHT, NKG2C, and B7-H3. In one example, the co-stimulatory signaling domain is derived from the cytoplasmic domain of CD28. In another instance, the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137). In yet another instance, the costimulatory signaling domain may be the intracellular domain of GITR as taught in U.S. Patent No. 9,175,308, the contents of which are incorporated herein by reference in their entirety.

[0291] In some embodiments, the intracellular signal transduction domains disclosed in International Patent Publication WO2014153270 may be used in this disclosure.

[0292] In some embodiments, the chimeric antigen receptor described herein may include a CD3ζ domain modified to regulate CAR activity. The CD3ζ domain may include one or more mutations in the immune receptor tyrosine activation motif (ITAM). In one aspect, tyrosine residues within the ITAM may be mutated, resulting in reduced phosphorylation and restricted downstream signaling. In some embodiments, one or more ITAMs may be deleted from the CD3ζ domain. In another aspect, CD3ζ may include one ITAM. This document may use either the CAR or the CD3ζ domain described by Feucht et al. 2019 (Calibration of CARactivation potential directs alternative T cell fates and therapeutic potency. Nature Medicine 25, 82-88 (2019); the contents of which are incorporated herein by reference in their entirety).

[0293] In some embodiments, the GITR co-stimulatory domain may be used in the CAR described herein. In some embodiments, the GITR domain may induce T cell effector function and activate T cells. In some aspects, the GITR domain described herein may inhibit inhibitory regulatory T cells that block immune responses. In some embodiments, CAR T cells containing the GITR intracellular domain may reduce cytokine production, which may alleviate cytokine release syndrome. Any of the GITR domains is described in International Patent Publication WO2018045034; the contents of which are incorporated herein by reference in their entirety.

[0294] Transmembrane structural domains and hinge regions

[0295] In some embodiments, the CAR of this disclosure may include a transmembrane domain. As used herein, the term "transmembrane domain (TM)" broadly refers to an amino acid sequence of approximately 15 residues in length that crosses the plasma membrane. More preferably, the transmembrane domain comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid residues and spans the plasma membrane. In some embodiments, the transmembrane domain of this disclosure may be derived from a natural or synthetic source. The transmembrane domain of the CAR may be derived from any natural membrane-binding protein or transmembrane protein. For example, the transmembrane region may be derived from the transmembrane domain of the α, β or ζ chain of the T cell receptor, CD3ε, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152 or CD154 (i.e., containing at least its transmembrane domain).

[0296] Alternatively, the transmembrane domains of this disclosure can be synthetic. In some respects, the synthetic sequences may primarily contain hydrophobic residues, such as leucine and valine.

[0297] In some embodiments, the transmembrane domains of this disclosure may be selected from the CD8a transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, and human IgG4 Fc region. As a non-limiting example, the transmembrane domain may be the CTLA-4 transmembrane domain comprising the amino acid sequences of SEQ ID NO. 1-5 of International Patent Publication No. WO2014 / 100385; and the PD-1 transmembrane domain comprising the amino acid sequences of SEQ ID NO. 6-8 of International Patent Publication No. WO2014100385; the contents of each patent are incorporated herein by reference.

[0298] In some embodiments, the CAR of this disclosure may include an optional hinge region (also referred to as a spacer). The hinge sequence is a short amino acid sequence that facilitates the flexibility of the extracellular targeting domain, allowing the target-binding domain to exit the effector cell surface to achieve proper cell / cell contact, target binding, and effector cell activation (Patel et al., Gene Therapy, 1999; 6:412-419). The hinge sequence may be positioned between the targeting portion and the transmembrane domain.

[0299] In some embodiments, the CAR of this disclosure may include one or more linkers between any domains of the CAR. The length of the linker may be between 1 and 30 amino acids.

[0300] In some embodiments, components including the targeting portion, transmembrane domain, and intracellular signal transduction domain of this disclosure can be constructed in a single fusion peptide. The fusion peptide can be the payload of an effector submodule of this disclosure. In some embodiments, the effector submodule may include more than one CAR fusion peptide; for example, the effector submodule may include two, three, or more CARs under the control of a single SRE (e.g., DD).

[0301] In one implementation, the CAR construct comprises CD19scFv (e.g., CAT13.1E10 or FMC63), a CD8α spacer or transmembrane domain, and intracellular domains of 4-1BB and CD3ζ. Compared to constructs with FMC63, these constructs with CAT13.1E10 exhibit increased proliferation, increased cytotoxicity against CD19+ targets, and increased effector-target interactions upon in vitro stimulation.

[0302] In some embodiments, the payload of this disclosure may be any co-stimulatory molecule and / or intracellular domain described herein. In some embodiments, one or more co-stimulatory molecules, each under the control of a different SRE, may be used in this disclosure. First-generation, second-generation, third-generation, fourth-generation, or any other CAR design described herein may also be used to express SRE-regulated co-stimulatory molecules.

[0303] Tandem CAR

[0304] In some embodiments, the CAR disclosed herein may be a tandem chimeric antigen receptor (TanCAR) capable of targeting two, three, four, or more tumor-specific antigens. In some aspects, the CAR is a bispecific TanCAR comprising two targeting domains that recognize two different TSAs on tumor cells. A bispecific CAR may be further defined as comprising an extracellular region containing a targeting domain (e.g., an antigen recognition domain) specific to a first tumor antigen and a targeting domain (e.g., an antigen recognition domain) specific to a second tumor antigen. In other aspects, the CAR is a multispecific TanCAR comprising three or more targeting domains arranged in tandem. The spacing length between the targeting domains in a TanCAR can be between about 5 and about 30 amino acids, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 amino acids.

[0305] Split CAR

[0306] In some embodiments, the components, including the targeting moiety, transmembrane domain, and intracellular signaling domain of this disclosure, can be split into two or more parts, making it dependent on multiple inputs that promote the assembly of a fully functional receptor. In one embodiment, a split synthetic CAR system can be constructed in which the assembly of the activated CAR receptor depends on the binding of a ligand to an SRE (e.g., a small molecule) and the binding of a specific antigen to the targeting moiety. As a non-limiting example, a split CAR consists of two parts assembled in a small molecule-dependent manner; one part of the receptor is characterized by an extracellular antigen-binding domain (e.g., scFv), and the other part has an intracellular signaling domain, such as the CD3ζ intracellular domain.

[0307] In other respects, the splitting portion of the CAR system can be further modified to enhance signaling. In one instance, the second part of the cytoplasmic fragment can be anchored to the plasma membrane by incorporating a transmembrane domain (e.g., the CD8α transmembrane domain) into the construct. Additional extracellular domains, such as those mediating homodimerization, can also be added to the second part of the CAR system. These modifications can enhance receptor export activity, i.e., T cell activation.

[0308] In some respects, the two parts of a split CAR system contain heterodimerization domains that conditionally interact upon binding to a heterodimerization small molecule. As such, the receptor component assembles in the presence of the small molecule to form a complete system, which can then be activated by antigen-binding. Any known heterodimerization component can be incorporated into a split CAR system. Other small molecule-dependent heterodimerization domains can also be used, including but not limited to gibberellin-induced dimerization systems (GID1-GAI), trimethoprim-SLF-induced dimerization of ecDHFR and FKBP (Czlapinski et al., J AmChem Soc., 2008, 130(40): 13186-13187), and ABA (abscisic acid)-induced dimerization of PP2C and PYL domains (Cutler et al., Annu Rev Plant Biol. 2010, 61: 651-679). Using the dual regulation of induced assembly (e.g., ligand-dependent dimerization) and degradation (e.g., CAR degradation induced by destabilizing domains) of the split CAR system can provide greater flexibility in controlling the activity of CAR-modified T cells.

[0309] Switchable CAR

[0310] In some embodiments, the CAR disclosed herein can be a switchable CAR. Juillerat et al. (Juilerat et al., Sci. Rep., 2016, 6:18950; the contents of which are incorporated herein by reference in their entirety) recently reported a controllable CAR that can be transiently activated in response to stimuli (e.g., small molecules). In such CAR designs, the system is integrated directly into the hinge domain, separating the scFv domain of the CAR from the cell membrane domain. Such a system may allow different key functions of the CAR to split or combine, such as activation and co-stimulation within different chains of the receptor complex, thereby mimicking the complexity of the native TCR architecture. This integrated system can switch the interaction between scFv and antigen between on / off states controlled by the absence / presence of a stimulus.

[0311] Reversible CAR

[0312] In other embodiments, the CAR of this disclosure can be a reversible CAR system. In such a CAR structure, a LID domain (ligand-induced degradation) is incorporated into the CAR system. By adding a ligand with a LID domain, CAR can be temporarily downregulated. The combination of LID and DD-mediated regulation provides tunable control over persistently activated CAR T cells, thereby reducing CAR-mediated tissue toxicity.

[0313] Activation conditional CAR

[0314] In some embodiments, the payload of this disclosure may be an activating conditional chimeric antigen receptor, which is expressed only in activated immune cells. CAR expression may be coupled to an activation conditional control region, which refers to one or more nucleic acid sequences that induce, for example, the transcription and / or expression of the CAR under their control. Such an activation conditional control region may be a promoter of a gene upregulated during effector immune cell activation, such as the IL2 promoter or an NFAT binding site. In some embodiments, immune cell activation may be achieved by constitutively expressed CAR (International Publication No. WO2016126608; the contents of which are incorporated herein by reference in their entirety).

[0315] Polynucleotides

[0316] Biological circuit components, including effector modules, their SREs, and payloads, can be nucleic acid-based. The term "nucleic acid," in its broadest sense, includes any compound and / or substance comprising a polymer of nucleotides (e.g., linked nucleosides). These polymers are commonly referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of this disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a [β-D-ribo] configuration, α-LNA having an aL-ribo configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-α-LNA with 2'-amino functionalization) or hybrids thereof.

[0317] In some embodiments, the nucleic acid molecule is messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide encoding a polypeptide of interest that can be translated to produce the polypeptide of interest in vitro, in vivo, in situ, or ex vivo. The polynucleotides disclosed herein can be mRNA or any nucleic acid molecule and may or may not be chemically modified.

[0318] Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5' UTR, a 3' UTR, a 5' cap, and a poly-A tail. Building upon this wild-type modular structure, this disclosure expands the functional range of conventional mRNA molecules by providing a payload to maintain modular organization, but these include one or more structural and / or chemical modifications or alterations that impart useful properties to the polynucleotide, such as maintainability of function. As used herein, a "structural" feature or modification is one in which two or more linked nucleosides are inserted, deleted, repeated, inverted, or randomized within the polynucleotide without significant chemical modification of the nucleosides themselves. Structural modifications are chemical in nature because chemical bonds will necessarily break and reform, thus achieving the structural modification. However, structural modifications will result in different nucleotide sequences. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modification of the polynucleotide.

[0319] In some embodiments, the polynucleotides of this disclosure may include a 5'UTR sequence that functions in translation initiation. The 5'UTR sequence may include several features, such as the Kozak sequence, which is generally known to be involved in ribosomal initiation of gene translation. The Kozak sequence has a common sequence XCCR(A / G)CCAUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), and X is any nucleotide. In one embodiment, the Kozak sequence is ACCGCC. By engineering features typically found in genes heavily expressed in target cells or tissues, the stability and protein production of the polynucleotides of this disclosure can be enhanced.

[0320] Furthermore, polynucleotides containing an internal ribosome entry site (IRES) are provided, which plays an important role in initiating protein synthesis in the absence of a 5' cap structure in the polynucleotide. The IRES can act as a single ribosome binding site, or it can serve as one of multiple binding sites. Polynucleotides of this disclosure containing more than one functional ribosome binding site can encode several peptides or polypeptides that are independently translated by ribosomes to produce bicistronic and / or polycistronic nucleic acid molecules.

[0321] In one embodiment, the polynucleotide of this disclosure may encode a variant polypeptide that has a certain degree of identity with a reference polypeptide sequence. As used herein, a “reference polypeptide sequence” refers to a starting polypeptide sequence. The reference sequence may be a wild-type sequence or any sequence referenced when designing another sequence.

[0322] As is known in the art, the term "identity" refers to the relationship between two or more sequences determined by comparing said sequences. In the art, identity also means the degree of sequence correlation between sequences, as determined by the number of matches between two or more residue (amino acid or nucleic acid) strings. Identity measures the percentage of identical matches between two or more sequences, whose vacancy alignment (if any) is solved by a specific mathematical model or computer program (i.e., an "algorithm"). The identity of related sequences can be readily calculated using known methods. Such methods include, but are not limited to, those described in the following literature: Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, edited by Gribskov, M. and Devereux, J., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0323] In some embodiments, the variant sequence may have the same or similar activity as the reference sequence. Alternatively, the variant may have altered activity (e.g., increased or decreased) relative to the reference sequence. Typically, a variant of a particular polynucleotide or polypeptide disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with a particular reference polynucleotide or polypeptide, as determined by sequence alignment procedures and parameters described herein and known to those skilled in the art. Such alignment tools include those from BLAST suites (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.)

[0324] Chemical modification of polynucleotides

[0325] According to this disclosure, the term "modification" or, where appropriate, "modified" polynucleotide refers to a modification relative to an A, G, U (T in DNA) or C nucleotide.

[0326] The modifications to the polynucleotides disclosed herein may be on the nucleoside bases and / or sugar moieties of the nucleosides comprising the polynucleotides. In some embodiments, multiple modifications are included in the modified nucleic acid or one or more individual nucleosides or nucleotides. For example, modifications to the nucleosides may include one or more modifications to the nucleobases and sugars. Modifications to the polynucleotides of this disclosure may include, for example, any modifications taught in International Publication WO2013052523, the contents of which are incorporated herein by reference in their entirety.

[0327] As used herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof and an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as a “nucleobase”). As used herein, “nucleotide” is defined as a nucleoside containing a phosphate group.

[0328] Modifying nucleotides that can be incorporated into polynucleotides can be applied to the internucleotide link (e.g., the phosphate backbone). In the context of the polynucleotide backbone described herein, the phrases “phosphate ester” and “phosphate diester” are used interchangeably. The backbone phosphate group can be modified by substituting one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides may include completely replacing the unmodified phosphate moiety with another internucleotide link. Examples of modified phosphate groups include, but are not limited to, thiophosphates, phosphonoselenoates, boranophosphates, boranophosphate esters, hydrogen phosphates, aminophosphates, phosphonodiamidite, alkyl or aryl phosphonates, and triphosphates. In dithiophosphates, both non-linked oxygen atoms are substituted with sulfur. Phosphate ester linkers can also be modified by replacing the linking oxygen atoms with nitrogen (bridged aminophosphate), sulfur (bridged thiophosphate), and carbon (bridged methylene phosphonate). Other modifiers that may be used are taught in, for example, International Application WO2013052523, the contents of which are incorporated herein by reference in their entirety.

[0329] Different sugar modifications, nucleotide modifications, and / or internucleotide linkages (e.g., backbone structure) can be present at different positions on a polynucleotide. Those skilled in the art will understand that nucleotide analogs or other modifications can be located at any position on a polynucleotide without significantly reducing its function. Modifications can also be 5' or 3' end modifications. Polynucleotides may contain from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C) or any intermediate percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%). %, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%.

[0330] In some embodiments, the polynucleotide contains a modified pyrimidine or purine. In some embodiments, the pyrimidine or purine in the polynucleotide molecule may be from about 1% to about 100% modified uracil or modified uridine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%). The following percentages of modified pyrimidine or purine are used for substitution: 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%.

[0331] In some embodiments, the polynucleotide may comprise two or more effector module component sequences having a pattern repeated once, twice, or more than three times, such as ABABAB, AABBBAABBAABB, or ABCABCABC, or variations thereof. In these patterns, each letter A, B, or C represents a different effector module component.

[0332] In yet another embodiment, the polynucleotide may comprise two or more effector module component sequences, each component having one or more sequences. As a non-limiting example, the sequences may be in a pattern repeated once, twice, or more than three times in each region, such as ABABAB, AABBBAABBAABB, or ABCABCABC, or variations thereof. As another non-limiting example, the sequences may be in a pattern repeated once, twice, or more than three times throughout the polynucleotide, such as ABABAB, AABBBAABBAABB, or ABCABCABC, or variations thereof. In these patterns, each letter A, B, or C represents a different sequence or component.

[0333] Codon selection

[0334] In some embodiments, one or more codons of the polynucleotide of this disclosure may be replaced with other codons encoding the natural amino acid sequence, thereby regulating the expression of SREs through a process called codon selection. Since the mRNA codon and tRNA anticodon libraries tend to vary with organism, cell type, subcellular location, and time, the codon selection described herein is spatiotemporal (ST) codon selection.

[0335] In some embodiments of this disclosure, certain polynucleotide features may undergo codon optimization. Codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell. This process involves replacing at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons of the natural sequence with the most frequently used codons in the genes of the host cell while maintaining the natural amino acid sequence. Codon usage can be measured using the codon fitness index (CAI), which measures the deviation of the encoding polynucleotide sequence from a reference gene set. Codon usage databases (…) are available. http: / / www.kazusa.or.jp / codon / The codon usage table can be obtained and can be accessed through the EMBOSS CAI program. http: / / emboss.sourceforge.net / The codon optimization method is known in the art and can be used to achieve one or more of several objectives. These objectives include matching codon frequencies in the target and host organisms to ensure correct folding; biasing nucleotide content to alter stability or reduce secondary structure; minimizing tandem repeat codons or base strings that may impair gene construction or expression; customizing transcription and translation control regions; inserting or removing protein signaling sequences; removing / adding post-translational modification sites (e.g., glycosylation sites) in encoded proteins; adding, removing, or reorganizing protein domains; inserting or deleting restriction sites; modifying ribosome binding and degradation sites to modulate translation rates so that individual protein domains fold correctly; or reducing or eliminating problematic secondary structures within polynucleotides. In one embodiment, an optimization algorithm is used for codon optimization of a polynucleotide sequence or portions thereof. Codon options for each amino acid are well known in the art, as are various species tables, to optimize expression in that particular species.

[0336] In some embodiments of this disclosure, certain polynucleotide features may undergo codon optimization. For example, preferred regions for codon optimization may be upstream (5') or downstream (3') of a region encoding a polypeptide. These regions may be incorporated into the polynucleotide before and / or after codon optimization of the payload coding region or open reading frame (ORF).

[0337] After optimization (if necessary), the polynucleotide components are recombined and transformed into vectors, such as, but not limited to, plasmids, viruses, granules, and artificial chromosomes.

[0338] In some implementations, certain regions of the polynucleotide may be preferred for codon selection. For example, preferred regions for codon selection may be upstream (5') or downstream (3') of the region encoding the polypeptide. These regions can be incorporated into the polynucleotide before and / or after codon selection of the payload coding region or open reading frame (ORF).

[0339] The stop codons of the polynucleotides disclosed herein can be modified to include sequences and motifs that alter the expression levels of the SRE, payload, and effector modules of this disclosure. Sequences such as stop codons can be incorporated to induce stop codon readthrough, wherein the stop codon can specify an amino acid, such as selenocysteine ​​or pyrrolidone. In other cases, the stop codon can be skipped entirely, and translation can continue by alternating open reading frames. Stop codon readthrough can be used to modulate the expression of effector module components at a specific ratio (e.g., determined by the stop codon context). Examples of preferred stop codon motifs include UGAN, UAAN, and UAGN, where N is C or U.

[0340] Translational repression occurs during the translation of many viral mRNAs as a means of generating a second protein with an extended C-terminus. In retroviruses, the gag and pol genes are encoded by a single mRNA and separated by the amber stop codon UAG. Translational repression of the amber codon allows the synthesis of the gag pol precursor. Translational repression is mediated by repressive tRNAs capable of recognizing the stop codon and inserting specific amino acids. In some embodiments, the effector modules described herein can bind to the amber stop codon. In bicistronic constructs, such codons can be used to replace or complement the IRES and p2A sequences. Stop codon readthrough can be combined with p2A to achieve low-level expression of downstream genes (e.g., IL12). In some embodiments, the amber stop codon can be combined with tRNA expression or an amino-acyl tRNA synthetase for further control. In one aspect, the payload can be a regulated tRNA synthetase.

[0341] Conjugate

[0342] This disclosure contemplates that the compositions of this disclosure can be complexed, conjugated, or combined with one or more homologous or heterologous molecules. As used herein, the term "homologous molecule" refers to a molecule that is similar to a starting molecule in at least one aspect of its structure or function, while "heterologous molecule" refers to a molecule that is different from a starting molecule in at least one aspect of its structure or function. Thus, structural homologs are molecules that are substantially structurally similar. In some embodiments, such homologs can be completely identical. Functional homologs are molecules that are substantially functionally similar. In some embodiments, such homologs can be completely identical.

[0343] The pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) disclosed herein may comprise conjugates. Such conjugates may include naturally occurring substances or ligands, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Conjugates may also be recombinant or synthetic molecules, such as synthetic polymers, for example synthetic polyamino acids or oligonucleotides (e.g., aptamers). Examples of polyamino acids may include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide mimicry polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0344] In some embodiments, the conjugate may also include a targeting group. As used herein, the term "targeting group" refers to a functional group or portion attached to an agent that facilitates the localization of the agent to a desired region, tissue, cell, and / or protein. Such targeting groups may include, but are not limited to, cell or tissue-targeting agents or groups (e.g., lectins, glycoproteins, lipids, proteins, antibodies that bind to specific cell types such as kidney cells or other cell types). In some embodiments, the targeting group may include...

[0345] Melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, RGD peptides, RGD peptide mimics or aptamers.

[0346] In some implementations, the targeting group can be a protein, such as a glycoprotein, or a peptide, such as a molecule with specific affinity for a coligand, or an antibody, such as an antibody that binds to a specific cell type (e.g., cancer cells, endothelial cells, or osteoblasts). The targeting group may also contain hormones and / or hormone receptors.

[0347] In some embodiments, the targeting group can be any ligand capable of targeting a specific receptor. Examples include, but are not limited to, folate, GalNAc, galactose, mannose, mannose-6-phosphate, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In some embodiments, the targeting group is an aptamer. Such an aptamer can be unmodified or any combination of modifications disclosed herein.

[0348] In other embodiments, the pharmaceutical compositions, biological circuits, biological circuit components, and effector submodules (including their SREs or payloads) of this disclosure may be covalently conjugated to cell-penetrating peptides. In some embodiments, the cell-penetrating peptides may also include a signaling sequence. In some embodiments, the conjugates described herein may be designed to have enhanced stability, enhanced cell transfection, and / or altered biodistribution (e.g., targeting to specific tissues or cell types).

[0349] In some embodiments, the conjugated portion may be added to the pharmaceutical compositions, biological circuits, biological circuit components, effector modules (including their SREs or payloads) of this disclosure, such that they allow detectable markers to attach to the target for clearance. Such detectable markers include, but are not limited to, biotinylate, ubiquitin, fluorescent molecules, human influenza hemagglutinin (HA), c-myc, histidine (His), flag, glutathione S-transferase (GST), V5 (a paramyxovirus with epitope 5 of simian virus), biotin, avidin, streptavidin, horseradish peroxidase (HRP), and digoxigenin.

[0350] In some embodiments, the pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) disclosed herein may be combined with each other or with other molecules in the treatment of diseases and / or conditions.

[0351] Other effects submodule functions

[0352] The effect submodule disclosed herein may further include signal sequences that modulate the distribution of the payload of interest, cutting and / or processing features that facilitate the cutting of the payload from the effect submodule construct, targeting and / or penetration signals that can modulate the cellular localization of the effect submodule, tags and / or one or more connector sequences that connect different components of the effect submodule.

[0353] In some implementations, additional effect submodule features of this disclosure include, but are not limited to, any features taught in Table 7.

[0354] Table 7: Characteristics of the Effect Submodule

[0355]

[0356] signal sequence

[0357] In addition to the SRE and payload region, the effect submodule of this disclosure may also include one or more other features, such as one or more signal sequences.

[0358] Signal sequences (sometimes referred to as signal peptides, targeting signals, target peptides, localization sequences, transport peptides, leader sequences, or leader peptides) direct proteins (e.g., the effector submodules of this disclosure) to their designated cellular and / or extracellular locations. Protein signal sequences play a central role in the targeting and translocation of virtually all secreted proteins and many integrated membrane proteins.

[0359] Signal sequences are short peptides (5-30 amino acids long) located at the N-terminus of most newly synthesized proteins destined for a specific location. These signal sequences can be recognized by signal recognition particles (SRPs) and cleaved by type I and type II signal peptidases. Signal sequences derived from human proteins can be incorporated as regulatory modules into effector modules to direct them to specific cellular and / or extracellular sites. These signal sequences have been experimentally validated and can be cleaved (Zhang Z. and Henzel WJ; "Signal peptide prediction based on analysis of experimentally verified cleavage sites."; Protein Sci. 2004, 13:2819-2824).

[0360] In some implementations, the signal sequence may (though not necessarily) be located at the N-terminus or C-terminus of the effect submodule, and may (though not necessarily) be cut off from the desired effect submodule to produce a “mature” payload.

[0361] In some implementations, the signal sequence used herein excludes methionine at position 1 of the amino acid sequence of the signal sequence. This can be referred to as an M1del mutation.

[0362] In addition to naturally occurring signal sequences, such as those from secretory proteins, the signal sequence can be a variant modified from a known signal sequence of the protein. For example, U.S. Patent Nos. 8,258,102 and 9,133,265 to Sleep disclose modified albumin signal sequences having a secretion signal and additional X1-X2-X3-X4-X5 motifs that can increase protein secretion; U.S. Patent No. 9,279,007 to Do discloses a signal sequence of a modified fragment of human immunoglobulin heavy chain binding protein (Bip) that can enhance protein expression and secretion; and U.S. Patent No. 8,148,494 to Leonhartsberger et al. discloses a signal peptide with a cleavage site that can fuse with a recombinant protein; the contents of these respective patents are incorporated herein by reference in their entirety.

[0363] In some cases, the secreted signal sequence can be a cytokine signal sequence, such as, but not limited to, the IL2 signal sequence or the p40 signal sequence.

[0364] In some cases, signaling sequences that direct the payload of interest to the surface membrane of target cells can be used. Expression of the payload on the target cell surface can be utilized to limit the diffusion of the payload into the non-target in vivo environment, potentially improving payload safety. Additionally, membrane presentation of the payload can allow for physiological and qualitative signaling, as well as payload stabilization and recycling, for a longer half-life. The membrane sequence can be an endogenous signaling sequence of the N-terminal component of the payload of interest. Optionally, it may be desirable to exchange this sequence for a different signaling sequence. The signaling sequence can be selected based on its compatibility with the secretory pathway of the cell type of interest to present the payload on the surface of T cells. In some embodiments, the signaling sequence can be an IgE signaling sequence, a CD8a signaling sequence (also known as a CD8a leader sequence), an IL15Ra signaling sequence (also known as an IL15Ra leader sequence), or a M1del CD8a signaling sequence (also known as a M1del CD8 leader sequence).

[0365] Other variations of signal sequences that may be used in this effect submodule may include those discussed in U.S. Patent Application Publication No. 2007 / 01416666 and PCT Patent Application Publication No. 1993 / 018181, the contents of which are incorporated herein by reference in their entirety.

[0366] Other examples of signal sequences include variations that may be modified signal sequences discussed in U.S. Patent Nos. 8,148,494, 8,258,102, 9,133,265, 9,279,007, and U.S. Patent Application Publication No. 20070141666; and International Patent Application Publication No. WO1993018181, the contents of which are incorporated herein by reference in their entirety.

[0367] In other instances, the signal sequence can be a heterogeneous signal sequence from other organisms such as viruses, yeast, and bacteria, which can direct the effector module to a specific cellular site, such as the cell nucleus (e.g., EP 1209450). Other examples may include aspartic protease (NSP24) signal sequences from Trichoderma, which can enhance the secretion of fusion proteins such as enzymes (e.g., U.S. Patent No. 8,093,016 to Cervin and Kim), bacterial lipoprotein signal sequences (e.g., PCT Application Publication No. WO 199109952 to Lau and Rioux), Escherichia coli enterotoxin II signal peptides (e.g., U.S. Patent No. 6,605,697 to Kwon et al.), Escherichia coli secretion signal sequences (e.g., U.S. Patent Publication No. US2016090404 to Mallley et al.), lipase signal sequences from methyltrophic yeasts (e.g., U.S. Patent No. 8,975,041), and DNase signal peptides from Corynebacterium (e.g., U.S. Patent No. 4,965,197), the contents of which are incorporated herein by reference in their entirety.

[0368] The signal sequence may also include nuclear localization signals (NLS), nuclear export signals (NES), polarized cellular tubule-vesicle localization signals (see, for example, U.S. Patent No. 8,993,742; Cour et al., Nucleic Acids Res. 2003, 31(1):393-396; the contents of which are incorporated herein by reference in their entirety), extracellular localization signals, and signals to subcellular locations (e.g., lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, plasma membrane, and peroxisomes, etc.) (see, for example, U.S. Patent No. 7,396,811; and Negi et al., Database, 2015, 1-7, the contents of which are incorporated herein by reference in their entirety).

[0369] Cutting site

[0370] In some implementations, the effect submodule includes cutting and / or processing features.

[0371] The effector submodule of this disclosure may include at least one protein cleavage signal / site. The protein cleavage signal / site may be located at the N-terminus, the C-terminus, any space between the N-terminus and the C-terminus, such as, but not limited to, midway between the N-terminus and the C-terminus, between the N-terminus and the midway point, and between the midway point and the C-terminus, as well as combinations thereof.

[0372] The effector submodule may include one or more cleavage signals / sites of any protease. The protease may be a serine protease, cysteine ​​protease, endopeptidase, dipeptidase, metalloproteinase, glutamate protease, threonine protease, or aspartic protease. In some respects, the cleavage site can be furin, actinidain, calpain-1, carboxypeptidase A, carboxypeptidase P, carboxypeptidase Y, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, cathepsin B, cathepsin C, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin S, cathepsin V, clostridium protease, chymotrypsin, chymotrypsin, elastase, endopeptidase, enterokinase, factor Xa, formic acid, granzyme B, matrix metallopeptidase-2, matrix metallopeptidase-3, pepsin, proteinase K, SUMO protease, subtilisin, TEV protease, thermophilic protease, thrombin, trypsin, and TAGZyme.

[0373] Label

[0374] In some implementations, the effector submodule includes a protein tag.

[0375] Protein tags can be used to detect and monitor the processes of effector modules. Effector modules may include one or more tags, such as epitope tags (e.g., FLAG or hemagglutinin (HA) tags). A wide range of protein tags can be used in this effector module. These include, but are not limited to, self-labeled peptide tags (e.g., halotag 2 or halotag 7, ACP tags, clip tags, MCP tags, snap tags), epitope tags (e.g., FLAG, HA, His, and Myc), fluorescent tags (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), and their variants), bioluminescent tags (e.g., luciferase and its variants), affinity tags (e.g., maltose-binding protein (MBP) tags, glutathione S-transferase (GST) tags), immunogen affinity tags (e.g., protein A / G, IRS, AU1, AU5, glu-glu, KT3, S-tag, HSV, VSV-G, Xpress, and V5), and other tags (e.g., biotin (small molecule), StrepTag (StrepII), SBP, biotin carboxyl carrier protein). Proteins (BCCP), eXact, CBP, CYD, HPC, CBD containing peptide-chitin binding domains, Trx, NorpA, and NusA.

[0376] In other embodiments, the label may also be selected from those disclosed in U.S. Patent Nos. 8,999,897, 8,357,511, 7,094,568, 5,011,912, 4,851,341, and 4,703,004; U.S. Patent Application Publications Nos. US2013115635 and US2013012687; and International Application Publication No. WO2013091661, the contents of which are incorporated herein by reference in their entirety.

[0377] In some cases, multiple protein tags can be used, which may be the same or different tags; each tag may be located at the same N or C end, while in other cases, these tags may be located at each end.

[0378] connector

[0379] In some implementations, the effect submodule includes a connector.

[0380] In some embodiments, the effector submodules of this disclosure may further include adapter sequences. The adapter region primarily serves as a spacer between two or more peptides within the effector submodule. As used herein, "adapter" or "spacer" refers to a molecule or group of molecules that connects two molecules or two parts of a molecule (e.g., two domains of a recombinant protein).

[0381] In some implementations, as used herein, "linker (L)," "linker domain," "linker region," "linker module," or "peptide linker" refers to an oligomeric or polypeptide region of about 1 to 100 amino acids in length that links any domain / region of an effector submodule together (also referred to as a peptide linker). Peptide linkers can have a length of 1-40 amino acids, 2-30 amino acids, 20-80 amino acids, or 50-100 amino acids. The linker length can also be optimized based on the type of payload used and the crystal structure of the payload. In some cases, a shorter linker length may be preferred. In some aspects, the peptide linker consists of amino acids linked together by peptide bonds, preferably 1-20 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), serine (S), cysteine ​​(C), threonine (T), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), and glutamine (Q). As those skilled in the art will understand, one or more of these amino acids may be glycosylated.

[0382] The adapter sequence can be a natural adapter derived from a multi-domain protein. A natural adapter is a short peptide sequence that separates two different domains or motifs within a protein.

[0383] In some respects, the adapter may be flexible or rigid. In other respects, the adapter may be cleavable or non-cleavable. As used herein, the terms "cleavable adapter domain or region" or "cleavable peptide adapter" are used interchangeably. In some embodiments, the adapter sequence may be enzymatically and / or chemically cleaved.

[0384] The linkers disclosed herein can also be non-peptide linkers. For example, alkyl linkers, such as —NH—(CH2)aC(O)—, where a = 2-20, can be used. These alkyl linkers can be further substituted with any non-sterically hindered group, such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc.

[0385] Targeted or penetrating peptides

[0386] In some implementations, the effector submodule contains targeting and / or penetrating peptides.

[0387] Effector modules can be targeted to desired organs, tissues, or cells using small targeting and / or penetrating peptides that selectively recognize cell surface markers (e.g., receptors, transmembrane proteins, and extracellular matrix molecules). Short peptides (5-50 amino acid residues) synthesized in vitro and naturally occurring peptides or their analogues, variants, or derivatives can be incorporated into effector modules to home them to desired organs, tissues, and cells and / or subcellular locations within cells.

[0388] In some embodiments, targeting sequences and / or penetrating peptides may be included in the effector submodule to drive the effector submodule to a target organ or tissue or cell (e.g., cancer cells). In other embodiments, targeting and / or penetrating peptides may direct the effector submodule to a specific subcellular location within the cell. As a non-limiting example, such targeting sequences and / or penetrating peptides may include those for targeting the effector submodule to a desired region belonging to the central nervous system (e.g., U.S. Patent No. 9,259,432; U.S. Application Publication No. 2015 / 259392); or adipose tissue (e.g., U.S. Patent Nos. 8,067,377 and 8,710,017); or the prostate (e.g., U.S. Patent Publication No. 2016 / 0046668); the contents of which are incorporated herein by reference in their entirety.

[0389] In other embodiments, targeting and / or penetrating peptides can direct the effector submodule to specific subcellular locations within the cell. As a non-limiting example, the effector submodule may include mitochondrial targeting peptides and / or mitochondrial membrane-penetrating peptides to drive the effector submodule to the mitochondria of the cell. See, for example, U.S. Patent Nos. 9,260,495; 9,173,952 and 9,132,198; and U.S. Application Publication No. 2015 / 361140; the contents of which are incorporated herein by reference in their entirety.

[0390] The targeting peptide has any number of amino acids from about 6 to about 30 (including the terminal value). The peptide may have 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. Typically, the targeting peptide may have 25 or fewer amino acids, for example 20 or fewer, such as 15 or fewer.

[0391] Small, naturally occurring, targeting and / or penetrating peptides that recognize specific tissues or cells bind with high affinity to cell surface molecules (e.g., receptors, transmembrane proteins), making them attractive transport moieties. Such peptides can include peptide toxins and their analogues, variants, and derivatives from microorganisms, insects (e.g., scorpions, bees, spiders), animals (e.g., snakes), and plants; as well as secreted peptide hormones, ligands, and signal peptides.

[0392] In some respects, analogues, variants, and derivatives of natural toxins that eliminate their cytotoxic activity can be used as targeting peptides. Exotoxins are toxins secreted by bacteria. Many exotoxins have been shown to bind to specific cellular molecules. For example, enterotoxins are a group of protein toxins produced and secreted by bacterial organisms that bind to the mucosal (epithelial) cells of the intestinal wall. Enterotoxins may include, but are not limited to, heat-stable enterotoxin (ST) from *Escherichia coli*, cholera toxin (CT), heat-labile enterotoxin (LT) from *Escherichia coli*, pertussis toxin (PT) from *Bordetella pertussis*, exotoxin A (ETA) from *Pseudomonas aeruginosa*, staphylococcal enterotoxin, diphtheria toxin from *Corynebacterium diphtheriae*, and enterotoxin NSP4 from the genus *Rotavirus*. Other exotoxins include neurotoxins affecting the nervous system, cardiotoxins affecting the heart, *Pseudomonas* exotoxins, botulinum neurotoxins, Shiga toxins, Shiga-like toxins 1 and 2, *Clostridium difficile* toxins, *Clostridium perfringens* ε toxin, and anthrax toxins.

[0393] Besides toxins, other toxins may include toxins isolated from plants, such as corn RIP, gelonin, American pokeweed antiviral protein, saponin, trichirosin, ricin, and absinthecin; scorpions, such as scorpion venom; spiders, such as PcTxl; conch shells, such as PcTxl; sea anemones, such as sea anemone lysozyme 1; bees, such as bee venom, a group of water-soluble, cationic, amphiphilic 26-amino acid α-helical peptides isolated from bee venom, said bees being honeybees (Apis mellifera) (Western honeybee or European honeybee or giant honeybee), honeybees (Apis florea) (small honeybee or dwarf honeybee), honeybees (Apis dorsata) (giant honeybee), and honeybees (Apis cerana) (Eastern honeybee); snake venom; and bufotoxin, originally isolated from toad skin, which binds to g protein-coupled gastrin-releasing peptide receptors (such as BBR-1 / 2 / 3) in the gastrointestinal tract and brain. See, for example, Suchanek, G. et al., PNAS (1978) 75:701-704; its contents are incorporated herein by reference in their entirety.

[0394] Peptide hormones and other signal peptides transmit important information in intercellular communication, selectively binding to cells that express their receptors with high affinity. In some aspects, effector submodules may include peptide hormones. Such small peptide hormones and signal peptides may include, but are not limited to, adiponectin, fat-derived hormones, muscarinic signal peptide, pharyngeal inhibin, amylin, angiotensin, atrial natriuretic peptide, bomben-like peptide, gastrin, betatrophin, bradykinin, calcitonin, corticotropin-releasing hormone, cosyntrophin, endothelin, glucagon, FGF, FNDC5, follicle-stimulating hormone, gastrin, ghrelin, glucagon and glucagon-like peptide, gonadotropins, granulocyte colony-stimulating factor, growth hormone, growth hormone-releasing hormone, hepcidin, human chorionic gonadotropin, human placental prolactin, incretin, insulin, and insulin-like substances. Similar substances, insulin-like growth factor, leptin, small gastrin, liraglutide, luteinizing hormone, melanocortin, small gastrin, α-melanocyte stimulating hormone, neuropeptide Y, nerve growth factor (NGF), neurotrophic factor-3 / 4, NPH insulin, orexin, obesity suppressor, osteocalcin, pancreatic hormones, parathyroid hormone, peptide hormones, peptide YY, prolactin, prohormone, relaxin, renin, salmon calcitonin, somatostatin (SST), secretin, substance P, sincallitase, teleostleptin, bullfrog antimicrobial peptide (temporin), tesamorelin, thyroid-stimulating hormone, urocortin, vasoactive intestinal peptide (VIP), VGF, and vitellogenin.

[0395] Targeting and penetrating peptides can also be engineered biomimetic peptides and / or chemically modified small peptides. Many peptides with specific motifs and sequences have been identified that target specific cells and tissues with high affinity and selectivity under normal or disease conditions. Synthetic targeting peptides can be 30 amino acids or longer. Targeting peptides typically have at least about 5 amino acids, but may have fewer, such as 4 or 3 amino acids. Typically, targeting peptides have any number of amino acids from about 6 to about 30 (including terminal values). The peptide may have 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. Typically, targeting peptides may have 25 or fewer amino acids, such as 20 or fewer, or even 15 or fewer.

[0396] Chimeric peptides can also be synthesized using fused amino acids derived from naturally occurring proteins and artificial amino acid sequences.

[0397] Stimulate

[0398] The biological circuits of this disclosure are triggered by one or more stimuli. Stimuli include ligands, externally added or endogenous metabolites, the presence or absence of a defined ligand, the presence or action of one or more effector submodules, or concentration gradients of ions or biomolecules, etc.

[0399] ligands

[0400] In some implementations, the stimulus is a ligand. The ligand can be nucleic acid-based, protein-based, lipid-based, organic, inorganic, or any combination of the foregoing.

[0401] In some implementations, the ligand may be, but is not limited to, proteins, peptides, nucleic acids, lipids, lipid derivatives, sterols, steroids, metabolites, metabolite derivatives, and small molecules.

[0402] In some implementations, the stimulant is a small molecule. In some implementations, the small molecule is cell-permeable. In some implementations, the small molecule is FDA-approved, safe, and orally administered.

[0403] In some embodiments, the ligand binds to carbonic anhydrase. In some embodiments, the ligand binds to and inhibits carbonic anhydrase function, and is referred to herein as a carbonic anhydrase inhibitor.

[0404] In some embodiments, the ligand is a small molecule that binds to carbonic anhydrase 2. In one embodiment, the small molecule is a CA2 inhibitor. Examples of CA2 inhibitors include, but are not limited to, celecoxib (also known as celecoxib), vardicoxib, rofecoxib, acetazolamide, methylphenidate, dazolamide, brinzolamide, diclofenac, itazolamide, zonisamide, dansylsulfonamide, and dichlorobenzenesulfonamide.

[0405] In some embodiments, the ligand may contain a portion of a small molecule known to mediate binding to CA2. The ligand may also be modified to reduce off-target binding to carbonic anhydrases other than CA2 and to increase specific binding to CA2.

[0406] Ligands can also be selected through structure-activity relationship (SAR) studies, analyzing the dependence of the activity of known CA2 ligands on their molecular / chemical structure. Stable ligands of this disclosure can be identified using any SAR-related methods known in the art. SAR can be used to improve ligand properties such as specificity, potency, pharmacokinetics, bioavailability, and safety. SAR analysis of known CA2 inhibitors can also be combined with high-resolution X-ray structures of CA2 complexed with ligands.

[0407] In one implementation, the stimulus of this disclosure may be an FDA-approved ligand capable of binding to a specific DD or a target region within the DD.

[0408] In some implementations, ligands that do not affect the activity of immune cells and / or chimeric antigen receptors in the absence of SREs can be preferably selected.

[0409] In some implementations, two or more ligands can be used to stabilize the same stimulus-response element.

[0410] ligand conjugates

[0411] In some embodiments, the ligand may complex or bind to another molecule, such as, but not limited to, another ligand, a protein, peptide, nucleic acid, lipid, lipid derivative, sterol, steroid, metabolite, metabolite derivative, or small molecule. In some embodiments, the ligand stimulus is complexed or bound to one or more other molecules. In some embodiments, the ligand stimulus is complexed or bound to one or more different kinds and / or numbers of other molecules. In some embodiments, the ligand stimulus is a polymer of the same ligand. In some embodiments, the ligand stimulus polymer comprises 2, 3, 4, 5, 6, or more monomers.

[0412] The regulatory role of ligands (such as small molecules that are well-known for binding candidate proteins) in protein responses can be tested. Small molecules have been clinically proven to be safe and possess appropriate pharmacokinetics and distribution. In some implementations, the stimulus is a ligand with a destabilizing domain (DD), for example, a small molecule that binds to the destabilizing domain and stabilizes a POI fused to it.

[0413] In some implementations, the stimulus is a small molecule. In some implementations, the small molecule is cell-permeable.

[0414] Embedded stimuli, signals or other modalities

[0415] In some implementations, the effector submodule of this disclosure may further include one or more microRNAs, microRNA binding sites, promoters, and tunable elements.

[0416] microRNA

[0417] In one embodiment, microRNAs can be used to support the creation of tunable biological circuits. Each aspect or mode of regulation can impart different regulatory characteristics to the effector submodule or biological circuit. For example, destabilizing domains can alter the cleavage site, dimerization properties, or half-life of the payload, and including one or more microRNAs or microRNA binding sites can confer cellular detargeting or transport characteristics. Thus, this disclosure includes biological circuits that are multifactorial in terms of their maintainability. Such biological circuits and effector submodules can be engineered to contain one, two, three, four, or more regulatory features.

[0418] MicroRNAs (or miRNAs) are 19-25 nucleotide long non-coding RNAs that bind to the 3'UTR of nucleic acid molecules and downregulate gene expression by reducing the stability of nucleic acid molecules or inhibiting translation. The polynucleotides disclosed herein may contain one or more microRNA target sequences, microRNA sequences, or microRNA seeds.

[0419] For example, if the polynucleotide is not intended to be delivered to the liver but eventually does reach the liver, the abundant miR-122 (a microRNA) in the liver can inhibit polynucleotide expression, provided that one or more target sites of miR-122 are engineered into the polynucleotide. The introduction of one or more binding sites of different microRNAs can be engineered to further reduce the lifetime, stability, and protein translation of the polynucleotide, thus providing an additional sustainable layer beyond stimulus selection, SRE design, and payload variation.

[0420] As used herein, the term “microRNA site” refers to a microRNA target site or microRNA recognition site, or any nucleotide sequence that binds to or associates with a microRNA. It should be understood that “binding” can follow the traditional Watson-Crick hybridization rule, or it can reflect any stable association between a microRNA and a target sequence located at or near a microRNA site.

[0421] Conversely, for the purposes of the polynucleotides disclosed herein, microRNA binding sites can be engineered to be removed (i.e., removed) from their naturally occurring sequences in order to increase protein expression in specific tissues. For example, the miR-122 binding site can be removed to improve protein expression in the liver.

[0422] Expression can be regulated in a variety of tissues by introducing or removing one or more microRNA binding sites.

[0423] Specifically, microRNAs are known to be differentially expressed in immune cells (also known as hematopoietic cells), such as antigen-presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc. Immune cell-specific microRNAs are involved in immunogenicity, autoimmunity, immune responses to infection, inflammation, and unwanted immune responses following gene therapy and tissue / organ transplantation. Immune cell-specific microRNAs also regulate many aspects of the development, proliferation, differentiation, and apoptosis of hematopoietic cells (immune cells). For example, miR-142 and miR-146 are expressed only in immune cells, particularly in myeloid dendritic cells. Introducing the miR-142 binding site into the 3'-UTR of the disclosed peptide can selectively inhibit gene expression in antigen-presenting cells through miR-142-mediated mRNA degradation, limiting antigen presentation in professional APCs (e.g., dendritic cells), thereby preventing antigen-mediated immune responses following gene delivery (see, Annoni A et al., blood, 2009, 114, 5152-5161, the contents of which are incorporated herein by reference in their entirety).

[0424] In one implementation, microRNA binding sites known to be expressed in immune cells, particularly antigen-presenting cells, can be engineered into polynucleotides to inhibit polynucleotide expression in APCs via microRNA-mediated RNA degradation, thereby attenuating antigen-mediated immune responses, while maintaining polynucleotide expression in non-immune cells that do not express immune cell-specific microRNAs.

[0425] Numerous studies on microRNA expression have been conducted and described in this field to characterize the differential expression of microRNAs in various cancer cells / tissues and other diseases. Some microRNAs are aberrantly overexpressed in certain cancer cells, while others are underexpressed. For example, microRNAs are differentially expressed in the following: cancer cells (WO2008 / 154098, US2013 / 0059015, US2013 / 0042333, WO2011 / 157294); cancer stem cells (US2012 / 0053224); pancreatic cancer and related diseases (US2009 / 0131348, US2011 / 0171646, US2010 / 0286232, US8389210); asthma and... Inflammation (US8415096); Prostate cancer (US2013 / 0053264); Hepatocellular carcinoma (WO2012 / 151212, US2012 / 0329672, WO2008 / 054828, US8252538); Lung cancer cells (WO2011 / 076143, WO2013 / 033640, WO2009 / 070653, US2010 / 0323357); Cutaneous T-cell lymphoma (WO2013) / 011378); colorectal cancer cells (WO2011 / 0281756, WO2011 / 076142); cancer-positive lymph nodes (WO2009 / 100430, US2009 / 0263803); nasopharyngeal carcinoma (EP2112235); chronic obstructive pulmonary disease (US2012 / 0264626, US2013 / 0053263); thyroid cancer (WO2013 / 066678); ovarian cancer cells (US2012 / ... ovarian cancer cells (WO2011 / 011378); ovarian cancer cells (WO2011 / 011378); ovarian cancer cells (WO2011 / 011378); ovarian cancer cells (WO2011 / 011378); ovarian cancer cells (WO2011 / 011378); ovarian cancer cells (WO2011 / 011378); ovarian cancer cells (WO2011 / 011378); ovarian cancer cells (WO2011 / 011378); ovarian cancer cells (WO2011 / 011378); ovarian cancer cells (WO2011 / 0113 0309645, WO2011 / 095623); breast cancer cells (WO2008 / 154098, WO2007 / 081740, US2012 / 0214699); leukemia and lymphoma (WO2008 / 073915, US2009 / 0092974, US2012 / 0316081, US2012 / 0283310, WO2010 / 018563; the contents of each are incorporated herein by reference in their entirety).

[0426] In one implementation, as described herein, microRNAs can be used to support the creation of tunable biological circuits.

[0427] In some implementations, effector modules can be designed to encode (as DNA, RNA, or mRNA) one or more payloads, SREs, and / or regulatory sequences, such as microRNAs or microRNA binding sites. In some implementations, any encoded payload or SRE can be stabilized or destabilized by mutation and then bound to one or more regulatory sequences, thereby creating effector modules or biological circuit systems with dual or multiple regulation.

[0428] Each aspect or mode of regulation can impart different regulatory characteristics to the effector submodule or biological circuit. For example, SRE can represent a destabilizing domain, while mutations in the protein payload can alter its cleavage site, dimerization properties, or half-life, and including one or more microRNAs or microRNA binding sites can confer cellular targeting or transport characteristics. Thus, this disclosure includes biological circuits that are multifactorial in terms of their maintainability.

[0429] Such biological circuits can be engineered to contain one, two, three, four or more regulatory features.

[0430] promoter

[0431] In some embodiments, the compositions disclosed herein contain a promoter.

[0432] As used herein, a promoter is defined as a DNA sequence recognized by the cell's transcription machinery that is required to initiate specific transcription of the polynucleotide sequence disclosed herein. The vector may contain a natural or non-natural promoter operatively linked to the polynucleotide sequence disclosed herein. The selected promoter may be a strong promoter, a weak promoter, a constitutive promoter, an inducible promoter, a tissue-specific promoter, a developmental stage-specific promoter, and / or a biology-specific promoter. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter, such as, but not limited to, SEQ ID NO:210476-210478. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of the polynucleotide sequence operatively linked to it. Another example of a promoter is elongation growth factor-1α (EF-1α), such as, but not limited to, SEQ ID NO:210479-210483. Other constitutive promoters may also be used, including but not limited to simian virus 40 (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV), long terminal repeat (LTR) promoters, avian leukosis virus promoters, Epstein-Barr virus immediate early promoters, Rous sarcoma virus promoters, and human gene promoters, including but not limited to phosphoglycerate kinase (PGK) promoters (non-limiting examples include SEQ ID NO: 210484-210491), actin promoters, myosin promoters, hemoglobin promoters, ubiquitin C (Ubc) promoters, human U6 small nucleoprotein promoters, and creatine kinase promoters. In some cases, inducible promoters may be used, such as, but not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0433] In some implementations, an optimal promoter can be selected based on the ability to achieve the SRE of this disclosure and minimal expression of the payload in the absence of a ligand, and detectable expression in the presence of a ligand.

[0434] The frequency of transcription initiation can be regulated using additional promoter elements, such as enhancers. Such regions can be located 10–100 base pairs upstream or downstream of the initiation site. In some cases, two or more promoter elements can be used to activate transcription cooperatively or independently.

[0435] In some embodiments, the promoter disclosed herein may be the Tet-ON promoter. The binding of the transcriptional regulation Tet system to DD allows for simultaneous control of gene expression and protein stability. By Pedone et al. (2018) doi: https: / / doi.org / 10.1101 / 404699 Any dual Tet ON-DD system described herein may be used (the contents of which are incorporated herein by reference in their entirety).

[0436] Other regulatory features

[0437] In some embodiments, the compositions of this disclosure may optionally include a proteasome adaptor. As used herein, the term "proteasome adaptor" refers to any nucleotide / amino acid sequence that targets an additional payload for degradation. In some aspects, the adaptor directly targets the payload for degradation, thereby circumventing the need for a ubiquitination reaction. The proteasome adaptor may be used in conjunction with a destabilizing domain to reduce the basal expression of the payload. Exemplary proteasome adaptors include the UbL domain of Rad23 or hHR23b, HPV E7, which binds with high affinity to the target protein Rb and the S4 subunit of the proteasome, allowing direct proteasome targeting and thus bypassing the ubiquitination machinery; and a gankyrin repeat sequence that binds to Rb and the S6 subunit of the proteasome.

[0438] Exemplary effect submodule build

[0439] The biological circuits of this disclosure may include at least one effector submodule, which may include at least one SRE derived from CA2 (referred to as "CA2 SRE") operatively connectable to at least one payload of interest. These types of biological circuits and effector submodules are referred to as "CA2 biological circuits" and "CA2 effector submodules." Additionally, CA2 effector submodules may include further features, including but not limited to signal sequences, connectors, spacers, tags, markers, cleavage sites, and IRES. Any exemplary SREs (e.g., DDs), payloads of interest, signal sequences, connectors, spacers, tags, markers, cleavage sites, and IRES taught herein or known in the art can be combined to produce the CA2 effector submodules of this disclosure.

[0440] Payload of interest

[0441] In one implementation, the CA2 effector submodule contains a payload of interest. The payload of interest may be a wild-type peptide, a fragment of a wild-type peptide, and / or contain one or more mutations relative to the wild-type peptide.

[0442] In one implementation, the CA2 effector submodule generates regulated interleukin-15 (IL15).

[0443] In one implementation, the CA2 effector submodule generates a regulated interleukin-15 receptor subunit α (IL15Ra).

[0444] In one embodiment, the CA2 effector submodule generates a regulated fluorescent protein. In one embodiment, at least one payload in the CA2 effector submodule is an mCherry protein. In one embodiment, at least one payload in the CA2 effector submodule is a wild-type sequence of *Rhizopus spp.* luciferase (SEQ ID NO: 210643, encoded by SEQ ID NO: 210644). In one embodiment, at least one payload in the CA2 effector submodule is a *Rhizopus spp.* luciferase sequence. In one embodiment, at least one payload in the CA2 effector submodule is a region of a *Firefly spp.* luciferase s...

Claims

1. A stimulus-response element (SRE) comprising human carbonic anhydrase 2 (CA2), wherein the SRE is: (i) The amino acid sequence of CA2 WT aa 2-260, L156H, SEQ ID NO: 210600, SEQ ID NO: 210600 is shown below: SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFG KAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK, (ii) The amino acid sequences of CA2, L156H, SEQ ID NO: 210602, and SEQ ID NO: 210602 are shown below: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFG or (iii) The amino acid sequence of CA2 WT, aa 2-260, L156H, S172C, F178Y, E186D, SEQ ID NO: 210756, is shown below: SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFG KAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKCADFTNYDPRGLLPDSLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK.

2. The SRE according to claim 1, wherein the SRE is aa2-260, L156H of CA2 WT, and the SRE comprises SEQ ID NO: 210600, the amino acid sequence of which is shown below: SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFG KAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK.

3. The SRE according to claim 1, wherein the SRE is CA2 L156H, and the SRE comprises SEQ ID NO:210602, the amino acid sequence of which is shown below: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDF GKAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK.

4. A CA2 biological circuit system comprising at least one effector submodule, said at least one effector submodule comprising: (a) A stimulus-response element SRE comprising human carbonic anhydrase 2 CA2, wherein the SRE is (i) The amino acid sequence of CA2 WT aa 2-260, L156H, SEQ ID NO: 210600, SEQ ID NO: 210600 is shown below: SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFG KAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK, (ii) The amino acid sequences of CA2, L156H, SEQ ID NO: 210602, and SEQ ID NO: 210602 are shown below: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFG or (iii) The amino acid sequence of CA2 WT, aa 2-260, L156H, S172C, F178Y, E186D, SEQ ID NO: 210756, is shown below: SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFG and (b) At least one payload, wherein the at least one payload is connected to the SRE, and wherein the at least one payload is a protein or nucleic acid whose function is to be altered.

5. The CA2 biological loop system according to claim 4, wherein the SRE is aa 2-260, L156H of CA2 WT, and the SRE contains SEQ ID NO: 210600, the amino acid sequence of which is shown below: SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFG KAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK.

6. The CA2 biological loop system according to claim 4, wherein the SRE is CA2 L156H, and the SRE contains SEQ ID NO: 210602, the amino acid sequence of which is shown below: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDF GKAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK.

7. The CA2 biological loop system according to claim 4, wherein the payload is a cytokine or CAR.

8. The CA2 biological circuit system according to claim 4, wherein the payload is IL-12, CD40L or CD19CAR.

9. The CA2 biological circuit system according to claim 8, wherein the IL-12 is membrane-bound IL-12.

10. The CA2 biological circuit system according to claim 4, wherein the effector submodule comprises the amino acid sequence SEQ ID NO: 210927, which is shown below: .

11. The CA2 biological circuit system according to claim 4, wherein the effector submodule comprises the amino acid sequence SEQ ID NO: 210694, SEQ ID NO: 211114 or SEQ ID NO: 210874. The amino acid sequence SEQ ID NO: 210694 is shown below: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK; The amino acid sequence SEQ ID NO: 211114 is as follows: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKCADFTNYDPRGLLPDSLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK; The amino acid sequence SEQ ID NO: 210874 is as follows: MDMRVPAQLLGLLLLWLSGARCNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTMKCFLLELQVISLESGDASIHDTVENLIILANNSLSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSSKQEHFPDNLPSWAITLISVNGIFVICCLTYCFAPRCRERRGSSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNNGEGEPEELMVDNWRPAQPLKNRQIKASFK.

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