Human carbonic anhydrase 2 compositions and methods for tunable regulation

Abstract

To provide human carbonic anhydrase 2 compositions and methods for tunable regulation.SOLUTION: The present disclosure provides regulatable biocircuit systems. Such systems provide modular and tunable protein expression systems in support of the discovery and development of therapeutic modalities. In particular, the present application is directed to fusion proteins comprising a fragment of human carbonic anhydrase 2 and a chimeric antigen receptor (CAR). The activity of the destabilizing domain can be regulated by externally administered agents.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of 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 in their entireties.

[0002] Sequence Listing Reference This application contains "redundant" sequence listings submitted on CD-Rs in lieu of printed paper copies, which are incorporated herein by reference in their entireties. Said CD-Rs, created and recorded on March 6, 2020, are designated "CRF," "Copy 1," and "Copy 2," respectively. Labeled "Copy 3" and "Copy 4," each contains only one identical 699,270,904 byte (measured in MS-WINDOWS) file named 268052-462540_SL.txt. The machine-readable format of each CD-R is IBM-PC, and the operating system on each compact disc is MS-Windows.

[0003] Field The present disclosure relates to a destabilizing domain (DD) derived from human carbonic anhydrase 2 (CA2) that can modulate protein stability for at least one payload, as well as compositions and methods of use thereof. Provided herein are polypeptides of the CA2 biological circuit system, CA2 effector modules, and stimulus response elements (SREs), polynucleotides encoding the same, and vectors and cells containing the polypeptides and / or polynucleotides for use in cancer immunotherapy. [Background technology]

[0004] background Gene and cell therapy have revolutionized drug therapy, offering new prospects for treating previously intractable disease states. However, most current technologies do not allow for control of the timing or level of target protein induction. This makes many potential gene and cell therapy applications difficult or impossible to deploy safely and effectively.

[0005] Inadequate exogenous and / or endogenous gene control is a significant problem in many gene and cell therapy settings, and this lack of tunability makes it difficult to safely express proteins with narrow or unstable therapeutic windows or those requiring more regulated or transient expression.

[0006] One approach to controlled protein expression or function is the use of destabilizing domains (DDs). Destabilizing domains are small protein domains that can be added to target proteins of interest. In the absence of a DD-binding ligand, the DD destabilizes the bound target protein, causing it to be rapidly degraded by the cellular ubiquitin-proteasome system. However, when a specific small molecule DD-binding ligand binds to the DD, the bound target protein is stabilized and protein function is achieved.

[0007] DD technology forms the basis of a new class of cell and gene therapies that can provide tunable and temporal control of gene expression and function, expanding the universe of protein therapeutics that can be safely and effectively incorporated into cell and gene therapy modalities. Summary of the Invention

[0008] The present disclosure provides a novel protein domain derived from human carbonic anhydrase 2 (CA2) that exhibits small molecule-dependent stability. Such a protein domain is termed a destabilizing domain (DD). In the absence of its binding ligand, the DD destabilizes a payload (e.g., a protein of interest (POI)) fused to the DD, causing its degradation, whereas in the presence of its binding ligand, the fused DD and payload can be stabilized, and the stability is dose-dependent.

[0009] In a first aspect, the present disclosure provides a composition comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region or the entirety 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.

[0010] In a second aspect, the disclosure provides compositions comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region or the entirety 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.

[0011] In a third aspect of the present disclosure, a composition is provided comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE comprises a destabilization domain (DD), wherein the DD comprises a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717), and further comprises a W208S mutation at position 208 of SEQ ID NO: 11717 (W208).

[0012] In a fourth aspect of the present disclosure, a composition is provided comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE comprises a destabilization domain (DD), wherein the DD comprises a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717), and further comprises an I59N mutation at position 59 of amino acid (I59) in SEQ ID NO: 11717.

[0013] In a fifth aspect of the present disclosure, a composition is provided comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717), and further comprises an L156H mutation at position 156 of SEQ ID NO: 11717 (L156). In embodiments related to the fifth aspect, DD further comprises (i) a W4Y mutation of amino acid (W4) at position 4 of SEQ ID NO: 11717; (ii) a F225L mutation of amino acid (F225) at position 225 of SEQ ID NO: 11717; (iii) a deletion of amino acids at positions 257-260 of SEQ ID NO: 11717; (iv) a deletion of amino acids at positions 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.

[0014] In a sixth aspect, the present disclosure provides compositions comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD) comprising a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717) and further comprising 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.

[0015] In a seventh aspect, a composition is provided comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE comprises a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717), and further comprises a destabilizing domain (DD) comprising an N or F substitution at amino acid position 56 (S56) of SEQ ID NO: 11717.

[0016] In an eighth aspect, a composition is provided that includes an effector module. The effector module includes a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE includes a destabilizing domain (DD) that includes a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717) and further includes one or more substitutions relative to SEQ ID NO: 11717, wherein at least one substitution is a D or N substitution at amino acid position 63 (G63) of SEQ ID NO: 11717, and the one or more substitutions correspond to G63D; G63D and M240L; G63D, E69V and N231I; or T55K, G63N and Q248N.

[0017] In a ninth aspect, a composition is provided comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD) comprising a region or all of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717) and further comprising two or more substitutions relative to SEQ ID NO: 11717, wherein one of the two or more substitutions is a substitution of L or K 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.

[0018] In a tenth aspect, a composition is provided comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD) comprising a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717) and further comprising 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 at amino acid position 241 (V241) of SEQ ID NO: 11717, or (ii) a substitution of F or L at amino acid position 249 (P249) of SEQ ID NO: 11717, and 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.

[0019] In an eleventh aspect, the present disclosure provides a composition comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload operably linked to the SRE. The SRE comprises a destabilizing domain (DD) comprising a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717) and further comprising one or more substitutions relative to SEQ ID NO: 11717 selected from Y51T, L183S, Y193I, L197P, and combinations of V134F and L228F.

[0020] In embodiments relating to all of the exemplified aspects provided above, the SRE is responsive to one or more irritants. In various embodiments, the irritant is a small molecule selected from acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclophenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. Further, the compositions described and exemplified herein include a DD, wherein the DD has at least one mutation or substitution in the DD that destabilizes the DD and at least one payload in the absence of the irritant, and the DD and payload are stabilized in the presence of the irritant.

[0021] In a twelfth aspect, the present disclosure provides a biological circuit system comprising any one or more of the compositions according to aspects 1 to 10; a pharmaceutical composition comprising the composition according to aspects 1 to 10 and a pharmaceutically acceptable excipient; a polynucleotide encoding the composition according to aspects 1 to 10; a vector comprising the polynucleotide encoding the composition according to aspects 1 to 10; a cell comprising the polynucleotide encoding the composition according to aspects 1 to 10; and a pharmaceutical composition comprising the cell comprising the polynucleotide encoding the composition according to aspects 1 to 10 and a pharmaceutically acceptable excipient.

[0022] In a thirteenth aspect, the present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising: (a) administering to the subject a therapeutically effective amount of the cells of aspect 11, wherein the cells comprise a payload that treats the disease; and (b) administering to the subject a therapeutically effective amount of a stimuli, wherein the SRE is responsive to the stimuli, wherein expression of the payload is modulated in response to the stimuli, thereby treating the disease.

[0023] In some embodiments, the present disclosure provides a stimulus response element (SRE) that can include a destabilization domain (DD) derived in whole or in part from human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717). In one embodiment, the DD can include the entire CA2 (SEQ ID NO: 11717).

[0024] In some embodiments, the disclosure provides a method for the detection of a nucleotide sequence comprising a region or all of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717) and including the amino acids 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, D1 61V, 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, E204A, E204D, E204G, E204N, E213*, E2 13G, E213N, E220K, E220R, E220S, E233D, E233G, E233R, E235*, E235G, E235 N, E237K, E237R, E238*, E238N, E238R, E26S, E69D, E69K, E69S, F130L, F146 V, F175I, F175L, F175S, F178L, F178S, F20L, F20S, F225I, F225L, F225S, F22 5Y, F230I, F230L, F230S, F259L, F259S, F66S, F70I, F70L, F95Y, G102D, G10 4R, G104V, G128R, G12D, G12E, G131E, G131R, G131W, G139D, G144D, G144V, G1 50A, G150S, G150W, G155A, G155C, G155D, G155S, G170A, G170D, G182A, G182 W, 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、The present invention provides a DD that further comprises a mutation relative to SEQ ID NO: 11717 selected from Y190*, Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, and S29A.

[0025] In some embodiments, the disclosure provides a DD that includes a region or all of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717) and further includes a mutation relative to SEQ ID NO: 11717 selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0026] In some embodiments, the present disclosure provides a DD that includes a region or all of human carbonic anhydrase 2 (CA2; SEQ ID NO:11717) and further includes two or more mutations relative to SEQ ID NO:11717. In some embodiments, DD is selected from the group consisting of CA2(aa2-260 of WT, R27L, H122Y), CA2(aa2-260 of WT, T87I, H122Y), CA2(aa2-260 of WT, H122Y, N252D), CA2(aa2-260 of WT, D72F, V241F), CA2(aa2-260 of WT, V241F, P249L), CA2(aa2-260 of WT, D72F, P249L), CA2(aa2-260 of WT, D71L, L250R), CA2(aa2-260 of WT, D7 2F, P249F), CA2 (aa2-260 of WT, T55K, G63N, Q248N), CA2 (aa2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa2-260 of WT, W4Y, L156H), CA2 (aa2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa2 of WT CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156H), CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210703). Sequence number 210503), CA2 (aa 2 to 260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2 to 260 of WT, L156H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, D71F, N231F) (SEQ ID NO: 210505), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, D71K, P249H) (SEQ ID NO: 210516),CA2(aa2-260 of WT, D72F, P249H) (SEQ ID NO: 210518), CA2(aa2-260 of WT, Q53N, N61Y) (SEQ ID NO: 210521), CA2(aa2-260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2(aa2-260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2(aa2-260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2(aa2-260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2(aa2-260 of WT, Y193L, K260L) (SEQ ID NO: 210540), CA2 (aa 2-260 of WT, D71F, V241F, P249L) (SEQ ID NO: 210544), CA2 (aa 2-260 of WT, L147F, Q248F) (SEQ ID NO: 210548), CA2 (aa 2-260 of WT, D52I, S258P) (SEQ ID NO: 210550), CA2 (aa 2-260 of WT, D72S, T192N) (SEQ ID NO: 210552), CA2 (aa 2-260 of WT, D179E, T192I) (SEQ ID NO: 210554), CA2 (aa 2-260 of WT, S56N, Q1 03K) (SEQ ID NO: 210558), CA2 (aa 2-260 of WT, D71Y, Q248L) (SEQ ID NO: 210560), CA2 (aa 2-260 of WT, S73N, R89F) (SEQ ID NO: 210562), CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F) (SEQ ID NO: 210564), CA2 (aa 2-260 of WT, D72F, P249I) (SEQ ID NO: 210568), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210572), CA2 (aa 2-260 of WT, A54X, S CA2 (aa 2 to 260 of WT, Y193V, K260F) (SEQ ID NO: 210576), CA2 (aa 2 to 260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2 to 260 of WT, V134F, L228F) (SEQ ID NO: 210580), CA2 (aa 2 to 260 of WT, D71G, N231K) (SEQ ID NO: 210582), CA2 (aa 2 to 260 of WT, S56F, D71S) (SEQ ID NO: 210584), CA2 (aa 2 to 260 of WT, D52L, G128R,CA2 (aa 2 to 260 of WT, S73X, R89X) (SEQ ID NO: 210588), CA2 (aa 2 to 260 of WT, Y51X, D72X, V241X, P249X) (SEQ ID NO: 210592), CA2 (aa 2 to 260 of WT, D72I, W97C) (SEQ ID NO: 210594), CA2 (aa 2 to 260 of WT, D71K, T192F, N231F) (SEQ ID NO: 210596), CA2 (aa 2 to 260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO: 21069 8), CA2 (aa 2 to 260 of WT, F70I, F146V) (SEQ ID NO: 210700), CA2 (aa 2 to 260 of WT, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO: 210704), CA2 (aa 2 to 260 of WT, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO: 210706), CA2 (aa 2 to 260 of WT, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO: 210708), CA2 (aa 2 to 260 of WT, G63D, W123R, E220K) (SEQ ID NO: 2 10712), CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO: 210714), CA2 (aa 2-260 of WT, I59N, G102R, A173T) (SEQ ID NO: 210716), CA2 (aa 2-260 of WT, L79F, P180S) (SEQ ID NO: 210718), CA2 (aa 2-260 of WT, A77P, G102R, D138N) (SEQ ID NO: 210724), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N23 1I) (SEQ ID NO: 210726), CA2 (aa 2 to 260 of WT, T199N, L202P, L228F) (SEQ ID NO: 210728), CA2 (aa 2 to 260 of WT, K9N, H122Y, T168K) (SEQ ID NO: 210730), CA2 (aa 2 to 260 of WT, Q53H, L90V, Q92H, G131E) (SEQ ID NO: 210732), CA2 (aa 2 to 260 of WT, L44M, L47V, N62K, E69D) (SEQ ID NO: 210734), CA2 (aa 2 to 260 of WT, D75V, K169N, F259L) (SEQ ID NO: 210738),CA2 (aa 2-260 of WT, T207S, V222A, N231D) (SEQ ID NO: 210740), CA2 (aa 2-260 of WT, I59F, V206M, G232R) (SEQ ID NO: 210742), CA2 (aa 2-260 of WT, P13A, A133T) (SEQ ID NO: 210744), CA2 (aa 2-260 of WT, I59N, R89I) (SEQ ID NO: 210750), CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, CA2 (aa 2-260 of WT, G12R, H15Y, D19V) (SEQ ID NO: 210754), CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) (SEQ ID NO: 210758), and / or CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210851 or 210847).

[0027] In some embodiments, DD is CA2(aa2-260 of WT, R27L, H122Y), CA2(aa2-260 of WT, T87I, H122Y), CA2(aa2-260 of WT, H122Y, N252D), CA2(aa2-260 of WT, D72F, V241F), CA2(aa2-260 of WT, V241F, P249L), CA2(aa2-260 of WT, D72F, P249L), CA2(aa2-260 of WT, D71L, L250R), CA2(aa2-260 of WT, D72F, P249F), CA2(aa2-260 of WT, , T55K, G63N, Q248N), CA2 (aa2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa2-260 of WT, W4Y, L156H), CA2 (aa2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa2-260 of WT, L156H, F225L), CA2 (aa2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2-260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2-260 of WT, L156 H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2 (aa 2 to 260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2 to 260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2 to 260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2 to 260 of WT, S73N,R89F) (SEQ ID NO: 210562), CA2 (aa 2-260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2-260 of WT, V134F, L228F) (SEQ ID NO: 210580), and / or CA2 (aa 2-260 of WT, S56F, D71S) (SEQ ID NO: 210584).

[0028] The SREs described herein may be responsive to one or more irritants. Such irritants may be small molecules, such as, but not limited to, acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide. In embodiments, the small molecule may be acetazolamide. In some aspects, the irritant may be celecoxib.

[0029] The present disclosure provides a CA2 biological circuit system that includes an effector module. Such an effector module may include a stimulus-response element (SRE). Provided herein is a biological circuit having an SRE that includes a region or the entirety of human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717).

[0030] The payload included in the CA2 biological circuit system can be a therapeutic agent, a natural protein, a fusion polypeptide, an antibody, or a variant or fragment thereof.

[0031] In some embodiments, the payload can be a therapeutic agent, hi some embodiments, the therapeutic agent can be a cytokine, a chimeric antigen receptor, a cytokine, or a cytokine-cytokine receptor fusion protein.

[0032] The CA2 biological circuit system can be responsive to one or more stimuli. In one aspect, the stimuli can be small molecules, such as, but not limited to, acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. In one embodiment, the small molecule can be acetazolamide. In another aspect, the small molecule can be celecoxib.

[0033] Also provided herein are polynucleotides encoding the SREs, biological circuit systems, and / or compositions described herein, as well as vectors containing the polynucleotides. The present disclosure also describes pharmaceutical compositions comprising the CA2 biological circuits and / or compositions described herein and a pharmaceutically acceptable excipient.

[0034] The foregoing and other objects, features, and advantages will become apparent from the following description of specific embodiments of the present disclosure, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the present disclosure. [Brief explanation of the drawings]

[0035] [Figure 1] 1 shows ligand-dependent regulation of the CA2 chimeric antigen receptor. [Figure 2] Response of the CA2 biological circuit to various doses of acetazolamide is shown. [Figure 3] 1 shows the acetazolamide response for OT-002347 (labeled as CA2-070) and OT-001978 (labeled as CA2-026). DETAILED DESCRIPTION OF THE INVENTION

[0036] The details of one or more embodiments of the present disclosure are set forth below in the accompanying specification. Although any materials and methods similar or equivalent to those described herein can be used in practicing or testing the present disclosure, the preferred materials and methods are now described. Other features, objects, and advantages of the present disclosure will become apparent from the specification. In the specification, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates 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 belongs. In case of conflict, the present specification will control.

[0037] I. Composition Biological circuits or biological circuit systems According to the present disclosure, biological circuit systems are provided that include, at their core, at least one effector module. Such effector module(s) are independently associated with or integrated with one or more stimulus response elements (SREs). Typically, the stimulus response element (SRE) can be operably linked to a payload, which can be any protein of interest (POI) (e.g., an immunotherapeutic agent), to form the effector module. When activated by a specific stimulus, such as a small molecule, the SRE can generate a signal or outcome to either up- or down-regulate the transcription and / or protein levels of the linked payload by sustaining a stabilizing or destabilizing signal or any other type of regulation. Many detailed descriptions of biological circuit systems are taught in commonly owned U.S. Provisional Patent Application Nos. 62 / 320,864, filed April 11, 2016, 62 / 466,596, filed March 3, 2017, and International Publication No. WO 2017 / 180587, the contents of each of which are incorporated herein by reference in their entirety. In accordance with the present disclosure, biological circuit systems, effector modules, SREs, and components are provided that modulate the expression levels and activity of any agent used for immunotherapy.

[0038] As used herein, a "biological circuit" or "biological circuit system" is defined as a circuit within or useful in a biological system that includes a stimuli and at least one effector module responsive to the stimuli, and the response to the stimuli produces at least one signal or outcome 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 is also understood that a biological circuit may be an artificial circuit that uses a stimuli or effector module taught by the present disclosure to produce a signal or outcome in a cell-free environment, such as a diagnostic, reporter system, device, assay, or kit. The artificial circuit may be associated with one or more electrical, magnetic, or radioactive components or moieties.

[0039] According to the present disclosure, the biological circuit system may 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 which may act as a signal for any other of these biological circuit systems.

[0040] Effector Module The biological circuits of the present disclosure include at least one effector module. As used herein, an "effector module" is a single- 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., proteins of interest (POIs)).

[0041] Effector modules can be designed to include one or more additional features, including the presence or absence of one or more payloads, one or more SREs, one or more cleavage sites, one or more signal sequences, and one or more linkers. Exemplary effector module embodiments of the present disclosure are shown in Figures 2-6 in International Publication No. WO 2017 / 180587 (the contents of which are incorporated herein by reference in their entirety). Biological circuits and components utilizing such effector molecules are provided in Figures 7-12 in International Publication No. WO 2017 / 180587 (the contents of which are incorporated herein by reference in their entirety).

[0042] As shown in Figure 2 of International Publication No. WO 2017 / 180587, a representative embodiment of an effector module containing one payload, i.e., one immunotherapeutic agent, is shown. The components of the effector module can be positioned or arranged in various configurations, either without (A-F) or with (G-Z and AA-DD) cleavage sites. Optional linkers can be inserted between the components of the effector module.

[0043] Figures 3-6 in International Publication No. WO2017 / 180587 show exemplary effector module embodiments containing two payloads, i.e., two immunotherapeutic agents. In some embodiments, more than two immunotherapeutic agents (payloads) can be included in the effector module under the control of the same SRE (e.g., the same DD). The two or more agents can be directly linked to each other or can be separated. The SRE can be located at the N-terminus of the construct, the C-terminus of the construct, or an internal location.

[0044] In some embodiments, the biological circuits of the present disclosure can be modified to reduce their immunogenicity. Immunogenicity is the result of a complex series of reactions to substances recognized as foreign, including the generation of neutralizing and non-neutralizing antibodies, immune complex formation, complement activation, mast cell activation, inflammation, hypersensitivity reactions, and anaphylaxis. Several factors, including but not limited to the protein sequence, route and frequency of administration, and patient population, can contribute to protein immunogenicity. In preferred embodiments, protein modifications can be used to reduce the immunogenicity of the compositions of the present disclosure. In some embodiments, modifications to reduce immunogenicity can include modifications that reduce binding of processed peptides derived from the parent sequence to MHC proteins. For example, amino acid modifications can be made to eliminate or minimize the number of immune epitopes predicted to bind with high affinity to any common MHC allele. Several methods for identifying MHC-binding epitopes in known protein sequences are known in the art and can be used to score epitopes in the compositions of the present disclosure. Such methods are disclosed in U.S. Patent Publication Nos. US20020119492, US20040230380, and US20060148009, the contents of each of which are incorporated by reference in their entirety.

[0045] 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 of the foregoing.

[0046] Effector modules (including their SREs and payloads) can individually, collectively, or independently comprise peptides, polypeptides, or proteins. At the protein level, such payloads can be any natural or artificial peptide or polypeptide, or fragments thereof. The natural peptide or polypeptide component of the payload can be derived from any known protein of any species.

[0047] Effector modules can be designed to work in groups of 1, 2, 3, 4 or more modules. When multiple effector modules are utilized in a biological circuit, it is known as the effector module system of that biological circuit.

[0048] Stimulus-Response Element (SRE) As used herein, a "stimulus response element" (SRE) is a component of an effector module that is conjugated, bound, linked, or associated with one or more payloads and, in some instances, is responsible for the effector module's responsiveness to one or more stimuli. As used herein, the "responsiveness" characteristic of an SRE to a stimuli may be characterized by a covalent or non-covalent interaction, a direct or indirect association, or a structural or chemical reaction with the stimuli. Furthermore, the response of any SRE to a stimuli may be a matter of degree or type. The response may be a partial response. The response may be a reversible response. The response may ultimately lead to a regulated signal or output. Such an output signal may be of a relative nature to the stimuli, for example, a regulatory effect between 1% and 100%, or the production of a fold increase or decrease, such as a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more. In some embodiments, the SRE is a polypeptide fused to a polypeptide payload.

[0049] In some embodiments, the present disclosure provides methods for modulating protein expression, function, or levels. In some aspects, modulation of protein expression, function, or levels is at least about 20%, e.g., 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%, 30-100%, This refers to modulation of expression, function or levels by 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%.

[0050] Destabilizing domain A destabilizing domain (DD) is a small protein domain that can be added to a target protein of interest. The term destabilizing domain (DD) is interchangeable with the term drug-responsive domain (DRD). DDs destabilize the bound target protein in the absence of a DD-binding ligand, causing the protein to be rapidly degraded by the cellular ubiquitin-proteasome system (Stankunas, K., et al., Mol. Cell, 2003, 12:1615-1624; Banaszynski, et al., Cell; 2006, 126(5):995-1004, reviewed in Banaszynski, L.A., and Wandless, T.J. 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 a rapid, non-perturbative switch from a stable protein to an unstable substrate for degradation. Moreover, its dependence on the concentration of the ligand further provides tunable control of the degradation rate.

[0051] In one embodiment, the SRE is a destabilizing domain (DD). The presence, absence, or amount of a small molecule ligand that binds to or interacts with the DD can modulate the stability and consequently the function of the payload(s) upon such binding or interaction. Depending on the degree of binding and / or interaction, the altered function of the payload can vary, thereby providing "tuning" of payload function.

[0052] In some embodiments, desirable properties of a DD may include, but are not limited to, low protein levels in the absence of the DD's ligand (e.g., low basal stability), a wide kinetic range, robust and predictable dose-response behavior, and rapid kinetics of degradation. A DD that binds to a desired ligand but not to endogenous molecules may be preferred.

[0053] In some embodiments, the DDs of the present disclosure can be developed from known proteins, referred to herein as parent proteins. In some embodiments, the CA2 destabilizing domains described herein or known in the art can be used as SREs in the biological circuit systems of the present disclosure in association with any of the payloads taught herein (e.g., proteins of interest or immunotherapeutic agents).

[0054] Regions or portions or domains of wild-type proteins (e.g., CA2) can be utilized in whole or in part as SREs / DDs, and they can be combined or rearranged to create new peptides, proteins, regions or domains, any of which can be used as SREs / DDs or as starting points for the design of additional SREs and / or DDs.

[0055] In one embodiment, the SRE is derived from a region of a parent protein (eg, CA2) or a variant protein. The parent protein regions are 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, 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, 7, 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, 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, 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, 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, 61, 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, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 4 408, 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 than 450 amino acids. A region of a parent protein 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 in length. As a non-limiting example, a region of a parent protein can be 250-270 amino acids in length. As a non-limiting example, a region of a parent protein can be 225-250 amino acids in length. As a non-limiting example,The region of the parent protein can be 225 to 260 amino acids in length.

[0056] In one embodiment, the SRE is derived from a parent protein (e.g., CA2) or a variant protein and comprises a region of the parent protein. The SRE may comprise 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%, 15-20%, 20-25%, 25-30%, 30-35%, or 40-45% of the parent protein or variant protein. 5%, 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%, 6 It may comprise a region of the parent protein that is 0-100%, 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%.

[0057] In one embodiment, the SRE is derived from a parent protein (e.g., CA2) or a variant protein and is 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%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. 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% 0%, 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% identity.

[0058] Candidate destabilization domain sequences identified from the protein domain of the parent protein (as a template) can be mutated to generate a library of variants based on the candidate template domain sequence. Mutagenesis strategies used to generate a DD library can include, for example, site-directed mutagenesis using structure-guided information; or random mutagenesis using, for example, error-prone PCR, or a combination of both. In some embodiments, the destabilization domains identified using random mutagenesis can be used to identify structural features of the candidate DD that may be required for destabilization, which can then be used to further generate a library of variants using site-directed mutagenesis.

[0059] In some embodiments, DD mutant libraries can be screened for mutations with altered, preferably higher, binding affinity to a ligand compared to the wild-type protein. DD libraries can also be screened using two or more ligands to preferentially select for DD mutations that are stabilized by some ligands but not others. DD mutations that preferentially bind to a ligand compared to naturally occurring proteins can also be selected. Such methods can be used to optimize the ligand selectivity and ligand binding affinity of DDs. Such approaches can also be used to minimize adverse effects caused by off-target ligand binding.

[0060] In some embodiments, suitable DDs can be identified by screening a mutant library using barcodes. Such methods can be used to detect, identify, and quantify individual mutant clones within a heterogeneous mutant library. Each DD mutant within the library can have a distinct barcode sequence. In other examples, polynucleotides can also have barcode sequences that differ by 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleobases. Each DD mutant within the library can also contain multiple barcode sequences. When multiple barcodes are used, each barcode can be used so that it is unique relative to any other barcode. Alternatively, each barcode used may not be unique, but the combination of barcodes used can generate a unique sequence that can be independently tracked. The barcode sequence can be placed upstream of the SRE, downstream of the SRE, or, in some examples, within the SRE. DD mutants can be identified by barcodes using sequencing approaches, such as Sanger sequencing and next-generation sequencing, but can also be identified by polymerase chain reaction and quantitative polymerase chain reaction. In some embodiments, polymerase chain reaction primers that amplify different size products for each barcode can be used to identify each barcode on an agarose gel. In other examples, each barcode can have a unique quantitative polymerase chain reaction probe sequence that allows for targeted amplification of each barcode.

[0061] In one embodiment, an effector module and / or SRE of the present disclosure can comprise at least one destabilizing domain (DD). An effector module and / or SRE can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DDs. When multiple DDs are present, each of the DDs can be derived from the same parent protein, different parent proteins, can be derived from a fusion of two different parent proteins, or can be artificial.

[0062] In one embodiment, an effector module and / or SRE of the present disclosure may comprise two DDs. In one embodiment, an effector module and / or SRE of the present disclosure may comprise three DDs. In one embodiment, an effector module and / or SRE of the present disclosure may comprise four DDs. In one embodiment, an effector module and / or SRE of the present disclosure may comprise five DDs. In one embodiment, an effector module and / or SRE of the present disclosure may comprise six DDs. In one embodiment, an effector module and / or SRE of the present disclosure may comprise seven DDs. In one embodiment, an effector module and / or SRE of the present disclosure may comprise eight DDs. In one embodiment, an effector module and / or SRE of the present disclosure may comprise nine DDs. In one embodiment, an effector module and / or SRE of the present disclosure may comprise ten DDs. The DDs may be derived from any parent protein known in the art and / or described herein. In some embodiments, the DDs are derived from the same parent protein. In some embodiments, the DDs are derived from different regions of the same parent protein. In some embodiments, the DDs are derived from different parent proteins.

[0063] CA2 destabilizing domain In some embodiments, the DD of the present disclosure can be derived from human carbonic anhydrase 2 (CA2), a member of carbonic anhydrase (CA, EC 4.2.1.1), a superfamily of metalloenzymes present in all living kingdoms. CAs equilibrate the reaction between three chemical species: CO, bicarbonate, and protons. CAs have evolved convergently, with seven genetically distinct CA families, α, β, γ, δ, ζ, η, and θ-CA, that evolved independently in bacteria, archaea, and eukaryotes. In some embodiments, the DDs described herein may be derived from at least one parent 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).

[0064] In one embodiment, the DD may be derived from a 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, the DD may be derived from a mitochondrial CA, such as, but not limited to, carbonic anhydrase 5A (CA5A) and carbonic anhydrase 5B (CA5B). In one embodiment, the DD may be derived from a secreted CA, such as, but not limited to, carbonic anhydrase 6 (CA6). In one embodiment, the DD may be derived from a membrane-associated 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, the DD may be derived from CA2. In another embodiment, the DD may be derived from CA9.

[0065] In some embodiments, the DD of the present disclosure may be derived from CA2 (SEQ ID NO: 11717; Uniprot ID: P00918), which may be stabilized by a ligand, for example, a small molecule inhibitor of CA2. As used herein, the term "CA2 WT" refers to the human wild-type CA2 protein sequence defined as SEQ ID NO: 11717, with GenBank accession number P00918, having the amino acid sequence: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK. In some aspects, the DD can be derived from CA2 of SEQ ID NO: 11718 (having the amino acid sequence: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAASLGSLEHQIWGFWESCAAT) or SEQ ID NO: 11719 (having the amino acid sequence: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAEKGISMLRKKDVKNIHSPDNACEE). In some embodiments, the DD of the present disclosure can be identified by utilizing a cocktail of CA2 inhibitors. In other examples, suitable DDs can be identified by first screening with one CA2 inhibitor and then screening with a second CA2 inhibitor.

[0066] The amino acid sequences of the destabilization domains encompassed by the present disclosure have at least about 40%, 50, or 60% identity, even 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 to the amino acid sequences set forth herein. Percent identity can be determined by comparing sequence information using, for example, advanced BLAST computer programs, including version Magic-BLAST 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 by reference in their entirety.

[0067] In some embodiments, the DD derived from CA2 can include amino acids 2-260 of the parent CA2 sequence, referred to herein as the M1del mutation. In one embodiment, the DD derived from CA2 can include amino acids 2-237 of the parent CA2 sequence.

[0068] Provided herein in Tables 1, 2, 3, 4, and 6 are CA2 mutants identified by mutagenesis, such as random mutagenesis screening or saturation mutagenesis, using a combination of nucleotide analog mutagenesis and error-prone PCR to generate a library of mutants. CA2 destabilizing mutants can also be identified by structure-guided mutagenesis and are 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. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0069] Additional CA2 destabilizing domains are provided in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0070] In some embodiments, the CA2 DD described herein can comprise any of the sequences provided in Table 3. In Table 3, 「*」 represents the translation of the stop codon. When an amino acid sequence in Table 3 contains one or more stop codons, the "AA SEQ ID NO" column provides the SEQ ID NOs of the individual components preceding and following the stop codon in the order in which they occur in the amino acid sequence. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

[0071] Additional CA2 destabilizing domains are provided in Table 4. The CA2 destabilizing mutants provided in Table 3B are identified, for example, by structure-guided mutagenesis or by combining single mutants, as described above. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0072] In some embodiments, a region or portion of CA2 WT can be used as a template to generate CA2 DD. In some embodiments, the CA2 DD can exclude the lysine at position 260 of SEQ ID NO: 11717. In some aspects, CA2 regions can include, but are not limited to, those listed in Table 5. [Table 5-1] [Table 5-2] [Table 5-3]

[0073] Any of the CA2 regions described herein can be utilized to generate CA2 DDs. Table 6 provides CA2 DDs derived from the CA2 region. [Table 6-1] [Table 6-2]

[0074] In some embodiments, the DD derived from CA2 may contain one, two, three, four, five or more of the mutations set forth in the preceding table.

[0075] In some embodiments, mutations can be conserved (having similar physicochemical properties to the amino acid at the mutation site), semi-conserved (e.g., a negatively to positively charged amino acid), 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, portions, or domains of wild-type proteins can be utilized in whole or in part as SREs / DDs. They can be combined or rearranged to create new peptides, proteins, regions, or domains, any of which can be used as SREs / DDs or as starting points for the design of additional SREs and / or DDs.

[0076] The destabilizing domains described herein can also include amino acid and nucleotide substitutions that do not affect stability, including conservative and non-conservative substitutions and / or polymorphisms. In some embodiments, the CA2 DDs described herein can also be fragments of the destabilizing domains, including fragments containing variant amino acid sequences. Preferred fragments are unstable in the absence of a stimulus and stabilized upon the addition of a stimulus. Preferred fragments retain the ability to interact with a stimulus as efficiently as the DDs described herein.

[0077] In one embodiment, the SRE comprises a region of the CA2 protein. The region of the CA2 protein is 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, 20, 21, 22, 23, 24, 25, 26 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, 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, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260 or more than 260 amino acids.The region of the parent protein can be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-260 amino acids in length.

[0078] In some embodiments, the CA2 DD described herein may contain one or more mutations relative to Uniprot ID: P00918 (SEQ ID NO: 11717). These mutations 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, D138G, D138M, D138N, and D161 * , D161M, D161V, D164G, D164I, D174*, D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34T, D 41T, D52I, D52L, D71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D72T, 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, E237R, E238 *、E238N、E238R、E26S、E69D、E69K、E69S、F130L、F146V、F175I、F175L、F175S、F178L、F178S、F20L、F20S、F225I、F225L、F225S、F225Y、F230I、F230L、F230S、F259L、F259S、F66S、F70I、F70L、F95Y、G102D、G104R、G104V、G128R、G12D、G12E、G131E、G131R、G131W、G139D、G144D、G144V、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. 「*」 indicates the translation of a stop codon, and X indicates any amino acid.

[0079] In one embodiment, the CA2 DD described herein comprises a mutation relative to Uniprot ID:P00918 (sequence number 11717) selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0080] In some embodiments, the CA2 DD described herein can include mutations relative to Uniprot ID: P00918 (SEQ ID NO: 11717), including CA2(aa 2-260 of WT, R27L, H122Y), CA2(aa 2-260 of WT, T87I, H122Y), CA2(aa 2-260 of WT, H122Y, N252D), CA2(aa 2-260 of WT, D72F, V241F), CA2(aa 2-260 of WT, V241F, P249L), CA2(aa 2-260 of WT, D72F, P249L), CA2(aa 2-260 of WT, D71L, L250R), CA2(aa 2-260 of WT, D72F, P249F), CA2(W CA2 (WT aa 2-260, T55K, G63N, Q248N), CA2 (WT aa 2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT aa 2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT aa 2-260, W4Y, L156H), CA2 (WT aa 2-260, L156H, G234del, E235del, P236del), CA2 (WT aa 2-260, L156H, F225L), CA2 (W CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2-260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2 to 260 of WT, L156H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, D71F, N231F) (SEQ ID NO: 210505), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, D71K, P249H) (SEQ ID NO: 210516), CA2 (aa 2 to 260 of WT, D72F, P249H) (SEQ ID NO: 210518),CA2 (aa 2-260 of WT, Q53N, N61Y) (SEQ ID NO: 210521), CA2 (aa 2-260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2 (aa 2-260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2-260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2-260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2-260 of WT, Y193L, K260L) (SEQ ID NO: 210540), CA2 (aa 2-260 of WT , D71F, V241F, P249L) (SEQ ID NO: 210544), CA2 (aa 2-260 of WT, L147F, Q248F) (SEQ ID NO: 210548), CA2 (aa 2-260 of WT, D52I, S258P) (SEQ ID NO: 210550), CA2 (aa 2-260 of WT, D72S, T192N) (SEQ ID NO: 210552), CA2 (aa 2-260 of WT, D179E, T192I) (SEQ ID NO: 210554), CA2 (aa 2-260 of WT, S56N, Q103K) (SEQ ID NO: 210558), CA2 (aa 2-260 of WT, D71Y, Q2 48L) (SEQ ID NO: 210560), CA2 (aa 2-260 of WT, S73N, R89F) (SEQ ID NO: 210562), CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F) (SEQ ID NO: 210564), CA2 (aa 2-260 of WT, D72F, P249I) (SEQ ID NO: 210568), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210572), CA2 (aa 2-260 of WT, A54X, S56X, L57X, T192X) (SEQ ID NO: 210574), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210575). 260, Y193V, K260F) (SEQ ID NO: 210576), CA2 (aa 2 to 260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2 to 260 of WT, V134F, L228F) (SEQ ID NO: 210580), CA2 (aa 2 to 260 of WT, D71G, N231K) (SEQ ID NO: 210582), CA2 (aa 2 to 260 of WT, S56F, D71S) (SEQ ID NO: 210584), CA2 (aa 2 to 260 of WT, D52L, G128R, Q248F) (SEQ ID NO: 210586), CA2 (aa 2 to 260 of WT, S73X,R89X) (SEQ ID NO: 210588), CA2 (aa 2 to 260 of WT, Y51X, D72X, V241X, P249X) (SEQ ID NO: 210592), CA2 (aa 2 to 260 of WT, D72I, W97C) (SEQ ID NO: 210594), CA2 (aa 2 to 260 of WT, D71K, T192F, N231F) (SEQ ID NO: 210596), CA2 (aa 2 to 260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO: 210698), CA2 (aa 2 to 260 of WT, F70I, F146V) (SEQ ID NO: 210700) , CA2 (aa 2-260 of WT, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO: 210704), CA2 (aa 2-260 of WT, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO: 210706), CA2 (aa 2-260 of WT, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO: 210708), CA2 (aa 2-260 of WT, G63D, W123R, E220K) (SEQ ID NO: 210712), CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M) , K113I, T125I, D138G, G155S) (SEQ ID NO: 210714), CA2 (aa 2-260 of WT, I59N, G102R, A173T) (SEQ ID NO: 210716), CA2 (aa 2-260 of WT, L79F, P180S) (SEQ ID NO: 210718), CA2 (aa 2-260 of WT, A77P, G102R, D138N) (SEQ ID NO: 210724), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO: 210726), CA2 (aa 2-260 of WT, T199N, L202P, L 228F) (SEQ ID NO: 210728), CA2 (aa 2 to 260 of WT, K9N, H122Y, T168K) (SEQ ID NO: 210730), CA2 (aa 2 to 260 of WT, Q53H, L90V, Q92H, G131E) (SEQ ID NO: 210732), CA2 (aa 2 to 260 of WT, L44M, L47V, N62K, E69D) (SEQ ID NO: 210734), CA2 (aa 2 to 260 of WT, D75V, K169N, F259L) (SEQ ID NO: 210738), CA2 (aa 2 to 260 of WT, T207S, V222A, N231D) (SEQ ID NO: 210740),CA2 (aa 2-260 of WT, I59F, V206M, G232R) (SEQ ID NO: 210742), CA2 (aa 2-260 of WT, P13A, A133T) (SEQ ID NO: 210744), CA2 (aa 2-260 of WT, I59N, R89I) (SEQ ID NO: 210750), CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L) (SEQ ID NO: 210752), CA2 (aa 2-260 of WT, G12R, H15Y, D19V) (SEQ ID NO: 210754), CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) (SEQ ID NO: 210758), and / or CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210851 or 210847).

[0081] In one embodiment, the CA2 DD described herein is selected from the group consisting of CA2(aa 2-260 of WT, R27L, H122Y), CA2(aa 2-260 of WT, T87I, H122Y), CA2(aa 2-260 of WT, H122Y, N252D), CA2(aa 2-260 of WT, D72F, V241F), CA2(aa 2-260 of WT, V241F, P249L), CA2(aa 2-260 of WT, D72F, P249L), CA2(aa 2-260 of WT, D71L, L250R), CA2(aa 2-260 of WT, D72F, P249F), CA2(aa 2-260 of WT, T55K, G6 3N, Q248N), CA2 (aa 2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa 2-260 of WT, W4Y, L156H), CA2 (aa 2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 of WT, L156H, F225L), CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156 H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2-260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2-260 of WT, L15 6H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2 (aa 2 to 260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2 to 260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2 to 260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2 to 260 of WT,CA2 (aa 2-260 of WT, S73N, R89F) (SEQ ID NO: 210562), CA2 (aa 2-260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2-260 of WT, V134F, L228F) (SEQ ID NO: 210580), and / or CA2 (aa 2-260 of WT, S56F, D71S) (SEQ ID NO: 210584),

[0082]

[0083] In some embodiments, CA2 can be derived from Homo sapiens carbonic anhydrase. In some embodiments, the CA2 DD described herein can have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity to a particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. In some embodiments, the reference polypeptide can be SEQ ID NO: 11717. Tools for alignment can include those in the BLAST suite (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).

[0084] In some embodiments, CA2 DD may be derived from species other than Homo sapiens carbon dioxide. canadensis、Cebus capucinus imitator、Ceratotherium simum、Cercocebus atys、Chinchilla lanigera、Chlorocebus sabaeus、Colobus angolensis palliatus、Delphinapterus leucas、Dipodomys ordii、Enhydra lutris kenyoni、Equus asinus、Equus caballus、Equus przewalskii、Erinaceus europaeus、Eumetopias jubatus、Felis catus、Galeopterus variegatus、Gorilla、Human wise, Ictidomys tridecemlineatus, Jaculus, Lagenorhynchus obliquidens, Lemur catta, Leptonychotes weddellii, Lipotes vexillifer, Loxodonta africana, Macaca fascicularis, Macaca mulatta, Macaca nemestrina, Mandrillus leucophaeus, Manis javanica, Marmota flaviventris, Marmota, Microcebus murinus, Mus caroli, Mus musculus, Mus pahari, Mustela putoriusfuro, Nannospalax galili, Neomonachus schauinslandi, Neophocaena asiaeorientalis, Nomascus leucogenys, Odobenus rosmarus divergens, Orcinus orca, Oryctolagus cuniculus, Otolemur garnettii, Pan paniscus, Pan troglodytes, Panthera pardus, Panthera tigris altaica, Papio anubis, Physeter catodon, Piliocolobus tephrosceles, Pongo abelii, Propithecus coquereli, Puma concolor, Rhinopithecus bieti, Rhinopithecus roxellana, Saimiri boliviensis, Sus scrofa, Theropithecus gelada, Trichechus manatus latirostris, Tupaia chinensis, Tursiops truncatus, Urocitellus parryii, Ursus It may be derived from carbonic anhydrases of species such as arctos horribilis, Ursus maritimus, Vulpes, and / or Zalophus californianus.

[0085] The SREs described herein may include, but are not limited to, 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, A24 7S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C20 5M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, 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、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、E237R、E238 *、E238N、E238R、E26S、E69D、E69K、E69S、F130L、F146V、F175I、F175L、F175S、F178L、F178S、F20L、F20S、F225I、F225L、F225S、F225Y、F230I、F230L、F230S、F259L、F259S、F66S、F70I、F70L、F95Y、G102D、G104R、G104V、G128R、G12D、G12E、G131E、G131R、G131W、G139D、G144D、G144V、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, V241 G, 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.

[0086] In one embodiment, an SRE described herein can comprise a CA2 DD comprising a mutation selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0087] SREs described herein include, but are not limited to, CA2(aa 2-260 of WT, R27L, H122Y), CA2(aa 2-260 of WT, T87I, H122Y), CA2(aa 2-260 of WT, H122Y, N252D), CA2(aa 2-260 of WT, D72F, V241F), CA2(aa 2-260 of WT, V241F, P249L), CA2(aa 2-260 of WT, D72F, P249L), CA2(aa 2-260 of WT, D71L, L250R), CA2(aa 2-260 of WT, D72F, P249F ... ~260, T55K, G63N, Q248N), CA2 (WT aa2~260, L156H, A257del, S258del, F259del, K260del), CA2 (WT aa2~260, L156H, S2del, H3del, H4del, W5del), CA2 (WT aa2~260, W4Y, L156H), CA2 (WT aa2~260, L156H, G234del, E235del, P236del), CA2 (WT aa2~260, L156H, F225L), CA2 (WT aa2~260, D70N, D74N, D10 0N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2-260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2-260 of WT, L1 56H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, D71F, N231F) (SEQ ID NO: 210505), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, D71K, P249H) (SEQ ID NO: 210516), CA2 (aa 2 to 260 of WT, D72F, P249H) (SEQ ID NO: 210518), CA2 (aa 2 to 260 of WT, Q53N, N61Y) (SEQ ID NO: 210521), CA2 (aa 2 to 260 of WT, E106D,C205S) (SEQ ID NO: 210523), CA2 (aa 2-260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2-260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2-260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2-260 of WT, Y193L, K260L) (SEQ ID NO: 210540), CA2 (aa 2-260 of WT, D71F, V241F, P249L) (SEQ ID NO: 210544), CA2 (aa 2-260 of WT, L147F, Q248F ) (SEQ ID NO: 210548), CA2 (aa 2 to 260 of WT, D52I, S258P) (SEQ ID NO: 210550), CA2 (aa 2 to 260 of WT, D72S, T192N) (SEQ ID NO: 210552), CA2 (aa 2 to 260 of WT, D179E, T192I) (SEQ ID NO: 210554), CA2 (aa 2 to 260 of WT, S56N, Q103K) (SEQ ID NO: 210558), CA2 (aa 2 to 260 of WT, D71Y, Q248L) (SEQ ID NO: 210560), CA2 (aa 2 to 260 of WT, S73N, R89F) (SEQ ID NO: 210562) , CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F) (SEQ ID NO: 210564), CA2 (aa 2-260 of WT, D72F, P249I) (SEQ ID NO: 210568), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210572), CA2 (aa 2-260 of WT, A54X, S56X, L57X, T192X) (SEQ ID NO: 210574), CA2 (aa 2-260 of WT, Y193V, K260F) (SEQ ID NO: 210576), CA2 (aa 2-260 of WT, G63D, M2 40L) (SEQ ID NO: 210578), CA2 (aa 2 to 260 of WT, V134F, L228F) (SEQ ID NO: 210580), CA2 (aa 2 to 260 of WT, D71G, N231K) (SEQ ID NO: 210582), CA2 (aa 2 to 260 of WT, S56F, D71S) (SEQ ID NO: 210584), CA2 (aa 2 to 260 of WT, D52L, G128R, Q248F) (SEQ ID NO: 210586), CA2 (aa 2 to 260 of WT, S73X, R89X) (SEQ ID NO: 210588), CA2 (aa 2 to 260 of WT, Y51X, D72X, V241X,P249X) (SEQ ID NO: 210592), CA2 (aa 2 to 260 of WT, D72I, W97C) (SEQ ID NO: 210594), CA2 (aa 2 to 260 of WT, D71K, T192F, N231F) (SEQ ID NO: 210596), CA2 (aa 2 to 260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO: 210698), CA2 (aa 2 to 260 of WT, F70I, F146V) (SEQ ID NO: 210700), CA2 (aa 2 to 260 of WT, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO: No. 210704), CA2 (aa 2-260 of WT, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO: 210706), CA2 (aa 2-260 of WT, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO: 210708), CA2 (aa 2-260 of WT, G63D, W123R, E220K) (SEQ ID NO: 210712), CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO: 21071 ... CA2 (aa 2-260 of WT, I59N, G102R, A173T) (SEQ ID NO: 210716), CA2 (aa 2-260 of WT, L79F, P180S) (SEQ ID NO: 210718), CA2 (aa 2-260 of WT, A77P, G102R, D138N) (SEQ ID NO: 210724), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO: 210726), CA2 (aa 2-260 of WT, T199N, L202P, L228F) (SEQ ID NO: 210728), CA2 (aa 2-260 of WT, K9N, H122Y, T16 8K) (SEQ ID NO: 210730), CA2 (aa 2 to 260 of WT, Q53H, L90V, Q92H, G131E) (SEQ ID NO: 210732), CA2 (aa 2 to 260 of WT, L44M, L47V, N62K, E69D) (SEQ ID NO: 210734), CA2 (aa 2 to 260 of WT, D75V, K169N, F259L) (SEQ ID NO: 210738), CA2 (aa 2 to 260 of WT, T207S, V222A, N231D) (SEQ ID NO: 210740), CA2 (aa 2 to 260 of WT, I59F, V206M, G232R) (SEQ ID NO: 210742),CA2 (aa 2-260 of WT, P13A, A133T) (SEQ ID NO: 210744), CA2 (aa 2-260 of WT, I59N, R89I) (SEQ ID NO: 210750), CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L) (SEQ ID NO: 210752), ... The CA2 DD may include mutations such as CA2 (aa 2 to 260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) (SEQ ID NO: 210758), and / or CA2 DD containing mutations such as CA2 (aa 2 to 260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210851 or 210847).

[0088] In one embodiment, the SRE described herein is selected from the group consisting of CA2(aa2-260 of WT, R27L, H122Y), CA2(aa2-260 of WT, T87I, H122Y), CA2(aa2-260 of WT, H122Y, N252D), CA2(aa2-260 of WT, D72F, V241F), CA2(aa2-260 of WT, V241F, P249L), CA2(aa2-260 of WT, D72F, P249L), CA2(aa2-260 of WT, D71L, L250R), CA2(aa2-260 of WT, D72F, P249F ... 260, T55K, G63N, Q248N), CA2 (aa2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa2-260 of WT, W4Y, L156H), CA2 (aa2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa2-260 of WT, L156H, F225L), CA2 (aa2-260 of WT, D70N, D74N, D100 N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2-260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2-260 of WT, L15 6H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2 (aa 2 to 260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2 to 260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2 to 260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2 to 260 of WT, S73N,The CA2 DD may comprise a mutation selected from CA2 (aa 2-260 of WT, R89F) (SEQ ID NO: 210562), CA2 (aa 2-260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2-260 of WT, V134F, L228F) (SEQ ID NO: 210580), and / or CA2 (aa 2-260 of WT, S56F, D71S) (SEQ ID NO: 210584).

[0089] Also provided herein is a biological circuit system comprising at least one effector module. The effector module of the biological circuit may comprise 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 comprise at least one payload that can be bound, attached, or associated with the SRE.

[0090] The SRE of a biological circuit system comprising, 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, A173R, A173C, A173D, A173G, A173L, A173R, A173D, A173G, A173H ... A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77 P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, D138G, D138M, D138N, D161 * , D161M, D161V, D164G, D164I, D174 * , D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34T, D41T, D52I, D52L, D 71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D72T, 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、E237R、E238 * 、E238N、E238R、E26S、E69D、E69K、E69S、F130L、F146V、F175I、F175L、F175S、F178L、F178S、F20L、F20S、F225I、F225L、F225S、F225Y、F230I、F230L、F230S、F259L、F259S、F66S、F70I、F70L、F95Y、G102D、G104R、G104V、G128R、G12D、G12E、G131E、G131R、G131W、G139D、G144D、G144V、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.

[0091] 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 comprise a mutation selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0092] SREs of biological circuit systems comprising, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717) can include multiple mutations, such as, but not limited to, CA2(aa 2-260 of WT, R27L, H122Y), CA2(aa 2-260 of WT, T87I, H122Y), CA2(aa 2-260 of WT, H122Y, N252D), CA2(aa 2-260 of WT, D72F, V241F), CA2(aa 2-260 of WT, V241F, P249L), CA2(aa 2-260 of WT, D72F, P249L ... CA2 (WT aa2-260, D71L, L250R), CA2 (WT aa2-260, D72F, P249F), CA2 (WT aa2-260, T55K, G63N, Q248N), CA2 (WT aa2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT aa2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT aa2-260, W4Y, L156H), CA2 (WT aa2-260, L156H, G234del, E235del, P236del ), CA2 (aa 2-260 of WT, L156H, F225L), CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA 2 (aa 2 to 260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2 to 260 of WT, L156H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, D71F, N231F) (SEQ ID NO: 210505), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, D71K, P249H) (SEQ ID NO: 210516), CA2 (aa 2 to 260 of WT, D72F, P249H) (SEQ ID NO: 210518),CA2 (aa 2-260 of WT, Q53N, N61Y) (SEQ ID NO: 210521), CA2 (aa 2-260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2 (aa 2-260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2-260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2-260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2-260 of WT, Y193L, K260L) (SEQ ID NO: 210540), CA2 (aa 2-260 of WT , D71F, V241F, P249L) (SEQ ID NO: 210544), CA2 (aa 2-260 of WT, L147F, Q248F) (SEQ ID NO: 210548), CA2 (aa 2-260 of WT, D52I, S258P) (SEQ ID NO: 210550), CA2 (aa 2-260 of WT, D72S, T192N) (SEQ ID NO: 210552), CA2 (aa 2-260 of WT, D179E, T192I) (SEQ ID NO: 210554), CA2 (aa 2-260 of WT, S56N, Q103K) (SEQ ID NO: 210558), CA2 (aa 2-260 of WT, D71Y, Q2 48L) (SEQ ID NO: 210560), CA2 (aa 2-260 of WT, S73N, R89F) (SEQ ID NO: 210562), CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F) (SEQ ID NO: 210564), CA2 (aa 2-260 of WT, D72F, P249I) (SEQ ID NO: 210568), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210572), CA2 (aa 2-260 of WT, A54X, S56X, L57X, T192X) (SEQ ID NO: 210574), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210575). 260, Y193V, K260F) (SEQ ID NO: 210576), CA2 (aa 2 to 260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2 to 260 of WT, V134F, L228F) (SEQ ID NO: 210580), CA2 (aa 2 to 260 of WT, D71G, N231K) (SEQ ID NO: 210582), CA2 (aa 2 to 260 of WT, S56F, D71S) (SEQ ID NO: 210584), CA2 (aa 2 to 260 of WT, D52L, G128R, Q248F) (SEQ ID NO: 210586), CA2 (aa 2 to 260 of WT, S73X,R89X) (SEQ ID NO: 210588), CA2 (aa 2 to 260 of WT, Y51X, D72X, V241X, P249X) (SEQ ID NO: 210592), CA2 (aa 2 to 260 of WT, D72I, W97C) (SEQ ID NO: 210594), CA2 (aa 2 to 260 of WT, D71K, T192F, N231F) (SEQ ID NO: 210596), CA2 (aa 2 to 260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO: 210698), CA2 (aa 2 to 260 of WT, F70I, F146V) (SEQ ID NO: 210700) , CA2 (aa 2-260 of WT, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO: 210704), CA2 (aa 2-260 of WT, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO: 210706), CA2 (aa 2-260 of WT, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO: 210708), CA2 (aa 2-260 of WT, G63D, W123R, E220K) (SEQ ID NO: 210712), CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M) , K113I, T125I, D138G, G155S) (SEQ ID NO: 210714), CA2 (aa 2-260 of WT, I59N, G102R, A173T) (SEQ ID NO: 210716), CA2 (aa 2-260 of WT, L79F, P180S) (SEQ ID NO: 210718), CA2 (aa 2-260 of WT, A77P, G102R, D138N) (SEQ ID NO: 210724), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO: 210726), CA2 (aa 2-260 of WT, T199N, L202P, L 228F) (SEQ ID NO: 210728), CA2 (aa 2 to 260 of WT, K9N, H122Y, T168K) (SEQ ID NO: 210730), CA2 (aa 2 to 260 of WT, Q53H, L90V, Q92H, G131E) (SEQ ID NO: 210732), CA2 (aa 2 to 260 of WT, L44M, L47V, N62K, E69D) (SEQ ID NO: 210734), CA2 (aa 2 to 260 of WT, D75V, K169N, F259L) (SEQ ID NO: 210738), CA2 (aa 2 to 260 of WT, T207S, V222A, N231D) (SEQ ID NO: 210740),CA2 (aa 2-260 of WT, I59F, V206M, G232R) (SEQ ID NO: 210742), CA2 (aa 2-260 of WT, P13A, A133T) (SEQ ID NO: 210744), CA2 (aa 2-260 of WT, I59N, R89I) (SEQ ID NO: 210750), CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L ) (SEQ ID NO: 210752), CA2 (aa 2-260 of WT, G12R, H15Y, D19V) (SEQ ID NO: 210754), CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) (SEQ ID NO: 210758), and / or CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210851 or 210847).

[0093] 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) is selected from the group consisting of CA2(aa2-260 of WT, R27L, H122Y), CA2(aa2-260 of WT, T87I, H122Y), CA2(aa2-260 of WT, H122Y, N252D), CA2(aa2-260 of WT, D72F, V241F), CA2(aa2-260 of WT, V241F, P249L), CA2(aa2-260 of WT, D72F, P249L), CA2(aa2-260 of WT, D71L, L250R), CA2(WT aa2-260, D72F, P249F), CA2(WT aa2-260, T55K, G63N, Q248N), CA2(WT aa2-260, L156H, A257del, S258del, F259del, K260del), CA2(WT aa2-260, L156H, S2del, H3del, H4del, W5del), CA2(WT aa2-260, W4Y, L156H), CA2(WT aa2-260, L156H, G234del, E235del, P236del), CA2( CA2 (aa 2-260 of WT, L156H, F225L), CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156H), CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (WT CA2 (aa2 to 260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa2 to 260 of WT, L156H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa2 to 260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2 (aa2 to 260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa2 to 260 of WT, S73N, R89Y) (SEQ ID NO: 210532),The mutations may include multiple mutations selected from CA2 (aa 2-260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2-260 of WT, S73N, R89F) (SEQ ID NO: 210562), CA2 (aa 2-260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2-260 of WT, V134F, L228F) (SEQ ID NO: 210580), and / or CA2 (aa 2-260 of WT, S56F, D71S) (SEQ ID NO: 210584).

[0094] Stabilization and destabilization ratio of SRE

[0095] In some embodiments, the present disclosure provides methods for modulating protein expression, function, or levels by measuring stabilization and destabilization ratios. As used herein, a stabilization ratio can be defined as the ratio of the expression, function, or level of a protein of interest in response to a stimulus to the expression, function, or level of the protein of interest in the absence of the stimulus specific for the SRE. In some aspects, the stabilization ratio is at least 1, e.g., 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, 30-1 The destabilization ratio may be 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. As used herein, a destabilization ratio may be defined as the ratio of expression, function, or levels of a protein of interest in the absence of a stimulus specific for the effector module to the expression, function, or levels of a constitutively expressed protein of interest in the absence of a stimulus specific for the SRE. As used herein, "constitutively" refers to the expression, function, or level of a protein of interest that is not linked to an SRE and is therefore expressed both in the presence and absence of a stimulus.In some embodiments, the destabilization ratio is at least 0, e.g., 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.

[0096] In some embodiments, the SRE of the effector module may stabilize the payload of interest at one or more stabilization ratios, which may include the ratio of expression, function, or level of the payload of interest in the presence of the stimulus to the expression, function, or level of the payload of interest in the absence of the stimulus.

[0097] In some embodiments, an SRE may destabilize a payload of interest with a destabilization ratio of between 0 and 0.09, which may include the ratio of expression, function, or level of a protein of interest in the absence of a stimulus specific for the SRE to the expression, function, or level of a constitutively expressed protein of interest in the absence of a stimulus specific for the SRE.

[0098] SRE protein-protein interactions In some embodiments, the present disclosure provides a stimulus response element (SRE) that can include a destabilization domain (DD) derived in whole or in part from human carbonic anhydrase 2 (CA2; SEQ ID NO: 11717). In one embodiment, the DD can include the entire CA2 (SEQ ID NO: 11717). In some embodiments, the DD can include a portion or region of human carbonic anhydrase.Portions or regions of CA2 include, but are not limited to, amino acids 2 to 260 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210492); amino acids 1 to 142 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210820); amino acids 2 to 142 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210821); amino acids 1 to 190 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210822 amino acids 2 to 190 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210823); amino acids 1 to 89 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210824); amino acids 2 to 89 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210825); amino acids 1 to 243 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210826); amino acids of CA2 (SEQ ID NO: 11717) amino acids 1 to 243 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210827); amino acids 1 to 166 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210828); amino acids 2 to 166 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210782); amino acids 1 to 116 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210830); amino acids 2 to 116 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: The DD may be selected from amino acids 1-152 of CA2 (SEQ ID NO: 11717), such as, 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, the DD may include amino acids 2-260 of CA2 (SEQ ID NO: 117 ...), for example, but not limited to, SEQ ID NO: 210492.

[0099] In one embodiment, DD can include amino acids 2-260 of CA2 (SEQ ID NO: 11717).

[0100] In some embodiments, the DD is 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, C205 M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, D138G, D138M, D138N, D161 * , D161M, D161V, D164G, D164I, D174 * , D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34T, D41T, D52I, D52L, D 71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D72T, 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, E237R, E238 *、E238N、E238R、E26S、E69D、E69K、E69S、F130L、F146V、F175I、F175L、F175S、F178L、F178S、F20L、F20S、F225I、F225L、F225S、F225Y、F230I、F230L、F230S、F259L、F259S、F66S、F70I、F70L、F95Y、G102D、G104R、G104V、G128R、G12D、G12E、G131E、G131R、G131W、G139D、G144D、G144V、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, V241 G, 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. 「*」 indicates the translation of a stop codon, and X indicates any amino acid.

[0101] In one embodiment, the DD may include an E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, or Y51T mutation.

[0102] In some embodiments, the DD comprises multiple mutations, such as, but not limited to, CA2(aa 2-260 of WT, R27L, H122Y), CA2(aa 2-260 of WT, T87I, H122Y), CA2(aa 2-260 of WT, H122Y, N252D), CA2(aa 2-260 of WT, D72F, V241F), CA2(aa 2-260 of WT, V241F, P249L), CA2(aa 2-260 of WT, D72F, P249L), CA2(aa 2-260 of WT, D71L, L250R), CA2(aa 2-260 of WT, D72F, P2 49F), CA2 (aa2-260 of WT, T55K, G63N, Q248N), CA2 (aa2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa2-260 of WT, W4Y, L156H), CA2 (aa2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa2-260 of WT, L156H, F225L), CA2 (aa2-260 of WT 260, D70N, D74N, D100N, L156H), (CA2 (aa 2 to 260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2 to 260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2 to 260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2 to 260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2 to 260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510 ), CA2 (aa2 to 260 of WT, L156H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa2 to 260 of WT, D71F, N231F) (SEQ ID NO: 210505), CA2 (aa2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa2 to 260 of WT, D71K, P249H) (SEQ ID NO: 210516), CA2 (aa2 to 260 of WT, D72F, P249H) (SEQ ID NO: 210518), CA2 (aa2 to 260 of WT, Q53N, N61Y) (SEQ ID NO: 210521),CA2 (aa 2-260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2 (aa 2-260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2-260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2-260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2-260 of WT, Y193L, K260L) (SEQ ID NO: 210540), CA2 (aa 2-260 of WT, D71F, V241F, P249L) (SEQ ID NO: 210544), CA2 (aa 2-260 of WT, D71F, V241F, P249L) (SEQ ID NO: 210545), CA2 (aa2-260 of WT, L147F, Q248F) (SEQ ID NO: 210548), CA2 (aa2-260 of WT, D52I, S258P) (SEQ ID NO: 210550), CA2 (aa2-260 of WT, D72S, T192N) (SEQ ID NO: 210552), CA2 (aa2-260 of WT, D179E, T192I) (SEQ ID NO: 210554), CA2 (aa2-260 of WT, S56N, Q103K) (SEQ ID NO: 210558), CA2 (aa2-260 of WT, D71Y, Q248L) (SEQ ID NO: 210560), CA2 (aa2-260 of WT, S73N, R CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F) (SEQ ID NO: 210564), CA2 (aa 2-260 of WT, D72F, P249I) (SEQ ID NO: 210568), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210572), CA2 (aa 2-260 of WT, A54X, S56X, L57X, T192X) (SEQ ID NO: 210574), CA2 (aa 2-260 of WT, Y193V, K260F) (SEQ ID NO: 210576), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210577). CA2 (aa 2-260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2-260 of WT, V134F, L228F) (SEQ ID NO: 210580), CA2 (aa 2-260 of WT, D71G, N231K) (SEQ ID NO: 210582), CA2 (aa 2-260 of WT, S56F, D71S) (SEQ ID NO: 210584), CA2 (aa 2-260 of WT, D52L, G128R, Q248F) (SEQ ID NO: 210586), CA2 (aa 2-260 of WT, S73X, R89X) (SEQ ID NO: 210588), CA2 (aa 2-260 of WT, Y51X,CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210592), CA2 (aa 2-260 of WT, D72I, W97C) (SEQ ID NO: 210594), CA2 (aa 2-260 of WT, D71K, T192F, N231F) (SEQ ID NO: 210596), CA2 (aa 2-260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO: 210698), CA2 (aa 2-260 of WT, F70I, F146V) (SEQ ID NO: 210700), CA2 (aa 2-260 of WT, K45N, V68L, H119Y, K16 CA2 (aa 2 to 260 of WT, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO: 210706), CA2 (aa 2 to 260 of WT, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO: 210708), CA2 (aa 2 to 260 of WT, G63D, W123R, E220K) (SEQ ID NO: 210712), CA2 (aa 2 to 260 of WT, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO: 21 0714), CA2 (aa 2-260 of WT, I59N, G102R, A173T) (SEQ ID NO: 210716), CA2 (aa 2-260 of WT, L79F, P180S) (SEQ ID NO: 210718), CA2 (aa 2-260 of WT, A77P, G102R, D138N) (SEQ ID NO: 210724), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO: 210726), CA2 (aa 2-260 of WT, T199N, L202P, L228F) (SEQ ID NO: 210728), CA2 (aa 2-260 of WT, T199N, L202P, L228F) (SEQ ID NO: 210729). 260, K9N, H122Y, T168K) (SEQ ID NO: 210730), CA2 (aa 2 to 260 of WT, Q53H, L90V, Q92H, G131E) (SEQ ID NO: 210732), CA2 (aa 2 to 260 of WT, L44M, L47V, N62K, E69D) (SEQ ID NO: 210734), CA2 (aa 2 to 260 of WT, D75V, K169N, F259L) (SEQ ID NO: 210738), CA2 (aa 2 to 260 of WT, T207S, V222A, N231D) (SEQ ID NO: 210740), CA2 (aa 2 to 260 of WT, I59F, V206M,G232R) (SEQ ID NO: 210742), CA2 (aa 2 to 260 of WT, P13A, A133T) (SEQ ID NO: 210744), CA2 (aa 2 to 260 of WT, I59N, R89I) (SEQ ID NO: 210750), CA2 (aa 2 to 260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L) (SEQ ID NO: 210752) , CA2 (aa 2-260 of WT, G12R, H15Y, D19V) (SEQ ID NO: 210754), CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) (SEQ ID NO: 210758), and / or CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210851 or 210847).

[0103] In one embodiment, DD is selected from the group consisting of CA2(aa2-260 of WT, R27L, H122Y), CA2(aa2-260 of WT, T87I, H122Y), CA2(aa2-260 of WT, H122Y, N252D), CA2(aa2-260 of WT, D72F, V241F), CA2(aa2-260 of WT, V241F, P249L), CA2(aa2-260 of WT, D72F, P249L), CA2(aa2-260 of WT, D71L, L250R), CA2(aa2-260 of WT, D72F, P249F), CA2(aa2-260 of WT, T5 5K, G63N, Q248N), CA2 (aa 2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa 2-260 of WT, W4Y, L156H), CA2 (aa 2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 of WT, L156H, F225L), CA2 (aa 2-260 of WT, D70N, D74N, D100N, L1 56H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2-260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2-260 of WT, L156H , S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2 (aa 2 to 260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2 to 260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2 to 260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2 to 260 of WT, S73N,R89F) (SEQ ID NO: 210562), CA2 (aa 2-260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2-260 of WT, V134F, L228F) (SEQ ID NO: 210580), and / or CA2 (aa 2-260 of WT, S56F, D71S) (SEQ ID NO: 210584).

[0104] In one embodiment, DD can include amino acids 2-260 of CA2 (SEQ ID NO: 11717), for example, but not limited to, SEQ ID NO: (210492).

[0105] In some embodiments, the SRE is selected from, but not limited to, CA2(aa 2-260 of WT, R27L, H122Y), CA2(aa 2-260 of WT, T87I, H122Y), CA2(aa 2-260 of WT, H122Y, N252D), CA2(aa 2-260 of WT, D72F, V241F), CA2(aa 2-260 of WT, V241F, P249L), CA2(aa 2-260 of WT, D72F, P249L), CA2(aa 2-260 of WT, D71L, L250R), CA2(aa 2-260 of WT, D72F, P249F), CA2(WT CA2 (aa2-260 of WT, T55K, G63N, Q248N), CA2 (aa2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa2-260 of WT, W4Y, L156H), CA2 (aa2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa2-260 of WT, L156H, F225L), CA2 (aa2-260 of WT, D70N, D74 N, D100N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2-260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702). 260, L156H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, D71F, N231F) (SEQ ID NO: 210505), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, D71K, P249H) (SEQ ID NO: 210516), CA2 (aa 2 to 260 of WT, D72F, P249H) (SEQ ID NO: 210518), CA2 (aa 2 to 260 of WT, Q53N, N61Y) (SEQ ID NO: 210521), CA2 (aa 2 to 260 of WT,E106D, C205S) (SEQ ID NO: 210523), CA2 (aa 2 to 260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2 to 260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2 to 260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2 to 260 of WT, Y193L, K260L) (SEQ ID NO: 210540), CA2 (aa 2 to 260 of WT, D71F, V241F, P249L) (SEQ ID NO: 210544), CA2 (aa 2 to 260 of WT, L147F, Q248F) (SEQ ID NO: 210548), CA2 (aa 2-260 of WT, D52I, S258P) (SEQ ID NO: 210550), CA2 (aa 2-260 of WT, D72S, T192N) (SEQ ID NO: 210552), CA2 (aa 2-260 of WT, D179E, T192I) (SEQ ID NO: 210554), CA2 (aa 2-260 of WT, S56N, Q103K) (SEQ ID NO: 210558), CA2 (aa 2-260 of WT, D71Y, Q248L) (SEQ ID NO: 210560), CA2 (aa 2-260 of WT, S73N, R89F) (SEQ ID NO: 2105 62), CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F) (SEQ ID NO: 210564), CA2 (aa 2-260 of WT, D72F, P249I) (SEQ ID NO: 210568), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210572), CA2 (aa 2-260 of WT, A54X, S56X, L57X, T192X) (SEQ ID NO: 210574), CA2 (aa 2-260 of WT, Y193V, K260F) (SEQ ID NO: 210576), CA2 (aa 2-260 of WT, G63D, M 240L) (SEQ ID NO: 210578), CA2 (aa 2 to 260 of WT, V134F, L228F) (SEQ ID NO: 210580), CA2 (aa 2 to 260 of WT, D71G, N231K) (SEQ ID NO: 210582), CA2 (aa 2 to 260 of WT, S56F, D71S) (SEQ ID NO: 210584), CA2 (aa 2 to 260 of WT, D52L, G128R, Q248F) (SEQ ID NO: 210586), CA2 (aa 2 to 260 of WT, S73X, R89X) (SEQ ID NO: 210588), CA2 (aa 2 to 260 of WT, Y51X, D72X, V241X,P249X) (SEQ ID NO: 210592), CA2 (aa 2 to 260 of WT, D72I, W97C) (SEQ ID NO: 210594), CA2 (aa 2 to 260 of WT, D71K, T192F, N231F) (SEQ ID NO: 210596), CA2 (aa 2 to 260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO: 210698), CA2 (aa 2 to 260 of WT, F70I, F146V) (SEQ ID NO: 210700), CA2 (aa 2 to 260 of WT, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO: No. 210704), CA2 (aa 2-260 of WT, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO: 210706), CA2 (aa 2-260 of WT, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO: 210708), CA2 (aa 2-260 of WT, G63D, W123R, E220K) (SEQ ID NO: 210712), CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO: 21071 ... CA2 (aa 2-260 of WT, I59N, G102R, A173T) (SEQ ID NO: 210716), CA2 (aa 2-260 of WT, L79F, P180S) (SEQ ID NO: 210718), CA2 (aa 2-260 of WT, A77P, G102R, D138N) (SEQ ID NO: 210724), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO: 210726), CA2 (aa 2-260 of WT, T199N, L202P, L228F) (SEQ ID NO: 210728), CA2 (aa 2-260 of WT, K9N, H122Y, T16 8K) (SEQ ID NO: 210730), CA2 (aa 2 to 260 of WT, Q53H, L90V, Q92H, G131E) (SEQ ID NO: 210732), CA2 (aa 2 to 260 of WT, L44M, L47V, N62K, E69D) (SEQ ID NO: 210734), CA2 (aa 2 to 260 of WT, D75V, K169N, F259L) (SEQ ID NO: 210738), CA2 (aa 2 to 260 of WT, T207S, V222A, N231D) (SEQ ID NO: 210740), CA2 (aa 2 to 260 of WT, I59F, V206M, G232R) (SEQ ID NO: 210742),CA2 (aa 2-260 of WT, P13A, A133T) (SEQ ID NO: 210744), CA2 (aa 2-260 of WT, I59N, R89I) (SEQ ID NO: 210750), CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L) (SEQ ID NO: 210752), ... CA2 (aa 2-260 of WT, G12R, H15Y, D19V) (SEQ ID NO: 210754), CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) (SEQ ID NO: 210758), and / or CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210851 or 210847).

[0106] In one embodiment, DD is selected from the group consisting of CA2(aa2-260 of WT, R27L, H122Y), CA2(aa2-260 of WT, T87I, H122Y), CA2(aa2-260 of WT, H122Y, N252D), CA2(aa2-260 of WT, D72F, V241F), CA2(aa2-260 of WT, V241F, P249L), CA2(aa2-260 of WT, D72F, P249L), CA2(aa2-260 of WT, D71L, L250R), CA2(aa2-260 of WT, D72F, P249F), CA2(aa2-260 of WT, T5 5K, G63N, Q248N), CA2 (aa 2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa 2-260 of WT, W4Y, L156H), CA2 (aa 2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 of WT, L156H, F225L), CA2 (aa 2-260 of WT, D70N, D74N, D100N, L1 56H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2-260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2-260 of WT, L156 H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, D71F, N231F) (SEQ ID NO: 210505), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, D71K, P249H) (SEQ ID NO: 210516), CA2 (aa 2 to 260 of WT, D72F, P249H) (SEQ ID NO: 210518), CA2 (aa 2 to 260 of WT, Q53N, N61Y) (SEQ ID NO: 210521), CA2 (aa 2 to 260 of WT, E106D,C205S) (SEQ ID NO: 210523), CA2 (aa 2-260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2-260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2-260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2-260 of WT, Y193L, K260L) (SEQ ID NO: 210540), CA2 (aa 2-260 of WT, D71F, V241F, P249L) (SEQ ID NO: 210544), CA2 (aa 2-260 of WT, L147F, Q248F ) (SEQ ID NO: 210548), CA2 (aa 2 to 260 of WT, D52I, S258P) (SEQ ID NO: 210550), CA2 (aa 2 to 260 of WT, D72S, T192N) (SEQ ID NO: 210552), CA2 (aa 2 to 260 of WT, D179E, T192I) (SEQ ID NO: 210554), CA2 (aa 2 to 260 of WT, S56N, Q103K) (SEQ ID NO: 210558), CA2 (aa 2 to 260 of WT, D71Y, Q248L) (SEQ ID NO: 210560), CA2 (aa 2 to 260 of WT, S73N, R89F) (SEQ ID NO: 210562) , CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F) (SEQ ID NO: 210564), CA2 (aa 2-260 of WT, D72F, P249I) (SEQ ID NO: 210568), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210572), CA2 (aa 2-260 of WT, A54X, S56X, L57X, T192X) (SEQ ID NO: 210574), CA2 (aa 2-260 of WT, Y193V, K260F) (SEQ ID NO: 210576), CA2 (aa 2-260 of WT, G63D, M2 40L) (SEQ ID NO: 210578), CA2 (aa 2 to 260 of WT, V134F, L228F) (SEQ ID NO: 210580), CA2 (aa 2 to 260 of WT, D71G, N231K) (SEQ ID NO: 210582), CA2 (aa 2 to 260 of WT, S56F, D71S) (SEQ ID NO: 210584), CA2 (aa 2 to 260 of WT, D52L, G128R, Q248F) (SEQ ID NO: 210586), CA2 (aa 2 to 260 of WT, S73X, R89X) (SEQ ID NO: 210588), CA2 (aa 2 to 260 of WT, Y51X, D72X, V241X,P249X) (SEQ ID NO: 210592), CA2 (aa 2 to 260 of WT, D72I, W97C) (SEQ ID NO: 210594), CA2 (aa 2 to 260 of WT, D71K, T192F, N231F) (SEQ ID NO: 210596), CA2 (aa 2 to 260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO: 210698), CA2 (aa 2 to 260 of WT, F70I, F146V) (SEQ ID NO: 210700), CA2 (aa 2 to 260 of WT, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO: No. 210704), CA2 (aa 2-260 of WT, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO: 210706), CA2 (aa 2-260 of WT, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO: 210708), CA2 (aa 2-260 of WT, G63D, W123R, E220K) (SEQ ID NO: 210712), CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO: 21071 ... CA2 (aa 2-260 of WT, I59N, G102R, A173T) (SEQ ID NO: 210716), CA2 (aa 2-260 of WT, L79F, P180S) (SEQ ID NO: 210718), CA2 (aa 2-260 of WT, A77P, G102R, D138N) (SEQ ID NO: 210724), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO: 210726), CA2 (aa 2-260 of WT, T199N, L202P, L228F) (SEQ ID NO: 210728), CA2 (aa 2-260 of WT, K9N, H122Y, T16 8K) (SEQ ID NO: 210730), CA2 (aa 2 to 260 of WT, Q53H, L90V, Q92H, G131E) (SEQ ID NO: 210732), CA2 (aa 2 to 260 of WT, L44M, L47V, N62K, E69D) (SEQ ID NO: 210734), CA2 (aa 2 to 260 of WT, D75V, K169N, F259L) (SEQ ID NO: 210738), CA2 (aa 2 to 260 of WT, T207S, V222A, N231D) (SEQ ID NO: 210740), CA2 (aa 2 to 260 of WT, I59F, V206M, G232R) (SEQ ID NO: 210742),CA2 (aa 2-260 of WT, P13A, A133T) (SEQ ID NO: 210744), CA2 (aa 2-260 of WT, I59N, R89I) (SEQ ID NO: 210750), CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L) (SEQ ID NO: 210752), ... CA2 (aa 2-260 of WT, G12R, H15Y, D19V) (SEQ ID NO: 210754), CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) (SEQ ID NO: 210758), and / or CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210851 or 210847).

[0107] Also provided herein are isolated polypeptide variants comprising at least one mutation relative to SEQ ID NO: 11717. Non-limiting examples of mutations relative to SEQ ID 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, C205 R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, D138G, D138M, D138N, D161 * , D161M, D161V, D164G, D164I, D174 * , D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34T, D41T, D52I, D52L, D 71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D72T, 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、E237R、E238 * 、E238N、E238R、E26S、E69D、E69K、E69S、F130L、F146V、F175I、F175L、F175S、F178L、F178S、F20L、F20S、F225I、F225L、F225S、F225Y、F230I、F230L、F230S、F259L、F259S、F66S、F70I、F70L、F95Y、G102D、G104R、G104V、G128R、G12D、G12E、G131E、G131R、G131W、G139D、G144D、G144V、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, V241 G, 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.

[0108] In some embodiments, the isolated polypeptide variants are CA2(aa 2-260 of WT, R27L, H122Y), CA2(aa 2-260 of WT, T87I, H122Y), CA2(aa 2-260 of WT, H122Y, N252D), CA2(aa 2-260 of WT, D72F, V241F), CA2(aa 2-260 of WT, V241F, P249L), CA2(aa 2-260 of WT, D72F, P249L), CA2(aa 2-260 of WT, D71L, L250R), CA2(aa 2-260 of WT, D72F, P249F ), CA2 (aa 2-260 of WT, T55K, G63N, Q248N), CA2 (aa 2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa 2-260 of WT, W4Y, L156H), CA2 (aa 2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 of WT, L156H, F225L), CA2 (aa 2-26 0, D70N, D74N, D100N, L156H), (CA2 (aa 2 to 260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2 to 260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2 to 260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2 to 260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2 to 260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510) , CA2 (aa2 to 260 of WT, L156H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa2 to 260 of WT, D71F, N231F) (SEQ ID NO: 210505), CA2 (aa2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa2 to 260 of WT, D71K, P249H) (SEQ ID NO: 210516), CA2 (aa2 to 260 of WT, D72F, P249H) (SEQ ID NO: 210518), CA2 (aa2 to 260 of WT, Q53N, N61Y) (SEQ ID NO: 210521),CA2 (aa 2-260 of WT, E106D, C205S) (SEQ ID NO: 210523), CA2 (aa 2-260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2-260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2-260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2-260 of WT, Y193L, K260L) (SEQ ID NO: 210540), CA2 (aa 2-260 of WT, D71F, V241F, P249L) (SEQ ID NO: 210544), CA2 (aa 2-260 of WT, D71F, V241F, P249L) (SEQ ID NO: 210545), CA2 (aa2-260 of WT, L147F, Q248F) (SEQ ID NO: 210548), CA2 (aa2-260 of WT, D52I, S258P) (SEQ ID NO: 210550), CA2 (aa2-260 of WT, D72S, T192N) (SEQ ID NO: 210552), CA2 (aa2-260 of WT, D179E, T192I) (SEQ ID NO: 210554), CA2 (aa2-260 of WT, S56N, Q103K) (SEQ ID NO: 210558), CA2 (aa2-260 of WT, D71Y, Q248L) (SEQ ID NO: 210560), CA2 (aa2-260 of WT, S73N, R CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F) (SEQ ID NO: 210564), CA2 (aa 2-260 of WT, D72F, P249I) (SEQ ID NO: 210568), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210572), CA2 (aa 2-260 of WT, A54X, S56X, L57X, T192X) (SEQ ID NO: 210574), CA2 (aa 2-260 of WT, Y193V, K260F) (SEQ ID NO: 210576), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210577). CA2 (aa 2-260 of WT, G63D, M240L) (SEQ ID NO: 210578), CA2 (aa 2-260 of WT, V134F, L228F) (SEQ ID NO: 210580), CA2 (aa 2-260 of WT, D71G, N231K) (SEQ ID NO: 210582), CA2 (aa 2-260 of WT, S56F, D71S) (SEQ ID NO: 210584), CA2 (aa 2-260 of WT, D52L, G128R, Q248F) (SEQ ID NO: 210586), CA2 (aa 2-260 of WT, S73X, R89X) (SEQ ID NO: 210588), CA2 (aa 2-260 of WT, Y51X,CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210592), CA2 (aa 2-260 of WT, D72I, W97C) (SEQ ID NO: 210594), CA2 (aa 2-260 of WT, D71K, T192F, N231F) (SEQ ID NO: 210596), CA2 (aa 2-260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO: 210698), CA2 (aa 2-260 of WT, F70I, F146V) (SEQ ID NO: 210700), CA2 (aa 2-260 of WT, K45N, V68L, H119Y, K16 CA2 (aa 2 to 260 of WT, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO: 210706), CA2 (aa 2 to 260 of WT, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO: 210708), CA2 (aa 2 to 260 of WT, G63D, W123R, E220K) (SEQ ID NO: 210712), CA2 (aa 2 to 260 of WT, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO: 21 0714), CA2 (aa 2-260 of WT, I59N, G102R, A173T) (SEQ ID NO: 210716), CA2 (aa 2-260 of WT, L79F, P180S) (SEQ ID NO: 210718), CA2 (aa 2-260 of WT, A77P, G102R, D138N) (SEQ ID NO: 210724), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO: 210726), CA2 (aa 2-260 of WT, T199N, L202P, L228F) (SEQ ID NO: 210728), CA2 (aa 2-260 of WT, T199N, L202P, L228F) (SEQ ID NO: 210729). 260, K9N, H122Y, T168K) (SEQ ID NO: 210730), CA2 (aa 2 to 260 of WT, Q53H, L90V, Q92H, G131E) (SEQ ID NO: 210732), CA2 (aa 2 to 260 of WT, L44M, L47V, N62K, E69D) (SEQ ID NO: 210734), CA2 (aa 2 to 260 of WT, D75V, K169N, F259L) (SEQ ID NO: 210738), CA2 (aa 2 to 260 of WT, T207S, V222A, N231D) (SEQ ID NO: 210740), CA2 (aa 2 to 260 of WT, I59F, V206M,G232R) (SEQ ID NO: 210742), CA2 (aa 2 to 260 of WT, P13A, A133T) (SEQ ID NO: 210744), CA2 (aa 2 to 260 of WT, I59N, R89I) (SEQ ID NO: 210750), CA2 (aa 2 to 260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L) (SEQ ID NO: 210752) , CA2 (aa 2-260 of WT, G12R, H15Y, D19V) (SEQ ID NO: 210754), CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) (SEQ ID NO: 210758), and / or CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210851 or 210847).

[0109] Also provided herein is a biological circuit system comprising at least one effector module. The effector module of the biological circuit may comprise 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 comprise at least one payload that can be bound to, attached to, or associated with the SRE.

[0110] The SRE of a biological circuit system comprising, 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, A173R, A173C, A173D, A173G, A173L, A173R, A173D, A173G, A173H ... A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77 P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, 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、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、E237R、E238 * 、E238N、E238R、E26S、E69D、E69K、E69S、F130L、F146V、F175I、F175L、F175S、F178L、F178S、F20L、F20S、F225I、F225L、F225S、F225Y、F230I、F230L、F230S、F259L、F259S、F66S、F70I、F70L、F95Y、G102D、G104R、G104V、G128R、G12D、G12E、G131E、G131R、G131W、G139D、G144D、G144V、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.

[0111] In some embodiments, the SRE may be, but is not limited to, CA2(aa 2-260 of WT, R27L, H122Y), CA2(aa 2-260 of WT, T87I, H122Y), CA2(aa 2-260 of WT, H122Y, N252D), CA2(aa 2-260 of WT, D72F, V241F), CA2(aa 2-260 of WT, V241F, P249L), CA2(aa 2-260 of WT, D72F, P249L), CA2(aa 2-260 of WT, D71L, L250R), CA2(aa 2-260 of WT, D72F, P249F ... CA2 (aa2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa2-260 of WT, W4Y, L156H), CA2 (aa2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa2-260 of WT, L156H, F225L), CA2 (aa2-260 of WT, D70N, D74N, D1 00N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R) (SEQ ID NO: 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210748), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) (SEQ ID NO: 210702), CA2 (aa 2-260 of WT, D72F, V241F, P249L) (SEQ ID NO: 210503), CA2 (aa 2-260 of WT, D71L, T87N, L250R) (SEQ ID NO: 210510), CA2 (aa 2-260 of WT, L 156H, S172C, F178Y, E186D) (SEQ ID NO: 210756), CA2 (aa 2 to 260 of WT, D71F, N231F) (SEQ ID NO: 210505), CA2 (aa 2 to 260 of WT, A77I, P249F) (SEQ ID NO: 210514), CA2 (aa 2 to 260 of WT, D71K, P249H) (SEQ ID NO: 210516), CA2 (aa 2 to 260 of WT, D72F, P249H) (SEQ ID NO: 210518), CA2 (aa 2 to 260 of WT, Q53N, N61Y) (SEQ ID NO: 210521), CA2 (aa 2 to 260 of WT, E106D,C205S) (SEQ ID NO: 210523), CA2 (aa 2-260 of WT, C205S, W208S) (SEQ ID NO: 210525), CA2 (aa 2-260 of WT, S73N, R89Y) (SEQ ID NO: 210532), CA2 (aa 2-260 of WT, D71K, T192F) (SEQ ID NO: 210534), CA2 (aa 2-260 of WT, Y193L, K260L) (SEQ ID NO: 210540), CA2 (aa 2-260 of WT, D71F, V241F, P249L) (SEQ ID NO: 210544), CA2 (aa 2-260 of WT, L147F, Q248F ) (SEQ ID NO: 210548), CA2 (aa 2 to 260 of WT, D52I, S258P) (SEQ ID NO: 210550), CA2 (aa 2 to 260 of WT, D72S, T192N) (SEQ ID NO: 210552), CA2 (aa 2 to 260 of WT, D179E, T192I) (SEQ ID NO: 210554), CA2 (aa 2 to 260 of WT, S56N, Q103K) (SEQ ID NO: 210558), CA2 (aa 2 to 260 of WT, D71Y, Q248L) (SEQ ID NO: 210560), CA2 (aa 2 to 260 of WT, S73N, R89F) (SEQ ID NO: 210562) , CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F) (SEQ ID NO: 210564), CA2 (aa 2-260 of WT, D72F, P249I) (SEQ ID NO: 210568), CA2 (aa 2-260 of WT, D72X, V241X, P249X) (SEQ ID NO: 210572), CA2 (aa 2-260 of WT, A54X, S56X, L57X, T192X) (SEQ ID NO: 210574), CA2 (aa 2-260 of WT, Y193V, K260F) (SEQ ID NO: 210576), CA2 (aa 2-260 of WT, G63D, M2 40L) (SEQ ID NO: 210578), CA2 (aa 2 to 260 of WT, V134F, L228F) (SEQ ID NO: 210580), CA2 (aa 2 to 260 of WT, D71G, N231K) (SEQ ID NO: 210582), CA2 (aa 2 to 260 of WT, S56F, D71S) (SEQ ID NO: 210584), CA2 (aa 2 to 260 of WT, D52L, G128R, Q248F) (SEQ ID NO: 210586), CA2 (aa 2 to 260 of WT, S73X, R89X) (SEQ ID NO: 210588), CA2 (aa 2 to 260 of WT, Y51X, D72X, V241X,P249X) (SEQ ID NO: 210592), CA2 (aa 2 to 260 of WT, D72I, W97C) (SEQ ID NO: 210594), CA2 (aa 2 to 260 of WT, D71K, T192F, N231F) (SEQ ID NO: 210596), CA2 (aa 2 to 260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) (SEQ ID NO: 210698), CA2 (aa 2 to 260 of WT, F70I, F146V) (SEQ ID NO: 210700), CA2 (aa 2 to 260 of WT, K45N, V68L, H119Y, K169R, D179E) (SEQ ID NO: No. 210704), CA2 (aa 2-260 of WT, H15L, A54V, K111E, E220K, F225I) (SEQ ID NO: 210706), CA2 (aa 2-260 of WT, P13S, P83A, D101G, K111N, F230I) (SEQ ID NO: 210708), CA2 (aa 2-260 of WT, G63D, W123R, E220K) (SEQ ID NO: 210712), CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) (SEQ ID NO: 21071 ... CA2 (aa 2-260 of WT, I59N, G102R, A173T) (SEQ ID NO: 210716), CA2 (aa 2-260 of WT, L79F, P180S) (SEQ ID NO: 210718), CA2 (aa 2-260 of WT, A77P, G102R, D138N) (SEQ ID NO: 210724), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I) (SEQ ID NO: 210726), CA2 (aa 2-260 of WT, T199N, L202P, L228F) (SEQ ID NO: 210728), CA2 (aa 2-260 of WT, K9N, H122Y, T16 8K) (SEQ ID NO: 210730), CA2 (aa 2 to 260 of WT, Q53H, L90V, Q92H, G131E) (SEQ ID NO: 210732), CA2 (aa 2 to 260 of WT, L44M, L47V, N62K, E69D) (SEQ ID NO: 210734), CA2 (aa 2 to 260 of WT, D75V, K169N, F259L) (SEQ ID NO: 210738), CA2 (aa 2 to 260 of WT, T207S, V222A, N231D) (SEQ ID NO: 210740), CA2 (aa 2 to 260 of WT, I59F, V206M, G232R) (SEQ ID NO: 210742),CA2 (aa 2-260 of WT, P13A, A133T) (SEQ ID NO: 210744), CA2 (aa 2-260 of WT, I59N, R89I) (SEQ ID NO: 210750), CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L) (SEQ ID NO: 210752), ... CA2 (aa 2-260 of WT, G12R, H15Y, D19V) (SEQ ID NO: 210754), CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) (SEQ ID NO: 210758), and / or CA2 (aa 2-260 of WT, G63D, E69V, N231I) (SEQ ID NO: 210851 or 210847).

[0112] The biological circuit systems described herein may include SREs that are responsive to more than one stimuli.

[0113] In some embodiments, the irritant may be a small molecule, and the small molecule may be celecoxib, valdecoxib, rofecoxib, acetazolamide, methazolamide, dorzolamide, brinzolamide, diclophenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide. In embodiments, the small molecule may be acetazolamide.

[0114] Also described herein are vectors encoding the biological circuit systems, and pharmaceutical compositions comprising the biological circuit systems and pharmaceutically acceptable excipients.

[0115] payload As used herein, "payload" or "target payload" or "payload of interest (POI)" is defined as any protein or nucleic acid whose function is to be altered.

[0116] The payload may comprise any coding or non-coding gene or any protein or fragment thereof.

[0117] Payloads are often associated with one or more SREs and can be encoded in the polynucleotides of the present disclosure, either alone or in combination with one or more SREs. The payload itself can be varied (at the protein or nucleic acid level) to provide an additional layer of protection for the effector module. For example, payloads can be modified or designed to contain single or multiple mutations that affect the stability of the payload or its susceptibility to degradation, cleavage, or trafficking. Combining an SRE that can have a diverse response to a stimulus with a payload that has been altered to exhibit a diverse response or gradual change in output signal, e.g., expression level, generates biological circuits superior to those in the art. For example, mutation or substitution designs, such as those created for IL12 in WO2016048903 (particularly Example 1 thereof), the contents of which are incorporated herein by reference in their entirety, can be used in any protein payload in conjunction with the SREs of the present disclosure to generate dual, tunable biological circuits. The ability to independently tune both the SRE and the payload greatly expands the scope of use for the effector modules of the present disclosure.

[0118] The artificial peptide or polypeptide component of the payload can be derived from any known polypeptide that does not occur in nature.

[0119] As used herein, the phrase "derived from," when referring to an effector module, SRE, or payload, means that the effector module, SRE, or payload originates, at least in part, from the described 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 then modified in any of the ways taught herein to optimize SRE function.

[0120] In one embodiment, the payload is derived from a region of a parent protein or a variant protein. The regions of the parent protein are 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, 8, 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, 25 08, 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, 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, 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, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, or more than 450 amino acids. The region of the parent protein 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 in length.

[0121] In one embodiment, the payload is derived from a region of a parent protein or a variant protein and comprises a region of the parent protein. The payload may comprise 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%, 15-20%, 20-25%, 25-30%, 30%, or 40% of the parent or variant protein. ~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%, 6 It may comprise a region of the parent protein that is 0-100%, 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%.

[0122] In one embodiment, the payload is derived from the parent or variant protein and is 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% of the parent or variant protein. , 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%, 6 0-70%, 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%, 4 It may have 0 to 80%, 50 to 90%, 60 to 100%, 10 to 60%, 20 to 70%, 30 to 80%, 40 to 90%, 50 to 100%, 10 to 70%, 20 to 80%, 30 to 90%, 40 to 100%, 10 to 80%, 20 to 90%, 30 to 100%, 10 to 90%, 20 to 100%, 25 to 50%, 50 to 75%, or 75 to 100% identity.

[0123] In one embodiment, the transmembrane domain region of the first payload may be replaced with a transmembrane domain, variant or fragment from a second parent protein.

[0124] Polypeptides and polypeptides as payloads The stimulants, biological circuit components, and effector modules (including their SREs and payloads) of the present disclosure may exist as whole polypeptides, multiple polypeptides, or fragments of polypeptides, which may be independently encoded by one or more nucleic acids, multiple nucleic acids, fragments of nucleic acids, or variants of any of the foregoing.

[0125] As used herein, the term "polypeptide" refers to a polymer of amino acid residues (natural or unnatural) linked together, most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, the encoded polypeptide is less than about 50 amino acids, and the polypeptide is then referred to as a peptide. When a polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residues in length. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides can be single molecules or multimolecular complexes, such as dimers, trimers, or tetramers. They can also include single-chain or multiple-chain polypeptides, which can be associated or linked. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0126] As used herein, the term "polypeptide variant" refers to molecules whose amino acid sequence differs from a native or reference sequence. Amino acid sequence variants may possess substitutions, deletions, and / or insertions at predetermined positions within the amino acid sequence compared to the native or reference sequence. Typically, variants possess at least about 50% identity (homology) to the native or reference sequence; preferably, they are at least about 80%, and more preferably at least about 90% identical (homological) to the native or reference sequence.

[0127] In some embodiments, "variant mimetics" are provided. As used herein, the term "variant mimetics" refers to variants containing one or more amino acids that will mimic an activation sequence. For example, glutamate can function as a mimic for phospho-threonine and / or phospho-serine. Alternatively, variant mimetics can result in deactivation or inactivated products containing the mimic. For example, phenylalanine can act as an inactivating substitution for tyrosine; or alanine can act as an inactivating substitution for serine. The amino acid sequences of the pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) of the present disclosure can include naturally occurring amino acids and, as such, can be considered proteins, peptides, polypeptides, or fragments thereof. Alternatively, the pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) can include both naturally occurring and non-naturally occurring amino acids.

[0128] As used herein, the term "amino acid sequence variant" refers to molecules that have some differences in their amino acid sequence compared to a native or starting sequence. Amino acid sequence variants may possess substitutions, deletions, and / or insertions at certain positions within the amino acid sequence. As used herein, the terms "native" or "starting," when referring to a sequence, are relative terms that refer to the original molecule to which a comparison can be made. A native or starting sequence should not be confused with a wild-type sequence. A native sequence or molecule can represent the wild type (the sequence found in nature), but need not be identical to the wild-type sequence.

[0129] Ordinarily, variants will retain at least about 70% homology to the native sequence; preferably, they will be at least about 80%, more preferably at least about 90% homologous to the native sequence.

[0130] 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 identical to the residues in the amino acid sequence of a second sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum homology. Methods and computer programs for alignment are well known in the art. It is understood that homology depends on the calculation of the percentage of identity, but the value may vary due to gaps and penalties introduced in the calculation.

[0131] As used herein, the term "homolog" when applied to an amino acid sequence means a corresponding sequence in another species that has substantial identity to a second sequence in the second species.

[0132] As used herein, the term "analog" is meant to include polypeptide variants that differ by one or more amino acid changes, e.g., substitutions, additions, or deletions of amino acid residues, that still retain the properties of the parent polypeptide.

[0133] As used herein, the term "derivative" is used interchangeably with the term "variant" and refers to a molecule that is modified or changed in any way relative to a reference or starting molecule.

[0134] The present disclosure contemplates several types of pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) that are amino acid-based, including variants and derivatives. These include substitution, insertion, deletion, and covalent variants and derivatives. Thus, pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) that contain substitutions, insertions, additions, deletions, and / or covalent modifications are within the scope of the present disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the present disclosure (e.g., at the N- or C-terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or enable biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to provide a truncated sequence. Certain amino acids (e.g., C-terminal or N-terminal residues) can alternatively be deleted, depending on the use of the sequence, e.g., expression of the sequence as part of a larger sequence that is soluble or linked to a solid support.

[0135] "Substitutional variants," when referring to proteins, are those in which at least one amino acid residue in a native or starting sequence has been removed and a different amino acid inserted in its place at the same position. The substitutions can be simple, where only one amino acid in the molecule has been substituted, or they can be multiple, where two or more amino acids have been substituted in the same molecule.

[0136] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with an amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue, such as isoleucine, valine, and leucine, for another non-polar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, the substitution of a basic residue, such as lysine, arginine, or histidine, for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid, for another, are additional examples of conservative substitutions. Examples of non-conservative substitutions include substitution of a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, with a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, methionine, and / or substitution of a non-polar residue with a polar residue.

[0137] As used herein, the term "insertion variant," when referring to a protein, is one that has one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. As used herein, the term "immediately adjacent" refers to adjacent amino acids connected to either the alpha-carboxy or alpha-amino functionality of the starting or reference amino acid.

[0138] As used herein, the term "deletion variant," when referring to a protein, refers to one in which one or more amino acids in the native or starting amino acid sequence have been removed. Typically, deletion variants have one or more amino acids deleted in a particular region of the molecule.

[0139] As used herein, the term "derivative," as referred to herein, includes variants of a native or starting protein containing one or more modifications with organic proteinaceous or non-proteinaceous derivatizing agents, as well as post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of a protein with organic derivatizing agents capable of reacting with selected side chains or terminal residues, or by utilizing post-translational modification mechanisms operative in a selected recombinant host cell. The resulting covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for preparing anti-protein antibodies for immunoaffinity purification of recombinant glycoproteins. Such modifications are within the ordinary skill of those in the art and are performed without undue experimentation.

[0140] As used herein, the term "site," when referring to amino acid-based embodiments, is used interchangeably with "amino acid residue" and "amino acid side chain." Sites represent locations within a peptide or polypeptide that can be modified, manipulated, changed, derivatized, or altered in the polypeptide-based molecules of the present disclosure.

[0141] As used herein, the term "terminus" or "terminus," when referring to a protein, refers to the end of a peptide or polypeptide. Such ends are not limited to only the first or last position of a peptide or polypeptide, but may include additional amino acids in the terminal region. Polypeptide-based molecules of the present disclosure may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)).

[0142] The polypeptides or proteins of the present disclosure are sometimes composed of multiple polypeptide chains (multimers, oligomers) held together by disulfide bonds or non-covalent forces. These types of proteins have multiple N- and C-termini. Alternatively, the ends of the polypeptides can optionally be modified to begin or end with non-polypeptide-based moieties, such as organic conjugates.

[0143] Once any of the features have been identified or defined as components of the disclosed biological circuit system components, stimulators, or effector modules (including SREs or payloads), manipulation and / or modification of some of these features can be performed by moving, swapping, inverting, deleting, randomizing, or duplicating. Furthermore, it is understood that manipulation of features can produce the same outcome as modifications to the disclosed compositions. For example, manipulations including deletion of domains will result in a change in the length of the molecule, just as modifying a nucleic acid to encode less than a full-length molecule would.

[0144] Modification and manipulation can be accomplished by methods known in the art, such as site-directed mutagenesis, and the resulting modified molecules can then be tested for activity using in vitro or in vivo assays, such as those described herein, or any other suitable screening assay known in the art.

[0145] In some embodiments, compositions of the present disclosure may contain one or more atoms that are isotopes. As used herein, the term "isotope" refers to a chemical element that has one or more additional neutrons. In some embodiments, compounds of the present disclosure may be deuterated. As used herein, the term "deuterated" refers to the process of replacing one or more hydrogen atoms in a substance with a deuterium isotope. A deuterium isotope is an isotope of hydrogen. A hydrogen nucleus contains one proton, whereas a deuterium nucleus contains both a proton and a neutron. Pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) of the present disclosure may be deuterated to alter one or more physical properties, such as stability, or to enable the pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) to be used in diagnostic and / or experimental applications.

[0146] At the protein level, any of the biological circuit components can contain one or more post-translational modifications (PTMs), which can occur intracellularly following administration of a protein-based biological circuit component or during or after translation of a biological circuit component administered as a nucleic acid encoding said biological circuit component.

[0147] Post-translational modifications (PTMs) of the present disclosure include, but are not limited to, acetylation, phosphorylation, ubiquitination, carboxylation, deamidation, deamination, deacetylation, dihydroxylation, dephosphorylation, formylation, gamma-carboxyglutamation, glutathionylation, glycation, hydroxylation, methylation, nitration, sumoylation, N- or O-transglutamation, glycosylation, and farnesylation.

[0148] Effector modules (including their SREs and payloads) can independently have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more PTMs that are the same or different.

[0149] Effector modules can be designed to contain one or more structural or functional domains, repeats, or motifs from protein families, which are categorized by protein family, and representative families are provided in the EMBL-EBI database at http: / / www.ebi.ac.uk / .

[0150] In some embodiments, protein modifications that modify the structure of the compositions of the present disclosure to prevent antigen processing and peptide loading, such as glycosylation and PEGylation, may also be useful in the present disclosure. The compositions of the present disclosure may also be modified to include non-classical amino acid side chains to design compositions with lower immunogenicity. Any of the methods discussed in International Patent Publication No. WO2005051975 (the contents of which are incorporated by reference in their entirety) for reducing immunogenicity may be useful in the present disclosure.

[0151] The SRE can be, but is not limited to, a peptide, peptide conjugate, peptide-protein conjugate, protein, fusion protein, protein conjugate, or protein-protein conjugate. The SRE can include one or more regions derived from any native or mutant protein or antibody. In this embodiment, the SRE is an element that can regulate the intracellular localization, intramolecular activation, and / or degradation of the payload in response to a stimulus.

[0152] In some embodiments, the effector modules of the present disclosure may include additional features that facilitate expression and control of the effector module, such as one or more signal 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 linkers. The effector modules of the present disclosure may also include other regulatory sites, such as an inducible promoter, an enhancer sequence, a microRNA site, and / or a microRNA targeting site. Each aspect or regulated modality may confer different regulated characteristics to the effector module or biological circuit. For example, an SRE may represent a destabilizing domain, while a mutation in the protein payload may alter its cleavage site, dimerization properties, or half-life, and the inclusion of one or more microRNAs or microRNA-binding sites may affect cellular detargeting or trafficking characteristics. Consequently, the present disclosure encompasses biological circuits that are multifaceted in their protective potential. Such biological circuits can be modified to contain one, two, three, four or more regulated features.

[0153] In some embodiments, the effector module of the present disclosure may contain one or more degrons to regulate expression. As used herein, "degron" refers to the minimal sequence within a protein sufficient for recognition and degradation by the protein degradation system. An important property of degrons is that they are transposable, i.e., addition of a degron to a sequence results in degradation of the sequence. In some embodiments, a degron may be added to a destabilizing domain, a payload, or both. Incorporation of a degron within an effector module of the present disclosure may be used to confer additional protein instability to the effector module and minimize basal expression. In some embodiments, the degron may be an N-degron, a phosphodegron, a heat-inducible degron, a light-sensitive degron, or an oxygen-dependent degron. As a non-limiting example, a degron can be an ornithine decarboxylase degron as 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 by reference in their entireties. Other examples of degrons useful in the present disclosure include degrons described in International Patent Publication Nos. WO2017004022, WO2016210343, and WO2011062962, the contents of each of which are incorporated by reference in their entireties.

[0154] immunotherapy agents The biological circuits described herein may include an immunotherapeutic agent. In some embodiments, the payload of the present disclosure may be an immunotherapeutic agent that induces an immune response in an organism. The immunotherapeutic agent may be, but is not limited to, an antibody and fragments and variants thereof, a chimeric antigen receptor (CAR), a chimeric switch receptor, a cytokine, a chemokine, a cytokine receptor, a chemokine receptor, a cytokine-cytokine receptor fusion polypeptide, or any agent that induces an immune response. In one embodiment, the immunotherapeutic agent induces an anti-cancer immune response in a cell or a subject.

[0155] Cytokines, chemokines and other factors According to the present disclosure, the payloads of the present disclosure can be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells, and other cell types that can act as chemical communicators between cells and tissues in the body. These proteins mediate a wide range of physiological functions, from effects on cell growth, differentiation, migration, and survival to numerous effector activities. For example, activated T cells produce a variety of cytokines for cytotoxic function to eliminate tumor cells.

[0156] In some embodiments, the payload of the present disclosure may be a cytokine, including, but not limited to, an interleukin, tumor necrosis factor (TNF), interferon (IFN), TGF-beta, and a chemokine, as well as fragments, variants, analogs, and derivatives thereof. It is understood in the art that a given gene and / or protein nomenclature for the same gene or protein may or may not include punctuation marks such as a dash (-) or symbols such as Greek letters. Whether or not these are included herein does not mean that the meaning is altered, as understood by those skilled in the art. For example, IL2, IL2, and IL-2 refer to the same interleukin. Similarly, TNF-alpha, TNFα, TNF-alpha, TNF-α, TNF-alpha, and TNFα all refer to the same protein. Similarly, CD40L, CD40L, and CD40LG refer to the same protein.

[0157] In some embodiments, the payload of the present disclosure can be a cytokine that stimulates an immune response, hi other embodiments, the payload of the present disclosure can be an antagonist of a cytokine that negatively impacts an anti-cancer immune response.

[0158] In some embodiments, the payload of the present disclosure can be a cytokine receptor, a recombinant receptor, variants, analogs and derivatives thereof; or a signaling component of a cytokine.

[0159] In some embodiments, cytokines of the present disclosure can be utilized to improve the proliferation, survival, persistence, and efficacy of immune cells used for immunotherapy, such as CD8+ TEM cells, natural killer cells, and tumor-infiltrating lymphocyte (TIL) cells. In other embodiments, T cells modified with two or more DD-regulating cytokines are utilized to provide kinetic control of T cell activation and tumor microenvironment remodeling. In one aspect, the present disclosure provides biological circuits and compositions for minimizing the toxicity associated with cytokine therapy. Despite its success in alleviating tumor burden, systemic cytokine therapy often results in the development of severe, dose-limiting side effects. Two factors contribute to the observed toxicity: (a) cytokines are pleiotropic, affecting different cell types and sometimes producing opposing effects on the same cells depending on the context; and (b) cytokines have short serum half-lives, thus requiring administration at high doses to achieve therapeutic benefit, exacerbating the pleiotropic effects. In one aspect, cytokines of the present disclosure can be utilized to regulate cytokine expression in the event of adverse effects. In some embodiments, cytokines of the present disclosure can be engineered to have a prolonged lifespan or improved specificity to minimize toxicity.

[0160] In some embodiments, the payload of the present disclosure can be an interleukin (IL) cytokine. Interleukins (IL) are a class of glycoproteins produced by white blood cells to control immune responses. As used herein, the term "interleukin (IL)" refers to an interleukin polypeptide from any species or source, including full-length proteins as well as fragments or portions of proteins. In some embodiments, the interleukin payload is selected from the group consisting of IL1, IL1 alpha (also called hematopoietin-1), IL1 beta (catabolic), IL1 delta, IL1 epsilon, IL1 eta, IL1 zeta, interleukin-1 family members 1-11 (IL1F1-IL1F11), interleukin-1 homologs 1-4 (IL1H1-IL1H4), IL1 related proteins 1-3 (IL1RP1-IL1RP3), IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL10C, IL10D, IL11, IL11a, IL11b, IL12, IL IL13, IL14, IL15, IL16, IL17, IL17A, IL17B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL20-like (IL20L), IL21, IL22, IL23, IL23A, IL23-p19, IL23-p40, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36alpha, IL36beta, IL36gamma, IL36RN, IL37, IL37a, IL37b, IL37c, IL37d, IL37e, and IL38. In other aspects, the payload of the present disclosure can 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 aspects, the payload of the present disclosure can be a member of the TNF superfamily, including but not limited to TNF alpha, CD40L, lymphotoxin (LTA) alpha, LTA beta, and OX40L.

[0161] Antibodies and antibody fragments and variants The biological circuits described herein can include one or more of the antibodies described herein. In some embodiments, one or more of the antibodies described herein can be a payload.

[0162] In some embodiments, antibody fragments and variants may comprise antigen-binding regions from intact antibodies. Examples of antibody fragments and variants may include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as single-chain variable fragments (scFv); and multispecific antibodies formed from antibody fragments. The pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) of the present disclosure may comprise one or more of these fragments.

[0163] For purposes of this specification, an "antibody" may comprise heavy and light variable domains as well as an Fc region. As used herein, the term "native antibody" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons, typically composed of two identical light (L) chains and two identical heavy (H) chains linked to each other by disulfide bonds. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is juxtaposed with the first constant domain of the heavy chain, and the light chain variable domain is juxtaposed with the variable domain of the heavy chain.

[0164] As used herein, the term "variable domain" refers to a specific antibody domain found in both antibody heavy and light chains whose sequences vary widely among antibodies and are responsible for the binding and specificity of each particular antibody for its particular antigen. Variable domains include hypervariable regions. As used herein, the term "hypervariable region" refers to a region within a variable domain that contains amino acid residues that contribute to antigen binding. The amino acids present within the hypervariable region determine the structure of the complementarity-determining region (CDR), which becomes part of the antigen-binding site of the antibody. As used herein, the term "CDR" refers to a region of an antibody that contains a structure complementary to its target antigen or epitope. The other portion of the variable domain that does not interact with the antigen is referred to as the framework (FW) region. The antigen-binding site (also known as the antigen integration site or paratope) contains the amino acid residues necessary for interaction with a specific antigen.

[0165] The VH and VL domains each comprise three CDRs. The VL CDRs are referred to herein, in order of occurrence when moving from N-terminus to C-terminus along the variable domain polypeptide, as CDR-L1, CDR-L2, and CDR-L3. The VH CDRs are referred to herein, in order of occurrence when moving from N-terminus to C-terminus along the variable domain polypeptide, as CDR-H1, CDR-H2, and CDR-H3.

[0166] As used herein, the term "Fv" refers to an antibody fragment that contains the minimum fragment of an antibody required to form a complete antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. Fv fragments can be generated by proteolytic cleavage and are generally unstable. Recombinant methods for generating stable Fv fragments are known in the art and typically involve the insertion of a flexible linker between the light and heavy chain variable domains (to form a single-chain Fv (scFv)) or the introduction of a disulfide bridge between the heavy and light chain variable domains (Strohl, WR Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p. 46-47, the contents of which are incorporated herein by reference in their entirety).

[0167] As used herein, the term "light chain" refers to the component of an antibody from any vertebrate species that is assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of the constant domain. Depending on the amino acid sequence of the constant domain of their heavy chain, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0168] As used herein, the term "single-chain Fv" or "scFv" refers to a fusion protein of VH and VL antibody domains joined together by a flexible peptide linker in a single polypeptide chain. In some embodiments, the Fv polypeptide linker allows the scFv to form the desired structure for antigen binding. In some embodiments, scFvs are utilized in conjunction with phage display, yeast display, or other display methods, where they are expressed in association with surface members (e.g., phage coat proteins) and can be used to identify high-affinity peptides against a given antigen.

[0169] Using molecular genetics, two scFvs can be tandemly modified into a single polypeptide separated by a linker domain, termed a "tandem scFv" (tascFv). Constructing a tascFv using genes for two different scFvs generates a "bispecific single-chain variable fragment" (bis-scFv). Only two tascFvs have been developed by commercial companies; both are bispecific agents under ongoing early-stage development by Micromet for cancer indications and are described as "bispecific T-cell engagers (BiTEs)." Blinatumomab is an anti-CD19 / anti-CD3 bispecific tascFv in phase 2 trials that enhances T-cell responses against B-cell non-Hodgkin's lymphoma. MT110 is an anti-EP-CAM / anti-CD3 bispecific tascFv in phase 1 trials that enhances T-cell responses against solid tumors. Bispecific tetravalent "TandAbs" have also been investigated by Affimed (Nelson, AL, MAbs., 2010, Jan-Feb;2(1):77-83). Maxibodies (bivalent scFvs fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG) may also be included.

[0170] As used herein, the term "bispecific antibody" refers to an antibody capable of binding to two different antigens. Such antibodies typically contain regions from at least two different antibodies. Bispecific antibodies may include any of those described in 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 each of which are incorporated herein by reference in their entirety).

[0171] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites. Diabodies are functional bispecific single-chain antibodies (bscAbs). Diabodies comprise a heavy chain variable domain VH connected to a light chain variable domain VL on the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and form two antigen-binding sites.

[0172] The term "intrabody" refers to a form of antibody that is not secreted from the cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies can be used to affect numerous cellular processes, including, but not limited to, intracellular trafficking, transcription, translation, metabolic processes, growth signaling, and cell division. In some embodiments, the methods of the present disclosure can involve intrabody-based therapy. In some such embodiments, the variable domain sequences and / or CDR sequences disclosed herein can be incorporated into one or more constructs for intrabody-based therapy.

[0173] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous cells (or clones). That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that may arise during monoclonal antibody generation (such variants are usually present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0174] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As used herein, monoclonal antibodies also include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies.

[0175] As used herein, the term "humanized antibody" refers to a chimeric antibody that contains minimal portions from one or more non-human (e.g., murine) antibody source(s), with the remainder derived from one or more human immunoglobulin sources. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient antibody are replaced by hypervariable region residues from an antibody of a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and / or capacity. In one embodiment, the antibody can be a humanized full-length antibody. As a non-limiting example, the antibody may 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.

[0176] As used herein, the term "antibody variant" refers to an antibody that has been modified (relative to the native or starting antibody) or a biomolecule (e.g., an antibody mimetic) that resembles the native or starting antibody in structure and / or function. Antibody variants may be altered in their amino acid sequence, composition, or structure compared to the native antibody. 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.

[0177] In some embodiments, the pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) of the present disclosure may be antibody mimics. As used herein, the term "antibody mimic" refers to any molecule that mimics the function or effect of an antibody and binds specifically and with high affinity to its molecular target. In some embodiments, the antibody mimic may be a monobody designed to incorporate a fibronectin type III domain (Fn3) as a protein scaffold (US Pat. No. 6,673,901; US ​​Pat. No. 6,348,584). In some embodiments, the antibody mimic may be any known in the art, including, but not limited to, affibody molecules, affilins, affitins, anticalins, avimers, centyrins, DARPINST™, Fynomers, and Kunitz domain peptides. In other embodiments, the antibody mimic may include one or more non-peptide regions.

[0178] In one embodiment, the antibody may comprise a modified Fc region. By way of non-limiting example, the modified Fc region may be produced by the methods described in U.S. Patent Publication No. US20150065690, the contents of which are incorporated herein by reference in their entirety, or may be any of the regions described therein.

[0179] In some embodiments, the payload of the present disclosure may encode a multispecific antibody that binds to multiple epitopes. 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 of 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 each of which are incorporated herein by reference in their entirety). These antibodies can also bind to multiple antigens with high specificity and high affinity.

[0180] In certain embodiments, a multispecific antibody is a "bispecific antibody" that recognizes two different epitopes on the same or different antigens. In one aspect, a bispecific antibody is capable of binding to two different antigens. Such an antibody typically comprises antigen-binding regions from at least two different antibodies. For example, a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies, allowing the BsAb to bind to two different types of antigens. Bispecific antibody frameworks include those described in Riethmuller, G., 2012. Cancer Immunity, 2012, 12:12-18; Marvin et al. al., Acta Pharmacologica Sinica. 2005, 26(6):649-658; and Schaefer et al., PNAS. 2011, 108(27):11187-11192 (the contents of each of which are incorporated herein by reference in their entirety). A new generation of BsMAbs called "trifunctional bispecific" antibodies has been developed. These consist of two heavy chains and two light chains, each derived from two different antibodies, with two Fab regions (arms) directed against two antigens and an Fc region (foot) containing the two heavy chains and forming a third binding site.

[0181] In some embodiments, the payload may encode an antibody comprising a single antigen-binding domain. These molecules are extremely small, with molecular weights approximately one-tenth that observed in full-size mAbs. Additional antibodies may include "nanobodies," which are derived from the antigen-binding variable heavy chain regions (VHHs) of heavy-chain antibodies found in camels and llamas, lacking light chains (Nelson, AL, MAbs. 2010. Jan-Feb;2(1):77-83).

[0182] In some embodiments, antibodies can be "miniaturized." The best examples of miniaturized mAbs include the small modular immunopharmaceuticals (SMIPs) from Trubion Pharmaceuticals. These molecules, which can be monovalent or bivalent, are recombinant single-chain molecules containing one VL, one VH antigen-binding domain, and one or two constant "effector" domains, all connected by a linker domain. Potentially, such molecules could offer the advantage of increased tissue or tumor penetration claimed by fragments while retaining the effector immune function provided by the constant domains. At least three "miniaturized" SMIPs are in clinical development.

[0183] An example of a miniaturized antibody is called a "unibody" in which the hinge region has been removed from an IgG4 molecule. Although IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with each other, deletion of the hinge region completely prevents heavy-heavy chain pairing, leaving highly specific monovalent light / heavy heterodimers while retaining the Fc region to ensure stability and half-life in vivo.

[0184] In some embodiments, the payload of the present disclosure may encode a single domain antibody (sdAb, or nanobody), which is an antibody fragment consisting of a single monomeric variable antibody domain. Like a complete antibody, it can selectively bind to a specific antigen. In one aspect, the sdAb may be a "camelid Ig" or "camelid VHH." As used herein, the term "camelid Ig" refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolté, et al., FASEB J., 2007, 21:3490-3498). A "heavy chain antibody" or "camelid antibody" refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al, J. Immunol. Methods, 1999, 231:25-38; International Patent Publication Nos. WO1994 / 04678 and WO1994 / 025591; and U.S. Patent No. 6,005,079). In another aspect, the sdAb may be an "immunoglobulin neoantigen receptor" (IgNAR). As used herein, the term "immunoglobulin neoantigen receptor" refers to a class of antibodies from the shark immune repertoire that consist of a homodimer of one variable neoantigen receptor (VNAR) domain and five constant neoantigen receptor (CNAR) domains.

[0185] In some embodiments, the payload of the present disclosure may encode an intrabody. Intrabodies are a form of antibody that are not secreted from the cell in which they are produced, but instead target one or more intracellular proteins. Intrabodies are expressed and function intracellularly and can be used to affect numerous cellular processes, including, but not limited to, intracellular trafficking, transcription, translation, metabolic processes, growth signaling, and cell division. In some embodiments, the methods described herein include intrabody-based therapy. In some such embodiments, the variable domain sequences and / or CDR sequences disclosed herein are incorporated into one or more constructs for intrabody-based therapy. For example, an intrabody may target one or more glycosylated intracellular proteins or modulate the interaction between one or more glycosylated intracellular proteins and alternative proteins.

[0186] In some embodiments, the payload of the present disclosure may encode a biosynthetic antibody as described in U.S. Patent No. 5,091,513, the contents of which are incorporated herein by reference in their entirety. Such antibodies may contain one or more sequences of amino acids that constitute a region that behaves as a biosynthetic antibody binding site (BABS). The site may include: 1) noncovalently associated or disulfide-linked synthetic VH and VL dimers; 2) VH-VL or VL-VH single chains in which the VH and VL are linked by a polypeptide linker; or 3) individual VH or VL domains. A binding domain comprises linked CDR and FR regions, which may be derived from separate immunoglobulins. Biosynthetic antibodies may also include other polypeptide sequences that function, for example, as an enzyme, a toxin, a binding site, or a binding site for a solidified medium or a radioactive atom. Methods are disclosed for generating biosynthetic antibodies, for designing BABS with any specificity that can be elicited by in vivo generation of antibodies, and for generating their analogs.

[0187] In some embodiments, the payload may encode an antibody having an antibody acceptor framework as taught in U.S. Patent No. 8,399,625. Such an antibody acceptor framework may be particularly well suited to accept CDRs from the antibody of interest.

[0188] In one embodiment, the antibody can be a conditionally active biological protein. The antibody can be used to generate a conditionally active biological protein that is reversibly or irreversibly inactivated under normal wild-type physiological conditions, and uses of such conditionally active biological proteins are provided. Such methods and conditionally active proteins are taught, for example, in International Publication Nos. WO2015175375 and WO2016036916 and U.S. Patent Publication No. US20140378660 (the contents of each of which are incorporated herein by reference in their entirety).

[0189] Antibodies used in immunotherapy In some embodiments, the payload of the present disclosure can be antibodies, fragments, and variants thereof specific for tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). Antibodies circulate throughout the body until they find and bind to TSAs / TAAs. Upon binding, they recruit other components of the immune system, increasing 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 elicit an anti-tumor immune response in the host organism. In one embodiment, a TSA can be a tumor neoantigen. Tumor antigen-specific antibodies mediate complement-dependent cytotoxicity against tumor cells expressing the same antigen.

[0190] In some embodiments, tumor-specific antigens (TSAs), tumor-associated antigens (TAAs), pathogen-associated antigens, or fragments thereof, can be expressed as peptides or as intact proteins or portions thereof. The intact proteins or portions thereof can be native or mutated. The cancer antigens associated with cancer or virus-induced cancer described herein are well known in the art. Such TSAs or TAAs may have previously been associated with cancer or may be identified by any method known in the art.

[0191] In one embodiment, 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 proliferation of B cell populations upon B cell receptor activation. CD19 is widely expressed on both normal and neoplastic B cells. B cell-derived malignancies, such as chronic lymphocytic leukemia, acute lymphocytic leukemia, and many non-Hodgkin's lymphomas, frequently retain CD19 expression. This near-ubiquitous expression and specificity for a single cell lineage makes 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 of CD19, and exons 6-14 encoding the cytoplasmic tail.

[0192] In one embodiment, the payload of the present disclosure may be an antibody, fragment or variant thereof specific for the CD19 antigen.

[0193] In some embodiments, the immunotherapeutic agent can be an antibody that is specifically immunoreactive with an antigen selected from a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), or an antigenic epitope.

[0194] In one aspect, the antigen can be an antigenic epitope. In some embodiments, the antigenic epitope can be CD19.

[0195] Tumor-specific antigens (TSAs) can be tumor neoantigens. Neoantigens are mutated antibodies expressed only by tumor cells due to genetic mutations or alterations in transcription that alter the protein coding sequence, thus generating new, foreign antigens. The genetic alterations result from gene replacement, insertion, deletion, or any other genetic alteration of the native cognate protein (i.e., a molecule expressed in normal cells).

[0196] Chimeric antigen receptor (CAR) The biological circuits described herein can include chimeric antigen receptors. In some embodiments, the payload of the present disclosure can be a chimeric antigen receptor (CAR) that, when transduced into immune cells (e.g., T cells and NK cells), can redirect the immune cells to targets (e.g., tumor cells) that express a molecule recognized by the extracellular targeting moiety of the CAR.

[0197] As used herein, the term "chimeric antigen receptor (CAR)" refers to a synthetic receptor that mimics the TCR on the surface of a T cell. Typically, a CAR is composed of an extracellular targeting domain, a transmembrane domain / region, and an intracellular signaling / activation domain. In a standard CAR receptor, the components: the extracellular targeting domain, the transmembrane domain, and the intracellular signaling / activation domain are linearly assembled into a single fusion protein. The extracellular region contains a targeting domain / moiety (e.g., scFv) that recognizes a specific tumor antigen or other tumor cell-surface molecule. The intracellular region may contain a signaling domain of the TCR complex (e.g., the signaling region of CD3ζ) and / or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137), and OX-40 (CD134). For example, "first-generation CARs" have only the CD3ζ signaling domain. In an effort to enhance T cell persistence and proliferation, a costimulatory intracellular domain has been added, resulting in the emergence of second-generation CARs, which have one costimulatory signaling domain in addition to the CD3ζ signaling domain, and third-generation CARs, which have two or more costimulatory signaling domains in addition to the CD3ζ signaling domain. When expressed by a T cell, a CAR generates a T cell with antigen specificity determined by the extracellular targeting site of the CAR. Recently, there has also been a desire to develop more effective and safer CAR structures by adding one or more elements, such as homing and suicide genes, which are referred to as fourth-generation CARs.

[0198] In some embodiments, the effector module immunotherapeutic agent is a chimeric antigen receptor (CAR). A chimeric antigen may include an extracellular targeting site, a transmembrane domain, an intracellular signaling domain, and, optionally, one or more costimulatory domains.

[0199] In some embodiments, the extracellular targeting domain is joined to the intracellular signaling domain via a hinge (also referred to as a spacer or spacer) and a transmembrane region. The hinge connects the extracellular targeting domain to the transmembrane domain, which crosses the cell membrane and connects to the intracellular signaling domain. Depending on the size of the target protein to which the targeting moiety binds, as well as the size and affinity of the targeting domain itself, the hinge may need to be altered to optimize the efficacy of CAR-expressing cells against cancer cells. Once the targeting moiety recognizes and binds to the target cell, the intracellular signaling domain provides an activation signal for the CAR T cell, which is further amplified by a "second signal" from one or more intracellular costimulatory domains. Once activated, the CAR T cell can destroy the target cell.

[0200] In some embodiments, the CARs of the present disclosure can be split into two parts, each linked to a dimerization domain, such that a dimerization-inducing input promotes assembly of an intact, functional receptor. Wu and Lim recently reported a split CAR in which the extracellular CD19-binding domain and intracellular signaling elements are separated, and the FKBP domain and FRB domain heterodimerize in the presence of the rapamycin analog AP21967. * The split receptor is linked to the FKBP-rapamycin binding domain (T2089L mutant). In the presence of AP21967, the split receptor assembles and activates T cells upon specific antigen binding (Wu et al., Science, 2015, 625(6258):aab4077).

[0201] In some embodiments, the CAR of the present disclosure can be designed as an inducible CAR. Sakemura et al. recently reported the incorporation of a Tet-On inducible system into 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 have equivalent cytotoxicity against CD19+ cell lines and equivalent cytokine production and proliferation upon CD19 stimulation compared to conventional CD19CAR T cells (Sakemura et al., Cancer Immuno. Res., 2016, Jun 21, Epub ahead of print). In one example, this Tet-CAR can be a payload for an effector module under the control of the SRE (e.g., DD) of the present disclosure. The dual system provides greater flexibility for turning CAR expression on and off in transduced T cells.

[0202] According to the present disclosure, the payload of the present disclosure can be a first-generation CAR, or a second-generation CAR, or a third-generation CAR, or a fourth-generation CAR. In some embodiments, the payload of the present disclosure can be a complete CAR construct consisting of an extracellular domain, a hinge and transmembrane domain, and an intracellular signaling region. In other embodiments, the payload of the present disclosure can be a component of a complete CAR construct including an extracellular targeting moiety, a hinge region, a transmembrane domain, an intracellular signaling domain, one or more costimulatory domains, and other additional elements that improve CAR structure and functionality, including, but not limited to, a leader sequence, a homing element, and a safety switch, or a combination of such components.

[0203] CARs controlled by the disclosed biological circuits and compositions are tunable, thereby offering several advantages. A reversible on-off switch mechanism allows for management of acute toxicity caused by excessive CAR-T cell proliferation. Pulsatile CAR expression using the disclosed SREs can be achieved by circulating ligand levels. Ligand-mediated CAR control can be effective in counteracting tumor escape induced by antigen loss, avoiding functional attrition caused by tonic signaling due to chronic antigen exposure, and improving the persistence of CAR-expressing cells in vivo.

[0204] In some embodiments, the biological circuits and compositions of the present disclosure can be utilized to downregulate CAR expression to limit on-target tissue toxicity caused by tumor lysis syndrome. Downregulating expression of a CAR of the present disclosure after anti-tumor efficacy can prevent (1) on-target off-tumor toxicity caused by antigen expression in normal tissues, and (2) antigen-independent activation in vivo.

[0205] Extracellular targeting domains / sites According to the present disclosure, the extracellular targeting moiety of a CAR can be any agent that recognizes and binds with high specificity and affinity to a given target molecule, e.g., a neoantigen on a tumor cell. The targeting moiety can be an antibody and variants thereof that specifically bind to a target molecule on a tumor cell, or a peptide aptamer selected from a random sequence pool based on its ability to bind to a target molecule on a tumor cell, or a variant or fragment thereof that can bind to a target molecule on a tumor cell, or an antigen recognition domain from a native T cell receptor (TCR) (e.g., the CD4 extracellular domain that recognizes HIV-infected cells), or a foreign recognition component, e.g., a tethered cytokine that results in recognition of a target cell bearing the cytokine receptor, or the receptor's natural ligand.

[0206] In some embodiments, the targeting domain of the CAR can be an Ig NAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, an Fv, a single-chain variable fragment (scFv), a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a unibody, a nanobody, or an antigen-binding region derived from an antibody that specifically recognizes a target molecule, such as a tumor-specific antigen (TSA). In one embodiment, the targeting moiety is an scFv. The scFv domain is expressed on the surface of the CAR T cell and, when subsequently bound to a target protein on the cancer cell, can maintain the CAR T cell in the vicinity of the cancer cell and induce T cell activation. scFvs can be generated using conventional recombinant DNA technology techniques and are discussed in this disclosure.

[0207] In one embodiment, the extracellular targeting moiety can be an scFv derived from an antibody. In one embodiment, the scFv can specifically bind to the CD19 antigen.

[0208] Intracellular signaling domains After binding to its target molecule, the intracellular domain of the CAR fusion polypeptide transmits a signal to the effector immune cell and activates at least one of the effector immune cell's normal effector functions, including cytolytic activity (e.g., cytokine secretion) or helper activity. Thus, the intracellular domain comprises the "intracellular signaling domain" of the T cell receptor (TCR).

[0209] In some embodiments, the entire intracellular signaling domain can be used, while in other embodiments, truncated portions of the intracellular signaling domain can be used in place of the intact chain, so long as they transmit the effector function signal.

[0210] In some embodiments, the intracellular signaling domain of the present disclosure can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one example, the intracellular signaling domain is a CD3 zeta (CD3ζ) signaling domain.

[0211] In some embodiments, the intracellular region of the present disclosure further comprises one or more costimulatory signaling domains that provide additional signals to effector immune cells. These costimulatory signaling domains combined with the signaling domains can further improve the proliferation, activation, memory, persistence, and tumor eradication efficiency of CAR-modified immune cells (e.g., CAR T cells). In some cases, the costimulatory signaling region contains one, two, three, or four cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules. The costimulatory signaling domains can be the intracellular / cytoplasmic domains of costimulatory molecules, 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 costimulatory signaling domain is derived from the cytoplasmic domain of CD28. In another example, the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137). In another example, the costimulatory signaling domain can 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.

[0212] In some embodiments, the intracellular signaling domains disclosed in International Patent Publication WO2014153270 may be useful in the present disclosure.

[0213] In some embodiments, the chimeric antigen receptors described herein can comprise a CD3 zeta domain altered to modulate CAR activity. The CD3 zeta domain can comprise one or more mutations in immunoreceptor tyrosine-based activation motifs (ITAMs). In one aspect, tyrosine residues within an ITAM can be mutated, resulting in reduced phosphorylation and limited downstream signaling. In some embodiments, one or more ITAMs can be deleted from the CD3 zeta domain. In one aspect, a CD3 zeta can comprise one ITAM. Any of the CAR and CD3 zeta domains described by Feucht et al. 2019 can be used herein (Calibration of CAR activation potential directs alternative T cell functions and therapeutic potency. Nature Medicine 25, 82-88 (2019) (the contents of which are incorporated herein by reference in their entirety)).

[0214] In some embodiments, a GITR costimulatory domain may be useful in the CARs described herein. In some embodiments, the GITR domain may be capable of inducing T cell effector function and activating T cells. In some aspects, the GITR domain described herein may be capable of suppressing inhibitory T regulatory cells that block immune responses. In some embodiments, GITR intracellular domain-containing CAR T cells may reduce cytokine production, which may alleviate cytokine release syndrome. Any of the GITR domains described in International Patent Publication WO2018045034 (the contents of which are incorporated herein by reference in their entirety).

[0215] Transmembrane domain and hinge region In some embodiments, the CAR of the present disclosure may comprise a transmembrane domain. As used herein, the term "transmembrane domain (TM)" broadly refers to an amino acid sequence of about 15 residues in length that spans the cell 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 cell membrane. In some embodiments, the transmembrane domain of the present disclosure may be derived from either natural or synthetic sources. The transmembrane domain of the CAR may be derived from any natural membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e., may include at least the transmembrane region(s) of) the alpha, beta, or zeta chain of the T cell receptor, CD3 epsilon, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, or CD154.

[0216] Alternatively, the transmembrane domains of the present disclosure may be synthetic. In some aspects, the synthetic sequences may comprise primarily hydrophobic residues, such as leucine and valine.

[0217] In some embodiments, the transmembrane domain of the present disclosure may be selected from the group consisting of a CD8α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, and an Fc region of human IgG4. As non-limiting examples, the transmembrane domain may be a CTLA-4 transmembrane domain comprising the amino acid sequences of SEQ ID NOS: 1-5 of International Patent Publication No. WO2014 / 100385; and a PD-1 transmembrane domain comprising the amino acid sequences of SEQ ID NOS: 6-8 of International Patent Publication No. WO2014100385, the contents of each of which are incorporated herein by reference in their entirety.

[0218] In some embodiments, the CARs of the present disclosure may comprise an optional hinge region (also referred to as a spacer). A hinge sequence is a short sequence of amino acids that facilitates flexibility of the extracellular targeting domain, moving the target binding domain away from the effector cell surface to allow proper cell / cell contact, target binding, and effector cell activation (Patel et al., 2002). (Et al., Gene Therapy, 1999;6:412-419). A hinge sequence can be positioned between the targeting moiety and the transmembrane domain.

[0219] In some embodiments, a CAR of the present disclosure may include one or more linkers between any of the domains of the CAR. The linkers may be 1 to 30 amino acids in length.

[0220] In some embodiments, components comprising the targeting moiety, transmembrane domain, and intracellular signaling domain of the present disclosure can be assembled into a single fusion polypeptide. The fusion polypeptide can be the payload of an effector module of the present disclosure. In some embodiments, multiple CAR fusion polypeptides can be included in an effector module, for example, two, three, or more CARs can be included in an effector module under the control of a single SRE (e.g., DD).

[0221] In one embodiment, the CAR construct comprises a CD19scFv (e.g., CAT13.1E10 or FMC63), a CD8α spacer or transmembrane domain, and a 4-1BB and CD3ζ endodomain. These constructs with CAT13.1E10 may have increased proliferation after in vitro stimulation, increased cytotoxicity against CD19+ targets, and increased effector-target interaction compared to constructs with FMC63.

[0222] In some embodiments, the payload of the present disclosure can be any of the costimulatory molecules and / or intracellular domains described herein. In some embodiments, one or more costimulatory molecules, each under the control of a different SRE, can be used in the present disclosure. SRE-controlled costimulatory molecules can also be expressed in conjunction with first-generation CARs, second-generation CARs, third-generation CARs, fourth-generation CARs, or any other CAR design described herein.

[0223] Tandem Car (TanCAR) In some embodiments, the CAR of the present disclosure may be a tandem chimeric antigen receptor (TanCAR) that can target two, three, four, or more tumor-specific antigens. In some aspects, the CAR is a bispecific TanCAR that includes two targeting domains that recognize two different TSAs on tumor cells. A bispecific CAR may be further defined as including an extracellular region that includes 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 that includes three or more targeting domains configured in a tandem arrangement. The spaces between targeting domains in a TanCAR can be between about 5 and about 30 amino acids in length, 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.

[0224] Split CAR In some embodiments, components comprising the targeting moiety, transmembrane domain, and intracellular signaling domain of the present disclosure can be split into two or more parts to rely on multiple inputs that promote assembly of an intact, functional receptor. In one embodiment, a split synthetic CAR system can be constructed in which assembly of an activated CAR receptor depends on binding of a ligand (e.g., a small molecule) to the SRE and binding of a specific antigen to the targeting moiety. As a non-limiting example, a split CAR consists of two parts that assemble in a small molecule-dependent manner, with one part of the receptor featuring an extracellular antigen-binding domain (e.g., scFv) and the other part having an intracellular signaling domain, e.g., a CD3ζ intracellular domain.

[0225] In other embodiments, the split part of the CAR system can be further modified to increase signal. In one example, the second part of the cytoplasmic fragment can be fixed to the cell membrane by incorporating a transmembrane domain (e.g., CD8α transmembrane domain) into the construct. Additional extracellular domains, such as extracellular domains that mediate homodimerization, can also be added to the second part of the CAR system. These modifications can increase receptor output activity, i.e., T cell activation.

[0226] In some embodiments, the two parts of the split CAR system contain heterodimerization domains that conditionally interact upon binding of a heterodimerization small molecule. In this way, the receptor components assemble in the presence of the small molecule to form an intact system, which can then be activated by antigen engagement. Any known heterodimerization component can be incorporated into the split CAR system. Other small molecule-dependent heterodimerization domains can also be used, including, but not limited to, the gibberellin-induced dimerization system (GID1-GAI), trimethoprim-SLF-induced ecDHFR and FKBP dimerization (Czlapinski et al., J Am Chem 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). Dual regulation using inducible assembly (e.g., ligand-dependent dimerization) and degradation (e.g., destabilization domain-induced CAR degradation) of split CAR systems may provide greater flexibility for controlling the activity of CAR-modified T cells.

[0227] Switchable Car In some embodiments, the CAR of the present disclosure may 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 switched on in response to a stimulus (e.g., a small molecule). In this CAR design, a system is directly integrated into the hinge domain that separates the scFv domain from the plasma membrane domain in the CAR. Such a system can separate or combine different important functions of the CAR, such as activation and costimulation, within different chains of the receptor complex, mimicking the complexity of the native structure of the TCR. This integrated system can switch the interaction between the scFv and antigen between on / off states controlled by the absence / presence of the stimulus.

[0228] Reversible CAR In another embodiment, the CAR of the present disclosure can be a reversible CAR system. In this CAR structure, a LID domain (ligand-induced degradation) is incorporated into the CAR system. The CAR can be temporarily downregulated by adding a ligand of the LID domain. The combination of LID- and DD-mediated regulation provides adjustable control of continuously activated CAR T cells, thereby reducing CAR-mediated tissue toxicity.

[0229] Activation-Conditional CAR In some embodiments, the payload of the present disclosure can be an activating conditional chimeric antigen receptor that is expressed only in activated immune cells. Expression of the CAR can be linked to a sequence, e.g., an activation conditional control region, which refers to one or more nucleic acid sequences that direct the transcription and / or expression of the CAR under its control. Such an activation conditional control region can be a promoter of a gene that is upregulated during activation of effector immune cells, e.g., an IL2 promoter or an NFAT binding site. In some embodiments, activation of immune cells can be achieved by a constitutively expressed CAR (International Publication No. WO2016126608, the contents of which are incorporated herein by reference in their entirety).

[0230] Polynucleotides 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 often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with a β-D-ribo configuration, α-LNA (a diastereomer of LNA) with an α-L-ribo configuration, 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-α-LNA with a 2′-amino functionalization), or hybrids thereof.

[0231] In some embodiments, the nucleic acid molecule is messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. The polynucleotide of the present disclosure can be mRNA or any nucleic acid molecule, and may or may not be chemically modified.

[0232] 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 on this wild-type modular structure, the present disclosure expands the scope of functionality of traditional mRNA molecules by providing payloads that maintain the modular organization but contain one or more structural and / or chemical modifications or alterations that confer useful properties to the polynucleotide, such as functional defensibility. As used herein, a "structural" feature or modification is one in which two or more linked nucleosides in a polynucleotide are inserted, deleted, duplicated, inverted, or randomized without significant chemical alteration to the nucleosides themselves. Because chemical bonds are necessarily broken and reformed to achieve the structural modification, the structural modification is chemical in nature and is therefore a chemical modification. However, the structural modification results in a different nucleotide sequence. 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" is inserted, resulting in a structural modification of the polynucleotide.

[0233] In some embodiments, polynucleotides of the present disclosure may possess a 5' UTR sequence that plays a role in translation initiation. The 5' UTR sequence may contain features, such as a Kozak sequence, commonly known to be involved in the process by which ribosomes initiate gene translation. The Kozak sequence has the consensus 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. Modifications of features typically found in abundantly expressed genes in target cells or tissues may improve the stability and protein production of polynucleotides of the present disclosure.

[0234] Additionally, polynucleotides are provided that may contain an internal ribosome entry site (IRES), which plays an important role in initiating protein synthesis in the absence of a 5' cap structure in the polynucleotide. The IRES may act as the sole ribosome binding site or may function as one of multiple binding sites. Polynucleotides of the present disclosure that contain multiple functional ribosome binding sites may encode several peptides or polypeptides that are independently translated by ribosomes, resulting in bicistronic and / or multicistronic nucleic acid molecules.

[0235] In one embodiment, the polynucleotides of the present disclosure can encode variant polypeptides having a predetermined identity with a reference polypeptide sequence. As used herein, "reference polypeptide sequence" refers to the starting polypeptide sequence. The reference sequence can be a wild-type sequence or any sequence that is referenced in the design of another sequence.

[0236] The term "identity," as known in the art, refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, identity also refers to the degree of sequence relatedness between sequences, as determined by the number of matches between two or more strings of residues (amino acids or nucleic acids). Identity measures the percentage of identical matches between two or more sequences, with gap alignments (if any) being addressed by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related sequences can be readily calculated by known methods. Such methods include Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1998; Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0237] In some embodiments, a variant sequence may have the same or similar activity as a reference sequence. Alternatively, a variant may have altered activity (e.g., increased or decreased) relative to the reference sequence. Typically, a variant of a particular polynucleotide or polypeptide of the present disclosure 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 that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. Such tools for alignment include the BLAST suite (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 These include database search programs, Nucleic Acids Res. 25:3389-3402).

[0238] Chemical modifications to polynucleotides According to the present disclosure, the term "modified" or, where appropriate, "modified" polynucleotide refers to modifications with respect to A, G, U (T in DNA) or C nucleotides.

[0239] Modifications to the polynucleotides of the present disclosure can be on the nucleoside base and / or sugar moieties of the nucleosides comprising the polynucleotide. In some embodiments, multiple modifications are included in the modified nucleic acid or one or more individual nucleosides or nucleotides. For example, a modification to a nucleoside can include one or more modifications to the nucleoside base and sugar. Modifications to the polynucleotides of the present disclosure can include, for example, any of those taught in International Publication WO2013052523 (the contents of which are incorporated herein by reference in their entirety).

[0240] As used herein, a "nucleoside" is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). As used herein, a "nucleotide" is defined as a nucleoside containing a phosphate group.

[0241] Modified nucleotides that can be incorporated into polynucleotides can be modified at the internucleoside linkage (e.g., the phosphate backbone). In the context of polynucleotide backbones, the terms "phosphate" and "phosphodiester" are used interchangeably herein. The phosphate backbone can be modified by replacing one or more of the oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can contain a bulk replacement of the unmodified phosphate site with a different internucleoside linkage. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphonoselenoates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphonodiamidites, alkyl or aryl phosphonates, and phosphotriesters. In phosphorodithioates, both non-linked oxygens are replaced by sulfur. Phosphate linkers can also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). Other modifications that may be used are taught, for example, in International Application WO2013052523, the contents of which are incorporated herein by reference in their entirety.

[0242] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in a polynucleotide. Those skilled in the art will appreciate that nucleotide analogs or other modification(s) can be placed at any position(s) of a polynucleotide so long as the function of the polynucleotide is not substantially impaired. Modifications can also be 5'- or 3'-terminal modifications. Polynucleotides may contain from about 1% to about 100% modified nucleotides (with respect to the overall nucleotide content, or with respect to one or more types of nucleotides, i.e., any one or more of A, G, U, or C) or any intervening percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 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%).

[0243] In some embodiments, the polynucleotide comprises modified pyrimidines or purines. In some embodiments, the pyrimidines or purines in the polynucleotide molecule are from about 1% to about 100% modified uracil or modified uridine (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50 ... 0%, 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% modified pyrimidines or purines).

[0244] In some embodiments, a polynucleotide may comprise two or more effector module component sequences that are repeated one, two, or more than three times in a pattern such as ABABAB or AABBABBAABB or ABCABCABC or variants thereof, where each letter A, B, or C in these patterns represents a different effector module component.

[0245] In yet another embodiment, the polynucleotide may contain two or more effector module component sequences, with each component having one or more sequences. As a non-limiting example, the sequence may be in a pattern such as ABABAB, or AABBABBAABB, or ABCABCABC, or a variant thereof, repeated once, twice, or more than three times in each region. As another non-limiting example, the sequence may be in a pattern such as ABABAB, or AABBABBAABB, or ABCABCABC, or a variant thereof, repeated once, twice, or more than three times throughout the polynucleotide. In these patterns, each letter A, B, or C represents a different sequence or component.

[0246] Codon Selection In some embodiments, one or more codons of the polynucleotides of the present disclosure can be replaced with other codons that encode the native amino acid sequence to modulate expression of the SRE, through a process referred to as codon selection. Because mRNA codon and tRNA anticodon pools tend to vary between organisms, cell types, subcellular locations, and over time, the codon selection described herein is spatiotemporal (ST) codon selection.

[0247] In some embodiments of the present disclosure, a predetermined polynucleotide feature may be codon-optimized. Codon optimization refers to the process of modifying a nucleic acid sequence for improved expression in a host cell by replacing at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons of the native sequence with the codons most frequently used in the host cell's genes while maintaining the native amino acid sequence. Codon usage may be measured using the codon compatibility index (CAI), which measures the deviation of a coding polynucleotide sequence from a reference gene set. Codon usage tables are available at the Codon Usage Database (http: / / www.kazusa.or.jp / codon / ), and CAI may be calculated using the EMBOSS CAI program (http: / / emboss.sourceforge.net / ). Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include matching the codon frequencies of the target and host organisms to ensure proper folding, biasing nucleotide content to alter stability or reduce secondary structure, minimizing tandem repeat codons or base runs that may impair gene assembly or gene expression, customizing transcriptional and translational control regions, inserting or removing protein signaling sequences, removing / adding sites for post-translational modification of the encoded protein (e.g., glycosylation sites), adding, removing, or shuffling protein domains, inserting or deleting restriction enzyme sites, modifying ribosome binding and degradation sites, adjusting translation rates to allow for proper folding of various domains of the protein, or reducing or eliminating problematic secondary structures within the polynucleotide. In one embodiment, a polynucleotide sequence, or portions thereof, can be codon-optimized using an optimization algorithm. Codon options for each amino acid are well known in the art, as can be found in tables for various species to optimize for expression in that particular species.

[0248] In some embodiments of the present disclosure, certain polynucleotide features may be codon-optimized. For example, preferred regions for codon optimization may be upstream (5') or downstream (3') of the polypeptide-encoding region. These regions may be incorporated into the polynucleotide before and / or after codon optimization of the payload-encoding region or open reading frame (ORF).

[0249] After optimization (if desired), the polynucleotide components are reconstituted and converted into vectors, including, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.

[0250] In some embodiments, certain regions of a polynucleotide may be preferred for codon selection. For example, preferred regions for codon selection 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 the codon selection of the payload-encoding region or open reading frame (ORF).

[0251] The stop codons of the polynucleotides of the present disclosure can be modified to include sequences and motifs to alter the expression levels of the SREs, payloads, and effector modules of the present disclosure. Such sequences can be incorporated to induce stop codon readthrough, where the stop codon can specify an amino acid, such as selenocysteine ​​or pyrrolysine. In other examples, the stop codon can be skipped entirely to resume translation via another open reading frame. Stop codon readthrough can be utilized to adjust the expression of the components of the effector module in a specific ratio (e.g., as indicated by the context of the stop codon). Examples of preferred stop codon motifs include UGAN, UAAN, and UAGN (where N is C or U).

[0252] Termination suppression occurs during translation of many viral mRNAs as a means of generating a second protein with an extended carboxy terminus. In retroviruses, the gag and pol genes are encoded by a single mRNA and separated by the amber stop codon UAG. Translational suppression of the amber codon allows synthesis of the gag pol precursor. Translational suppression is mediated by suppressor tRNAs that can recognize the stop codon and insert specific amino acids. In some embodiments, the effector modules described herein can incorporate an amber stop codon. Such codons can be used instead of or in addition to IRES and p2A sequences in bicistronic constructs. Stop codon readthrough can be combined with P2A to obtain low-level expression of downstream genes (e.g., IL12). In some embodiments, the amber stop codon can be combined with tRNA expression or amino-acyl-tRNA synthetases for further control. In one aspect, the payload can be a regulated tRNA synthetase.

[0253] Conjugates It is contemplated by the present disclosure that the compositions of the present 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 of structure or function, while "heterologous molecule" refers to a molecule that is different from a starting molecule in at least one of structure or function.Thus, a structural homolog is a molecule that can be substantially structurally similar.In some embodiments, such a homolog can be identical.A functional homolog is a molecule that can be substantially functionally similar.In some embodiments, such a homolog can be identical.

[0254] The pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) of the present disclosure may comprise conjugates. Such conjugates of the present disclosure may comprise 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, e.g., synthetic polyamino acids, or oligonucleotides (e.g., aptamers). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) 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, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0255] In some embodiments, the conjugate may also include a targeting group. As used herein, the term "targeting group" refers to a functional group or moiety attached to a drug that facilitates localization of the drug 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, e.g., kidney cells or other cell types). In some embodiments, the targeting group is

[0256] The polypeptide may include melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptidomimetic, or aptamer.

[0257] In some embodiments, the targeting group can be a protein, e.g., a glycoprotein, or a peptide, e.g., a molecule with a specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a specific cell type, e.g., cancer cells, endothelial cells, or bone cells. Targeting groups can also include hormones and / or hormone receptors.

[0258] In some embodiments, the targeting group can be any ligand capable of targeting a specific receptor. Examples include, but are not limited to, folic acid, 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 aptamers can be unmodified or can include any combination of modifications disclosed herein.

[0259] In still other embodiments, the pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) of the present disclosure may be covalently conjugated to cell-penetrating polypeptides. In some embodiments, the cell-penetrating peptide may also include a signal sequence. In some embodiments, the conjugates described herein may be designed to have increased stability, increased cell transfection, and / or altered biodistribution (e.g., targeted to specific tissues or cell types).

[0260] In some embodiments, conjugation moieties can be added to the pharmaceutical compositions, biological circuits, biological circuit components, and effector modules (including their SREs or payloads) of the present disclosure, allowing them to be attached with a detectable label to a target for clearance. Such detectable labels include, but are not limited to, biotin, ubiquitin, fluorescent molecules, human influenza hemagglutinin (HA), c-myc, histidine (His), Flag, glutathione S-transferase (GST), V5 (a paramyxovirus with a simian virus 5 epitope), biotin, avidin, streptavidin, horseradish peroxidase (HRP), and digoxigenin.

[0261] In some embodiments, the pharmaceutical compositions, biological circuits, biological circuit components, effector modules (including their SREs or payloads) of the present disclosure may be combined with each other or with other molecules in the treatment of diseases and / or pathologies.

[0262] Additional Effects Module Features Effector modules of the present disclosure may further comprise a signal sequence that controls distribution of the payload of interest, cleavage and / or processing features that facilitate cleavage of the payload from the effector module construct, targeting and / or permeability signals that may control cellular localization of the effector module, tags, and / or one or more linker sequences that link different components of the effector module.

[0263] In some embodiments, additional effector module features of the present disclosure include, but are not limited to, any of those taught in Table 7. [Table 7]

[0264] signal sequence In addition to the SRE and payload region, the effector modules of the present disclosure may further comprise one or more additional features, for example, one or more signal sequences.

[0265] Signal sequences (sometimes referred to as signal peptides, targeting signals, targeting peptides, localization sequences, transit peptides, leader sequences, or leader peptides) direct proteins (e.g., effector modules of the present disclosure) to their designated cellular and / or extracellular location. Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins.

[0266] Signal sequences are short (5-30 amino acids long) peptides present at the N-terminus of most newly synthesized proteins that direct them to specific locations. Signal sequences are recognized by the signal recognition particle (SRP) and can be cleaved using type I and type II signal peptide peptidases. Signal sequences from human proteins can be incorporated as regulatory modules of effector modules to direct the effector modules to specific cellular and / or extracellular locations. 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).

[0267] In some embodiments, the signal sequence may, but is not necessarily, located at the N- or C-terminus of the effector module and may, but is not necessarily, be cleaved from the desired effector module to generate the "mature" payload.

[0268] In some embodiments, the signal sequence used herein may omit the methionine at position 1 of the amino acid sequence of the signal sequence. This may be referred to as an M1del mutation.

[0269] In addition to naturally occurring signal sequences, such as those derived from secreted proteins, signal sequences can be modified variants of known signal sequences of proteins. For example, U.S. Patent Nos. 8,258,102 and 9,133,265 to Sleep disclose a modified albumin signal sequence with an additional X1-X2-X3-X4-X5-motif that can increase secretory signal and protein secretion; U.S. Patent No. 9,279,007 to Do discloses a modified fragment signal sequence of human immunoglobulin heavy chain binding protein (Bip) that can improve 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 be fused to a recombinant protein; the contents of each of these are incorporated by reference in their entirety.

[0270] In some examples, the secreted signal sequence can be a cytokine signal sequence, such as, but not limited to, an IL2 signal sequence or a p40 signal sequence.

[0271] In some instances, a signal sequence can be used to direct the payload of interest to the surface membrane of the target cell. Expression of the payload on the surface of the target cell can be useful for limiting the diffusion of the payload to non-target in vivo environments, potentially improving the safety profile of the payload. Furthermore, membrane presentation of the payload can enable physiological and quantitative signaling as well as stabilization and recycling of the payload for a longer half-life. The membrane sequence can be the endogenous signal sequence of the N-terminal component of the payload of interest. Optionally, it may be desirable to replace this sequence with a different signal sequence. Signal sequences can be selected based on their compatibility with the secretory pathway of the cell type of interest so that the payload is presented on the surface of T cells. In some embodiments, the signal sequence is an IgE signal sequence, a CD8a signal sequence (also referred to as the CD8a leader), an IL15Ra signal sequence (also referred to as the IL15Ra leader), or an M1del signal sequence. It may be the CD8a signal sequence (also called the M1del CD8 leader sequence).

[0272] Other signal sequence variants that may be used in the present effector modules may include U.S. Patent Application Publication No. 2007 / 0141666; PCT Patent Application Publication No. 1993 / 018181, the contents of each of which are incorporated herein by reference in their entirety.

[0273] Other examples of signal sequences include variants 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 each of which are incorporated herein by reference in their entirety.

[0274] In other examples, the signal sequence may be a heterologous signal sequence from other organisms, such as viruses, yeast and bacteria, which may direct the effector module to a specific cellular location, such as the nucleus (e.g., EP1209450). Other examples may include the aspartic protease (NSP24) signal sequence from Trichoderma (e.g., U.S. Patent No. 8,093,016 to Cervin and Kim), which may increase secretion of fusion proteins such as enzymes; bacterial lipoprotein signal sequences (e.g., PCT Application Publication No. WO199109952 to Lau and Rioux); E. coli enterotoxin II signal peptide (e.g., U.S. Patent No. 6,605,697 to Kwon et al.); E. coli secretion signal sequences (e.g., U.S. Patent Publication No. US2016090404 to Malley et al.); lipase signal sequences from methylotrophic yeast (e.g., U.S. Patent No. 8,975,041); and signal peptides for DNases from Corynebacterium bacteria (e.g., U.S. Patent No. 4,965,197), the contents of each of which are incorporated herein by reference in their entirety.

[0275] Signal sequences may also include nuclear localization signals (NLS), nuclear export signals (NES), polarized tubule-vesicle structure localization signals (see, e.g., U.S. Patent No. 8,993,742; Cour et al., Nucleic Acids Res. 2003, 31(1):393-396, the contents of each of which are incorporated herein by reference in their entirety), extracellular localization signals, signals for intracellular locations (e.g., lysosomes, endoplasmic reticulum, Golgi, mitochondria, plasma membrane, peroxisomes, etc.) (see, e.g., U.S. Patent No. 7,396,811; and Negi et al., Database, 2015, 1-7, the contents of each of which are incorporated herein by reference in their entirety).

[0276] Cutting site In some embodiments, the effector module comprises a cleavage and / or processing feature.

[0277] The effector module of the present disclosure may comprise at least one protein cleavage signal / site, which may be located at the N-terminus, the C-terminus, any space between the N-terminus and the C-terminus, including, but not limited to, midway between the N-terminus and the C-terminus, between the N-terminus and a midpoint, between a midpoint and the C-terminus, and combinations thereof.

[0278] The effector module may contain one or more cleavage signal(s) / site(s) of any proteinase. The proteinase may be a serine proteinase, a cysteine ​​proteinase, an endopeptidase, a dipeptidase, a metalloproteinase, a glutamic acid proteinase, a threonine proteinase, or an aspartic acid proteinase. In some embodiments, the cleavage site is 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, clostripain, chymase, chymotrypsin, elastase, endoproteinase, enterokinase, factor Xa, formate, granzyme B, matrix metallopeptidase-2, matrix metallopeptidase-3, pepsin, proteinase K, SUMO protease, subtilisin, TEV protease, thermolysin, thrombin, trypsin, and TAGZyme signal sequences.

[0279] tag In some embodiments, the effector module comprises a protein tag.

[0280] Protein tags can be used to detect and monitor the process of the effector module. The effector module can include one or more tags, for example, epitope tags (e.g., FLAG or hemagglutinin (HA) tags). Numerous protein tags can be used for the effector module, including self-labeling polypeptide tags (e.g., haloalkane dehalogenase (HaloTag2 or HaloTag7), 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 variants thereof), bioluminescent tags (e.g., luciferase and variants thereof), affinity tags (e.g., maltose binding protein (MBP) tags, glutamic acid ... These include, but are not limited to, guanine-S-transferase (GST) tags), immunogenic 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 (BCCP), eXact, CBP, CYD, HPC, CBD intein-chitin binding domain, Trx, NorpA, and NusA).

[0281] In other embodiments, tags 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 Publication Nos. US2013115635 and US2013012687; and International Application Publication No. WO2013091661, the contents of each of which are incorporated herein by reference in their entirety.

[0282] In some embodiments, multiple protein tags (the same or different tags) may be used; each of the tags may be located at the same N- or C-terminus, while in other cases the tags may be located at each end.

[0283] Linker In some embodiments, the effector module comprises a linker.

[0284] In some embodiments, the effector module of the present disclosure may further comprise a linker sequence. The linker region primarily functions as a spacer between two or more polypeptides within the effector module. As used herein, "linker" or "spacer" refers to a molecule or group of molecules that connects two molecules or two parts of a molecule, such as two domains of a recombinant protein.

[0285] In some embodiments, "linker" (L) or "linker domain" or "linker region" or "linker module" or "peptide linker," as used herein, refers to an oligo- or polypeptide region of about 1-100 amino acids in length that connects any of the domains / regions of an effector module to one another (also referred to as a peptide linker). Peptide linkers can be 1-40 amino acids in length, or 2-30 amino acids in length, or 20-80 amino acids in length, or 50-100 amino acids in length. Linker length can also be optimized based on the crystal structure of the payload, depending on the type of payload utilized. In some instances, shorter linker lengths may be preferred. In some embodiments, the peptide linker is composed of amino acids linked together by peptide bonds, preferably 1 to 20 amino acids linked by peptide bonds, selected from the 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). One or more of these amino acids may be glycosylated, as will be understood by those of skill in the art.

[0286] The linker sequence may be a natural linker derived from a multidomain protein, which is a short peptide sequence that separates two different domains or motifs within a protein.

[0287] In some aspects, a linker can be flexible or rigid. In other aspects, a linker can be cleavable or non-cleavable. As used herein, the terms "cleavable linker domain or region" or "cleavable peptide linker" are used interchangeably. In some embodiments, the linker sequence can be cleaved enzymatically and / or chemically.

[0288] The linker of the present disclosure may also be a non-peptide linker. For example, an alkyl linker such as -NH-(CH)C(O)- (a = 2 to 20) may be used. These alkyl linkers may be further substituted with any non-sterically hindering group, such as lower alkyl (e.g., C to C), lower acyl, halogen (e.g., Cl, Br), CN, NH, phenyl, etc.

[0289] Targeting or Penetrating Peptides In some embodiments, the effector module comprises a targeting and / or penetrating peptide.

[0290] Small targeting and / or penetrating peptides that selectively recognize cell surface markers (e.g., receptors, transmembrane proteins, and extracellular matrix molecules) can be used to target effector modules to desired organs, tissues, or cells. Short peptides (5-50 amino acid residues) synthesized in vitro and naturally occurring peptides, or analogs, variants, and derivatives thereof, can be incorporated into effector modules to home the effector modules to desired organs, tissues, and cells, and / or subcellular locations within cells.

[0291] In some embodiments, a targeting sequence and / or penetrating peptide can be included in the effector module to deliver the effector module to a target organ, tissue, or cell (e.g., a cancer cell). In other embodiments, the targeting and / or penetrating peptide can direct the effector module to a specific intracellular location within a cell. By way of non-limiting example, such targeting sequences and / or penetrating peptides can include those for targeting the effector module to a desired region of 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 each of which are incorporated herein by reference in their entirety.

[0292] In other embodiments, a targeting and / or penetrating peptide may direct the effector module to a specific subcellular location within a cell. By way of non-limiting example, a mitochondrial targeting peptide and / or a mitochondrial membrane-penetrating peptide may be included in the effector module to deliver the effector module to the mitochondria of a cell. See, e.g., 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 each of which are incorporated herein by reference in their entirety).

[0293] Targeting peptides can have any number of amino acids, from about 6 to about 30 (inclusive). Peptides can 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 can have 25 or fewer amino acids, e.g., 20 or fewer, e.g., 15 or fewer.

[0294] Naturally occurring small 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 trafficking sites. Such peptides can include peptide toxins from microorganisms, insects (e.g., scorpions, bees, spiders), animals (e.g., snakes), and plants, and their analogs, variants, and derivatives; as well as secreted peptide hormones, ligands, and signal peptides.

[0295] In some embodiments, analogs, variants, and derivatives derived from natural toxins that neutralize 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, a group of protein toxins produced and secreted by bacterial organisms, bind to mucosal (epithelial) cells of the intestinal wall. Enterotoxins can include, but are not limited to, E. coli heat-stable enterotoxin (ST), cholera toxin (CT), E. coli heat-labile enterotoxin (LT), Bordetella pertussis pertussis toxin (PT), Pseudomonas aeruginosa exotoxin A (ETA), Staphylococcus enterotoxin, diphtheria toxin from Corynebacterium diphtheria, and enterotoxin NSP4 from rotavirus. Other exotoxins include neurotoxins that affect the nervous system, cardiotoxins that affect the heart, Pseudomonas exotoxins, Botulinum neurotoxins, Shiga toxins, Shiga-like toxins 1 and 2, Clostridium difficile toxin, Clostridium perfringens epsilon toxin, and anthrax toxin.

[0296] In addition to exotoxins, other toxins include those isolated from plants, e.g., corn RIP, gelonin, firefly antiviral protein, saporin, trixanthin, ricin, abrin; scorpions, e.g., charybdotoxin; spiders, e.g., PcTx1; cone snails, e.g., PcTx1; sea anemones, e.g., gigantoxin 1; honeybees, e.g., Apis mellifera (Western or European or giant honeybee), Apis florea (small honeybee or small honeybee), Apis dorsata (giant honeybee), and Apis These include melittin, a group of water-soluble, cationic, amphipathic 26-amino acid alpha-helical peptides isolated from the venom of Apis cerana (the Oriental honeybee); and bombesin, originally isolated from toad skin, which binds to g-protein-coupled gastrin-releasing peptide receptors (e.g., BBR-1 / 2 / 3) in snake venom toxins, the gastric tract, and the brain. See, e.g., Suchanek, G., et al., PNAS (1978) 75:701-704, the contents of which are incorporated by reference in their entirety.

[0297] Peptide hormones and other signal peptides transmit important messages for cell-to-cell communication and selectively bind with high affinity to cells expressing their receptors. In some embodiments, peptide hormones can be included in effector modules. Such small peptide hormones and signal peptides include adiponectin, adipose-derived hormone, agouti signaling peptide, allatostatin, amylin, angiotensin, atrial natriuretic peptide, bomben-like peptide, big gastrin, betatrophin, bradykinin, calcitonin, corticotropin-releasing hormone, cosyntrophin, endothelin, enteroglucagon, FGF, FNDC5, follicle-stimulating hormone, gastrin, ghrelin, glucagon and glucagon-like peptides, gonadotropins, granulocyte colony-stimulating factor, growth hormone, growth hormone-releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, incretins, insulin, and insulin adenosine triphosphate (AGT). These may include, but are not limited to, analogs, insulin-like growth factors, leptin, mini-gastrin, liraglutide, luteinizing hormone, melanocortin, mini-gastrin, alpha-melanocyte-stimulating hormone, neuropeptide Y, nerve growth factor (NGF), neurotrophin-3 / 4, NPH insulin, orexin, obestatin, osteocalcin, pancreatic hormones, parathyroid hormone, peptide hormones, peptide YY, prolactin, preprohormones, relaxin, renin, salcatonin, somatostatin (SST), secretin, substance P, syncalide, teleost leptin, temporin, tesamorelin, thyroid-stimulating hormone, urocortin, vasoactive intestinal peptide (VIP), VGF, and vitellogenin.

[0298] Targeting and penetrating peptides can also be modified biomimetic peptides and / or chemically modified small peptides. Numerous peptides with specific motifs and sequences have been identified that target specific cells and tissues with high affinity and selectivity under normal or diseased conditions. Synthetic targeting peptides can be up to 30 amino acids in length, or longer. Targeting peptides typically have at least about 5 amino acids, but can have fewer, e.g., 4 or 3 amino acids. Typically, targeting peptides have any number of amino acids, from about 6 to about 30 (inclusive). Peptides can 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, a targeting peptide may have 25 or fewer amino acids, such as 20 or fewer, for example 15 or fewer.

[0299] Chimeric peptides can also be synthesized using fused amino acids from naturally occurring proteins and artificial amino acid sequences.

[0300] stimulant The biological circuits of the present disclosure are triggered by one or more stimuli, including ligands, exogenously added or endogenous metabolites, the presence or absence of a defined ligand, the presence or action of one or more effector modules, or concentration gradients of ions or biomolecules.

[0301] Ligand In some embodiments, the stimulus is a ligand. The ligand can be nucleic acid-based, protein-based, lipid-based, organic, inorganic, or any combination of the foregoing.

[0302] In some embodiments, the ligand can be, but is not limited to, a protein, a peptide, a nucleic acid, a lipid, a lipid derivative, a sterol, a steroid, a metabolite, a metabolite derivative, and a small molecule.

[0303] In some embodiments, the irritant is a small molecule. In some embodiments, the small molecule is cell-permeable. In some embodiments, the small molecule is FDA-approved, safe, and orally administered.

[0304] 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.

[0305] 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 Celebrex), valdecoxib, rofecoxib, acetazolamide, methazolamide, dorzolamide, brinzolamide, diclophenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide.

[0306] In some embodiments, the ligand may comprise a small molecule moiety known to mediate binding to CA2. The ligand may also be modified to reduce off-target binding to carbonic anhydrases other than CA2 and increase specific binding to CA2.

[0307] Ligands can also be selected through structure-activity relationship (SAR) studies to analyze the dependence of the activity of known CA2 ligands on their molecular / chemical structure. Any SAR-related method known in the art can be used to identify the stabilizing ligands of the present disclosure. SAR can be used to improve the properties of the ligand, 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 the ligand.

[0308] In one embodiment, the stimulant of the present disclosure may be an FDA-approved ligand capable of binding to a particular DD or target region within a DD.

[0309] In some embodiments, a ligand that does not affect the activity of immune cells and / or chimeric antigen receptors in the absence of an SRE may be preferably selected.

[0310] In some embodiments, two or more ligands may be utilized to stabilize the same stimulus-response element.

[0311] Ligand Conjugates In some embodiments, a ligand may be conjugated or bound to another molecule, such as, but not limited to, another ligand, a protein, a peptide, a nucleic acid, a lipid, a lipid derivative, a sterol, a steroid, a metabolite, a metabolite derivative, or a small molecule. In some embodiments, a ligand stimulus is conjugated or bound to one or more other molecules. In some embodiments, a ligand stimulus is conjugated or bound to one or more different types and / or numbers of other molecules. In some embodiments, a ligand stimulus is a multimer of the same type of ligand. In some embodiments, a ligand stimulus multimer includes two, three, four, five, six, or more monomers.

[0312] Ligands, such as small molecules, known to bind to candidate proteins can be tested for their regulation of protein responses. Small molecules may be clinically approved as safe and may have suitable pharmaceutical kinetics and distribution. In some embodiments, the stimulus is a ligand of a destabilizing domain (DD), e.g., a small molecule that binds to the destabilizing domain and stabilizes a POI fused to the destabilizing domain.

[0313] In some embodiments, the stimulus is a small molecule. In some embodiments, the small molecule is cell-permeable.

[0314] Implanted stimulators, signals, or other regulatory sites In some embodiments, the effector module of the present disclosure may further comprise one or more microRNAs, microRNA binding sites, promoters, and regulatable elements.

[0315] microRNA In one embodiment, microRNAs can be used to aid in the creation of tunable biological circuits. Each aspect or modality can provide a different tunable characteristic to the effector module or biological circuit. For example, a destabilizing domain can alter the cleavage site or dimerization characteristics or half-life of the payload, and the inclusion of one or more microRNAs or microRNA-binding sites can affect cellular detargeting or trafficking characteristics. Consequently, the present disclosure encompasses biological circuits that are multifactorial in their protective potential. Such biological circuits and effector modules can be modified to contain one, two, three, four, or more tunable characteristics.

[0316] 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 either by reducing nucleic acid molecule stability or by inhibiting translation. Polynucleotides of the present disclosure may contain one or more microRNA target sequences, microRNA sequences, or microRNA seeds.

[0317] For example, if a polynucleotide is not intended to be delivered to the liver but ends up there, then miR-122, a microRNA that is abundant in the liver, may inhibit expression of the polynucleotide if one or more target sites for miR-122 are modified within the polynucleotide. Introduction of one or more binding sites for different microRNAs may be modified to further reduce polynucleotide longevity, stability, and protein translation, thereby providing an additional layer of potential protection beyond irritant selection, SRE design, and payload variation.

[0318] As used herein, the term "microRNA site" refers to a microRNA target site or microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that "binding" may follow traditional Watson-Crick hybridization rules or may refer to any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.

[0319] Conversely, for purposes of the polynucleotides of the present disclosure, the microRNA binding site may be modified (i.e., removed) away from the naturally occurring sequence to increase protein expression in a particular tissue. For example, the miR-122 binding site may be removed to improve protein expression in the liver.

[0320] Control of expression in multiple tissues can be achieved through the introduction or removal of one or several microRNA binding sites.

[0321] Specifically, microRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen-presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, and natural killer cells. Immune cell-specific microRNAs are involved in immunogenicity, autoimmunity, immune responses to infection, inflammation, and unwanted immune responses after gene therapy and tissue / organ transplantation. Immune cell-specific microRNAs also regulate many aspects of hematopoietic cell (immune cell) development, proliferation, differentiation, and apoptosis. For example, miR-142 and miR-146 are exclusively expressed in immune cells and are particularly abundant in myeloid dendritic cells. Introduction of a miR-142 binding site into the 3'-UTR of a polypeptide of the present disclosure can selectively suppress gene expression in antigen-presenting cells via miR-142-mediated mRNA degradation, limiting antigen presentation in professional APCs (e.g., dendritic cells), thereby preventing antigen-mediated immune responses after gene delivery (see Annoni A et al., Blood, 2009, 114, 5152-5161, the contents of which are incorporated herein by reference in their entirety).

[0322] In one embodiment, a microRNA binding site known to be expressed in immune cells, particularly antigen-presenting cells, is modified within the polynucleotide to suppress expression of the polynucleotide in APCs via microRNA-mediated RNA degradation, thereby suppressing antigen-mediated immune responses, while expression of the polynucleotide is maintained in non-immune cells that do not express the immune cell-specific microRNA.

[0323] Many microRNA expression studies have been conducted to profile the differential expression of microRNAs in various cancer cells / tissues and other diseases and have been described in the art. Some microRNAs are abnormally overexpressed in certain cancer cells, while others are underexpressed. For example, microRNAs have been shown to be involved in cancer cells (WO2008 / 154098, US2013 / 0059015, US2013 / 0042333, WO2011 / 157294); cancer stem cells (US2012 / 0053224); pancreatic cancer and disease (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); colon cancer cells (WO2 011 / 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 / 0309645, WO2011 / 095623); breast It is differentially expressed in cancer cells (WO2008 / 154098, WO2007 / 081740, US2012 / 0214699), leukemias and lymphomas (WO2008 / 073915, US2009 / 0092974, US2012 / 0316081, US2012 / 0283310, WO2010 / 018563, the contents of each of which are incorporated herein by reference in their entirety).

[0324] In one embodiment, microRNAs may be used as described herein to aid in the generation of tunable biological circuits.

[0325] In some embodiments, an effector module can be designed to encode one or more payloads, SREs, and / or regulatory sequences, e.g., microRNAs or microRNA-binding sites (as DNA, RNA, or mRNA). In some embodiments, either the encoded payload or SRE can be stabilized or destabilized by mutation and then combined with one or more regulatory sequences to generate dual- or multi-regulated effector modules or biological circuit systems.

[0326] Each embodiment or modality may bring differently modulated characteristics to the effector module or biological circuit. For example, an SRE may represent a destabilizing domain, while a mutation in the protein payload may alter its cleavage site, dimerization properties, or half-life, and the inclusion of one or more microRNAs or microRNA-binding sites may affect cellular targeting or trafficking characteristics. Consequently, the present disclosure encompasses biological circuits that are multifactorial in their protective potential.

[0327] Such biological circuits can be modified to contain one, two, three, four or more regulated features.

[0328] promoter In some embodiments, the compositions of the present disclosure comprise a promoter.

[0329] As used herein, a promoter is defined as a DNA sequence recognized by the cellular transcriptional machinery required to initiate specific transcription of a polynucleotide sequence of the present disclosure. A vector may contain a native or non-native promoter operably linked to a polynucleotide of the present disclosure. The selected promoter may be strong, weak, constitutive, inducible, tissue-specific, developmental stage-specific, and / or organism-specific. One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter, for example, but not limited to, SEQ ID NOs: 210476-210478. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of a polynucleotide sequence operably linked to it. Another example of a promoter is the elongation growth factor-1 alpha (EF-1 alpha) promoter, for example, but not limited to, SEQ ID NOs: 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, the phosphoglycerate kinase (PGK) promoter (non-limiting examples include SEQ ID NOS: 210484-210491), actin promoter, myosin promoter, hemoglobin promoter, ubiquitin C (Ubc) promoter, human U6 small nucleoprotein promoter, and creatine kinase promoter. In some instances, inducible promoters may be used, such as, but not limited to, the metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0330] In some embodiments, an optimal promoter can be selected based on its ability to achieve minimal expression of the disclosed SRE and payload in the absence of a ligand and detectable expression in the presence of a ligand.

[0331] Additional promoter elements, such as enhancers, can be used to control the frequency of transcription initiation. Such regions can be located 10 to 100 base pairs upstream or downstream of the initiation site. In some instances, two or more promoter elements can be used, either cooperatively or independently, to activate transcription.

[0332] In some embodiments, the promoter of the present disclosure can be a Tet-ON promoter. The combination of the transcriptional regulatory Tet system with DD allows for simultaneous control of gene expression and protein stability. Any of the dual Tet ON-DD systems described by Pedone et al. (2018) doi:https: / / doi.org / 10.1101 / 404699 can be useful in the present disclosure (the contents of which are incorporated herein by reference in their entirety).

[0333] Other controllability features In some embodiments, the compositions of the present disclosure may include any proteasome adaptor. As used herein, the term "proteasome adaptor" refers to any nucleotide / amino acid sequence that targets an attached payload for degradation. In some aspects, the adaptor targets the payload for degradation, thereby directly bypassing the need for ubiquitination. Proteasome adaptors may be used in conjunction with a destabilizing domain to reduce basal expression of the payload. Exemplary proteasome adaptors include the UbL domain of Rad23 or hHR23b, and gankyrin, a protein that binds with high affinity to both the target protein Rb and the S4 subunit of the proteasome, HPV E7; Rb; and the proteasome subunit S6, allowing direct proteasome targeting and bypassing the ubiquitination machinery.

[0334] Exemplary Effector Module Constructs The biological circuits of the present disclosure can include at least one effector module, which can include at least one SRE derived from CA2 (referred to as a "CA2 SRE") that can be operably linked to at least one payload of interest. These types of biological circuits and effector modules are referred to as "CA2 biological circuits" and "CA2 effector modules." CA2 effector modules can also include additional features, including, but not limited to, signal sequences, linkers, spacers, tags, flags, cleavage sites, and IRESs. Any of the exemplary SREs taught herein (e.g., DDs), payloads of interest, signal sequences, linkers, spacers, tags, flags, cleavage sites, and IRESs known in the art can be combined to generate a CA2 effector module of the present disclosure.

[0335] Targeted payloads In one embodiment, the CA2 effector module comprises a payload of interest, which may be a wild-type polypeptide, a fragment of a wild-type polypeptide, and / or may comprise one or more mutations relative to the wild-type polypeptide.

[0336] In one embodiment, the CA2 effector module produces regulated interleukin-15 (IL15).

[0337] In one embodiment, the CA2 effector module produces regulated interleukin-15 receptor subunit alpha (IL15Ra).

[0338] In one embodiment, the CA2 effector module produces a regulated fluorescent protein. In one embodiment, at least one payload in the CA2 effector module is an mCherry protein. In one embodiment, at least one payload in the CA2 effector module is a Renilla luciferase wild-type sequence (SEQ ID NO: 210643 encoded by SEQ ID NO: 210644). In one embodiment, at least one payload in the CA2 effector module is a Renilla luciferase sequence. In one embodiment, at least one payload in the CA2 effector module is a firefly luciferase sequence. In one embodiment, at least one payload in the CA2 effector module is a region of the firefly luciferase sequence. In one embodiment, at least one payload in the CA2 effector module is an Aequorea coerulescens GFP (AcGFP) sequence. In one embodiment, at least one payload in the CA2 effector module is a region of the AcGFP sequence.

[0339] In one embodiment, the CA2 effector module generates a regulated CD19 scFv. The CA2 effector module can include a transmembrane and / or cytoplasmic domain payload from another parent protein and a CD19 scFv payload. In one embodiment, at least one payload in the CA2 effector module includes at least one mutation compared to the wild-type sequence.

[0340] In one embodiment, the CA2 effector module generates a regulated CAR.

[0341] In some aspects, the payloads described herein may be co-expressed with a chimeric antigen receptor.

[0342] In one embodiment, the CA2 effector module produces regulated interleukin-12 (IL12).

[0343] In one embodiment, the effector module generates a control

[0344] .

[0345] The CA2 biological circuits and / or CA2 effector modules of the present disclosure can be monocistronic or multicistronic, meaning that one (monocistronic) or more than one (multicistronic) message (e.g., payload of interest) is generated. If two messages are generated, the CA2 biological circuit or CA2 effector module is considered bicistronic.

[0346] In one embodiment, at least one CA2 effector module of the present disclosure is monocistronic.

[0347] In one embodiment, at least one CA2 effector module of the present disclosure is multicistronic.

[0348] In one embodiment, at least one CA2 effector module of the present disclosure is bicistronic.

[0349] In one embodiment, the CA2 biological circuit of the present disclosure is monocistronic.

[0350] In one embodiment, the CA2 biological circuit of the present disclosure is multicistronic.

[0351] In one embodiment, the CA2 biological circuit of the present disclosure is bicistronic.

[0352] CA2 GFP effector module In one embodiment, the CA2 DD is fused to AcGFP via a linker sequence at either the N- or C-terminus of the fusion construct. These are referred to as "CA2 GFP effector modules." The destabilization and ligand-dependent stabilization properties of the fusion proteins can be assessed by methods such as Western blotting and FACS. Examples of CA2 mutants fused to GFP are provided in Table 8. The constructs can be cloned into any vector known in the art, including, but not limited to, the pLVX.IRES.Puro vector. In Table 8, an asterisk indicates translation of the stop codon. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7]

[0353] Additional examples of CA2 mutants fused to GFP are provided in Table 9. Constructs can be cloned into any vector known in the art, including but not limited to, the pLVX.IRES.Puro vector. [Table 9-1] [Table 9-2] [Table 9-3]

Table 9-4

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

Table 9-12

Table 9-13

Table 9-14

Table 9-15

Table 9-16

Table 9-17

Table 9-18

Table 9-19

Table 9-20

[0354] CA2 CAR and CA2 IL15 effector modules In one embodiment, the CA2 DD described herein can be added to one or more of the CAR payloads of interest. These are referred to as "CA2 CAR effector modules." Table 10 provides CA2 CAR constructs. [Table 10-1] [Table 10-2]

[0355] Table 11 provides additional CA2 CAR constructs. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

[0356] CA2 CD40L effector module In some embodiments, the CA2 DD described herein can be attached to CD40L. Table 13 provides exemplary CA2 DDs attached to CD40L. Table 12 provides construct components for preparing the regulated CD40L CA2 constructs listed in Table 13. In Table 13, " * " represents the translation of the stop codon. [Table 12] [Table 13]

[0357] CA2 mbIL12 effector module with CAR In some embodiments, the CA2 DD described herein can be added to a membrane-bound IL12, referred to herein as "mbIL12." Table 15 provides a CA2 DD added to an mbIL12 payload. Such an effector module can further be operably linked to any of the CARs described herein, with membrane-associated IL12 constructs tandemed with a CD19 CAR provided in Table 15. Any of the DDs described herein can be combined with the construct components in Table 14 to prepare the regulated membrane-bound IL12 constructs listed in Table 15. In Table 15, " * " represents the translation of the stop codon. [Table 14-1] [Table 14-2] [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6]

[0358] II. Pharmaceutical Compositions and Formulations The present teachings further include pharmaceutical compositions comprising one or more of the disclosed stimulants, CA2 biological circuits, CA2 effector modules or systems, and optionally at least one pharmaceutically acceptable excipient or inactive ingredient.

[0359] As used herein, the term "pharmaceutical composition" refers to a preparation of one or more of the CA2 biological circuits or components described herein, or pharmaceutically acceptable salts thereof, optionally with other chemical components, such as physiologically suitable carriers and excipients.

[0360] The term "excipient" or "inactive ingredient" refers to an inactive or non-active substance added to a pharmaceutical composition to further facilitate administration of a compound. Non-limiting examples of such inactive ingredients are disclosed herein under formulations.

[0361] In some embodiments, the compositions are administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to any one or more of the CA2 biological circuit components described herein that are delivered.

[0362] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, agricultural animals, e.g., cows, horses, chickens, and pigs, domestic animals, e.g., cats, dogs, or research animals, e.g., mice, rats, rabbits, dogs, and non-human mammals, including non-human primates.

[0363] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is typically equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0364] The relative amounts of active ingredient, pharmaceutically acceptable excipient or inactive ingredient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route by which the composition is administered. By way of example, the composition may contain between 0.1% and 100%, e.g., between 0.5 and 50%, between 1 and 30%, between 5 and 80%, or at least 80% (w / w) active ingredient.

[0365] The effectiveness of disease treatment or improvement can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the drug dose required to maintain the treatment effect, the level of disease markers, or any other measurable parameter appropriate for the given disease being treated or targeted for prevention.It is well within the capabilities of those skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one of these parameters or any combination of parameters.In relation to the administration of the compositions of the present disclosure, for example, "effective against" cancer indicates that administration in a clinically relevant manner will result in a beneficial effect for at least a statistically significant proportion of patients, such as symptom improvement, cure, reduction in disease burden, reduction in tumor mass or cell count, prolongation of life, improvement in quality of life, or other effects that are generally recognized as positive by physicians familiar with the treatment of a particular type of cancer.

[0366] Treatment or prevention effects are evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no worsening or onset of symptoms that would otherwise be expected. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more of a measurable parameter of the disease can indicate effective treatment. The efficacy of a given composition or formulation of the present disclosure can also be determined using an experimental animal model for a given disease known in the art. When using an experimental animal model, the effectiveness of treatment is proven when a statistically significant change is observed.

[0367] formulation The compositions of the present disclosure may be formulated in any manner suitable for delivery, including, but not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, and combinations thereof.

[0368] In one embodiment, the formulation is a nanoparticle that can include at least one lipid. The lipid can be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, and PEGylated lipids. In another embodiment, the lipid can be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, and DODMA.

[0369] For the polynucleotides of the present disclosure, formulations may be selected from, for example, any of those taught in International Application No. PCT / US2012 / 069610, the contents of which are incorporated herein by reference in their entirety.

[0370] inactive ingredients In some embodiments, pharmaceutical or other formulations may contain at least one excipient that is an inactive ingredient.As used herein, the term "inactive ingredient" refers to one or more inactive ingredients contained in the formulation.In some embodiments, all of the inactive ingredients that can be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA), or none of them may be approved, or only some of them may be approved.

[0371] III. Dosage, Delivery and Administration The compositions of the present disclosure can be delivered to cells or subjects via one or more routes and modalities. Viral vectors containing one or more of the CA2 biological circuits, CA2 effector modules, SREs, payloads, and other components described herein can be used to deliver them to cells and / or subjects. Other modalities, such as mRNA, plasmids, and recombinant proteins, can also be used.

[0372] delivery Naked Delivery The pharmaceutical compositions, CA2 biological circuits, CA2 biological circuit components, and CA2 effector modules (including their SREs or payloads) of the present disclosure can be delivered to cells, tissues, organs, and / or organisms in a naked form. As used herein, the term "naked" refers to pharmaceutical compositions, CA2 biological circuits, CA2 biological circuit components, and CA2 effector modules (including their SREs or payloads) delivered without agents or modifications that promote transfection or permeability. Naked pharmaceutical compositions, CA2 biological circuits, CA2 biological circuit components, and CA2 effector modules (including their SREs or payloads) can be delivered to cells, tissues, organs, and / or organisms using administration routes known in the art and described herein. In some embodiments, naked delivery can involve formulation in a simple buffer, such as saline or PBS.

[0373] Formulated Delivery In some embodiments, the pharmaceutical compositions, CA2 biological circuits, CA2 biological circuit components, and CA2 effector modules (including their SREs or payloads) of the present disclosure can be formulated using the methods described herein. The formulations can include modified and / or unmodified pharmaceutical compositions, CA2 biological circuits, CA2 biological circuit components, and CA2 effector modules (including their SREs or payloads). The formulations can further include, but are not limited to, cell-permeating agents, pharmaceutically acceptable carriers, delivery agents, biodegradable or biocompatible polymers, solvents, and / or sustained-release delivery depots. The formulations of the present disclosure can be delivered to cells using routes of administration known in the art and described herein.

[0374] Pharmaceutical compositions, CA2 biological circuits, CA2 biological circuit components, CA2 effector modules (including their SREs or payloads) can also be formulated for direct delivery to an organ or tissue by any of several methods known in the art, including, but not limited to, direct immersion or bathing, via a catheter, by gels, powders, ointments, creams, gels, lotions, and / or droplets, by using a substrate such as a fabric or biodegradable material coated or impregnated with the composition.

[0375] Cellular delivery In another aspect of the present disclosure, polynucleotides encoding the CA2 biological circuits, CA2 effector modules, SREs (e.g., CA2 DDs), payloads of interest (e.g., immunotherapeutic agents), and compositions of the present disclosure, as well as vectors containing the polynucleotides, can be introduced into cells. As a non-limiting example, the cells can be effector immune cells.

[0376] In one aspect of the present disclosure, polynucleotides encoding the CA2 biological circuit, CA2 effector module, SRE (e.g., CA2 DD), payload of interest (e.g., immunotherapeutic agent), and compositions of the present disclosure can be packaged into a viral vector or integrated into the viral genome, allowing for transient or stable expression of the polynucleotide. Preferred viral vectors are retroviral vectors, including lentiviral vectors. To construct a retroviral vector, a polynucleotide molecule encoding the CA2 biological circuit, CA2 effector module, CA2 DD, or payload of interest (e.g., immunotherapeutic agent) is inserted into the viral genome in place of a predetermined viral sequence to generate a replication-deficient virus. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol, and env genes but lacking the long-term repeat (LTR) and packaging components. The recombinant retroviral particles are secreted into the culture medium, then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are particularly preferred because they can contact both dividing and non-dividing cells.

[0377] Vectors can also be introduced into cells by non-viral methods, such as physical methods, e.g., needles, electroporation, ultrasound, hydroporation; chemical carriers, e.g., inorganic particles (e.g., calcium phosphate, silica, gold), and / or chemical methods. In some embodiments, synthetic or natural biodegradable agents, such as cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based vectors, or polymer-based vectors, can be used for delivery.

[0378] In some embodiments, polypeptides of the present disclosure can be delivered directly to cells. In one embodiment, polypeptides of the present disclosure can be delivered using a synthetic peptide containing an endosomal leakage domain (ELD) fused to a cell membrane-penetrating domain (CLD). The polypeptides of the present disclosure are co-transfected into cells with an ELD-CLD-synthetic peptide. The ELD facilitates the release of endosome-trapped proteins into the cytosol. Such domains are derived from proteins of microbial and viral origin and have been described in the art. CPDs enable transport of proteins across the cell membrane and have also been described in the art. ELD-CLD fusion proteins synergistically improve transduction efficiency compared to co-transduction of either domain alone. In some embodiments, a histidine-rich domain can optionally be added to the shuttle construct as an additional method to enable release of the payload from the endosome into the cytosol. The shuttle can also contain cysteine ​​residues at the N- or C-terminus to generate fusion peptide multimers. Multimers of ELD-CLD fusion peptides, generated by the addition of cysteine ​​residues to the termini of the peptides, exhibit even higher transduction efficiency when compared to single fusion peptide constructs. Polypeptides of the present disclosure can also be appended to appropriate localization signals to direct the payload to the appropriate subcellular location, e.g., the nucleus. In some embodiments, any of the ELD, CLD, or fusion ELD-CLD synthetic peptides taught in International Patent Publications WO2016161516 and WO2017175072 may be useful in the present disclosure (the contents of each of which are incorporated herein by reference in their entirety).

[0379] Delivery Modality and / or Vector The CA2 biological circuit system, CA2 effector module, SRE, and / or payload of the present disclosure can be delivered using one or more modalities. The present disclosure also provides vectors packaging the disclosed polynucleotides encoding the CA2 biological circuit, CA2 effector module, SRE (e.g., CA2 DD), and payloads of interest, as well as combinations thereof. The vectors of the present disclosure can also be used to deliver the packaged polynucleotides to cells, local tissue sites, or subjects. These vectors can be of any type, including DNA vectors, RNA vectors, plasmids, viral vectors, and particles. Viral vector technology is well known and is described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Viruses useful as vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes simplex viral vectors, retroviral vectors, oncolytic viruses, and the like.

[0380] Vectors usually contain an origin of replication functional in at least one organism, a promoter sequence and convenient restriction endonuclease sites, as well as one or more selectable markers, such as drug resistance genes.

[0381] In some embodiments, recombinant expression vectors can include regulatory sequences, such as transcriptional and translational initiation and termination codons, that are specific to the type of host cell into which the vector is introduced.

[0382] In some embodiments, a vector of the present disclosure can include one or more payloads taught herein, and two or more payloads can be included in one CA2 effector module. In this case, the two or more payloads are simultaneously regulated by the same stimuli. In other embodiments, a vector of the present disclosure can include two or more CA2 effector modules, and each CA2 effector module includes a different payload. In this case, the two or more CA2 effector modules and payloads are regulated by different stimuli, separately providing independent control of two or more components. In other embodiments, a vector of the present disclosure can include one or more CA2 effector modules and one or more non-CA2 effector modules, and each CA2 effector module includes a different payload. In this case, the CA2 effector module and payload are regulated by different stimuli, separately providing independent control of two or more components.

[0383] Lentiviral vehicles / particles In some embodiments, lentiviral vehicles / particles can be used as a delivery modality. Lentiviruses are a subgroup of viruses in the Retroviridae family, named for the need for reverse transcription of the viral RNA genome into DNA prior to integration into the host genome. Thus, the most important feature of lentiviral vehicles / particles is the integration of their genetic material into the genome of target / host cells. Some examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), junburana disease virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna maedi, and caprine arthritis-encephalitis virus (CAEV).

[0384] Typically, lentiviral particles constituting gene delivery vehicles are themselves replication-deficient (also referred to as "self-inactivating"). Lentiviruses can infect both dividing and non-dividing cells by entering through the intact host's nuclear envelope (Naldini L et al., Curr. Opin. Biotechnol, 1998, 9:457-463). Recombinant lentiviral vehicles / particles have been generated by multiple attenuation of HIV pathogenicity genes. For example, the genes Env, Vif, Vpr, Vpu, Nef, and Tat are deleted, making the vector biologically safe. Correspondingly, for example, lentiviral vehicles derived from HIV-1 / HIV-2 can mediate efficient delivery, integration, and long-term expression of transgenes in non-dividing cells. As used herein, the term "recombinant" refers to vectors or other nucleic acids containing both lentiviral and non-lentiviral retroviral sequences.

[0385] Lentiviral particles can be produced by coexpressing viral packaging elements and the vector genome itself in producer cells, such as human HEK293T cells. These elements are usually provided in three or four separate plasmids. Producer cells are cotransfected with plasmids encoding lentiviral components, including the viral core (i.e., structural proteins) and enzymatic components, as well as the envelope protein(s) (referred to as the packaging system), and a plasmid encoding the genome containing the foreign transgene to be transferred to target cells, i.e., the vehicle itself (also referred to as the transfer vector). Typically, the plasmid or vector is contained in a producer cell line. The plasmid / vector is introduced into the producer cell line via transfection, transduction, or infection. Methods for transfection, transduction, or infection are well known to those skilled in the art. As non-limiting examples, packaging and transfer constructs can be introduced into producer cell lines by calcium phosphate transfection, lipofection, or electroporation, usually along with a dominant selectable marker such as neo, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.

[0386] The producer cells produce recombinant viral particles containing a foreign gene, for example, the CA2 effector module of the present disclosure. The recombinant viral particles are recovered from the culture medium and titered by standard methods used by those skilled in the art. The recombinant lentiviral vehicle can be used to infect target cells.

[0387] Cells that can be used to generate high-titer lentiviral particles can include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol. Ther., 2005, 11:452-459), the FreeStyle™ 293 Expression System (ThermoFisher, Waltham, MA), and other HEK293T-based producer cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 22(3):357-369; Lee et al., Biotechnol Bioeng, 2012, 10996):1551-1560; Throm et al., Blood. 2009, 113(21):5104-5110, the contents of each of which are incorporated herein by reference in their entirety).

[0388] In some embodiments, the envelope protein can be a heterologous envelope protein from another virus, such as the G protein of vesicular stomatitis virus (VSV G) or a baculovirus gp64 envelope protein. VSV-G glycoprotein is produced by species classified in the genus Vesiculovirus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis virus Indiana (VSIV) and Vesicular stomatitis New Jersey. virus(VSNJV) and / or Grass carp rhabdovirus, BeAn 157575 virus(BeAn 157575), Boteke virus(BTKV), Calchaqui virus(CQIV), Eel virus American(EVA), Gray Lodge virus(GLOV), Jurona virus(JURY), Klamath virus(KLAV), Kwatta virus(KWAV), La Joya virus(LJV), Malpais Spring virus (MSPV), Mount They may in particular be selected from among the strains provisionally classified in the genus Vesiculovirus as Elgon bat virus (MEBV), Perinet virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tupaia virus (TUPV), Ulcerative disease rhabdovirus (UDRV) and Yug Bogdanovac virus (YBV). The gp64 or other baculovirus env protein can be derived from Autographa californica nuclear polyhedrosis virus (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fumiferana nuclear polyhedrosis virus, Orgyia pseudotsugata monocapsid nuclear polyhedrosis virus, Epiphyas postvittana nuclear polyhedrosis virus, Hyphantria cunea nuclear polyhedrosis virus, Galleria mellonella nuclear polyhedrosis virus, Dori virus, Thogoto virus, Antheraea pemyi nuclear polyhedrosis virus, or Batken virus.

[0389] Other elements provided in the lentiviral particle may include a retroviral LTR (long terminal repeat) at either the 5' or 3' end, a retroviral export element, optionally a lentiviral reverse response element (RRE), a promoter or an active portion thereof, and a locus control region (LCR) or an active portion thereof. The CA2 effector module is ligated into the vector.

[0390] Methods for producing recombinant lentiviral particles are discussed in the art, e.g., U.S. Patent Nos. 8,846,385; 7,745,179; 7,629,153; 7,575,924; 7,179,903; and 6,808,905 (the contents of each of which are incorporated by reference herein in their entirety).

[0391] The lentiviral vector used may be selected from, but is not limited to, pLVX, pLenti, pLenti6, pLJM1, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJM1-EGFP, pULTRA, pInducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionII.

[0392] Lentiviral vehicles can be plasmid-based or virus-based and are known in the art (see U.S. Patent Nos. 9,260,725; 9,068,199; 9,023,646; 8,900,858; 8,748,169; 8,709,799; 8,420,104; 8,329,462; 8,076,106; 6,013,516; and 5,994,136, the contents of each of which are incorporated herein by reference in their entirety).

[0393] Lentiviral vectors and cell modification Lentiviral vectors are used to introduce transgenes into T cells (e.g., primary human T cells or Jurkat cells) for preclinical studies and clinical applications, including recently approved products such as tisagenlecleucel (KYMRIAH®) for relapsed / refractory B-cell lymphoma. VSV-G pseudotyped third-generation lentiviral vectors offer high titers, high transduction efficiency, and safety, making them the vector of choice for T cell modification. Without wishing to be bound by theory, T cell modification typically involves T cell activation with CD3 / CD28 antibodies, followed by lentiviral transduction, followed by cell expansion, which can last for 5–30 days (e.g., 9–14 or 9–15 days). Typically, lentiviral transgene integration can require more than 7 days to become fully stable in T cells (e.g., primary human T cells or Jurkat cells). While longer culture can increase cell numbers, longer culture can also shift the T cell phenotype toward a more differentiated state. Therefore, the duration of ex vivo culture can affect the persistence and efficacy of CAR T cells. For example, cells cultured for shorter periods of time may exhibit a less differentiated phenotype and may be highly effective in preclinical models.

[0394] Without wishing to be bound by theory, the state of T cell differentiation can affect T cell engraftment and persistence after adoptive transfer. Ghassemi et al. (Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells. Cancer Immunol Res; 6(9) September 2018, the contents of which are incorporated herein by reference in their entirety) described the differentiation of primary human T cells over time and determined that early harvested CAR T cells exhibited improved effector function and p...

Claims

[Claim 1] The invention described in the specification.

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