Human carbonic anhydrase 2 compositions and methods for tunable regulation

Abstract

20 26 21 41 11 10 A ug 2 02 6 A B S T R A C T 2 0 2 6 2 1 4 1 1 1 1 0 A u g 2 0 2 6

Description

[0001] This application is a divisional of Australian Patent Application No. 2020235865, the entire content of which is incorporated herein by reference. REFERENCE TO THE SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is presented in ST.26 format and is based on the Sequence Listing filed for the parent application entitled "Human carbonic anhydrase 2 compositions and methods for tunable regulation". FIELD

[0003] The present disclosure relates to destabilizing domains (DDs) derived from human carbonic anhydrase 2 (CA2) which can tune protein stability for at least one payload, and compositions and methods of use thereof. Provided in the present disclosure include polypeptides of CA2 biocircuit systems, CA2 effector modules, stimulus response elements (SREs), polynucleotides encoding the same, vectors and cells containing the polypeptides and / or polynucleotides for use in cancer immunotherapy. BACKGROUND

[0004] Gene and cell therapies are revolutionizing medicine and offering new promise for the treatment of previously intractable conditions. However, most current technologies do not allow titration of the timing or levels of target protein induction. This has rendered many potential gene and cell therapy applications difficult or impossible to safely and effectively deploy.

[0005] Inadequate exogenous and / or endogenous gene control is a critical issue in many gene and cell therapy settings. This lack of tunability also makes it difficult to safely express proteins with narrow or uncertain therapeutic windows or those requiring more titrated or transient expression.

[0006] One approach to regulated protein expression or function is the use of Destabilizing Domains (DDs). Destabilizing domains are small protein domains that can be appended to a target protein of interest. DDs render the attached protein of interest unstable in the absence of a DD-binding ligand and the protein of interest is rapidly degraded by the ubiquitin-proteasome system of the cell. However, when a specific small molecule DD-binding ligand binds to the DD, the attached protein of interest is stabilized, and protein function is achieved. 2026214111   10 Aug 2026

[0007] DD technology forms the basis of a new class of cell and gene therapies that can deliver 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

[0008] The present disclosure provides novel protein domains derived from human carbonic anhydrase 2 (CA2) displaying small molecule dependent stability. Such protein domains are called destabilizing domains (DDs). In the absence of its binding ligand, the DD is destabilizing and causes degradation of a payload fused to the DD (e.g., a protein of interest (POI), while in the presence of its binding ligand, the fused DD and pay load can be stabilized, and its 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 which is operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises a H122Y mutation in the amino acid at position 122 (H122) of SEQ ID NO. 11717.

[0010] In a second 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 which is operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises a E106D mutation in the amino acid at position 106 (E106) of SEQ ID NO. 11717.

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

[0012] In a fourth aspect of the present disclosure, a composition comprising an effector module is provided. The effector module comprises a stimulus response element (SRE) and at least one payload which is operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises a I59N mutation in the amino acid at position 59 (159) of SEQ ID NO. 11717.

[0013] In a fifth aspect of the present disclosure, a composition comprising an effector module is provided. The effector module comprises a stimulus response element (SRE) and at least one payload which is operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), wherein the DD comprises a region of 2026214111   10 Aug 2026 or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises a L156H mutation in the amino acid at position 156 (L156) of SEQ ID NO. 11717. In related embodiments to the fifth aspect, the DD further comprises: (i) aW4Y mutation in the amino acid at position 4 (W4) of SEQ ID NO. 11717; (ii) a F225L mutation in the amino acid at position 225 (F225) of SEQ ID NO. 11717; (iii) deletion of amino acids at positions 257-260 of SEQ ID NO. 11717; (iv) deletion of amino acids at positions 1-5 of SEQ ID NO. 11717; or (v) deletion of amino acids G234, E235 andP236 of SEQ ID NO. 11717; orthe 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 a composition comprising an effector module. The effector module comprises a stimulus response element (SRE) and at least one payload which is operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises a first mutation and a second mutation relative to SEQ ID NO. 11717, wherein: (i) the first mutation is a S73N mutation in the amino acid at position 73 (S73) of SEQ ID NO. 11717; and (ii) the second mutation is a substitution of F or Y at the amino acid position 89 (R89) of SEQ ID NO. 11717.

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

[0016] In an eighth aspect, a composition comprising an effector module is provided. The effector module comprises a stimulus response element (SRE) and at least one pay load which is operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises one or more substitutions relative to SEQ ID NO. 11717, wherein at least one substitution is a substitution of D or N at the amino acid position 63 (G63) of SEQ ID NO. 11717, and wherein the one or more substitutions correspond to: G63D; G63D and M240L; G63D, E69V and N23II; or T55K, G63N and Q248N.

[0017] In a ninth aspect, a composition comprising an effector module is provided. The effector module comprises a stimulus response element (SRE) and at least one pay load which is operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), comprising a region of or the whole human carbomc anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises 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 the 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. 2026214111   10 Aug 2026

[0018] In a tenth aspect, a composition comprising an effector module is provided. The effector module comprises a stimulus response element (SRE) and at least one pay load which is operably linked to the SRE. In various embodiments, the SRE comprises a destabilizing domain (DD), comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises two or more substitutions relative to SEQIDNO. 11717. The at least one of the two or more substitutions is: (i) a substitution of F at the amino acid position 241 (V241) of SEQ ID NO. 11717; or (ii) a substitution of F or L at the amino acid position 249 (P249) of SEQ ID NO. 11717; and wherein the two or more substitutions correspond to: D72F and V241F; D72F and P249L; D72F and P249F; D72F, V241F and P249L; A77I and P249F; or V241F and P249L.

[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 which is operably linked to the SRE. The SRE comprises a destabilizing domain (DD), comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQIDNO. 11717), and further comprises one or more substitutions relative to SEQIDNO. 11717, selected from Y51T, L183S, Y193I, L197P and the combination of V134F andL228F.

[0020] In related embodiments to all of the exemplified aspects provided above, the SRE is responsive to one or more stimuli. In various embodiments the stimuli is a small molecule selected from Acetazolamide, Celecoxib, Valdecoxib, Rofecoxib, Methazolamide, Dorzolamide, Brinzolamide, Diclofenamide, Ethoxzolamide, Zonisamide, Dansylamide, or Dichlorphenamide. Furthermore, the compositions described and exemplified herein comprise a DD having at least one mutation or substitution in the DD, which destabilizes the DD and the at least one pay load in the absence of a stimulus and wherein the DD and the pay load are stabilized in the presence of the stimulus.

[0021] In a twelfth aspect, the present disclosure provides: a biocircuit system comprising any one or more of the compositions described in the aspects 1-10; a pharmaceutical composition comprising a compositions described in aspects 1-10, and a pharmaceutically acceptable excipient; a polynucleotide encoding a composition described in aspects 1-10; a vector comprising a polynucleotide which encodes a composition described in aspects 1-10; a cell comprising a polynucleotide which encodes a composition described in aspects 1-10; a pharmaceutical composition comprising a cell which comprises a polynucleotide which encodes a composition described in aspects 1-10, 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 comprises: (a) administering to the subject a therapeutically effective amount of a cell of aspect, 11, wherein the cell comprises a pay load that treats the disease; and (b) administering to the subject, a therapeutically effective amount of a stimulus, wherein the SRE is responsive to the stimulus and wherein expression of the pay load is modulated in response to the stimulus to thereby treat the disease. 2026214111   10 Aug 2026

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

[0024] In some embodiments, the present disclosure provides a DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprising a mutation relative to SEQ ID NO. 11717 selected from Al 15L, 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, DI 101, 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, El 17*, E117N, EUN, E186*, E186N, E204A, E204D, E204G, E204N, E213 *, E213G, E213N, E220K, E220R, E220S, E233D, E233G, E233R, E23 5 *, E235G, E235N, E237K, E237R, E23 8*, 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, G13IE, 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, Hl 19T, H119Y, H122T, H122Y, H15L, H15T, H15Y, HUD, HUI, H36I, H36Q, H64M, H94T, H96T, I145F, I145M, I166H, I166L, I209D, I209L, I215H, I215S, I22L, I255N, I255S, I33S, I59F, I59N, I59S, I91F, KI 1 IE, K111N, K112R, KI 131, 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, LI 18F, 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, L21P, 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, P13 S, 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, 2026214111   10 Aug 2026 S258F, S258P, S29C, S29P, S43P, S43T, S48L, S50P, S56F, S56N, S56P, S56X, S73L, S73N, S73X, S99H, T108L, T125I, T125P, T168K, T168N, T168Q, T176H, T176L, T192D, T192F, T192I, T192N, T192P, T192X, T198D, T198I, T198P, T199A, T199H, T199P, T207D, T207I, T207P, T207S, T35I, T35L, T37Q, T55L, T87L, V109M, V109W, V121F, V134C, V134F, V142F, V149G, V149L, V159L, V159S, V160C, V160L, V162A, V162C, V206*, V206C, V206M, V210C, V217L, V217R, V217S, V222A, V222C, V222G, V241G, V241W, V241X, V31L, V49F, V68L, V68W, V78C, W123G, W123R, W16G, W191*, W191G, W191L, W208G, W208L, W208S, W244*, W244G, W244L, W97C, W97G, Y114H, Y114M, Y127M, Y190*, Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, and S29A.

[0025] In some embodiments, the present disclosure provides a DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQID NO. 11717), and further comprising a mutation relative to SEQIDNO. 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 comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprises two or more mutations relative to SEQ ID NO. 11717. In some embodiments, aDD may comprise CA2 (aa 2-260 ofWT, 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 ofWT, T55K, G63N, Q248N), CA2 (aa2-260 ofWT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 ofWT, L156H, S2del, H3del, H4del, W5del), CA2 (aa 2-260 ofWT, W4Y, L156H), CA2 (aa 2-260 ofWT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 ofWT, L156H, F225L), CA2 (aa2-260 ofWT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 ofWT, I59N, G102R)(SEQ ID NO. 210598), CA2 (aa 2-260 ofWT, G63D, E69V, N231I)(SEQ ID NO. 210748), CA2 (aa 2-260 ofWT, R27L, T87I, H122Y, N252D)(SEQ ID NO. 210702), CA2 (aa 2-260 ofWT, D72F, V241F, P249L)(SEQ ID NO. 210503), CA2 (aa 2-260 ofWT, D71L, T87N, L250R)(SEQ ID NO. 210510), CA2 (aa 2-260 ofWT, L156H, S172C, F178Y, E186D)(SEQ ID NO. 210756), CA2 (aa 2-260 ofWT, D71F, N231F)(SEQ ID NO. 210505), CA2 (aa 2-260 ofWT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 ofWT, D71K, P249H)(SEQ ID NO. 210516), CA2 (aa 2-260 ofWT, D72F, P249H)(SEQ ID NO. 210518), CA2 (aa 2-260 ofWT, Q53N, N61Y)(SEQ ID NO. 210521), CA2 (aa 2-260 ofWT, E106D, C205S)(SEQ ID NO. 210523), CA2 (aa 2-260 ofWT, C205S, W208S)(SEQ ID NO. 210525), CA2 (aa 2-260 ofWT, S73N, R89Y)(SEQ ID NO. 210532), CA2 (aa 2-260 ofWT, D71K, T192F)(SEQ ID NO. 210534), CA2 (aa 2-260 ofWT, Y193L, K260L)(SEQ ID NO. 210540), CA2 (aa 2-260 ofWT, D71F, V241F, P249L)(SEQ ID NO. 210544), CA2 (aa 2-260 ofWT, L147F, Q248F)(SEQ ID NO. 210548), CA2 (aa 2-260 ofWT, D52I, S258P)(SEQ ID NO. 210550), CA2 (aa 2-260 ofWT, D72S, T192N)(SEQ ID NO. 210552), CA2 (aa 2-260 ofWT, D179E, T192I)(SEQ ID NO. 210554), CA2 (aa 2-260 of WT, S56N, Q103K)(SEQ ID NO. 210558), CA2 (aa 2-260 ofWT, D71Y, Q248L)(SEQ ID NO. 210560), CA2 (aa 2-260 ofWT, S73N, R89F)(SEQ ID NO. 210562), CA2 (aa 2-260 ofWT, D71K, N231L, E235G, L239F)(SEQ ID NO. 2026214111   10 Aug 2026 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 2260 of WT, Y193V, K260F)(SEQ ID NO. 210576), 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, 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, 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, KI UN, 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, KI 131, T125I, D138G, G155S)(SEQ ID NO. 210714), CA2 (aa 2-260 of WT, I59N, G102R, A173T)(SEQ ID NO. 210716), CA2 (aa 2260 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, T168K)(SEQ ID NO. 210730), CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E)(SEQ ID NO. 210732), CA2 (aa 2-260 of WT, L44M, L47V, N62K, E69D)(SEQ ID NO. 210734), CA2 (aa 2-260 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, 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).

[0027] In some embodiments, a DD may comprise 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 ofWT, T55K, G63N, Q248N), CA2 (aa 2-260 ofWT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 ofWT, L156H, S2del, H3del, H4del, W5del), CA2 (aa 2-260 ofWT, W4Y, L156H), CA2 (aa 2-260 ofWT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 ofWT, L156H, F225L), CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 ofWT, I59N, G102R)(SEQ ID NO. 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I)(SEQ ID NO. 210748), CA2 (aa 2-260 ofWT, R27L, T87I, H122Y, N252D)(SEQ ID NO. 210702), CA2 (aa 2-260 ofWT, D72F, V241F, P249L)(SEQ ID NO. 210503), CA2 (aa 2-260 ofWT, D71L, T87N, L250R)(SEQ ID NO. 210510), CA2 (aa 2-260 ofWT, L156H, S172C, F178Y, E186D)(SEQ ID NO. 210756), CA2 (aa 2026214111   10 Aug 2026 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), 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, 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 stimuli. Such stimuli may be small molecule such as but not limited to Acetazolamide, Celecoxib, Valdecoxib, Rofecoxib, Methazolamide, Dorzolamide, Brinzolamide, Diclofenamide, Ethoxzolamide, Zonisamide, Dansylamide, and Dichlorphenamide. In embodiment, the small molecule may be Acetazolamide. In some aspects, the stimulus may Celecoxib.

[0029] The present disclosure provides CA2 biocircuit systems which include at least one effector module. Such effector modules may include a stimulus response element (SRE). Provided herein are biocircuits with an SRE which includes a region or the complete human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717).

[0030] Payloads included in the CA2 biocircuit systems may be a therapeutic agent, a natural protein, a fusion polypeptide, antibody or a variant or a fragment thereof.

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

[0032] CA2 biocircuit systems may be responsive to one or more stimuli. In one aspect, the stimulus may be a small molecule 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 may be Acetazolamide. In another aspect, the small molecule may be Celecoxib.

[0033] Also provided herein are polynucleotides encoding the SREs, biocircuit systems and / or compositions described herein as well as vectors that include the polynucleotides. The present disclosure also describes pharmaceutical compositions that include CA2 biocircuit and / or the compositions described herein and a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The foregoing and other features, and advantages will be apparent from the following description of particular 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.

[0035] Figure 1 shows the ligand dependent regulation of CA2 chimeric antigen receptors.

[0036] Figure 2 shows the response of CA2 biocircuits to varying doses of Acetazolamide.

[0037] Figure 3 shows Acetazolamide responses for OT-002347 (labeled as CA2-070) and OT-001978 (labeled as CA2-026). 2026214111   10 Aug 2026 DETAILED DESCRIPTION

[0038] The details of one or more embodiments of the present disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are now described. Other features, and advantages of the present disclosure will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. In the case of conflict, the present description will control. I. COMPOSITIONS Biocircuits or Biocircuit Systems

[0039] According to the present disclosure, biocircuit systems are provided which comprise, at their core, at least one effector module. Such effector module(s) are independently associated, or integral therewith, one or more stimulus response elements (SREs). In general, a stimulus response element (SRE) may be operably linked to a payload which could be any protein of interest (POI) (e.g., an immunotherapeutic agent), to form an effector module. The SRE, when activated by a particular stimulus, e.g., a small molecule, can produce a signal or outcome, to regulate transcription and / or protein levels of the linked payload either up or down by perpetuating a stabilizing signal or destabilizing signal, or any other types of regulation. A much-detailed description of a biocircuit system are taught in co-owned U.S. Provisional Patent Application No. 62 / 320,864 filed April 11, 2016, 62 / 466,596 filed March 3, 2017 and the International Publication WO2017 / 180587 (the contents each of which are herein incorporated by reference in their entirety). In accordance with the present disclosure, biocircuit systems, effector modules, SREs and components that tune expression levels and activities of any agents used for immunotherapy are provided.

[0040] As used herein, a “biocircuit” or “biocircuit system” is defined as a circuit within or useful in biologic systems comprising a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces at least one signal or outcome within, between, as an indicator of, or on a biologic system. Biologic systems are generally understood to be any cell, tissue, organ, organ system or organism, whether animal, plant, fungi, bacterial, or viral. It is also understood that biocircuits may be artificial circuits which employ the stimuli or effector modules taught by the present disclosure and effect signals or outcomes in acellular environments such as with diagnostic, reporter systems, devices, assays or kits. The artificial circuits may be associated with one or more electronic, magnetic, or radioactive components or parts.

[0041] In accordance with the present disclosure, a biocircuit system may be a destabilizing domain (DD) biocircuit system, a chimeric antigen receptor (CAR) biocircuit systems (e.g., I / O biocircuit systems), a dimerization biocircuit system, a receptor biocircuit system, and a cell biocircuit system. Any of these systems may act as a signal to any other of these biocircuit systems. 2026214111   10 Aug 2026 Effector modules

[0042] The biocircuits 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 comprising at least (a) one or more stimulus response elements (SREs) and (b) one or more pay loads (e.g., proteins of interest (POIs)).

[0043] Effector modules may be designed to include one or more pay loads, one or more SREs, one or more cleavage sites, one or more signal sequences and one or more additional features including the presence or absence of one or more linkers. Representative effector module embodiments of the present disclosure are illustrated in Figures 2-6 in International Publication No. WO2017 / 180587, the contents of which are herein incorporated by reference in their entirety. Biocircuits and components utilizing such effector molecules are given in Figures 7-12 in International Publication No. WO2017 / 180587, the contents of which are herein incorporated by reference in their entirety.

[0044] As shown in Figure 2 in International Publication No. WO2017 / 180587, representative effector module embodiments comprising one pay load, i.e. one immunotherapeutic agent are illustrated. Each components of the effector module may be located or positioned in various arrangements without (A to F) or with (G to Z, and AA to DD) a cleavage site. An optional linker may be inserted between each component of the effector module.

[0045] Figures 3 to 6 in International Publication No. WO2017 / 180587, illustrate representative effector module embodiments comprising two payloads, i.e. two immunotherapeutic agents. In some aspects, more than two immunotherapeutic agents (payloads) may be included in the effector module under the regulation of the same SRE (e.g., the same DD). The two or more agents may be either directly linked to each other or separated. The SRE may be positioned at the N terminus of the construct, or the C terminus of the construct, or in the internal location.

[0046] In some embodiments, biocircuits of the present disclosure may be modified to reduce their immunogenicity. Immunogenicity is the result of a complex series of responses to a substance that is perceived as foreign and may include the production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity responses, and anaphylaxis. Several factors can contribute to protein immunogenicity, including, but not limited to protein sequence, route and frequency of administration and patient population. In a preferred embodiment, protein engineering may be used to reduce the immunogenicity of the compositions of the present disclosure. In some embodiments, modifications to reduce immunogenicity may include modifications that reduce binding of the processed peptides derived from the parent sequence to MHC proteins. For example, amino acid modifications may be engineered such that there are no or a mimmal of number of immune epitopes that are predicted to bind with high affinity, to any prevalent MHC alleles. Several methods of identifying MHC binding epitopes of known protein sequences are known in the art and may be used to score epitopes in the compositions of the present disclosure. Such methods are disclosed in US Patent Publication No. US 20020119492, US20040230380, and US 20060148009; the contents of each of which are incorporated by reference in their entirety. 2026214111   10 Aug 2026

[0047] Effector modules, including their SREs and pay loads, may be nucleic acid-based, protein-based or a combination thereof. They may be in the form of DNA, RNA, mRNA, proteins, fusion proteins, or any combination of the foregoing.

[0048] Effector modules, including their SREs and payloads may individually, collectively or independently comprise peptides, polypeptides or proteins. At the protein level, such pay load may be any natural or artificial peptide or polypeptide or fragment thereof. Natural peptides or polypeptide components of the pay load may be derived from any known protein of any species.

[0049] Effector modules may be designed to operate in groups of one, two, three, four or more modules. When more than one effector module is utilized in a biocircuit, it is known as an effector module system of that biocircuit. Stimulus response element (SRE)

[0050] As used herein a “stimulus response element” (SRE) is a component of an effector module which is joined, attached, linked to or associated with one or more pay loads and in some instances, is responsible for the responsive nature of the effector module to one or more stimuli. As used herein, the “responsive” nature of an SRE to a stimulus may be characterized by a covalent or non-covalent interaction, a direct or indirect association or a structural or chemical reaction to the stimulus. Further, the response of any SRE to a stimulus may be a matter of degree or kind. 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 output signal may be of a relative nature to the stimulus, e.g., producing a modulatory effect of between 1% and 100% or a factored increase or decrease such as 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.

[0051] In some embodiments, the present disclosure provides methods for modulating protein expression, function or level. In some aspects, the modulation of protein expression, function or level refers to modulation of expression, function or level by at least about 20%, such as by 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%, 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%. Destabilizing Domains

[0052] Destabilizing domains (DDs) are small protein domains that can be appended to a target protein of interest. The term destabilizing domain (DD) is interchangeable with the term drug responsive domain (DRD). DDs render the attached protein of interest unstable in the absence of a DD-binding ligand such that the protein is rapidly degraded by the ubiquitin-proteasome system of the cell (Stankunas, K., et al., Mol. Cell, 2003,12: 1615-1624; Banaszynski, et al., Cell; 2006,126(5): 995-1004; reviewed inBanaszynski, L.A., andWandless, T.J. Chern. Biol:, 2006,13:11-21 and Rakhit R et al., Chern Biol. 2014; 21(9): 1238-1252). However, when a specific small molecule ligand binds its intended 2026214111   10 Aug 2026 DD as a ligand binding partner, the instability is reversed, and protein function is restored. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable substrate for degradation. Moreover, its dependency on the concentration of its ligand further provides tunable control of degradation rates.

[0053] In one embodiment, the SRE is a destabilizing domain (DD). The presence, absence or an amount of a small molecule ligand that binds to or interacts with the DD, can, upon such binding or interaction modulate the stability of the payload(s) and consequently the function of the payload. Depending on the degree of binding and / or interaction the altered function of the payload may vary, hence providing a “tuning” of the payload function.

[0054] In some embodiments, the desired characteristics of the DDs may include, but are not limited to, low protein levels in the absence of a ligand of the DD (e.g., low basal stability), large dynamic range, robust and predictable doseresponse behavior, and rapid kinetics of degradation. DDs that bind to a desired ligand, but not endogenous molecules may be preferred.

[0055] In some embodiments, the DDs of the present disclosure may be developed from known proteins herein referred to as the parent protein. In some embodiments, the CA2 destabilizing domains described herein or known in the art may be used as SREs in the biocircuit systems of the present disclosure in association with any of the pay loads (e.g., proteins of interest or immunotherapeutic agents) taught herein.

[0056] Regions or portions or domains of wild type proteins (e.g., CA2) may be utilized as SREs / DDs in whole or in part. They may be combined or rearranged to create new peptides, proteins, regions or domains of which any may be used as SREs / DDs or the starting point for the design of further SREs and / or DDs.

[0057] In one embodiment, the SRE is derived from a region of parent protein (e.g., CA2) or from a mutant protein. The region of the parent protein may be 10,11,12,13,14,15,16,17,18,19, 20, 21, 22, 23,24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46,47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69,70, 71, 72, 73,74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100,101,102,103,104,105,106,107,108,109,110, 111, 112,113,114,115,116,117,118,119, 120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143, 144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167, 168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191, 192,193,194,195,196,197,198,199, 200, 201, 202, 203,204, 205,206, 207, 208, 209, 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, 2026214111   10 Aug 2026 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404,405, 406,407, 408, 409, 410, 411,412, 413, 414,415, 416, 417, 418, 419,420, 421,422, 423, 424, 425, 426,427, 428,429, 430,431, 432, 433, 434, 435,436, 437, 438,439, 440, 441, 442, 443,444, 445,446, 447, 448, 449, 450, or more than 450 ammo acids in length. The region of the parent protein may be 5-50, 25-75, 50-100, 75-125,100-150,125-175,150-200,175225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 ammo acids in length. As a non-limiting example, the region of the parent protein may be 250-270 amino acids in length. As a non-limiting example, the region of the parent protein may be 225-250 amino acids in length. As a non-limiting example, the region of the parent protein may be 225-260 amino acids in length.

[0058] In one embodiment, the SRE is derived from a parent protein (e.g., CA2) or from a mutant protein and includes a region of the parent protein. The SRE may include a region of the parent protein which is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, 5-10%, 1015%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 7580%, 80-85%, 85-90%, 90-95%, 95-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90100%, 10-30%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 10-40%, 20-50%, 30-60%, 40-70%, 5080%, 60-90%, 70-100%, 10-50%, 20-60%, 30-70%, 40-80%, 50-90%, 60-100%, 10-60%, 20-70%, 30-80%, 40-90%, 50100%, 10-70%, 20-80%, 30-90%, 40-100%, 10-80%, 20-90%, 30-100%, 10-90%, 20-100%, 25-50%, 50-75%, or 75100% of the parent protein or mutant protein.

[0059] In one embodiment, the SRE is derived from a parent protein (e.g., CA2) or from a mutant protein and may have 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%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 6065%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 6070%, 70-80%, 80-90%, 90-100%, 10-30%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 10-40%, 2050%, 30-60%, 40-70%, 50-80%, 60-90%, 70-100%, 10-50%, 20-60%, 30-70%, 40-80%, 50-90%, 60-100%, 10-60%, 2070%, 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 to the parent protein or mutant protein.

[0060] Candidate destabilizing domain sequence identified from protein domains of parent proteins (as a template) may be mutated to generate libraries of mutants based on the template candidate domain sequence. Mutagenesis strategies used to generate DD libraries may include site-directed mutagenesis e.g. by using structure guided information; or random mutagenesis e.g. using error-prone PCR, or a combination of both. In some embodiments, destabilizing domains identified using random mutagenesis may be used to identify structural properties of the candidate DDs that may be required for destabilization, which may then be used to further generate libraries of mutations using site directed mutagenesis. 2026214111   10 Aug 2026

[0061] In some embodiments, DD mutant libraries may be screened for mutations with altered, preferably higher binding affinity to the ligand, as compared to the wild type protein. DD libraries may also be screened using two or more ligands and DD mutations that are stabilized by some ligands but not others may be preferentially selected. DD mutations that bind preferentially to the ligand compared to a naturally occurring protein may also be selected. Such methods may be used to optimize ligand selection and ligand binding affinity of the DD. Additionally, such approaches can be used to minimize deleterious effects caused by off-target ligand binding.

[0062] In some embodiments, suitable DDs may be identified by screening mutant libraries using barcodes. Such methods may be used to detect, identify and quantify individual mutant clones within the heterogeneous mutant library. Each DD mutant within the library may have distinct barcode sequences (with respect to each other). In other instances, the polynucleotides can also have different barcode sequences with respect to 2,3,4, 5,6,7, 8,9,10 or more nucleic acid bases. Each DD mutant within the library may also comprise a plurality of barcode sequences. When used in plurality may be used such that each barcode is unique to any other barcode. Alternatively, each barcode used may not be unique, but the combination of barcodes used may create a unique sequence that can be individually tracked. The barcode sequence may be placed upstream of the SRE, downstream of the SRE, or in some instances may be placed within the SRE. DD mutants may be identified by barcodes using sequencing approaches such as Sanger sequencing, and next generation sequencing, but also by polymerase chain reaction and quantitative polymerase chain reaction. In some embodiments, polymerase chain reaction primers that amplify a different size product for each barcode may be used to identify each barcode on an agarose gel. In other instances, each barcode may have a unique quantitative polymerase chain reaction probe sequence that enables targeted amplification of each barcode.

[0063] In one embodiment, the effector modules and / or SREs of the present disclosure may include at least one destabilizing domain (DD). The effector modules and / or SRE may include 1,2, 3, 4, 5, 6, 7, 8, 9,10 or more than 10 DDs. When there are more than one DDs, each of the DDs may be derived from the same parent protein, from different parent proteins, may be a fusion of two different parent proteins, or may be artificial.

[0064] In one embodiment, the effector modules and / or SREs of the present disclosure may include 2 DDs. In one embodiment, the effector modules and / or SREs of the present disclosure may include 3 DDs. In one embodiment, the effector modules and / or SREs of the present disclosure may include 4 DDs. In one embodiment, the effector modules and / or SREs of the present disclosure may include 5 DDs. In one embodiment, the effector modules and / or SREs of the present disclosure may include 6 DDs. In one embodiment, the effector modules and / or SREs of the present disclosure may include 7 DDs. In one embodiment, the effector modules and / or SREs of the present disclosure may include 8 DDs. In one embodiment, the effector modules and / or SREs of the present disclosure may include 9 DDs. In one embodiment, the effector modules and / or SREs of the present disclosure may include 10 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 2026214111   10 Aug 2026 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. CA2 Destabilizing Domains

[0065] In some embodiments, the DDs of the present disclosure may be derived from human carbonic anhydrase 2 CA2, which is a member of the Carbonic anhydrases (CAs, EC 4.2.1.1) a superfamily of metalloenzymes present in all life kingdoms. CAs equilibrate the reaction between three chemical species: CO2, bicarbonate, and protons. CAs have convergently evolved, with seven genetically distinct CA families that evolved independently in Bacteria, Archaea, and Eukarya, the a-, 0-, y-, 5-, £-, q-, and 9-CAs. 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 (CAI), 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 (CAIO), Carbonic Anhydrase 11 (CA11), Carbonic Anhydrase 12 (CA12), Carbonic Anhydrase 13 (CAI3). and Carbonic Anhydrase 14 (CA14).

[0066] In one embodiment, the DDs may be derived from cytosolic CAs such as but not limited to Carbonic Anhydrase 2 (CA2), Carbonic Anhydrase 1 (CAI), Carbonic Anhydrase 3 (CA3), Carbonic Anhydrase 7 (CA7), and Carbonic Anhydrase 13 (CA 13). In one embodiment, the DDs may be derived from mitochondrial CAs such as but not limited to Carbonic Anhydrase 5A (CA5A), and Carbonic Anhydrase 5B (CA5B). In one embodiment, the DDs may be derived from secreted CAs such as but not limited to Carbonic Anhydrase 6 (CA6). In one embodiment, the DDs may be derived from membrane associated CAs 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 is derived from CA2. In another aspect, the DD may be derived from CA9.

[0067] In some embodiments, the DDs of the present disclosure may be derived from CA2 (SEQ ID NO. 11717; Uniprot ID: P00918) which may be stabilized by ligands such as small molecule inhibitors of CA2. As used herein the term “CA2 WT”, refers to the human wildtype CA2 protein sequence, which is defined as SEQ ID NO. 11717, with the GenBank Access NO. P00918, having the amino acid sequence: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDS QDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLG IFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSE QVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK. In some aspects the DDs may be derived from CA2 of SEQ ID NO. 11718 (having the amino acid sequence: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDS QDKAASLGSLEHQIWGFWESCAAT) or SEQ ID NO. 11719 (having the amino acid sequence: MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFDDS 2026214111   10 Aug 2026 QDKAEKGISMLRKKDVKNIHSPDNACEE). In some embodiments, DDs of the present disclosure may be identified by utilizing a cocktail of CA2 inhibitors. In other instances, the suitable DDs may be identified by screening first with one CA2 inhibitor and subsequently screening with a second CA2 inhibitor.

[0068] The amino acid sequences of the destabilizing domains encompassed in the disclosure have at least about 40%, 50 or 60% identity, further 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 further preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth therein. Percent identity may be determined, for example, by comparing sequence information using the advanced BLAST computer program, 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).

[0069] In some embodiments, DDs derived from CA2 may comprise amino acids 2-260 of the parent CA2 sequence. This is referred to herein as an Ml del mutation. In one embodiment, the DDs derived from CA2 may comprise amino acids 2-237 of the parent CA2 sequence.

[0070] Provided herein in Table 1, Table 2, Table 3, Table 4, and Table 6 are CA2 mutants identified by mutagenesis such as random mutagenesis screening, using a combination of nucleotide analog mutagenesis and error-prone PCR, to generate libraries of mutants; or saturation mutagenesis. CA2 destabilizing mutants may also be identified by structure guided mutagenesis and are provided in Table 1. The position of the mutated amino acids listed in Table 1, Table 2, Table 3, Table 4, Table 5 and Table 6 is relative to the full length CA2 of SEQ ID NO. 11717. 2026214111   10 Aug 2026 Table 1: CA2 DDs Library ID Description AA SEQUENCE AA SEQID NO. NA SEQ ID NO. CA2 (aa 2260 ofWT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210492 210493 CA2 (aa 2260 ofWT, W208S) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTSIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210494 210495 LibC000103 CA2 (aa 2260 ofWT, Y51N) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSNDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210496 210497 LibCOOOlOl CA2 (aa 2260 ofWT, S56N) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATNLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210498 210499 LibC000098 CA2 (aa 2260 ofWT, Y51T) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSTDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210500 210501 LibC000097 210502 LibC000095 CA2 (aa 2260 ofWT, D72F, V241F, P249L) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDFSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMFDNWRPAQLLKNRQIKASFK 210503 210504 LibC000084 CA2 (aa 2260 ofWT, D71F, N231F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFFDSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFFGEGEPEELMVDNWRPAQPLKNRQIKASFK 210505 210506 LibC000079 CA2 (aa 2260 ofWT, S56F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATFLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210507 210508 LibC000078 210509 LibC000073 CA2 (aa 2260 ofWT, D71L, T87N, L250R) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFLDSQDKAVLKGGPLDGNYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPRKNRQIKASFK 210510 210511 2026214111   10 Aug 2026 LibC000090 CA2 (aa 2260 ofWT, L183S) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGSLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210512 210513 LibC000076 CA2 (aa 2260 ofWT, A77I, P249F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKIVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQFLKNRQIKASFK 210514 210515 LibC000099 CA2 (aa 2260 ofWT, D71K, P249H) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFKDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQHLKNRQIKASFK 210516 210517 LibC000081 CA2 (aa 2260 ofWT, D72F, P249H) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDFSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQFLKNRQIKASFK 210518 210519 LibC000065 210520 LibC000082 CA2 (aa 2260 ofWT, Q53N, N61Y) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDNATSLRILYNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210521 210522 CA2 (aa 2260 ofWT, E106D, C205S) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSDHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLESVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210523 210524 CA2 (aa 2260 ofWT, C205S, W208S) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLESVTSIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210525 210526 CA2 (aa 2260 ofWT, C205S) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLESVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210527 210528 LibC000066 CA2 (aa 2260 ofWT, Y193I) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTIPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210529 210530 LibC000069 210531 LibC000056 210532 210533 2026214111   10 Aug 2026 CA2 (aa 2260 ofWT, S73N, R89Y) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDNQDKAVLKGGPLDGTYYLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK LibC000057 CA2 (aa 2260 ofWT, D71K, T192F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFKDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWFYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210534 210535 LibC000061 CA2 (aa 2260 ofWT, E238*) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPE*LMVDNWRPAQPLKNRQIKASFK 210536; 210696 210537 LibC000061 CA2 (aa 2260 ofWT, G144D) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLDIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210538 210539 LibC000092 CA2 (aa 2260 ofWT, Y193L, K260L) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTLPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFL 210540 210541 LibC000053 CA2 (aa 2260 ofWT, V206M) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECMTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210542 210543 LibC000054 CA2 (aa 2260 ofWT, D71F, V241F, P249L) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFFDSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMFDNWRPAQLLKNRQIKASFK 210544 210545 LibC000055 CA2 (aa 2260 ofWT, Y193F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTFPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210546 210547 LibC000058 CA2 (aa 2260 ofWT, L147F, Q248F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFFKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAFPLKNRQIKASFK 210548 210549 LibC000059 210550 210551 2026214111   10 Aug 2026 CA2 (aa 2260 ofWT, D52I, S258P) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYIQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKAPFK LibC000060 CA2 (aa 2260 ofWT, D72S, T192N) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDSSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWNYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210552 210553 LibC000062 CA2 (aa 2260 ofWT, D179E, T192I) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFEP RGLLPESLDYWIYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210554 210555 LibC000063 CA2 (aa 2260 ofWT, Y193L) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTLPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210556 210557 LibC000064 CA2 (aa 2260 ofWT, S56N, Q103K) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATNLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGKGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210558 210559 LibC000067 CA2 (aa 2260 ofWT, D71Y, Q248L) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFYDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPALPLKNRQIKASFK 210560 210561 LibC000068 CA2 (aa 2260 ofWT, S73N, R89F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDNQDKAVLKGGPLDGTYFLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210562 210563 LibC000070 CA2 (aa 2260 ofWT, D71K, N231L, E235G, L239F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFKDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFLGEGGPEEFMVDNWRPAQPLKNRQIKASFK 210564 210565 LibC000071 CA2 (aa 2260 ofWT, D71F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFFDSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210566 210567 2026214111   10 Aug 2026 LibC000072 CA2 (aa 2260 ofWT, D72F, P249I) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDFSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQILKNRQIKASFK 210568 210569 LibC000074 CA2 (aa 2260 ofWT, T192N) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWNYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210570 210571 LibC000075 CA2 (aa 2260 ofWT, D72X, V241X, P249X) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDXSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMXDNWRPAQXLKNRQIKASFK 210572 210573 LibC000077 CA2 (aa 2260 ofWT, A54X, S56X, L57X, T192X) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQXTXXRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWXYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210574 210575 LibC000080 CA2 (aa 2260 ofWT, Y193V, K260F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTVPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFF 210576 210577 LibC000085 CA2 (aa 2260 ofWT, G63D, M240L) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNDHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELLVDNWRPAQPLKNRQIKASFK 210578 210579 LibC000086 CA2 (aa 2260 ofWT, V134F, L228F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAF QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KFNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210580 210581 LibC000087 CA2 (aa 2260 ofWT, D71G, N231K) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFGDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFKGEGEPEELMVDNWRPAQPLKNRQIKASFK 210582 210583 LibC000088 210584 210585 2026214111   10 Aug 2026 CA2 (aa 2260 ofWT, S56F, D71S) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATFLRILNNGHAFNVEFSDSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK LibC000089 CA2 (aa 2260 ofWT, D52L, G128R, Q248F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYLQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQ FHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYRDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAFPLKNRQIKASFK 210586 210587 LibC000091 CA2 (aa 2260 ofWT, S73X, R89X) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDXQDKAVLKGGPLDGTYXLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210588 210589 LibC000093 CA2 (aa 2260 ofWT, Y193X) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTXPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210590 210591 LibC000096 CA2 (aa 2260 ofWT, Y51X, D72X, V241X, P249X) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSXDQATSLRILNNGHAFNVEFDXSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMXDNWRPAQXLKNRQIKASFK 210592 210593 LibCOOOlOO CA2 (aa 2260 ofWT, D72I, W97C) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDISQDKAVLKGGPLDGTYRLIQ FHFHCGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQ QPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDP RGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210594 210595 LibC000102 CA2 (aa 2260 ofWT, D71K, T192F, N231F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFKDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWFYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFFGEGEPEELMVDNWRPAQPLKNRQIKASFK 210596 210597 - CA2 (aa 2260 ofWT, I59N, G102R) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRNLNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDRQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210598 210599 LibC000210 210847 LibC000184 210848 LibC000187 210849 2026214111   10 Aug 2026 CA2 (aa 2260 ofWT, L156H) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210600 210601 LibC000208 210850 CA2 (L156H) MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSL KPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYR LIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGK AVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTKGKSADFTN FDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLK FRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 210602 210603 LibC000061 truncated CA2 (aa 2237 ofWT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKP LSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLI QFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAV QQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFD PRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFR KLNFNGEGEPE 210536 210697

[0071] Additional CA2 destabilizing domains are provided in Table 2. Table 2: CA2DDs Library ID Description AA SEQUENCE AA SEQID NO. NA SEQID NO. LibC000229 CA2 (aa 2-260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTQTAKYDPT LKPLSVSFDQATSLRILNNGHAFDVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFWKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFHKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210698 210699 LibC000228 CA2 (aa 2-260 of WT, F70I, F146V) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEIDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIVLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210700 210701 LibC000226 CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D) SHHWGYGKHNGPEHWHKDFPIAKGELQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG IYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVYWNTKYG DFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKG KSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEP ISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKDRQIKA SFK 210702 210703 LibC000225 CA2 (aa 2-260 of WT, K45N, V68L, H119Y, K169R, D179E) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LNPLSVSYDQATSLRILNNGHAFNLEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELYLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTR GKSADFTNFEPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210704 210705 LibC000224 210706 210707 2026214111   10 Aug 2026 CA2 (aa 2-260 of WT, H15L, A54V, K111E, E220K, F225I) SHHWGYGKHNGPELWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQVTSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDEKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSKQVLKIRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK LibC000221 CA2 (aa 2-260 of WT, PBS, P83A, D101G, KI UN, F230I) SHHWGYGKHNGSEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGALD GTYRLIQFHFHWGSLGGQGSEHTVDNKKYAAELHLVHWNTK YGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVL KEPISVSSEQVLKFRKLNINGEGEPEELMVDNWRPAQPLKNRQI KASFK 210708 210709 LibC000220 CA2 (aa 2-260 of WT, L47R) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPRSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLD GTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTK YGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVL KEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQ IKASFK 210710 210711 LibC000219 CA2 (aa 2-260 of WT, G63D, W123R, E220K) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNDHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHRNTKYG DFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKG KSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEP ISVSSKQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKA SFK 210712 210713 LibC000217 CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S) SHHWGYGKHDGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVKFDDSQDKAVLKGGPLD DTYRLIQFHFHWGSLDGQGSEHTMDKKIYAAELHLVHWNIKY GDFGKAVQQPGGLAVLGIFLKVGSAKPSLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210714 210715 LibC000214 CA2 (aa 2-260 of WT, I59N, G102R, A173T) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRNLNNGHAFNVEFDDSQDKAVLKGGPLD GTYRLIQFHFHWGSLDRQGSEHTVDKKKYAAELHLVHWNTK YGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSTDFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLK EPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQI KASFK 210716 210717 LibC000213 CA2 (aa 2-260 of WT, L79F, P180S) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVFKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDSRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210718 210719 LibC000212 CA2 (aa 2-260 of WT, S73F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDFQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210720 210721 2026214111   10 Aug 2026 LibC0002U CA2 (aa 2-260 of WT, G12R) SHHWGYGKHNRPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210722 210723 LibC000209 CA2 (aa 2-260 of WT, A77P, G102R, D138N) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKPVLKGGPLDG TYRLIQFHFHWGSLDRQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPNGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210724 210725 LibC000183 CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I) SHHWGYGKHNGPEHWHKDLPIAKGERQSPVDIDTHTAKYDPS LNPLSVSYDQATSLRILNNDHAFNVVFDDSQDKAVLKGGPLD GTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTK YGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVL KEPISVSSEQVLKFRKLNFIGEGEPEELMVDNWRPAQPLKNRQI KASFK 210726 210727 LibC000207 CA2 (aa 2-260 of WT, T199N, L202P, L228F) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTNPPPLECVTWIVLK EPISVSSEQVLKFRKFNFNGEGEPEELMVDNWRPAQPLKNRQI KASFK 210728 210729 LibC000206 CA2 (aa 2-260 of WT, K9N, H122Y, T168K) SHHWGYGNHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVYWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKKK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210730 210731 LibC000205 CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDHATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRVIHFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFEKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210732 210733 LibC000204 CA2 (aa 2-260 of WT, L44M, L47V, N62K, E69D) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS MKPVSVSYDQATSLRILNKGHAFNVDFDDSQDKAVLKGGPLD GTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTK YGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVL KEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQ IKASFK 210734 210735 LibC000203 CA2 (aa 2-260 of WT, F20L, K45N, G104R, Al 16V) SHHWGYGKHNGPEHWHKDLPIAKGERQSPVDIDTHTAKYDPS LNPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQRSEHTVDKKKYAVELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210736 210737 LibC000202 CA2 (aa 2-260 of WT, D75V, K169N, F259L) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQVKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY 210738 210739 2026214111   10 Aug 2026 GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTN GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASLK LibC000182 CA2 (aa 2-260 of WT, T207S, V222A, N231D) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVSWIVLKE PISVSSEQALKFRKLNFDGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210740 210741 LibC000201 CA2 (aa 2-260 of WT, I59F, V206M, G232R) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRFLNNGHAFNVEFDDSQDKAVLKGGPLD GTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTK YGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECMTWIVL KEPISVSSEQVLKFRKLNFNREGEPEELMVDNWRPAQPLKNRQ IKASFK 210742 210743 LibC000199 CA2 (aa 2-260 of WT, P13A, A133T) SHHWGYGKHNGAEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKTVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210744 210745 LibC000198 CA2 (aa 2-260 of WT, N61R, K80M, K212N, N231T, L250P) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILRNGHAFNVEFDDSQDKAVLMGGPLD GTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTK YGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVL NEPISVSSEQVLKFRKLNFTGEGEPEELMVDNWRPAQPPKNRQ IKASFK 210746 210747 LibC000196 CA2 (aa 2-260 of WT, G63D, E69V, N231I) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNDHAFNVVFDDSQDKAVLKGGPLD GTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTK YGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVL KEPISVSSEQVLKFRKLNFIGEGEPEELMVDNWRPAQPLKNRQI KASFK 210748 210749 LibC000181 CA2 (aa 2-260 of WT, I59N, R89I) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRNLNNGHAFNVEFDDSQDKAVLKGGPLD GTYILIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210750 210751 LibC000194 CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHNFNVEFDDSQDKAVLKGGPLDD TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFRKAVQQPDGLAVLGIFLKVGSAKPDLQNVVDALDSIKTK DKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGELEELMVDNWRPAQPLKNRQIK ASFK 210752 210753 LibC000192 CA2 (aa 2-260 of WT, G12R, H15Y,D19V) SHHWGYGKHNRPEYWHKVFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK 210754 210755 2026214111   10 Aug 2026 GKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK LibC000193 CA2 (aa 2-260 of WT, L156H, S172C, F178Y, E186D) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGHQKVVDVLDSIKTK GKCADFTNYDPRGLLPDSLDYWTYPGSLTTPPLLECVTWIVLK EPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQI KASFK 210756 210757 LibC000189 CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHVFNVEFDDSQDKAVLKGGPLDG TYRLIQFHYHWGSLDGQGSGQTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGMFLKVGSAKPGLQKVVDVLDSIKT KGKSADITNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLK EPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQI KASFK 210758 210759 LibC000186 CA2 (aa 2-260 of WT, L197P) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDG TYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKY GDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTK GKSADFTNFDPRGLLPESLDYWTYPGSPTTPPLLECVTWIVLKE PISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIK ASFK 210760 210761 CA2 (aa 2-260 of WT, G63D, E69V, N231I) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS LKPLSVSYDQATSLRILNNDHAFNVVFDDSQDKAVLKGGPLD GTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTK YGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVL KEPISVSSEQVLKFRKLNFIGEGEPEELMVDNWRPAQPLKNRQI KASFK 210851 210852

[0072] In some embodiments, the CA2 DDs described herein may include any of the sequences provided in Table 3. In Table 3 represents the translation of stop codon. When the amino acid sequence in Table 3 contains one or more stop codons, the “AA SEQ ID” column provides the SEQ ID NO. of the individual components preceding and following the stop codon in the order in which they occur in the amino acid sequence. Table 3: CA2DDs Library ID Description AA SEQUENCE AA SEQ ID NO. NA SEQ ID NO. LibC000223 CA2 (aa 2-260 of WT, GUE, A38V, A65V, G98V, S99H,D101M, G102D, Q103K, G104V, S105Q, E106S, H107I, T108L, V109W, DI 101, KI 12R, KI 13N, Y114M, Al 15L, Al 16Q, E117N, L118F, H119T, L120W, V121F,H122T, W123G, N124T, T125P, K126N, Y127M, D1291, F130L, K132E, A133L, V134C, Q135S, Q136N, P137L, D138M, G139D, L140W, A141P, V142F, L143*, G144V, I145F, L156Q, V162A) SHHWGYGKHNEPEHWHKDFPIAKGE RQSPVDIDTHTVKYDPSLKPLSVSYDQ ATSLRILNNGHVFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWVHLMDKV QSILWIKRNMLQNFTWFTGTPNMGIL GELCSNLMDWPFWFFLKVGSAKPGQ QKVVDALDSIKTKGKSADFTNFDPRG LLPESLDYWTYPGSLTTPPLLECVTWI VLKEPISVSSEQVLKFRKLNFNGEGEP EELMVDNWRPAQPLKNRQIKASFK 210762; 210763 210764 LibC000185 210767 2026214111   10 Aug 2026 CA2 (aa 2-260 of WT, F20L, K45N, G104R, A116V, AU3G, W191*) SHHWGYGKHNGPEHWHKDLPIAKGE RQSPVDIDTHTAKYDPSLNPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWGSLDGQRS EHTVDKKKYAVELHLVHWNTKYGDF GKAVQQPDGLAVLGIFLKVGSAKPGL QKVVDVLDSIKTKGKSGDFTNFDPRG LLPESLDY*TYPGSLTTPPLLECVTWIV LKEPISVSSEQVLKFRKLNFNGEGEPEE LMVDNWRPAQPLKNRQIKASFK 210765; 210766 LibC000215 CA2 (aa 2-260 of WT, HUD, P30S, G81V, K132R, S151I, A152D, A173G, W19U) SHHWGYGKHNGPEHWDKDFPIAKGE RQSSVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KVGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDF GRAVQQPDGLAVLGIFLKVGIDKPGL QKVVDVLDSIKTKGKSGDFTNFDPRG LLPESLDY*TYPGSLTTPPLLECVTWIV LKEPISVSSEQVLKFRKLNFNGEGEPEE LMVDNWRPAQPLKNRQIKASFK 210768; 210766 210769 LibC000191 CA2 (aa 2-260 of WT, HUD, P30S, G81V, K132R, S151I, A152D, AU3G, W19U) SHHWGYGKHNGPEHWDKDFPIAKGE RQSSVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KVGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDF GRAVQQPDGLAVLGIFLKVGIDKPGL QKVVDVLDSIKTKGKSGDFTNFDPRG LLPESLDY*TYPGSLTTPPLLECVTWIV LKEPISVSSEQVLKFRKLNFNGEGEPEE LMVDNWRPAQPLKNRQIKASFK 210768; 210766 210770 LibC000200 CA2 (aa 2-260 of WT, I59N, LOO*, G102R) SHHWGYGKHNGPEHWHKDFPIAKGE RQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRNLNNGHAFNVEFDDSQDKAVL KGGPLDGTYR*IQFHFHWGSLDRQGS EHTVDKKKYAAELHLVHWNTKYGDF GKAVQQPDGLAVLGIFLKVGSAKPGL QKVVDVLDSIKTKGKSADFTNFDPRG LLPESLDYWTYPGSLTTPPLLECVTWI VLKEPISVSSEQVLKFRKLNFNGEGEP EELMVDNWRPAQPLKNRQIKASFK 210771; 210772 210773 LibC000197 CA2 (aa 2-260 of WT, T35I, Y114H, P154L, D161V, P200A, Q221R, F225L, E233D, W244*) SHHWGYGKHNGPEHWHKDFPIAKGE RQSPVDIDIHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKHAAELHLVHWNTKYGDF GKAVQQPDGLAVLGIFLKVGSAKLGL QKVVVVLDSIKTKGKSADFTNFDPRG LLPESLDYWTYPGSLTTAPLLECVTWI VLKEPISVSSERVLKLRKLNFNGDGEP EELMVDNWRPAQPLKNRQIKASFK 210774; 210775 210776 LibC000195 CA2 (aa 2-260 of WT, S105L, L140V, G155C, Y190*) SHHWGYGKHNGPEHWHKDFPIAKGE RQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWGSLDGQGL EHTVDKKKYAAELHLVHWNTKYGDF GKAVQQPDGVAVLGIFLKVGSAKPCL QKVVDVLDSIKTKGKSADFTNFDPRG LLPESLD*WTYPGSLTTPPLLECVTWIV 210777; 210778 210779 2026214111   10 Aug 2026 LKEPISVSSEQVLKFRKLNFNGEGEPEE LMVDNWRPAQPLKNRQIKASFK LibC000188 CA2 (aa 2-260 of WT, T35I, Y114H, P154L, D161V, P200A, F225L, E233D, W244*) SHHWGYGKHNGPEHWHKDFPIAKGE RQSPVDIDIHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKHAAELHLVHWNTKYGDF GKAVQQPDGLAVLGIFLKVGSAKLGL QKVVVVLDSIKTKGKSADFTNFDPRG LLPESLDYWTYPGSLTTAPLLECVTWI VLKEPISVSSEQVLKLRKLNFNGDGEP EELMVDN*RPAQPLKNRQIKASFK 210780; 210775 210781 LibC000180 CA2 (aa 2-260 of WT, K167*, R181H, E213*, V217L, L228H) SHHWGYGKHNGPEHWHKDFPIAKGE RQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDF GKAVQQPDGLAVLGIFLKVGSAKPGL QKVVDVLDSI*TKGKSADFTNFDPHGL LPESLDYWTYPGSLTTPPLLECVTWIV LK*PISLSSEQVLKFRKHNFNGEGEPEE LMVDNWRPAQPLKNRQIKASFK 210782; 210783; 210784 210785 LibC000190 CA2 (aa 2-259 of WT, K167*, R181H, P194Q, G195A, S196H, L197*, T198P, T199P, P200L, P201L, L202F, L203W, E204N, C205V, V206*, T207P, W208G, I209L, V210C, L21 IS, K212R, E213N, I215S, S216A, V217S, S218A, S219A, E220S, Q221R, V222C, L223*, K224N, F225S, R226V, K227N, N229T, F230S, N23 IM, E233R, G234L, E235N, E237K, E238N, L239*, M240W, V241W, D242T, N243T, W244G, R245A, P246Q, A247L, Q248S, P249H, L250*, K251R, N252T, R253G, Q254K, I255S, A257L, S258P, F259S) SHHWGYGKHNGPEHWHKDFPIAKGE RQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDF GKAVQQPDGLAVLGIFLKVGSAKPGL QKVVDVLDSI*TKGKSADFTNFDPHGL LPESLDYWTYQAH*PPLLFWNV*PGLC SRNPSASAASRC*NSVNLTSMGRLNPK N*WWTTGAQLSH*RTGKSKLPS 210782; 210786; 210787; 210788; 210789; 210790; 210791 210792 LibC000227 CA2 (aa 2-259 of WT, P13H, El 17*, G150S, L184F, P185L, E186N, S187P, L188W, D189I, Y190T, W191G, T192P, Y193T, P194Q, G195A, S196H, L197*, T198P, T199P, P200L, P201L, L202F, L203W, E204N, C205V, V206*, T207P, W208G, I209L, V210C, L211*, K212R, E213N, I215S, S216A, V217S, S218A, S219A, E220S, Q221R, V222C, L223 *, K224N, F225S, R226V, K227N, N229T, F230S, N231M, E233R, G234V, E235N, E237K, E238N, L239*, M240W, V241W, D242T, N243T, W244G, R245A, P246Q, A247L, Q248S, P249H, L250*, K251R, N252T, R253G, Q254K, I255S, A257L, S258P, F259S) SHHWGYGKHNGHEHWHKDFPIAKGE RQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAA*LHLVHWNTKYGDF GKAVQQPDGLAVLGIFLKVSSAKPGL QKVVDVLDSIKTKGKSADFTNFDPRG LFLNPWITGPTQAH*PPLLFWNV*PGL C*RNPSASAASRC*NSVNLTSMGRVNP KN*WWTTGAQLSH*RTGKSKLPS 210793; 210794; 210787; 210795; 210796; 210797; 210790; 210791 210798 LibC000218 CA2 (aa 2-260 of WT, A133T, L147F, K148E, V149G, G150W, S151Q, A152R, K153*, P154T, L156P, Q157S, K158E, V159S, V160C, D161*, V162C, L163A, D164G, S165F, I166H, K167*, T168N, K17 IQ, S172E, A173C, D174*, F175L, SHHWGYGKHNGPEHWHKDFPIAKGE RQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDF GKTVQQPDGLAVLGIFFEGWQR*TGPS 210799; 210800; 210801; 210802; LH; 210803; 210807 2026214111   10 Aug 2026 T176H, N177*, F178L, D179R, P180S, R181S, G182W, L183P, L184P, P185S, E186*, S187I, L188P, D189G, Y190L, W191L, T192D, Y193L, G195R, S196L, L197T, T198D, T199H, P201S, L202S, L203S, E204G, C205M, V206C, T207D, W208L, I209D, V210C, L211A, K212Q, E213G, P214T, I215H, S216Q, V217R, S218Q, S219Q, E220R, Q221A, V222G, L223V, K224E, F225I, R226P, K227*, L228T, N229*, F230L, N23 IQ, G232W, E233G, E235 *, P236T, E237R, E238R, L239T, M240D, V241G, D242G, N243Q, W244L, R245A, A247S, Q248S, P249A, L250T, K251E, N252E, R253Q, Q254A, I255N, K256Q, A257S, S258F, F259L, K260Q) ESC*CAGFH*NKGQEC*LH*LRSSWPP SHPGLLDLPRLTDHPSSSGMCDLDCA QGTHQRQQRAGVEIP*T*LQWGG*TR RTDGGQLAPSSATEEQANQSFLQ 210804; T; 210805; 210806 LibC000216 CA2 (aa 2-260 of WT, F20L, K45N, G104R, Al 16V, L147F, K148E, V149G, G150W, S151Q, A152R, K153 *, P154T, L156P, Q157S, K158E, V159S, V160C, DieP, V162C, L163A, D164G, S165F, I166H, K167*, T168N, K171Q, S172E, A173C, D174*, F175L, T176H, N177*, F178L, D179R, P180S, R181S, G182W, L183P, L184P, P185S, E186*, S1871, L188P, D189G, Y190L, W191L, T192D, Y193L, G195R, S196L, L197T, T198D, T199H, P201S, L202S, L203S, E204G, C205M, V206C, T207D, W208L, I209D, V210C, L211A, K212Q, E213G, P214T, I215H, S216Q, V217R, S218Q, S219Q, E220R, Q221A, V222G, L223I, K224E, F225I, R226P, K227*, L228T, N229*, F230L, N231Q, G232W, E233G, E235*, P236T, E237R, E238R, L239T, M240D, V241G, D242G, N243Q, W244L, R245A, A247S, Q248S, P249A, L250T, K251E, N252E, R253Q, Q254A, I255N, K256Q, A257S, S258F, F259L, K260Q) SHHWGYGKHNGPEHWHKDLPIAKGE RQSPVDIDTHTAKYDPSLNPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVL KGGPLDGTYRLIQFHFHWGSLDGQRS EHTVDKKKYAVELHLVHWNTKYGDF GKAVQQPDGLAVLGIFFEGWQR*TGP SESC*CAGFH*NKGQEC*LH*LRSSWP PSMPGLLDLPRLTDHPSSSGMCDLDCA QGTHQRQQRAGIEIP*T*LQWGG*TRR TDGGQLAPSSATEEQANQSFLQ 210808; 210800; 210801; 210802; LH; 210803; 210809; T; 210805; 210806 210810 LibC000222 CA2 (aa 2-259 of WT, N11K, G12D, P13L, E14N, H15T, W16G, H17I, K18R, D19T, F20S, I22L, A23P, K24R, G25E, E26S, R27A, Q28S, S29P, P30L, V3 IL, D32T, I33S, D34T, T35L, H36I, T37Q, A38P, K39S, Y40M, D41T, P42L, S43P, L44*, K45S, L47C, S48L, V49F, S50P, Y51M, D52I, Q53K, A54Q, T55L, S56P, L57*, R58G, I59S, L60S, N61T, N62M, G63V, H64M, A65L, F66S, N67T, V68W, E69S, F70L, D71M, D72T, S73L, Q74R, D75T, A77Q, V78C, L79S, K80R, G81E, G82D, L84W, D85M, G86A, T87L, Y88T, R89D, L90*, 19 IF, Q92S, H94T, H96T, W97G, G98V, S99H, DIO IM, G102D, Q103K, G104V, S105Q, E106S, H107I, T108L, V109W, DI 101, K112R, K113N, Y114M, Al 15L, Al 16Q, El 17N, LI 18F, Hl 19T, L120W, V121F, H122T, W123G, N124T, SHHWGYGKHKDLNTGIRTSPLPRESAS PLLTSTLIQPSMTLP*SPCLFPMIKQLP* GSSTMVMLSTWSLMTLRTKQCSREDP WMALTD*FSFTFTGVHLMDKVQSILW IKRNMLQNFTWFTGTPNMGILGKLCS NLMDWPF*VFF*RLAALNRAFRKLLM CWIPLKQRARVLTSLTSILVASFVNPWI TGPTQAH*PPLLFWNV*PGLCSWNPSA SAASRC*NYVNLTSMGRVNPKN*RWT TGALLSH*RTGKSKLPS 210811; 210812; 210813; 210814; VFF; 210815; 210787; 210816; 210817; 210818; 210791; 210819 2026214111   10 Aug 2026 T125P, K126N, Y127M, D129I, F130L, A133L, V134C, Q135S, Q136N, P137L, D138M, G139D, L140W, A141P, V142F, L143*, G144V, I145F, L147*, K148R, V149L, G150A, S151A, A152L, K153N, P154R, G155A, L156F, Q157R, V159L, V160L, D161M, V162C, L163W, D164I, S165P, I166L, T168Q, K169R, G170A, K171R, S172V, A173L, D174T, F175S, T176L, N177T, F178S, D179I, P180L, R181V, G182A, L183S, L184F, P185V, E186N, S187P, L188W, D1891, Y190T, W191G, T192P, Y193T, P194Q, G195A, S196H, L197*, T198P, T199P, P200L, P201L, L202F, L203W, E204N, C205V, V206*, T207P, W208G, I209L, V210C, L21 IS, K212W, E213N, I215S, S216A, V217S, S218A, S219A, E220S, Q221R, V222C, L223 *, K224N, F225Y, R226V, K227N, N229T, F230S, N231M, E233R, G234V, E235N, E237K, E238N, L23 9*, M240R, V241W, D242T, N243T, W244G, R245A, P246L, A247L, Q248S, P249H, L250*, K251R, N252T, R253G, Q254K, I255S, A257L, S258P, F259S)

[0073] Additional CA2 destabilizing domains are provided in Table 4. CA2 destabilizing mutants provided in Table 3B are identified as described above, such as by structure guided mutagenesis or by combining single mutants. Table 4: CA2DDs Description AA Sequence AASEQ ID NO. NA SEQ ID NO. CA2 (Mldel, E106D) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSDHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210968 211004 CA2 (Mldel, I59N, G102R) -variant 2 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRNLNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDRQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210969 211005 CA2 (Mldel, L197P) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSPTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210970 211006 CA2 (Mldel, L156H, S172C, SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW 210971 211007 2026214111   10 Aug 2026 F178Y, E186D) - variant 1 GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGHQKWDVLDSIKTKGKCADFTNYDPRGLLPDSLDYW TYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMV DNWRPAQPLKNRQIKASFK CA2 (Mldel, L156H) - variant 2 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGHQKWDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210972 211008 CA2 (Mldel, R27L, T87I, H122Y, N252D) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGELQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGIYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVYWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKDRQIKASFK 210973 211009 CA2 (I59N) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRNLNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210974 211010 CA2 (G63D) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNDHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210975 211011 CA2 (Mldel, H122Y) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVYWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210976 211012 CA2 (Mldel, G63D, M240L) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNDHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELLVD NWRPAQPLKNRQIKASFK 210977 211013 CA2 (Mldel, A77I, P249F) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKIVLKGGPLDGTYRLIQFHFHWG SLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGI FLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTY PGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDN WRPAQFLKNRQIKASFK 210978 211014 CA2 (Mldel, D71K, T192F) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFKDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL 210979 211015 2026214111   10 Aug 2026 GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWF YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK CA2 (Mldel, L156H) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGHQKWDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210980 211016 CA2 (Mldel, R27L, H122Y) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGELQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVYWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210981 211017 CA2 (Mldel, T87I, H122Y) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGIYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVYWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210982 211018 CA2 (Mldel, H122Y, N252D) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVYWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKDRQIKASFK 210983 211019 CA2 (Mldel, D72F, V241F) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDFSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMFD NWRPAQPLKNRQIKASFK 210984 211020 CA2 (Mldel, V241F, P249L) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMFD NWRPAQLLKNRQIKASFK 210985 211021 CA2 (Mldel, D72F, P249L) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDFSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQLLKNRQIKASFK 210986 211022 CA2 (Mldel, D71L, T87N) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFLDSQDKAVLKGGPLDGNYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT 210987 211023 2026214111   10 Aug 2026 YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK CA2 (Mldel, D71L, L250R) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFLDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPRKNRQIKASFK 210988 211024 CA2(Y51T)-variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV STDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210989 211025 CA2 (S73N, R89F) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDNQDKAVLKGGPLDGTYFLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210990 211026 CA2 (D72F, P249F) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDFSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210991 211027 CA2 (T55K, G63N, Q248N) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQAKSLRILNNNHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPANPLKNRQIKASFK 210992 211028 CA2 (Y193I) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT IPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210993 211029 CA2 (S56F) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATFLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210994 211030 CA2 (S56F, D7IS) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATFLRILNNGHAFNVEFSDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT 210995 211031 2026214111   10 Aug 2026 YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK CA2 (S73N, R89Y) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDNQDKAVLKGGPLDGTYYLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210996 211032 CA2 (V134F, L228F) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAFQQPDGLAVL GIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKFNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 210997 211033 CA2 (Mldel, L156H, A256del, S257del, F258del, K259del) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGHQKWDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIK 210998 211034 CA2 (Mldel, S2del, H3del, H4del, W5del, L156H) - variant 1 GYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLD GQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLK VGSAKPGHQKWDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGS LTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWR PAQPLKNRQIKASFK 210999 211035 CA2 (Mldel, W5Y, L156H) -variant 1 SHHYGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGHQKWDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 211000 211036 CA2 (Mldel, L156H, G234del, E235del, P236del) -variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGHQKWDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEEELMVDNW RPAQPLKNRQIKASFK 211001 211037 CA2 (Mldel, L156H, F225L) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHW GSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGHQKWDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKLRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK 211002 211038 CA2 (Mldel, D71N, D75N, DIO IN, L156H) - variant 1 SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSV SYDQATSLRILNNGHAFNVEFNDSQNKAVLKGGPLDGTYRLIQFHFHW GSLNGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVL GIFLKVGSAKPGHQKWDVLDSIKTKGKSADFTNFDPRGLLPESLDYWT 211003 211039 2026214111   10 Aug 2026 YPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVD NWRPAQPLKNRQIKASFK

[0074] In some embodiments a region or a portion of the CA2 WT may be used as template for generating CA2 DDs. In some embodiments, the CA2 DDs may exclude the lysine at position 260 of SEQ ID NO. 11717. In some aspects, the CA2 regions may include but are not limited to those described in Table 5. Table 5: CA2 regions Description AA SEQUENCE AA SEQ ID NO. NA SEQ ID NO. CA2 (aa 1-142 of WT) MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYD QATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAV 210820 - CA2 (aa 2-142 of WT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSE HTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAV 210821 - CA2 (aa 1-190 of WT) MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYD QATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGL QKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDY 210822 CA2 (aa 2-190 of WT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSE HTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQ KVVDVLDSIKTKGKSADFTNFDPRGLLPESLDY 210823 CA2 (aa 1-89 of WT) MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYD QATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYR 210824 - CA2 (aa 2-89 of WT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYR 210825 - CA2 (aa 1-243 of WT) MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYD QATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGL QKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIV LKEPISVSSEQVLKFRKLNFNGEGEPEELMVDN 210826 CA2 (aa 2-243 of WT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSE HTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQ KVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVL KEPISVSSEQVLKFRKLNFNGEGEPEELMVDN 210827 CA2 (aa 1-166 of WT) MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYD QATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGL QKVVDVLDSI 210828 CA2 (aa 2-166 of WT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSE HTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQ KVVDVLDSI 210782 210829 CA2 (aa 1-116 of WT) MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYD QATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAA 210830 - 2026214111   10 Aug 2026 CA2 (aa 2-116 of WT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSE HTVDKKKYAA 210831 - CA2 (aa 1-152 of WT) MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYD QATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGS EHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSA 210832 - CA2 (aa 2-152 of WT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQ ATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSE HTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSA 210833 - CA2 (aa 1-43 of WT) MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS 210834 - CA2 (aa 2-43 of WT) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPS 210835 -

[0075] Any of the CA2 regions described herein may be utilized to generate CA2 DD. Table 6 provides CA2 DDs derived from CA2 regions. Table 6: CA2 DDs derived from CA2 regions Description AA SEQUENCE AA SEQID NO. NA SEQ ID NO. CA2 (aa 2-142 of WT, G12E, A38V, A65V, G98V, S99H,D101M, G102D, Q103K, G104V, S105Q, E106S, H107I, T108L, V109W, DI 101, KI 12R, KI 13N, Y114M, A115L,A116Q,E117N,L118F, H119T, L120W, V121F, H122T, W123G, N124T, T125P, K126N, Y127M, D129I, F130L, K132E, A133L, V134C, Q135S, Q136N, P137L, D138M, G139D, L140W, A141P, V142F) SHHWGYGKHNEPEHWHKDFPIAKGERQSPVDIDTHTVKYDPSL KPLSVSYDQATSLRILNNGHVFNVEFDDSQDKAVLKGGPLDGT YRLIQFHFHWVHLMDKVQSILWIKRNMLQNFTWFTGTPNMGIL GELCSNLMDWPF 210762 210836 CA2 (aa 2-190 of WT, F20L, K45N, G104R, Al 16V, A173G) SHHWGYGKHNGPEHWHKDLPIAKGERQSPVDIDTHTAKYDPSL NPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGT YRLIQFHFHWGSLDGQRSEHTVDKKKYAVELHLVHWNTKYGD FGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKS GDFTNFDPRGLLPESLDY 210765 210837 CA2 (aa 2-190 of WT, HUD, P30S, G81V, K132R, S151I,A152D, A173G) SHHWGYGKHNGPEHWDKDFPIAKGERQSSVDIDTHTAKYDPSL KPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKVGPLDGT YRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGD FGRAVQQPDGLAVLGIFLKVGIDKPGLQKVVDVLDSIKTKGKSG DFTNFDPRGLLPESLDY 210768 210838 CA2 (aa 2-89 of WT, I59N) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSL KPLSVSYDQATSLRNLNNGHAFNVEFDDSQDKAVLKGGPLDGT YR 210771 210839 CA2 (aa 2-243 ofWT, T35I, Y114H,P154L,D161V,P200A, Q221R, F225L, E233D) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDIHTAKYDPSL KPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGT YRLIQFHFHWGSLDGQGSEHTVDKKKHAAELHLVHWNTKYGD FGKAVQQPDGLAVLGIFLKVGSAKLGLQKVVVVLDSIKTKGKS ADFTNFDPRGLLPESLDYWTYPGSLTTAPLLECVTWIVLKEPISV SSERVLKLRKLNFNGDGEPEELMVDN 210774 210840 CA2 (aa 2-189 of WT,S105L, L140V, G155C) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSL KPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGT YRLIQFHFHWGSLDGQGLEHTVDKKKYAAELHLVHWNTKYGD FGKAVQQPDGVAVLGIFLKVGSAKPCLQKVVDVLDSIKTKGKS ADFTNFDPRGLLPESLD 210777 210841 210780 210842 2026214111   10 Aug 2026 CA2 (aa 2-243 ofWT, T35I, Y114H, P154L, D161V, P200A, F225L, E233D) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDIHTAKYDPSL KPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGT YRLIQFHFHWGSLDGQGSEHTVDKKKHAAELHLVHWNTKYGD FGKAVQQPDGLAVLGIFLKVGSAKLGLQKVVVVLDSIKTKGKS ADFTNFDPRGLLPESLDYWTYPGSLTTAPLLECVTWIVLKEPISV SSEQVLKLRKLNFNGDGEPEELMVDN CA2 (aa 2-116 of WT, P13H) SHHWGYGKHNGHEHWHKDFPIAKGERQSPVDIDTHTAKYDPSL KPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGT YRLIQFHFHWGSLDGQGSEHTVDKKKYAA 210793 210843 CA2 (aa 2-152 of WT,A133T, L147F, K148E, V149G, G150W, S151Q, A152R) SHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSL KPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGT YRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGD FGKTVQQPDGLAVLGIFFEGWQR 210799 210844 CA2 (aa 2-152 of WT, F20L, K45N, G104R, A116V, L147F, K148E, V149G, G150W, S151Q, A152R) SHHWGYGKHNGPEHWHKDLPIAKGERQSPVDIDTHTAKYDPSL NPLSVSYDQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGT YRLIQFHFHWGSLDGQRSEHTVDKKKYAVELHLVHWNTKYGD FGKAVQQPDGLAVLGIFFEGWQR 210808 210845 CA2(aa2-43 of WT, N11K, G12D, P13L,E14N,H15T, W16G,H17I, K18R, D19T, F20S, I22L, A23P, K24R, G25E, E26S, R27A, Q28S, S29P, P30L, V31L, D32T, I33S, D34T, T35L, H36I, T37Q, A38P, K39S, Y40M, D41T, P42L, S43P) SHHWGYGKHKDLNTGIRTSPLPRESASPLLTSTLIQPSMTLP 210811 210846

[0076] In some embodiments, DDs derived from CA2 may include one, two, three, four, five, or more of the mutations described in the previous Tables.

[0077] In some embodiments, the mutation may be a conserved (with similar physicochemical properties as the amino acid at the mutation site), a semi conserved (e.g., negatively to positively charge amino acid) or a non-conserved (amino acid with different physicochemical properties than the amino acid at the mutation site). In some embodiments, the amino acid lysine may be mutated to glutamic acid or arginine; the amino acid phenylalanine may be mutated to leucine; the amino acid leucine may be mutated to phenylalanine; or the amino acid asparagine may be mutated to serine. Regions or portions or domains of wild type proteins may be utilized as SREs / DDs in whole or in part. They may be combined or rearranged to create new peptides, proteins, regions or domains of which any may be used as SREs / DDs or the starting point for the design of further SREs and / or DDs.

[0078] The destabilization domains described herein may also include amino acid and nucleotide substitutions that do not affect stability, including conservative, non-conservative substitutions and or polymorphisms. In some embodiments, CA2 DDs described herein may also be fragments of the above destabilizing domains, including fragments containing variant amino acid sequences. Preferred fragments are unstable in the absence of the stimulus and stabilized upon addition of the stimulus. Preferred fragments retain the ability to interact with the stimulus with similar efficiency as the DDs described herein.

[0079] In one embodiment, the SRE comprises a region of the CA2 protein. The region of the CA2 protein may be 10,11,12,13,14,15,16,17,18,19, 20,21, 22, 23, 24, 25,26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69,70, 71, 72, 73, 2026214111   10 Aug 2026 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, 250, 251, 252,253, 254, 255,256, 257, 258, 259, 260 or more than 260 amino acids in length. The region of the parent protein may be 5-50, 25-75, 50-100, 75-125,100-150,125-175,150-200,175-225,200-250, 225-260 ammo acids in length.

[0080] In some embodiments, CA2 DDs described herein may include one or more mutations that are relative to Uniprot ID: P00918 (SEQ ID NO. 11717). Those mutations may include, but are not limited to, Al 15L, Al 16Q, Al 16V, 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, DI 101, 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, El 17*, E117N, EUN, 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, Hl 19T, H119Y, H122T, H122Y, H15L, H15T, H15Y, HUD, HUI, H36I, H36Q, H64M, H94T, H96T, I145F, I145M, I166H, I166L, I209D, I209L, I215H, I215S, I22L, I255N, I255S, I33S, I59F, I59N, I59S, I91F, KI 1 IE, K111N, K112R, KI 131, 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, LI 18F, 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, L21 IS, 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, 2026214111   10 Aug 2026 N252E, N252T, N61R, N61T, N61Y, N62K, N62M, N67D, N67T, P137L, P13A, P13H, P13L, Pl 3 S, P154L, P154R, P154T, P180L, P180S, P185L, P185S, P185V, P194Q, P200A, P200L, P200S, P200T, P201A, P201L, P201R, P201S, P214T, P236L, P236T, P246L, P246Q, P249A, P249F, P249H, P249I, P249X, P30L, P30S, P42L, P83A, Q103K, Q135S, Q136N, Q157R, Q157S, Q221A, Q221R, Q248F, Q248L, Q248S, Q254A, Q254K, Q28S, Q53H, Q53K, Q53N, Q74R, Q92H, Q92S, R181H, R181S, R181V, R226H, R226P, R226V, R245A, R253G, R253Q, R27A, R58G, R89D, R89F, R89I, R89X, R89Y, S105L, S105Q, S151A, S151I, S151Q, S165F, S165P, S172E, S172V, S187I, S187P, S196H, S196L, S216A, S216Q, S218A, S218Q, S219A, S219Q, S258F, S258P, S29C, S29P, S43P, S43T, S48L, S50P, S56F, S56N, S56P, S56X, S73L, S73N, S73X, S99H, T108L, T125I, T125P, T168K, T168N, T168Q, T176H, T176L, T192D, T192F, T192I, T192N, T192P, T192X, T198D, T198I, T198P, T199A, T199H, T199P, T207D, T207I, T207P, T207S, T35I, T35L, T37Q, T55L, T87L, V109M, V109W, V121F, V134C, V134F, V142F, V149G, V149L, V159L, V159S, V160C, V160L, V162A, V162C, V206*, V206C, V206M, V210C, V217L, V217R, V217S, V222A, V222C, V222G, V241G, V241W, V241X, V31L, V49F, V68L, V68W, V78C, W123G, W123R, W16G, W191*, W191G, W191L, W208G, W208L, W208S, W244*, W244G, W244L, W97C, W97G, Y114H, Y114M, Y127M, Y190*, Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, S29A. As used herein    indicates the translation of the stop codon and X indicates any amino acid.

[0081] In one embodiment, a CA2 DD described herein comprises a mutation that is relative to Uniprot ID: P00918 (SEQ ID NO. 11717), selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, andY51T.

[0082] In some embodiments, CA2 DDs described herein may include mutations that are relative to Uniprot ID: P00918 (SEQ ID NO. 11717). Those mutations may 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), 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 2260 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, 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 2260 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 2-260 of WT, D71F, N231F)(SEQ ID NO. 210505), CA2 (aa 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 of WT, D71K, P249H)(SEQ ID NO. 210516), CA2 (aa 2-260 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- 2026214111   10 Aug 2026 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, 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, 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, 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, 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, K169R, D179E)(SEQ ID NO. 210704), CA2 (aa 2-260 of WT, H15L, A54V, KI 1 IE, 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, KI 131, 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, Pl80S)(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, T168K)(SEQ ID NO. 210730), CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E)(SEQ ID NO. 210732), CA2 (aa 2260 of WT, L44M, L47V, N62K, E69D)(SEQ ID NO. 210734), CA2 (aa 2-260 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, 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).

[0083] In one embodiment, a CA2 DD described herein comprises multiple mutations that are relative to Uniprot ID: P00918 (SEQ ID NO. 11717), selected from 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, 2026214111   10 Aug 2026 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 ofWT, T55K, G63N, Q248N), CA2 (aa2-260 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, 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, L156H, S172C, F178Y, E186D)(SEQ ID NO. 210756), CA2 (aa 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), 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, 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).

[0084]

[0085] In some embodiments, the CA2 may be derived from carbonic anhydrases of Homo sapiens. In some embodiments, the CA2 DDs described herein may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to a particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. In some embodiments the reference polypeptide may be SEQ ID NO. 11717. Tools for alignment may include those of 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).

[0086] In some embodiments, the CA2 DDs may be derived from carbonic anhydrases of species other than Homo sapiens. In some embodiments, the CA2 DDs may be derived from carbonic anhydrases of species such but not limited to Acinonyxjubatus, Ailuropoda melanoleuca, Balaenoptera acutorostrata scammoni, Callithrixjacchus, Callorhinus ursinus, Camelus bactrianus, Camelus dromedarius, Camelus ferns, Canis lupus dingo, Canis lupus familiaris, Carlito syrichta, Castor 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, Homo sapiens, Ictidomys tridecemlineatus, Jaculus, Lagenorhynchus obhquidens, 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 putorius furo, Narmospalax galili, Neomonachus schauinslandi, Neophocaena asiaeorientalis, Nomascus leucogenys, Odobenus rosmarus divergens, Orcinus orca, Oiyctolagus cuniculus, Otolemur 2026214111   10 Aug 2026 gamettii, 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 arctos horribilis, Ursus maritimus, Vulpes, and / or Zalophus californianus.

[0087] The SRE described herein may include CA2 DDs which include but are not limited to one, two, three or more mutations such as, but not limited to, A115L, A116Q, Al 16V, 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, DI 101, 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, El 17*, E117N, EUN, 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, G13IE, G131R, G131W, G139D, G144D, G144V, G150A, GI50S. 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, Hl 19T, H119Y, H122T, H122Y, H15L, HI5T. HI5Y. HUD, HUI, H36I, H36Q, H64M, H94T, H96T, I145F, I145M, I166H, I166L, I209D, I209L, I215H, I215S, I22L, I255N, I255S, I33S, I59F, I59N, I59S, I91F, KI 1 IE, K111N, K112R, KI 131, 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, LI 18F, 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, PI3L. 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, S1511, S15 IQ, S165F, S165P, S172E, S172V, S187I, S187P, S196H, 2026214111   10 Aug 2026 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.

[0088] In one embodiment, an SRE described herein may include a CA2 DD which comprises a mutation selected fromE106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0089] The SRE described herein may include CA2 DDs which include 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, 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 2260 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 (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 2-260 of WT, D71F, N231F)(SEQ ID NO. 210505), CA2 (aa 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 of WT, D71K, P249H)(SEQ ID NO. 210516), CA2 (aa 2-260 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 (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 (aa2-260 of WT, D72S, T192N)(SEQ ID NO. 210552), CA2 (aa2-260 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. 210562), CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F)(SEQ ID NO. 210564), CA2 (aa2-260 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, 2026214111   10 Aug 2026 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, 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, K169R, D179E)(SEQ ID NO. 210704), CA2 (aa 2-260 of WT, H15L, A54V, KI 1 IE, 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, KI 131, 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, Pl80S)(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, T168K)(SEQ ID NO. 210730), CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E)(SEQ ID NO. 210732), CA2 (aa 2260 of WT, L44M, L47V, N62K, E69D)(SEQ ID NO. 210734), CA2 (aa 2-260 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, 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).

[0090] In one embodiment, an SRE described herein may include a CA2 DD which comprises mutations selected from 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 2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2260 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 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 of WT, E106D, C205S)(SEQ ID NO. 210523), CA2 (aa 2-260 of WT, C205S, W208S)(SEQ ID NO. 2026214111   10 Aug 2026 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, S73N, R89F)(SEQ ID NO. 210562), CA2 (aa 2-260 of WT, G63D, M240L)(SEQ ID NO. 210578), CA2 (aa2-260 of WT, V134F, L228F)(SEQ ID NO. 210580), and / or CA2 (aa2-260 of WT, S56F, D71S)(SEQ ID NO. 210584).

[0091] Also provided herein are biocircuit systems that include at least one effector module. The effector module of the biocircuit may include a stimulus response element (SRE), that includes in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717). The biocircuits may also include at least one payload, which may be attached, appended or associated with the SRE.

[0092] The SRE of the biocircuit system which includes, 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 Al 15L, Al 16Q, Al 16V, 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, DI 101, 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, El 17*, E117N, EUN, 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, GI50S. 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, Hl 19T, H119Y, H122T, H122Y, H15L, H15T, H15Y, HUD, HUI, H36I, H36Q, H64M, H94T, H96T, I145F, I145M, I166H, I166L, I209D, I209L, I215H, I215S, I22L, I255N, I255S, I33S, I59F, I59N, I59S, I91F, KI 1 IE, K111N, K112R, KI 131, 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, LI 18F, 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, L21 IS, 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, PUL, PUS, P154L, P154R, P154T, P180L, P180S, P185L, P185S, P185V, P194Q, P200A, P200L, P200S, P200T, P201A, P201L, P201R, P201S, 2026214111   10 Aug 2026 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.

[0093] In one embodiment, the SRE of the biocircuit system which includes, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), may include a mutation selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0094] The SRE of the biocircuit system which includes, in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), may include multiple mutations such as but not limited toCA2 (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 2-260 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 2260 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 2-260 of WT, D71F, N231F)(SEQ ID NO. 210505), CA2 (aa 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 of WT, D71K, P249H)(SEQ ID NO. 210516), CA2 (aa 2-260 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 2260 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 2026214111   10 Aug 2026 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. 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, 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, 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, K169R, D179E)(SEQ ID NO. 210704), CA2 (aa 2-260 of WT, H15L, A54V, KI 1 IE, 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, KI 131, 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, Pl80S)(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, T168K)(SEQ ID NO. 210730), CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E)(SEQ ID NO. 210732), CA2 (aa 2260 of WT, L44M, L47V, N62K, E69D)(SEQ ID NO. 210734), CA2 (aa 2-260 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, 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).

[0095] In one embodiment, the SRE of the biocircuit system which includes, in whole or in part, human carbonic anhydrase 2 (CA2; SEQID NO. 11717), may include multiple mutations selected from CA2 (aa 2-260 ofWT, 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- 2026214111   10 Aug 2026 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, 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 2260 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 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), 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, 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).

[0096] Stabilization and Destabilization Ratio of SRE

[0097] In some embodiments, the present disclosure provides methods for modulating protein, expression, function or level by measuring the stabilization ratio and destabilization ratio. As used herein, the stabilization ratio may be defined as the ratio of expression, function or level of a protein of interest in response to the stimulus to the expression, function or level of the protein of interest in the absence of the stimulus specific to the SRE. In some aspects, the stabilization ratio is at least 1, such as by at least 1-10,1-20,1-30,1-40,1-50,1-60,1-70,1-80,1-90,1-100, 20-30,2040, 20-50, 20-60,20-70, 20-80,20-90, 20-95, 20-100, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-95, 30-100,40-50, 40-60,40-70, 40-80, 40-90,40-95, 40-100, 50-60, 50-70, 50-80, 50-90, 50-95, 50-100, 60-70,60-80, 60-90, 60-95,60100, 70-80,70-90, 70-95,70-100, 80-90, 80-95, 80-100, 90-95, 90-100 or 95-100. As used herein, the destabilization ratio may be defined as the ratio of expression, function or level of a protein of interest in the absence of the stimulus specific to the effector module to the expression, function or level of the protein of interest, that is expressed constitutively and in the absence of the stimulus specific to 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 the stimulus. In some aspects, the destabilization ratio is at least 0, such as by 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, 0.10.4,0.1-0.5, 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, 0.60.8,0.6-0.9,0.7-0.8,0.7-0.9 or 0.8-0.9.

[0098] In some embodiments, the SRE of the effector module may stabilize the pay load of interest by a stabilization ratio of 1 or more, wherein the stabilization ratio may comprise the ratio of expression, function or level of the pay load 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. 2026214111   10 Aug 2026

[0099] In some embodiments, the SRE may destabilize the pay load of interest by a destabilization ratio between 0, and 0.09, wherein the destabilization ratio may comprise the ratio of expression, function or level of the pay load of interest in the absence of the stimulus specific to the SRE to the expression, function or level of the pay load of interest that is expressed constitutively, and in the absence of the stimulus specific to the SRE. Protein-Protein Interactions of SRE

[0100] In some embodiments, the present disclosure provides stimulus response element (SRE) which may comprise a destabilizing domain (DD) derived from human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717) in whole or in part. In one embodiment, the DD may include the whole CA2 (SEQ ID NO. 11717). In some embodiments, the DD may include a portion or region of the human carbonic anhydrase. The portion or region of CA2 may be selected from but not limited to amino acids 2-260 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210492); amino acids 1-142 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210820); amino acids 2-142 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210821); amino acids 1-190 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210822); amino acids 2-190 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210823); amino acids 1-89 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210824); amino acids 2-89 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210825); amino acids 1-243 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210826); amino acids 2-243 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210827); amino acids 1-166 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210828); amino acids 2-166 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210782); amino acids 1-116 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210830); amino acids 2-116 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210831); ammo acids 1-152 of CA2 (SEQ ID NO. 11717) such as, but not limited to, SEQ ID NO. (210832); amino acids 2-152 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210833); amino acids 1-43 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210834); or amino acids 2-43 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210835). In one embodiment, the DD may include amino acids 2 to 260 of CA2 (SEQ ID NO. 11717). In one embodiment, the DD may include amino acids 2 to 260 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. 210492.

[0101] In one embodiment, the DD may include amino acids 2 to 260 of CA2 (SEQ ID NO. 11717).

[0102] In some embodiments, the DD may include one, two, three or more mutations such as, but not limited to, Al 15L, 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, DI 101, 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, 2026214111   10 Aug 2026 E106D, E106G, E106S, El 17*, E117N, EUN, 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, G13IE, G131R, G131W, G139D, G144D, G144V, G150A, GUOS, 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, Hl 19T, H119Y, H122T, H122Y, H15L, HUT, HUY, HUD, HUI, H36I, H36Q, H64M, H94T, H96T, I145F, I145M, I166H, I166L, I209D, I209L, I215H, I215S, I22L, I255N, I255S, I33S, I59F, I59N, I59S, I91F, Kll IE, K111N, K112R, KI 131, 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, LI 18F, 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, PUL, PUS, P154L, P154R, P154T, P180L, P180S, P185L, P185S, P185V, P194Q, P200A, P200L, P200S, P200T, P201A, P201L, P201R, P201S, P214T, P236L, P236T, P246L, P246Q, P249A, P249F, P249H, P249I, P249X, P30L, P30S, P42L, P83A, Q103K, Q135S, Q136N, Q157R, Q157S, Q221A, Q221R, Q248F, Q248L, Q248S, Q254A, Q254K, Q28S, Q53H, Q53K, Q53N, Q74R, Q92H, Q92S, R181H, R181S, R181V, R226H, R226P, R226V, R245A, R253G, R253Q, R27A, R58G, R89D, R89F, R89I, R89X, R89Y, S105L, S105Q, S151A, S151I, S151Q, S165F, S165P, S172E, S172V, S187I, S187P, S196H, S196L, S216A, S216Q, S218A, S218Q, S219A, S219Q, S258F, S258P, S29C, S29P, S43P, S43T, S48L, S50P, S56F, S56N, S56P, S56X, S73L, S73N, S73X, S99H, T108L, T125I, T125P, T168K, T168N, T168Q, T176H, T176L, T192D, T192F, T192I, T192N, T192P, T192X, T198D, T198I, T198P, T199A, T199H, T199P, T207D, T207I, T207P, T207S, T35I, T35L, T37Q, T55L, T87L, V109M, V109W, V121F, V134C, V134F, V142F, V149G, V149L, V159L, V159S, V160C, V160L, V162A, V162C, V206*, V206C, V206M, V210C, V217L, V217R, V217S, V222A, V222C, V222G, V241G, V241W, V241X, V31L, V49F, V68L, V68W, V78C, W123G, W123R, W16G, W191*, W191G, W191L, W208G, W208L, W208S, W244*, W244G, W244L, W97C, W97G, Y114H, Y114M, Y127M, Y190*, Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, S29A. As used herein indicates the translation of the stop codon and X indicates any amino acid.

[0103] In one embodiment, the DD may include aE106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, or Y51T mutation. 2026214111   10 Aug 2026

[0104] In some embodiments, the DD may 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 (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 2260 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 2260 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 (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 2-260 of WT, D71F, N231F)(SEQ ID NO. 210505), CA2 (aa 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 of WT, D71K, P249H)(SEQ ID NO. 210516), CA2 (aa 2-260 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 (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 (aa2-260 of WT, D72S, T192N)(SEQ ID NO. 210552), CA2 (aa2-260 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. 210562), CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F)(SEQ ID NO. 210564), CA2 (aa2-260 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, 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, 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, K169R, D179E)(SEQ ID NO. 210704), CA2 (aa 2-260 of WT, H15L, A54V, KI 1 IE, 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, KI 131, T125I, D138G, G155S)(SEQ ID NO. 210714), CA2 (aa 2-260 of WT, I59N, G102R, 2026214111   10 Aug 2026 A173T)(SEQ ID NO. 210716), CA2 (aa 2-260 of WT, L79F, Pl80S)(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, T168K)(SEQ ID NO. 210730), CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E)(SEQ ID NO. 210732), CA2 (aa 2260 of WT, L44M, L47V, N62K, E69D)(SEQ ID NO. 210734), CA2 (aa 2-260 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, 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).

[0105] In one embodiment, the DD may include 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 ofWT, T55K, G63N, Q248N), CA2 (aa 2-260 ofWT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 ofWT, L156H, S2del, H3del, H4del, W5del), CA2 (aa 2-260 ofWT, W4Y, L156H), CA2 (aa 2-260 ofWT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 ofWT, L156H, F225L), CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 ofWT, I59N, G102R)(SEQ ID NO. 210598), CA2 (aa 2-260 of WT, G63D, E69V, N231I)(SEQ ID NO. 210748), CA2 (aa 2-260 ofWT, R27L, T87I, H122Y, N252D)(SEQ ID NO. 210702), CA2 (aa 2-260 ofWT, D72F, V241F, P249L)(SEQ ID NO. 210503), CA2 (aa 2-260 ofWT, D71L, T87N, L250R)(SEQ ID NO. 210510), CA2 (aa 2-260 ofWT, L156H, S172C, F178Y, E186D)(SEQ ID NO. 210756), CA2 (aa 2-260 ofWT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 ofWT, E106D, C205S)(SEQ ID NO. 210523), CA2 (aa 2-260 ofWT, C205S, W208S)(SEQ ID NO. 210525), CA2 (aa 2-260 ofWT, S73N, R89Y)(SEQ ID NO. 210532), CA2 (aa 2-260 ofWT, D71K, T192F)(SEQ ID NO. 210534), CA2 (aa 2-260 ofWT, S73N, R89F)(SEQ ID NO. 210562), CA2 (aa 2-260 ofWT, G63D, M240L)(SEQ ID NO. 210578), CA2 (aa 2-260 ofWT, V134F, L228F)(SEQ ID NO. 210580), and / or CA2 (aa 2-260 ofWT, S56F, D71S)(SEQ ID NO. 210584).

[0106] In one embodiment, the DD may include amino acids 2 to 260 of CA2 (SEQ ID NO. 11717), such as, but not limited to, SEQ ID NO. (210492).

[0107] In some embodiments, the SRE may be, but is not limited to, CA2 (aa 2-260 of WT, R27L, H122Y), CA2 (aa 2-260 ofWT, T87I, H122Y), CA2 (aa 2-260 ofWT, H122Y, N252D), CA2 (aa2-260 ofWT, D72F, V241F), CA2 (aa2-260 ofWT, V241F, P249L), CA2 (aa2-260 ofWT, D72F, P249L), CA2 (aa2-260 ofWT, D71L, L250R), CA2 (aa 2-260 ofWT, D72F, P249F), CA2 (aa 2-260 ofWT, T55K, G63N, Q248N), CA2 (aa 2-260 ofWT, L156H, A257del, S258del, F259del, K260del), CA2 (aa2-260 ofWT, L156H, S2del, H3del, H4del, W5del), CA2 (aa2-260 ofWT, W4Y, 2026214111   10 Aug 2026 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, L156H), (CA2 (aa 2-260 of WT, I59N, G102R)(SEQ ID NO. 210598), CA2 (aa 2260 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 2-260 of WT, D71F, N231F)(SEQ ID NO. 210505), CA2 (aa 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 of WT, D71K, P249H)(SEQ ID NO. 210516), CA2 (aa 2-260 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, 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, S56X, L57X, T192X)(SEQ ID NO. 210574), CA2 (aa 2260 of WT, Y193V, K260F)(SEQ ID NO. 210576), 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, 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, 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, KI UN, 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, KI 131, T125I, D138G, G155S)(SEQ ID NO. 210714), CA2 (aa 2-260 of WT, I59N, G102R, A173T)(SEQ ID NO. 210716), CA2 (aa 2260 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, T168K)(SEQ ID NO. 210730), CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E)(SEQ ID NO. 210732), CA2 (aa 2-260 of WT, L44M, L47V, N62K, E69D)(SEQ ID NO. 210734), CA2 (aa 2-260 of WT, D75V, K169N, F259L)(SEQ ID NO. 210738), CA2 (aa 2-260 of WT, T207S, 2026214111   10 Aug 2026 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)..

[0108] In one embodiment, the DD may be 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 ofWT, T55K, G63N, Q248N), CA2 (aa2-260 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, 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, L156H, S172C, F178Y, E186D)(SEQ ID NO. 210756), CA2 (aa 2-260 of WT, D71F, N231F)(SEQ ID NO. 210505), CA2 (aa 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 of WT, D71K, P249H)(SEQ ID NO. 210516), CA2 (aa 2-260 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, 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, S56X, L57X, T192X)(SEQ ID NO. 210574), CA2 (aa 2260 of WT, Y193V, K260F)(SEQ ID NO. 210576), 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, 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 2026214111   10 Aug 2026 ID NO. 210698), CA2 (aa 2-260 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, Pl3 S, P83A, D101G, KI UN, 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, KI 131, T125I, D138G, G155S)(SEQ ID NO. 210714), CA2 (aa 2-260 of WT, I59N, G102R, A173T)(SEQ ID NO. 210716), CA2 (aa 2260 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, T168K)(SEQ ID NO. 210730), CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E)(SEQ ID NO. 210732), CA2 (aa 2-260 of WT, L44M, L47V, N62K, E69D)(SEQ ID NO. 210734), CA2 (aa 2-260 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, 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 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, C205R, C205V, C205W, C205Y, D101G, D101M, DI 101, 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, El 17*, E117N, EUN, 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, G13IE, 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, Hl 19T, H119Y, H122T, H122Y, H15L, H15T, H15Y, HUD, H17I, H36I, H36Q, H64M, H94T, H96T, I145F, I145M, I166H, I166L, I209D, I209L, I215H, I215S, I22L, I255N, I255S, I33S, I59F, I59N, I59S, 19IF, KI HE, KI UN, 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, LI 18F, L120W, L140V, L140W, L143 *, L147*, 2026214111   10 Aug 2026 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, L21 IS, 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, Pl 3 S, 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

[0110] In some embodiments, the isolated polypeptide variant may be 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 2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R)(SEQ ID NO. 210598), CA2 (aa 2260 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 2-260 of WT, D71F, N231F)(SEQ ID NO. 210505), CA2 (aa 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 of WT, D71K, P249H)(SEQ ID NO. 210516), CA2 (aa 2-260 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 2026214111   10 Aug 2026 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, 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, S56X, L57X, T192X)(SEQ ID NO. 210574), CA2 (aa 2260 of WT, Y193V, K260F)(SEQ ID NO. 210576), 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, 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, 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, KI UN, 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, KI 131, T125I, D138G, G155S)(SEQ ID NO. 210714), CA2 (aa 2-260 of WT, I59N, G102R, A173T)(SEQ ID NO. 210716), CA2 (aa 2260 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, T168K)(SEQ ID NO. 210730), CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E)(SEQ ID NO. 210732), CA2 (aa 2-260 of WT, L44M, L47V, N62K, E69D)(SEQ ID NO. 210734), CA2 (aa 2-260 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, 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).

[0111] Also provided herein are biocircuit systems that include at least one effector module. The effector module of the biocircuit may include a stimulus response element (SRE), and the SRE may include in whole or in part, human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717). The biocircuits may also include at least one pay load, which may be attached, appended or associated with the SRE. 2026214111   10 Aug 2026

[0112] The SRE of the biocircuit system which includes, 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, Al 15L, Al 16Q, Al 16V, 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, DI 101, 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, El 17*, E117N, EUN, 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, GI50S. 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, Hl 19T, H119Y, H122T, H122Y, H15L, HI5T. HI5Y. HUD, HUI, H36I, H36Q, H64M, H94T, H96T, I145F, I145M, I166H, I166L, I209D, I209L, I215H, I215S, I22L, I255N, I255S, I33S, I59F, I59N, I59S, I91F, KI 1 IE, K111N, K112R, KI 131, 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, LI 18F, 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, L21 IS, 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, PUL, PUS, 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, 2026214111   10 Aug 2026 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.

[0113] In some aspects, the SRE may be 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 (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 2-260 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 2260 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 2-260 of WT, D71F, N231F)(SEQ ID NO. 210505), CA2 (aa 2-260 of WT, A77I, P249F)(SEQ ID NO. 210514), CA2 (aa 2-260 of WT, D71K, P249H)(SEQ ID NO. 210516), CA2 (aa 2-260 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 2260 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, 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, 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, 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, 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 2026214111   10 Aug 2026 (aa 2-260 of WT, K45N, V68L, H119Y, K169R, D179E)(SEQ ID NO. 210704), CA2 (aa 2-260 of WT, H15L, A54V, KI 1 IE, 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, KI 131, 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, Pl80S)(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, T168K)(SEQ ID NO. 210730), CA2 (aa 2-260 of WT, Q53H, L90V, Q92H, G131E)(SEQ ID NO. 210732), CA2 (aa 2260 of WT, L44M, L47V, N62K, E69D)(SEQ ID NO. 210734), CA2 (aa 2-260 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, 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).

[0114] The biocircuit system described herein may include SREs that are responsive to one or more stimuli.

[0115] In some embodiments, the stimulus may be a small molecule, wherein the small molecule is Celecoxib, Valdecoxib, Rofecoxib, Acetazolamide, Methazolamide, Dorzolamide, Brinzolamide, Diclofenamide, Ethoxzolamide, Zonisamide, Dansylamide, and Dichlorphenamide. In embodiment, the small molecule may be Acetazolamide.

[0116] Also described herein are vectors encoding the biocircuit systems, and pharmaceutical compositions that include the biocircuit systems and pharmaceutically acceptable excipients. Payloads

[0117] As used herein a “payload "or “target payload” or “payload of interest (POI)” is defined as any protein or nucleic acid whose function is to be altered.

[0118] Payloads may include any coding or non-coding gene or any protein or fragment thereof.

[0119] Pay loads are often associated with one or more SREs and may be encoded alone or in combination with one or more SRE in a polynucleotide of the present disclosure. Payloads themselves may be altered (at the protein or nucleic acid level) thereby providing for an added layer of tenability of the effector module. For example, payloads may be engineered or designed to contain mutations, single or multiple, which affect the stability of the pay load or its susceptibility to degradation, cleavage or trafficking. The combination of an SRE which can have a spectrum of responses to a stimulus with a pay load which is altered to exhibit a variety of responses or gradations of output signals, e.g., expression levels, produce biocircuits which are superior to those in the art. For example, mutations or substitutional designs such as those created for IL 12 in WO2016048903 (specifically in Example 1 therein), the contents of which are 2026214111   10 Aug 2026 incorporated herein by reference in their entirety, may be used in any protein pay load in conjunction with an SRE of the present disclosure to create dual tunable biocircuits. The ability to independently tune both the SRE and the payload greatly increases the scope of uses of the effector modules of the present disclosure.

[0120] Artificial peptides or polypeptide components of the pay load may be derived from any known polypeptide which is not naturally occurring.

[0121] As used herein, the phrase “derived from” as it relates to effector modules, SRE’s or payloads means that the effector module, SRE or pay load originates at least in part from the stated parent molecule or sequence. For example, in designing an SRE, such SRE may be derived from an epitope or region of a naturally occurring protein but then have been modified in any of the ways taught herein to optimize the SRE function.

[0122] In one embodiment, the pay load is derived from a region of parent protein or from a mutant protein. The region of the parent protein may be 10,11,12,13,14,15,16,17,18,19, 20, 21, 22,23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43,44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71,72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100,101,102,103,104,105,106,107,108,109,110, 111, 112,113,114,115,116,117,118,119,120, 121, 122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145, 146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169, 170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193, 194,195,196,197,198,199, 200,201, 202, 203, 204, 205,206, 207,208, 209, 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, or more than 450 ammo acids in length. The region of the parent protein may be 5-50,25-75, 50-100,75-125,100-150,125-175,150-200,175-225,200250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 ammo acids in length.

[0123] In one embodiment, the pay load is derived from a region of parent protein or from a mutant protein and includes a region of the parent protein. The pay load may include a region of the parent protein which is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, 5- 2026214111   10 Aug 2026 10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 7075%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 8090%, 90-100%, 10-30%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 10-40%, 20-50%, 30-60%, 4070%, 50-80%, 60-90%, 70-100%, 10-50%, 20-60%, 30-70%, 40-80%, 50-90%, 60-100%, 10-60%, 20-70%, 30-80%, 4090%, 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% of the parent protein or mutant protein.

[0124] In one embodiment, the pay load is derived from a parent protein or from a mutant protein and may have 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%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 6570%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 7080%, 80-90%, 90-100%, 10-30%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 10-40%, 20-50%, 3060%, 40-70%, 50-80%, 60-90%, 70-100%, 10-50%, 20-60%, 30-70%, 40-80%, 50-90%, 60-100%, 10-60%, 20-70%, 3080%, 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 to the parent protein or mutant protein.

[0125] In one embodiment, the transmembrane domain region of a first pay load may be replaced with a transmembrane domain, variant or fragment thereof, from a second parent protein. Polypeptides and Polypeptides as Payloads

[0126] The stimuli, biocircuit components, effector modules, including their SREs and pay loads of the present disclosure may exist as a whole polypeptide, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or variants of any of the aforementioned.

[0127] 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 polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2,3,4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides and may be associated or linked. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0128] As used herein, the term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and / or 2026214111   10 Aug 2026 insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.

[0129] In some embodiments “variant mimics” are provided. As used herein, the term “variant mimic” refers to a variant which contains one or more amino acids which would mimic an activated sequence. For example, glutamate may serve as a mimic for phospho-threonine and / or phospho-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic, e.g., phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine. The amino acid sequences of the pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or pay loads of the present disclosure may comprise naturally occurring amino acids and as such may be considered to be proteins, peptides, polypeptides, or fragments thereof. Alternatively, the pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads may comprise both naturally and non-naturally occurring amino acids.

[0130] As used herein, the term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences as compared to a native or starting sequence. The 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 sequences are relative terms referring to an original molecule against which a comparison may be made. Native or starting sequences should not be confused with wild type sequences. Native sequences or molecules may represent the wild-type (that sequence found in nature) but do not have to be identical to the wild-type sequence.

[0131] Ordinarily, variants will possess at least about 70% homology to a native sequence, and preferably, they will be at least about 80%, more preferably at least about 90% homologous to a native sequence.

[0132] As used herein, the term "homology" as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.

[0133] As used herein, the term "homolog" as it applies to amino acid sequences is meant the corresponding sequence of other species having substantial identity to a second sequence of a second species.

[0134] As used herein, the term "analog" is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain the properties of the parent polypeptide.

[0135] As used herein, the term "derivative" is used synonymously with the term "variant" and refers to a molecule that has been modified or changed in any way relative to a reference molecule or starting molecule. 2026214111   10 Aug 2026

[0136] The present disclosure contemplates several types of pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or pay loads which are amino acid based including variants and derivatives. These include substitutional, insertional, deletional and covalent variants and derivatives. As such, included within the scope of this disclosure are pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads comprising substitutions, insertions, additions, deletions and / or covalent modifications. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences of the disclosure (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support.

[0137] "Substitutional variants" when referring to proteins are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[0138] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in the sequence with a different 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. Likewise, 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. Additionally, 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 acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue.

[0139] As used herein, the term "insertional variants" when referring to proteins are those with 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 an adjacent ammo acid that is connected to either the alpha-carboxy or alpha-amino functional group of a starting or reference amino acid.

[0140] As used herein, the term "deletional variants" when referring to proteins, are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule. 2026214111   10 Aug 2026

[0141] As used herein, the term “derivatives," as referred to herein includes variants of a native or starting protein comprising one or more modifications with organic proteinaceous or non-proteinaceous derivatizing agents, and post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.

[0142] As used herein, the term "site," as it pertains to amino acids-based embodiments is used synonymously with "amino acid residue" and "amino acid side chain". A site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide-based molecules of the present disclosure.

[0143] As used herein, the terms "termini” or “terminus," when referring to proteins refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions. The 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 (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)).

[0144] Polypeptides or proteins of the disclosure are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N-and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide-based moiety such as an organic conjugate.

[0145] Once any of the features have been identified or defined as a component of a biocircuit system component, stimulus, effector module including the SREs or pay loads of the disclosure, any of several manipulations and / or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the compositions of the disclosure. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full-length molecule would.

[0146] Modifications and manipulations can be accomplished by methods known in the art such as site directed mutagenesis. The resulting modified molecules may 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.

[0147] In some embodiments, compositions of the present disclosure may comprise 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 2026214111   10 Aug 2026 some embodiments, compounds of the present disclosure may be deuterated. As used herein, the term “deuterate” refers to the process of replacing one or more hydrogen atoms in a substance with deuterium isotopes. Deuterium isotopes are isotopes of hydrogen. The nucleus of hydrogen contains one proton while deuterium nuclei contain both a proton and a neutron. The pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present disclosure may be deuterated in order to change one or more physical property, such as stability, or to allow pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads to be used in diagnostic and / or experimental applications.

[0148] At the protein level, any of the biocircuit components may comprise one or more post-translational modifications (PTM). Such PTMs may occur intracellularly after administration of a protein-based biocircuit component or upon or after translation of a biocircuit component administered as a nucleic acid encoding said biocircuit component.

[0149] Post translational modifications (PTMs) of the present disclosure include, but are not limited to acetylation, phosphorylation, ubiquitination, carboxylation, deamidation, deamination, deacetylation, dihydroxylation, dephosphoiylation, formylation, gamma-carboxyglutamation, glutathionylation, glycation, hydroxylation, methylation, nitration, sumoylation, N-or O-transglutamination, glycosylation and farnesylation.

[0150] Effector modules, including their SREs and payloads, may independently have 1,2, 3, 4, 5, 6,7, 8, 9,10 or more PTMs which are the same or different.

[0151] Effector modules may be designed to include one or more structural or functional domain, repeat, or motif of a protein family. Such domains, repeats and motifs are categorized by protein family; and representative families are given in the EMBL-EBI database, located at http: / / www.ebi.ac.uk / .

[0152] In some embodiments, protein modifications engineered into the structure of the compositions of the disclosure to interfere with antigen processing and peptide loading such as glycosylation and PEGylation, may also be useful in the present disclosure. Compositions of the disclosure may also be engineered to include non-classical amino acid sidechains to design less immunogenic compositions. Any of the methods discussed in International Patent Publication No. WO2005051975 for reducing immunogenicity may be useful in the present disclosure (the contents of which are incorporated by reference in their entirety).

[0153] The SRE may be, but is not limited to, a peptide, peptide complex, peptide-protein complex, protein, fusion protein, protein complex, protein-protein complex. The SRE may include one or more regions derived from any natural or mutated protein, or antibody. In this aspect, the SRE is an element, when responding to a stimulus, can tune intracellular localization, intramolecular activation, and / or degradation of pay loads.

[0154] In some embodiments, effector modules of the present disclosure may comprise additional features that facilitate the expression and regulation of the effector module, such as one or more signal sequences (SSs), 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. Additionally, effector modules of the present disclosure may further comprise other regulatory moieties such as 2026214111   10 Aug 2026 inducible promoters, enhancer sequences, microRNA sites, and / or microRNA targeting sites. Each aspect or tuned modality may bring to the effector module or biocircuit a differentially tuned feature. For example, an SRE may represent a destabilizing domain, while mutations in the protein pay load may alter its cleavage sites or dimerization properties or half-life and the inclusion of one or more microRNA or microRNA binding site may impart cellular detargeting or trafficking features. Consequently, the present disclosure embraces biocircuits which are multifactorial in their tenability. Such biocircuits may be engineered to contain one, two, three, four or more tuned features.

[0155] In some embodiments, effector modules of the present disclosure may include one or more degrons to tune expression. As used herein, a "degron" refers to a minimal sequence within a protein that is sufficient for the recognition and the degradation by the proteolytic system. An important property of degrons is that they are transferrable, that is, appending a degron to a sequence confers degradation upon the sequence. In some embodiments, the degron may be appended to the destabilizing domains, the pay load or both. Incorporation of the degron within the effector module of the disclosure, confers additional protein instability to the effector module and may be used to minimize basal expression. In some embodiments, the degron may be an N degron, a phospho degron, a heat inducible degron, a photosensitive degron, an oxygen dependent degron. As a non-limiting example, the degron may be an Ornithine decarboxylase degron as described by Takeuchi et al. (Takeuchi J etal. (2008). Biochem J. 2008 Mar 1; 410(2):401-7; the contents of which are incorporated by reference in their entirety). 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 entirety. Immunotherapeutic Agents

[0156] Biocircuits described herein may include an immunotherapeutic agent. In some embodiments, pay loads of the present disclosure may be immunotherapeutic agents that induce immune responses 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, 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 in a subject. Cytokines, chemokines and other factors

[0157] In accordance with the present disclosure, pay loads of the present disclosure may be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells and other cell types, which act as chemical commumcators between cells and tissues within the body. These proteins mediate a wide range of physiological functions, from effects on cell growth, differentiation, migration and survival, to a number of effector activities. For example, activated T cells produce a variety of cytokines for cytotoxic function to eliminate tumor cells.

[0158] In some embodiments, pay loads of the present disclosure may be cytokines, and fragments, variants, analogs and derivatives thereof, including but not limited to interleukins, tumor necrosis factors (TNFs), interferons 2026214111   10 Aug 2026 (IFNs), TGF beta and chemokines. It is understood in the art that certain gene and / or protein nomenclature for the same gene or protein may be inclusive or exclusive of punctuation such as a dash or symbolic such as Greek letters. Whether these are included or excluded herein, the meaning is not meant to be changed as would be understood by one of skill in the art. For example, IL2, IL2 and IL-2 refer to the same interleukin. Likewise, TNF alpha, TNFa, TNF-alpha, TNF-a, TNF alpha and TNF a all refer to the same protein.. Likewise, CD40L, CD40 L and CD40LG refer to the same protein.

[0159] In some embodiments, pay loads of the present disclosure may be cytokines that stimulate immune responses. In other embodiments, pay loads of the disclosure may be antagonists of cytokines that negatively impact anticancer immune responses.

[0160] In some embodiments, pay loads of the present disclosure may be cytokine receptors, recombinant receptors, variants, analogs and derivatives thereof; or signal components of cytokines.

[0161] In some embodiments, cytokines of the present disclosure may be utilized to improve expansion, survival, persistence, and potency of immune cells such as CD8+TEM, natural killer cells and tumor infiltrating lymphocytes (TIL) cells used for immunotherapy. In other embodiments, T cells engineered with two or more DD regulated cytokines are utilized to provide kinetic control of T cell activation and tumor microenvironment remodeling. In one aspect, the present disclosure provides biocircuits and compositions to minimize toxicity related to cytokine therapy. Despite its success in mitigating tumor burden, systemic cytokine therapy often results in the development of severe dose limiting side effects. Two factors contribute to the observed toxicity (a) Pleiotropism, wherein cytokines affect different cells types and sometimes produce opposing effects on the same cells depending on the context (b) Cytokines have short serum half-life and thus need to be administered at high doses to achieve therapeutic effects, which exacerbates the pleiotropic effects. In one aspect, cytokines of the present disclosure may be utilized to modulate cytokine expression in the event of adverse effects. In some embodiments, cytokines of the present disclosure may be designed to have prolonged life span or enhanced specificity to minimize toxicity.

[0162] In some embodiments, the pay load of the present disclosure may be an interleukin (IL) cytokine. Interleukins (ILs) are a class of glycoproteins produced by leukocytes for regulating immune responses. As used herein, the term “interleukin (IL)” refers to an interleukin polypeptide from any species or source and includes the full-length protein as well as fragments or portions of the protein. In some aspects, the interleukin pay load is selected from IL 1, ILlalpha (also called hematopoietin-1), ILIbeta (catabolic), IL 1 delta, ILlepsilon, ILleta, IL1 zeta, interleukin-1 family member 1 to 11 (IL1F1 to IL1F11), interleukin-1 homolog 1 to 4 (IL1H1 to IL1H4), IL1 related protein 1 to 3 (IL1RP1 to IL1RP3), IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL10C, IL10D, IL11, ILlla, IL1 lb, IL12, IL13, IL14, IL15, IL16, IL17, IL17A, I117B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL20 like (IL20L), 1121, IL22, IL23, IL23A, IL23-pl9, IL23-p40, IL24,1125, IL26, IL27, IL28A, IL28B, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36 alpha, IL36 beta, IL36 gamma, IL36RN, IL37, IL37a, IL37b, IL37c, IL37d, IL37e and IL38. In other aspects, the payload of the present 2026214111   10 Aug 2026 disclosure may be an interleukin receptor selected from CD121a, CDwl21b, IL2Rot / CD25, IL2R0 / CD122, IL2Ry / CD132, CDwl31, CD124, CD131, CDwl25, CD126, CD130, CD127, CDw210, IL8RA, ILllRa, CD212, CD213al, CD213a2, IL 14R, IL15Ra, CDw217, IL18Ra, IL18R0, IL20Ra, and IL2OR0. In other aspects, the payload of the present disclosure may be a member of the TNF superfamily, including, without limitation, TNF alpha, CD40L, lymphotoxin (LTA) alpha, LTA beta and OX40L. Antibodies and Antibody fragments and variants

[0163] Biocircuits described herein may include one or more antibodies described herein. In some embodiments, the one or more of the antibodies described herein may be a pay load.

[0164] 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, Fab1, F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as single chain variable fragment (scFv); and multispecific antibodies formed from antibody fragments. Pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or pay loads of the present disclosure may comprise one or more of these fragments.

[0165] For the purposes herein, an "antibody" may comprise a heavy and light variable domain as well as an Fc region. As used herein, the term "native antibody" usually refers to a heterotetrameric glycoprotein of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains linked to each other by disulfide bonds. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

[0166] As used herein, the term "variable domain" refers to specific antibody domains found on both the antibody heavy and light chains that differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. Variable domains comprise hypervariable regions. As used herein, the term "hypervariable region" refers to a region within a variable domain comprising amino acid residues responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining regions (CDRs) that become part of the antigen-binding site of the antibody. As used herein, the term “CDR” refers to a region of an antibody comprising a structure that is complimentary to its target antigen or epitope. Other portions of the variable domain, not interacting with the antigen, are referred to as framework (FW) regions. The antigen-binding site (also known as the antigen combining site or paratope) comprises the amino acid residues necessary to interact with a particular antigen.

[0167] VH and VL domains have three CDRs each. VL CDRs are referred to herein as CDR-L1, CDR-L2 and CDR-L3, in order of occurrence when moving from N to C terminus along the variable domain polypeptide. VH CDRs 2026214111   10 Aug 2026 are referred to herein as CDR-H1, CDR-H2 and CDR-H3, in order of occurrence when moving from N to C terminus along the variable domain polypeptide.

[0168] As used herein, the term “Fv” refers to an antibody fragment comprising the minimum fragment on an antibody needed to form a complete antigen-binding site. These regions consist 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 but are largely unstable. Recombinant methods are known in the art for generating stable Fv fragments, typically through insertion of a flexible linker between the light chain variable domain and the heavy chain variable domain (to form a single chain Fv (scFv)) or through the introduction of a disulfide bridge between heavy and light chain variable domains (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p46-47, the contents of which are herein incorporated by reference in their entirety).

[0169] As used herein, the term "light chain" refers to a component of an antibody from any vertebrate species assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.

[0170] As used herein, the term "single chain Fv" or "scFv" refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together into a single polypeptide chain by a flexible peptide linker. In some embodiments, the Fv polypeptide linker enables 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 may be expressed in association with a surface member (e.g. phage coat protein) and used in the identification of high affinity peptides for a given antigen.

[0171] Using molecular genetics, two scFvs can be engineered in tandem into a single polypeptide, separated by a linker domain, called a “tandem scFv” (tascFv). Construction of a tascFv with genes for two different scFvs yields a “bispecific single-chain variable fragments” (bis-scFvs). Only two tascFvs have been developed clinically by commercial firms; both are bispecific agents in active early phase development by Micromet for oncologic indications and are described as “Bispecific T-cell Engagers (BiTE).” Blinatumomab is an anti-CD19 / anti-CD3 bispecific tascFv that potentiates T-cell responses to B-cell non-Hodgkin lymphoma in Phase 2. MT110 is an anti-EP-CAM / anti-CD3 bispecific tascFv that potentiates T-cell responses to solid tumors in Phase 1. Bispecific, tetravalent “TandAbs” are also being researched by Affimed (Nelson, A. L., MAbs., 2010, Jan-Feb; 2(1):77-83). maxibodies (bivalent scFv fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG may also be included.

[0172] As used herein, the term “bispecific antibody” refers to an antibody capable of binding two different antigens. Such antibodies typically comprise 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 2026214111   10 Aug 2026 Pharmacologica Sinica. 2005,26(6): 649-658 and Schaefer et al., PNAS. 2011,108(27):11187-11192, the contents of each of which are herein incorporated by reference in their entirety.

[0173] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites. Diabodies are functional bispecific single-chain antibodies (bscAb). Diabodies comprise a heavy chain variable domain VH connected to a light chain variable domain VL in 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 the complementary domains of another chain and create two antigen-binding sites.

[0174] The term “intrabody” refers to a form of antibody that is not secreted from a cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies may be used to affect a multitude of cellular processes including, but not limited to intracellular trafficking, transcription, translation, metabolic processes, proliferative signaling and cell division. In some embodiments, methods of the present disclosure may include intrabody-based therapies. In some such embodiments, variable domain sequences and / or CDR sequences disclosed herein may be incorporated into one or more constructs for intrabody-based therapy.

[0175] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibodies, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0176] 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. The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

[0177] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising a minimal portion from one or more non-human (e.g., murine) antibody source(s) with the remainder derived from one or more human immunoglobulin sources. For the most part, humamzed antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and / or capacity. In one embodiment, the antibody may be a humanized 2026214111   10 Aug 2026 full-length antibody. As a non-limiting example, the antibody may have been humanized using the methods taught in US Patent Publication NO. US20130303399, the contents of which are herein incorporated by reference in its entirety.

[0178] As used herein, the term “antibody variant” refers to a modified antibody (in relation to a native or starting antibody) or a biomolecule resembling a native or starting antibody in structure and / or function (e.g., an antibody mimetic). Antibody variants may be altered in their amino acid sequence, composition or structure as compared to a native antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.

[0179] In some embodiments, pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or pay loads of the present disclosure may be antibody mimetics. As used herein, the term “antibody mimetic” refers to any molecule which mimics the function or effect of an antibody and which binds specifically and with high affinity to their molecular targets. In some embodiments, antibody mimetics may be monobodies, designed to incorporate the fibronectin type III domain (Fn3) as a protein scaffold (US 6,673,901; US 6,348,584). In some embodiments, antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, Centyrins, DARPINSTM, Fynomers and Kunitz and domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide regions.

[0180] In one embodiment, the antibody may comprise a modified Fc region. As a non-limiting example, the modified Fc region may be made by the methods or may be any of the regions described in US Patent Publication NO. US20150065690, the contents of which are herein incorporated by reference in its entirety.

[0181] In some embodiments, pay loads of the disclosure may encode multispecific antibodies that bind more than one epitope. As used herein, the terms “multibody” or “multispecific antibody” refer to an antibody wherein two or more variable regions bind to different epitopes. The epitopes may be on the same or different targets. In one embodiment, the multispecific antibody may be generated and optimized by the methods described in International Patent Publication NO. WO2011109726 and US Patent Publication NO. US20150252119, the contents of which each of which are herein incorporated by reference in their entirety. These antibodies are able to bind to multiple antigens with high specificity and high affinity.

[0182] In certain embodiments, a multi-specific antibody is a "bispecific antibody" which recognizes two different epitopes on the same or different antigens. In one aspect, bispecific antibodies are capable of binding two different antigens. Such antibodies typically comprise 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, thus allowing the BsAb to bind to two different types of antigen. Bispecific antibody frameworks may include any of those described in Riethmuller, G., 2012. Cancer Immunity, 2012,12:12-18; Marvin et al., Acta Pharmacologica Sinica. 2005, 26(6):649-658; and Schaefer et al., PNAS. 2011,108(27): 11187-11192, the contents of 2026214111   10 Aug 2026 each of which are herein incorporated by reference in their entirety. New generations of BsMAb, called “trifunctional bispecific” antibodies, have been developed. These consist of two heavy and two light chains, one each from two different antibodies, where the two Fab regions (the arms) are directed against two antigens, and the Fc region (the foot) comprises the two heavy chains and forms the third binding site.

[0183] In some embodiments, payloads may encode antibodies comprising a single antigen-binding domain. These molecules are extremely small, with molecular weights approximately one-tenth of those observed for full-sized mAbs. Further antibodies may include “nanobodies” derived from the antigen-binding variable heavy chain regions (VHHs) of heavy chain antibodies found in camels and llamas, which lack light chains (Nelson, A. L., MAbs.2010. Jan-Feb; 2(1):77-83).

[0184] In some embodiments, the antibody may be “miniaturized”. Among the best examples of mAb miniaturization are 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 antigenbinding domain, and one or two constant “effector” domains, all connected by linker domains. Presumably, such a molecule might offer the advantages of increased tissue or tumor penetration claimed by fragments while retaining the effector immune functions conferred by constant domains. At least three “miniaturized” SMIPs have entered clinical development.

[0185] One example of miniaturized antibodies is called “unibody” in which the hinge region has been removed from IgG4 molecules. While IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with one another, deletion of the hinge region prevents heavy chain-heavy chain pairing entirely, leaving highly specific monovalent light / heavy heterodimers, while retaining the Fc region to ensure stability and half-life in vivo.

[0186] In some embodiments, pay loads of the disclosure may encode single-domain antibodies (sdAbs, or nanobodies) which are antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. In one aspect, a sdAb may be a “Camel Ig or "camelid VHH". As used herein, the term “camel Ig” refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-No Ite, et al, FASEB J., 2007,21:3490-3498). A "heavy chain antibody" or a "camelid antibody" refers to an antibody that contains two VH domains and no light chains (RiechmannL. etal, J. Immunol. Methods, 1999, 231: 25-38; International patent publication NOs. WO1994 / 04678 and W01994 / 025591; and U.S. PatentNo. 6,005,079). In another aspect, a sdAb may be a “immunoglobulin new antigen receptor” (IgNAR). As used herein, the term "immunoglobulin new antigen receptor" refers to class of antibodies from the shark immune repertoire that consist of homodimers of one variable new antigen receptor (VNAR) domain and five constant new antigen receptor (CNAR) domains.

[0187] In some embodiments, pay loads of the disclosure may encode intrabodies. Intrabodies are a form of antibody that is not secreted from a cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies are expressed and function intracellularly and may be used to affect a multitude of cellular processes 2026214111   10 Aug 2026 including, but not limited to intracellular trafficking, transcription, translation, metabolic processes, proliferative signaling and cell division. In some embodiments, methods described herein include intrabody-based therapies. In some such embodiments, variable domain sequences and / or CDR sequences disclosed herein are incorporated into one or more constructs for intrabody-based therapy. For example, intrabodies may target one or more glycated intracellular proteins or may modulate the interaction between one or more glycated intracellular proteins and an alternative protein.

[0188] In some aspects, pay loads of the disclosure may encode biosynthetic antibodies as described in U.S. Patent No. 5,091,513, the contents of which are herein incorporated by reference in their entirety. Such antibody may include one or more sequences of amino acids constituting a region which behaves as a biosynthetic antibody binding site (BABS). The sites comprise 1) non-covalently associated or disulfide bonded synthetic VH and VL dimers, 2) VH-VL or VL-VH single chains wherein the VH and VL are attached by a polypeptide linker, or 3) individuals VH or VL domains. The binding domains comprise linked CDR and FR regions, which may be derived from separate immunoglobulins. The biosynthetic antibodies may also include other polypeptide sequences which function, e.g., as an enzyme, toxin, binding site, or site of attachment to an immobilization media or radioactive atom. Methods are disclosed for producing the biosynthetic antibodies, for designing BABS having any specificity that can be elicited by in vivo generation of antibody, and for producing analogs thereof.

[0189] In some embodiments, pay loads may encode antibodies with antibody acceptor frameworks taught in U.S. Patent No. 8,399,625. Such antibody acceptor frameworks may be particularly well suited accepting CDRs from an antibody of interest.

[0190] In one embodiment, the antibody may be a conditionally active biologic protein. An antibody may be used to generate a conditionally active biologic protein which are reversibly or irreversibly inactivated at the wild type normal physiological conditions as well as to such conditionally active biologic proteins and uses of such conditional active biologic proteins are provided. Such methods and conditionally active proteins are taught in, for example, International Publication No. WO2015175375 and WO2016036916 andUS Patent Publication No. US20140378660, the contents of each of which are incorporated herein by reference in their entirety. Antibodies used for immunotherapy

[0191] In some embodiments, pay loads of the present disclosure may be antibodies, fragments and variants thereof which are specific to tumor specific antigens (TSAs) and tumor associated antigens (TAAs). Antibodies circulate throughout the body until they find and attach to the TSA / TAA. Once attached, they recruit other parts 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)” means an antigenic substance produced in tumor cells, which can trigger an anti-tumor immune response in a host organism. In one embodiment, a TSA may be a tumor neoantigen. The tumor antigen specific antibody mediates complement-dependent cytotoxic response against tumor cells expressing the same antigen. 2026214111   10 Aug 2026

[0192] In some embodiments, the tumor specific antigens (TSAs), tumor associated antigens (TAAs), pathogen associated antigens, or fragments thereof can be expressed as a peptide or as an intact protein or portion thereof. The intact protein or a portion thereof can be native or mutagenized. Antigens associated with cancers or virus-induced cancers as described herein are well-known in the art. Such a TSA or TAA may be previously associated with a cancer or may be identified by any method known in the art.

[0193] In one embodiment, the antigen is CD19, a B-cell surface protein expressed throughout B-cell development. CD 19 is a well-known B cell surface molecule, which upon B cell receptor activation enhances B-cell antigen receptor induced signaling and expansion of B cell populations. CD19 is broadly expressed in both normal and neoplastic B cells. Malignancies derived from B cells such as chronic lymphocytic leukemia, acute lymphocytic leukemia and many nonHodgkin lymphomas frequently retain CD19 expression. This near universal expression and specificity for a single cell lineage has made CD19 an attractive target for immunotherapies. Human CD19 has 14 exons wherein exon 1-4 encode the extracellular portion of the CD19, exon 5 encodes the transmembrane portion of CD19 and exons 6-14 encode the cytoplasmic tail.

[0194] In one embodiment, pay loads of the present disclosure may be antibodies, fragments and variants thereof which are specific to CD 19 antigen.

[0195] In some embodiments, the immunotherapeutic agent may be an antibody that is specifically immunoreactive to an antigen selected from a tumor specific antigen (TSA), a tumor associated antigen (TAA), or an antigenic epitope.

[0196] In one aspect, the antigen may be an antigenic epitope. In some embodiments, the antigenic epitope may be CD19.

[0197] A tumor specific antigen (TSA) may be a tumor neoantigen. A neoantigen is a mutated antigen that is only expressed by tumor cells because of genetic mutations or alterations in transcription which alter protein coding sequences, therefore creating novel, foreign antigens. The genetic changes result from genetic substitution, insertion, deletion or any other genetic changes of a native cognate protein (i.e. a molecule that is expressed in normal cells). Chimeric antigen receptors (CARs)

[0198] Biocircuits described herein may include a chimeric antigen receptor. In some embodiments, pay loads of the present disclosure may be a chimeric antigen receptors (CARs) which when transduced into immune cells (e.g., T cells and NK cells), can re-direct the immune cells against the target (e.g., a tumor cell) which expresses a molecule recognized by the extracellular target moiety of the CAR.

[0199] As used herein, the term “chimeric antigen receptor (CAR)” refers to a synthetic receptor that mimics the TCR on the surface of T cells. In general, 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, transmembrane domain and intracellular signaling / activation domain, are linearly constructed as a single fusion protein. The extracellular region comprises a targeting domain / moiety (e.g., a scFv) that 2026214111   10 Aug 2026 recognizes a specific tumor antigen or other tumor cell-surface molecules. The intracellular region may contain a signaling domain of TCR complex (e.g., the signal region of CD3Q, and / or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX-40 (CD134). For example, a “first-generation CAR” only has the CD3 / signaling domain. In an effort to augment T-cell persistence and proliferation, costimulatory intracellular domains are added, giving rise to second generation CARs having a CD3 / signal domain plus one costimulatoiy signaling domain, and third generation CARs having CD3 / signal domain plus two or more costimulatory signaling domains. A CAR, when expressed by a T cell, endows the T cell with antigen specificity determined by the extracellular targeting moiety of the CAR. Recently, it is also desirable to add one or more elements such as homing and suicide genes to develop a more competent and safer architecture of CAR, so called the fourth-generation CAR.

[0200] In some embodiments, the immunotherapeutic agent of the effector module is a chimeric antigen receptor (CAR). The chimeric antigen may comprise an extracellular target moiety; a transmembrane domain; an intracellular signaling domain; and optionally, one or more co-stimulatoiy domains.

[0201] In some embodiments, the extracellular targeting domain is joined through the hinge (also called space domain or spacer) and transmembrane regions to an intracellular signaling domain. The hinge connects the extracellular targeting domain to the transmembrane domain which transverses the cell membrane and connects to the intracellular signaling domain. The hinge may need to be varied to optimize the potency of CAR expressing cells toward cancer cells due to the size of the target protein where the targeting moiety binds, and the size and affinity of the targeting domain itself. Upon recognition and binding of the targeting moiety to the target cell, the intracellular signaling domain leads to an activation signal for the CAR T cell, which is further amplified by the “second signal” from one or more intracellular costimulatory domains. The CAR T cell, once activated, can destroy the target cell.

[0202] In some embodiments, the CAR of the present disclosure may be split into two parts, each part is linked a dimerizing domain, such that an input that triggers the dimerization promotes assembly of the intact functional receptor. Wu and Lim recently reported a split CAR in which the extracellular CD 19 binding domain and the intracellular signaling element are separated and linked to the FKBP domain and the FRB* (T2089L mutant of FKBP-rapamycin binding) domain that heterodimerize in the presence of the rapamycin analog AP21967. The split receptor is assembled in the presence of AP21967 and together with the specific antigen binding, activates T cells (Wu et al., Science, 2015, 625(6258): aab4077).

[0203] In some embodiments, the CAR of the present disclosure may be designed as an inducible CAR. Sakemura et al recently reported the incorporation of a Tet-On inducible system to the 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 were equivalently cytotoxic against CD 19+ cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells (Sakemura et al., Cancer Immuno. Res., 2016, Jun 21, Epub ahead of print). In one example, this Tet-CAR may be the pay load of the effector module under the control of SREs 2026214111   10 Aug 2026 (e.g., DDs) of the disclosure. The dual systems provide more flexibility to turn-on and off the CAR expression in transduced T cells.

[0204] According to the present disclosure, the pay load of the present disclosure may be a first-generation CAR, or a second-generation CAR, or a third-generation CAR, or a fourth-generation CAR. In some embodiments, the pay load of the present disclosure may be a full CAR construct composed of the extracellular domain, the hinge and transmembrane domain and the intracellular signaling region. In other embodiments, the pay load of the present disclosure may be a component of the full CAR construct including an extracellular targeting moiety, a hinge region, a transmembrane domain, an intracellular signaling domain, one or more co-stimulatory domain, and other additional elements that improve CAR architecture and functionality including but not limited to a leader sequence, a homing element and a safety switch, or the combination of such components.

[0205] CARs regulated by biocircuits and compositions of the present disclosure are tunable and thereby offer several advantages. The reversible on-off switch mechanism allows management of acute toxicity caused by excessive CAR-T cell expansion. Pulsatile CAR expression using SREs of the present disclosure may be achieved by cycling ligand level. The ligand conferred regulation of the CAR may be effective in offsetting tumor escape induced by antigen loss, avoiding functional exhaustion caused by tonic signaling due to chronic antigen exposure and improving the persistence of CAR expressing cells in vivo.

[0206] In some embodiments, biocircuits and compositions of the disclosure may be utilized to down regulate CAR expression to limit on target on tissue toxicity caused by tumor lysis syndrome. Down regulating the expression of the CARs of the present disclosure following anti-tumor efficacy may prevent (1) On target off tumor toxicity caused by antigen expression in normal tissue, (2) antigen independent activation in vivo. Extracellular targeting domain / moiety

[0207] In accordance with the disclosure, the extracellular target moiety of a CAR may be any agent that recognizes and binds to a given target molecule, for example, a neoantigen on tumor cells, with high specificity and affinity. The target moiety may be an antibody and variants thereof that specifically binds to a target molecule on tumor cells, or a peptide aptamer selected from a random sequence pool based on its ability to bind to the target molecule on tumor cells, or a variant or fragment thereof that can bind to the target molecule on tumor cells, or an antigen recognition domain from native T-cell receptor (TCR) (e.g. CD4 extracellular domain to recognize HIV infected cells), or exotic recognition components such as a linked cytokine that leads to recognition of target cells bearing the cytokine receptor, or a natural ligand of a receptor.

[0208] In some embodiments, the targeting domain of a CAR may be a Ig NAR, a Fab fragment, a Fab1 fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, Fv, a single chain variable fragment (scFv), a bis-scFv, a (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, for example a tumor specific antigen 2026214111   10 Aug 2026 (TSA). In one embodiment, the targeting moiety is a scFv. The scFv domain, when it is expressed on the surface of a CAR T cell and subsequently binds to a target protein on a cancer cell, is able to maintain the CAR T cell in proximity to the cancer cell and to trigger the activation of the T cell. A scFv can be generated using routine recombinant DNA technology techniques and is discussed in the present disclosure.

[0209] In one aspect, the extracellular target moiety may be an scFv derived from an antibody. In one aspect, the scFv may specifically bind to a CD 19 antigen. Intracellular signaling domains

[0210] The intracellular domain of a CAR fusion polypeptide, after binding to its target molecule, transmits a signal to the effector immune cell, activating at least one of the normal effector functions of effector immune cells, including cytolytic activity (e.g., cytokine secretion) or helper activity. Therefore, the intracellular domain comprises an “intracellular signaling domain" of a T cell receptor (TCR).

[0211] In some aspects, the entire intracellular signaling domain can be employed. In other aspects, a truncated portion of the intracellular signaling domain may be used in place of the intact chain as long as it transduces the effector function signal.

[0212] In some embodiments, the intracellular signaling domain of the present disclosure may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (IT AMs). Examples of IT AM containing cytoplasmic signaling sequences include those derived from TCR CD3zeta, 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 (CD3Q signaling domain.

[0213] In some embodiments, the intracellular region of the present disclosure further comprises one or more costimulatory signaling domains which provide additional signals to the effector immune cells. These costimulatoiy signaling domains, in combination with the signaling domain can further improve expansion, activation, memory, persistence, and tumor-eradicating efficiency of CAR engineered immune cells (e.g., CAR T cells). In some cases, the costimulatory signaling region contains 1, 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules. The costimulatory signaling domain may be the intracellular / cytoplasmic domain of a costimulatory molecule, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), GITR (glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function-associated antigen-1), LIGHT, NKG2C, B7-H3. In one example, the costimulatory signaling domain is derived from the cytoplasmic domain of CD28. In another example, the costimulatoiy signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137). In another example, the co-stimulatoiy signaling domain may be an intracellular domain of GITR as taught in U.S. Pat. NO. 9,175, 308; the contents of which are incorporated herein by reference in its entirety.

[0214] In some embodiments, the intracellular signaling domains disclosed in International Patent Publication, WO2014153270 may be useful in the present disclosure. 2026214111   10 Aug 2026

[0215] In some embodiments, the chimeric antigen receptors described herein may include CD3 zeta domains altered to tune CAR activity. The CD3 zeta domains may include one or more mutations in the immunoreceptor tyrosinebased activation motifs (ITAMs). In one aspect, the tyrosine residues within the ITAMs may be mutated resulting in reduced phosphorylation and limited downstream signaling. In some embodiments, one or more of the ITAMs may be deleted from the CD3 zeta domain. In one aspect, the CD3 zeta may include one ITAM. Any of the CARs and CD3 zeta domains described by Feucht et al. 2019 may be used herein (Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nature Medicine 25, 82-88 (2019); the contents of which are herein incorporated by reference in their entirety).

[0216] In some embodiments, the GITR co-stimulatory domains may be useful in the CAR described herein. In some embodiments, the GITR domains may be capable of inducing T cell effector function and activating T cells. In some aspects, GITR domains described herein may be able to suppress inhibitory T regulatory cells that block immune response. In some embodiments, GITR intracellular domain containing CAR T cells can decrease the production of cytokines, which may reduce the cytokine release syndrome. Any of the GITR domains described in International Patent Publication, WO2018045034; the contents of which are herein incorporated by reference in their entirety. Transmembrane domains and hinge regions

[0217] In some embodiments, the CAR of the present disclosure may comprise a transmembrane domain. As used herein, the term “Transmembrane domain (TM)” refers broadly to an amino acid sequence of about 15 residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes at least 20, 21, 22, 23, 24,25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43,44, or 45 amino acid residues and spans the plasma membrane. In some embodiments, the transmembrane domain of the present disclosure may be derived either from a natural or from a synthetic source. The transmembrane domain of a CAR may be derived from any naturally membranebound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, or CD154.

[0218] Alternatively, the transmembrane domain of the present disclosure may be synthetic. In some aspects, the synthetic sequence may comprise predominantly hydrophobic residues such as leucine and valine.

[0219] In some embodiments, the transmembrane domain of the present disclosure may be selected from the group consisting of a CD8a transmembrane domain, a CD4 transmembrane domain, a CD 28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, and a human IgG4 Fc region. 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. 2026214111   10 Aug 2026

[0220] In some embodiments, the CAR of the present disclosure may comprise an optional hinge region (also called spacer). A hinge sequence is a short sequence of amino acids that facilitates flexibility of the extracellular targeting domain that moves the target binding domain away from the effector cell surface to enable proper cell / cell contact, target binding and effector cell activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge sequence may be positioned between the targeting moiety and the transmembrane domain.

[0221] In some embodiments, the CAR of the present disclosure may comprise one or more linkers between any of the domains of the CAR. The linker may be between 1-30 amino acids long.

[0222] In some embodiments, the components including the targeting moiety, transmembrane domain and intracellular signaling domains of the present disclosure may be constructed in a single fusion polypeptide. The fusion polypeptide may be the pay load of an effector module of the disclosure. In some embodiments, more than one CAR fusion polypeptides may be included in an effector module, for example, two, three or more CARs may be included in the effector module under the control of a single SRE (e.g., a DD).

[0223] In one embodiment, the CAR construct comprises a CD19 scFv (e.g., CAT13.1E10 orFMC63), a CD8a spacer or transmembrane domain, anda 4-1BB and CD3( endodomain. These constructs with CAT13.1E10 may have increased proliferation after stimulation in vitro, increased cytotoxicity against the CD19+ targets, and increased effector and target interactions as compared to constructs with FMC63.

[0224] In some embodiments, the pay load of the disclosure may be any of the co-stimulatoiy molecules and / or intracellular domains described herein. In some embodiments, one or more co-stimulatoiy molecules, each under the control of different SRE may be used in the present disclosure. SRE regulated co-stimulatory molecules may also be expressed in conjunction with a first-generation CAR, a second-generation CAR, a third generation CAR, a fourth generation, or any other CAR design described herein. Tandem CAR (TanCAR)

[0225] In some embodiments, the CAR of the present disclosure may be a tandem chimeric antigen receptor (TanCAR) which is able to target two, three, four, or more tumor specific antigens. In some aspects, The CAR is a bispecific TanCAR including two targeting domains which recognize two different TSAs on tumor cells. The bispecific CAR may be further defined as comprising an extracellular region comprising a targeting domain (e.g., an antigen recognition domain) specific for a first tumor antigen and a targeting domain (e.g., an antigen recognition domain) specific for 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 space between the targeting domains in the TanCAR may 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. Split CAR 2026214111   10 Aug 2026

[0226] In some embodiments, the components including the targeting moiety, transmembrane domain and intracellular signaling domains of the present disclosure may be split into two or more parts such that it is dependent on multiple inputs that promote assembly of the intact functional receptor. In one embodiment, the split synthetic CAR system can be constructed in which the assembly of an activated CAR receptor is dependent on the binding of a ligand to the SRE (e.g. a small molecule) and a specific antigen to the targeting moiety. As a non-limiting example, the split CAR consists of two parts that assemble in a small molecule-dependent manner; one part of the receptor features an extracellular antigen binding domain (e.g. scFv) and the other part has the intracellular signaling domains, such as the CD3( intracellular domain.

[0227] In other aspects, the split parts of the CAR system can be further modified to increase signal. In one example, the second part of cytoplasmic fragment may be anchored to the plasma membrane by incorporating a transmembrane domain (e.g., CD8a transmembrane domain) to the construct. An additional extracellular domain may also be added to the second part of the CAR system, for instance an extracellular domain that mediates homodimerization. These modifications may increase receptor output activity, i.e., T cell activation.

[0228] In some aspects, the two parts of the split CAR system contain heterodimerization domains that conditionally interact upon binding of a heterodimerizing small molecule. As such, the receptor components are assembled in the presence of the small molecule, to form an intact system which can then be activated by antigen engagement. Any known heterodimerizing components can be incorporated into a split CAR system. Other small molecule dependent heterodimerization domains may also be used, including, but not limited to, 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). The dual regulation using inducible assembly (e.g., ligand dependent dimerization) and degradation (e.g., destabilizing domain induced CAR degradation) of the split CAR system may provide more flexibility to control the activity of the CAR modified T cells. Switchable CAR

[0229] In some embodiments, the CAR of the 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 controllable CARs 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 in the hinge domain that separate the scFv domain from the cell membrane domain in the CAR. Such system is possible to split or combine different key functions of a CAR such as activation and costimulation within different chains of a receptor complex, mimicking the complexity of the TCR native architecture. This integrated system can switch the scFv and antigen interaction between on / off states controlled by the absence / presence of the stimulus. Reversible CAR 2026214111   10 Aug 2026

[0230] In other embodiments, the CAR of the disclosure may be a reversible CAR system. In this CAR architecture, a LID domain (ligand-induced degradation) is incorporated into the CAR system. The CAR can be temporarily down-regulated by adding a ligand of the LID domain. The combination of LID and DD mediated regulation provides tunable control of continuingly activated CAR T cells, thereby reducing CAR mediated tissue toxicity. Activation-conditional CAR

[0231] In some embodiments, pay loads of the disclosure may be an activation-conditional chimeric antigen receptor, which is only expressed in an activated immune cell. The expression of the CAR may be coupled to activation conditional control region which refers to one or more nucleic acid sequences that induce the transcription and / or expression of a sequence e.g. a CAR under its control. Such activation conditional control regions may be promoters of genes that are upregulated during the activation of the effector immune cell e.g. IL2 promoter or NF AT binding sites. In some embodiments, activation of the immune cell may be achieved by a constitutively expressed CAR (International Publication NO. WO2016126608; the contents of which are incorporated herein by reference in their entirety). Polynucleotides

[0232] Biocircuit components including effector modules, their SREs and pay loads, may be nucleic acid-based. The term “nucleic acid,” in its broadest sense, includes any compound and / or substance that comprise a polymer of nucleotides, e.g., linked nucleosides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization) or hybrids thereof.

[0233] In some embodiments, the nucleic acid molecule is a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. Polynucleotides of the disclosure may be mRNA or any nucleic acid molecule and may or may not be chemically modified.

[0234] 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 pay load which maintain a modular organization, but which comprise one or more structural and / or chemical modifications or alterations which impart useful properties to the polynucleotide, for example tenability of function. As used herein, a “structural” feature or modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleosides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical 2026214111   10 Aug 2026 modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.

[0235] In some embodiments, polynucleotides of the present disclosure may harbor 5'UTR sequences which play a role in translation initiation. 5'UTR sequences may include features such as Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of genes, Kozak sequences have the consensus XCCR(A / G) CCAUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) and X is any nucleotide. In one embodiment, the Kozak sequence is ACCGCC. By engineering the features that are typically found in abundantly expressed genes of target cells or tissues, the stability and protein production of the polynucleotides of the disclosure can be enhanced.

[0236] Further provided are polynucleotides, which may contain an internal ribosome entry site (IRES) which play an important role in initiating protein synthesis in the absence of 5' cap structure in the polynucleotide. An IRES may act as the sole ribosome binding site or may serve as one of the multiple binding sites. Polynucleotides of the disclosure containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes giving rise to bicistronic and / or multicistronic nucleic acid molecules.

[0237] In one embodiment, polynucleotides of the present disclosure may encode variant polypeptides which have a certain identity with a reference polypeptide sequence. As used herein, a “reference polypeptide sequence” refers to a starting polypeptide sequence. Reference sequences may be wild type sequences or any sequence to which reference is made in the design of another sequence.

[0238] The term “identity” as known in the art, refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between sequences, as determined by the number of matches between strings of two or more residues (amino acid or nucleic acid). Identity measures the percent of identical matches between two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, 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).

[0239] In some embodiments, the variant sequence may have the same or a similar activity as the reference sequence. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to a reference 2026214111   10 Aug 2026 sequence. Generally, variants of a particular polynucleotide or polypeptide of the 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 to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of 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 database search programs", Nucleic Acids Res. 25:3389-3402.) Chemical modifications to polynucleotides

[0240] According to the present disclosure, the terms “modification” or, as appropriate, “modified” polynucleotides refer to modification with respect to A, G, U (T in DNA) or C nucleotides.

[0241] Modifications of the polynucleotides of the disclosure may be on the nucleoside base and / or sugar portion of the nucleosides which comprise the polynucleotide. In some embodiments, multiple modifications are included in the modified nucleic acid or in one or more individual nucleoside or nucleotide. For example, modifications to a nucleoside may include one or more modifications to the nucleobase and the sugar. Modifications to the polynucleotides of the present disclosure may include any of those taught in, for example, International Publication WO2013052523, the contents of which are incorporated herein by reference in its entirety.

[0242] As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a 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 “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group.

[0243] The modified nucleotides, which may be incorporated into a polynucleotide can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphonoselenoates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphonodiamidites, alkyl or ary 1 phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with mtrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). Other modifications which may be used are taught in, for example, International Application WO2013052523, the contents of which are incorporated herein by reference in their entirety. 2026214111   10 Aug 2026

[0244] Different sugar modifications, nucleotide modifications, and / or intemucleoside linkages (e.g., backbone structures) may exist at various positions in the polynucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. A modification may also be a 5' or 3' terminal modification. The polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

[0245] In some embodiments, the polynucleotide includes a modified pyrimidine or purine. In some embodiments, the pyrimidine or purine in the polynucleotide molecule may be replaced with from about 1% to about 100% of a modified uracil or modified uridine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100% of a modified pyrimidine or purine.

[0246] In some embodiments, the polynucleotides may comprise two or more effector module component sequences which are in a pattern such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different effector module component.

[0247] In yet another embodiment, the polynucleotides may comprise two or more effector module component sequences with each component having one or more sequences. As a non-limiting example, the sequences may be in a pattern such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times in each of the regions. As another non-limiting example, the sequences may be in a pattern such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times across the entire polynucleotide. In these patterns, each letter, A, B, or C represent a different sequence or component. 2026214111   10 Aug 2026 Codon Selection

[0248] In some embodiments, one or more codons of the polynucleotides of the present disclosure may be replaced with other codons encoding the native amino acid sequence to tune the expression of the SREs, through a process referred to as codon selection. Since mRNA codon, and tRNA anticodon pools tend to vary among organisms, cell types, sub cellular locations and over time, the codon selection described herein is a spatiotemporal (ST) codon selection.

[0249] In some embodiments of the disclosure, certain polynucleotide features may be codon optimized. Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cell by replacing at least 1,2, 3, 4, 5,10,15, 20,25, 50 or more codons of the native sequence with codons that are most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Codon usage may be measured using the Codon Adaptation 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 the CAI can be calculated by 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 to match codon frequencies in target and host organisms to ensure proper folding, bias nucleotide content to alter stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein signaling sequences, remove / add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. In one embodiment, a polynucleotide sequence or portion thereof is codon optimized using optimization algorithms. Codon options for each amino acid are well-known in the art as are various species table for optimizing for expression in that particular species.

[0250] In some embodiments of the disclosure, certain polynucleotide features may be codon optimized. For example, a preferred region for codon optimization may be upstream (5’) or downstream (3 ’) to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and / or after codon optimization of the payload encoding region or open reading frame (ORF).

[0251] After optimization (if desired), the polynucleotides components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.

[0252] In some embodiments, certain regions of the polynucleotide may be preferred for codon selection. For example, a preferred region for codon selection may be upstream (5 ’) or downstream (3 ’) to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and / or after codon selection of the payload encoding region or open reading frame (ORF). 2026214111   10 Aug 2026

[0253] The stop codon of the polynucleotides of the present disclosure may be modified to include sequences and motifs to alter the expression levels of the SREs, pay loads and effector modules of the present disclosure. Such sequences may be incorporated to induce stop codon readthrough, wherein the stop codon may specify amino acids e.g. selenocysteine or pyrrolysine. In other instances, stop codons may be skipped altogether to resume translation through an alternate open reading frame. Stop codon read through may be utilized to tune the expression of components of the effector modules at a specific ratio (e.g.as dictated by the stop codon context). Examples of preferred stop codon motifs include UGAN, UAAN, and UAGN, where N is either C or U.

[0254] Suppression of termination occurs during translation of many viral mRNAs as a means of generating a second protein with extended carboxy terminus. In retroviruses, gag and pol genes are encoded by a single mRNA and separated by an amber termination codon UAG. Translational suppression of the amber codon allows synthesis of the gag pol precursor. Translation suppression is mediated by suppressor tRNAs that can recognize termination codons and insert a specific amino acid. In some embodiments, effector modules described herein may incorporate amber termination codons. Such codons may be used in lieu of or in addition to IRES and p2A sequences in bicistronic constructs. Stop codon read through may be combined with P2A to obtain low level expression of downstream gene (e.g. IL 12). In some embodiments, the amber stop codons may be combined with tRNA expression or amino-acyl tRNA synthetase for further control. In one aspect, the payload may be a regulated tRNA synthetase. Conjugates

[0255] It is contemplated by the present disclosure that the compositions of the present disclosure may be complexed, conjugated or combined with one or more homologous or heterologous molecules. As used herein, the term “homologous molecule” refers to a molecule which is similar in at least one of structure or function relative to a starting molecule while a “heterologous molecule” is one that differs in at least one of structure or function relative to a starting molecule. Structural homologs are therefore molecules which may be substantially structurally similar. In some embodiments, such homologs may be identical. Functional homologs are molecules which may be substantially functionally similar. In some embodiments, such homologs may be identical.

[0256] Pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or pay loads of the present disclosure may comprise conjugates. Such conjugates of the disclosure may include naturally occurring substances or ligands, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); carbohydrates (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluromc acid); or lipids. Conjugates may also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic poly amino acids, an oligonucleotide (e.g. an aptamer). Examples of polyamino acids may include poly lysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacryIlic acid), N- 2026214111   10 Aug 2026 isopropylaciylamide polymers, or polyphosphazine. Example of poly amines include: polyethylenimine, poly lysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0257] In some embodiments, conjugates may also include targeting groups. As used herein, the term “targeting group” refers to a functional group or moiety attached to an agent that facilitates localization of the agent to a desired region, tissue, cell and / or protein. Such targeting groups may include, but are not limited to cell or tissue targeting agents or groups (e.g. lectins, glycoproteins, lipids, proteins, an antibody that binds to a specified cell type such as a kidney cell or other cell type). In some embodiments, targeting groups may comprise

[0258] , melanotropins, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, poly glutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer.

[0259] In some embodiments, targeting groups may be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Targeting groups may also comprise hormones and / or hormone receptors.

[0260] In some embodiments, targeting groups may be any ligand capable of targeting specific receptors. Examples include, without limitation, folate, GalNAc, galactose, mannose, marmose-6-phosphate, apatamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In some embodiments, targeting groups are aptamers. Such aptamers may be unmodified or comprise any combination of modifications disclosed herein.

[0261] In still other embodiments, pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or pay loads of the present disclosure may be covalently conjugated to cell penetrating polypeptides. In some embodiments, cell-penetrating peptides may also include signal sequences. In some embodiments, 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.)

[0262] In some embodiments, conjugating moieties may be added to pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or pay loads of the present disclosure such that they allow the attachment of detectable labels to targets for clearance. Such detectable labels include, but are not limited to biotin labels, ubiquitins, fluorescent molecules, human influenza hemagglutinin (HA), c-myc, histidine (His), flag, glutathione S-transferase (GST), V5 (a paramyxovirus of simian virus 5 epitope), biotin, avidin, streptavidin, horse radish peroxidase (HRP) and digoxigenin. 2026214111   10 Aug 2026

[0263] In some embodiments, pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or pay loads of the present disclosure may be combined with one another or other molecules in the treatment of diseases and / or conditions. Additional Effector Module Features

[0264] The effector module of the present disclosure may further comprise a signal sequence which regulates the distribution of the pay load of interest, a cleavage and / or processing feature which facilitate cleavage of the pay load from the effector module construct, a targeting and / or penetrating signal which can regulate the cellular localization of the effector module, a tag, and / or one or more linker sequences which link different components of the effector module.

[0265] In some embodiments, additional effector module features of the present disclosure include, without limitation, any of those taught in Table 7. Table 7: Effector Module Features Feature AA sequence AASEQ ID NO. NA sequence or SEQ ID NO. EGFP VSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDAT YGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPD HMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVK FEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYI MADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIG DGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTA AGITLGMDELYK 211128 211133 Flag tag DYKDDDDK 211129 211134 Linker (GSGSG) GSGSG 211130 211135 HA tag YPYDVPDYA 211131 211136 Linker (GS) GS - ggatcc Linker (GSLDG) GSLDG 211132 211137 Signal sequences

[0266] In addition to the SRE and pay load region, effector modules of the disclosure may further comprise one or more additional features such as one or more signal sequences.

[0267] Signal sequences (sometimes referred to as signal peptides, targeting signals, target peptides, localization sequences, transit peptides, leader sequences or leader peptides) direct proteins (e.g., the effector module of the present disclosure) to their designated cellular and / or extracellular locations. Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins.

[0268] A signal sequence is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards a particular location. Signal sequences can be recognized by signal recognition particles (SRPs) and cleaved using type I and type II signal peptide peptidases. Signal sequences derived 2026214111   10 Aug 2026 from human proteins can be incorporated as a regulatory module of the effector module to direct the effector module to a particular cellular and / or extracellular location. These signal sequences are experimentally verified and can be cleaved (Zhang Z. and Henzel W.J.; "Signal peptide prediction based on analysis of experimentally verified cleavage sites."; Protein Sci. 2004,13:2819-2824).

[0269] In some embodiments, a signal sequence may be, although not necessarily, located at the N-terminus or C-terminus of the effector module, and may be, although not necessarily, cleaved off the desired effector module to yield a “mature” pay load.

[0270] In some embodiments, the signal sequence used herein may exclude the methionine at the position 1 of amino acid sequence of the signal sequence. This may be referred to as an Mldel mutation.

[0271] In addition to signal sequences naturally occurring such as from a secreted protein, a signal sequence may be a variant modified from a known signal sequence of a protein. For example, U.S. Pat. NOs.: 8,258,102 and 9,133,265 to Sleep disclose a modified albumin signal sequence having a secretion signal and an additional X1-X2-X3-X4-X5-motif which can increase protein secretion; U.S. Pat. NO.: 9,279,007 to Do discloses signal sequences of modified fragments of human immunoglobulin heavy chain binding protein (Bip) that can enhance protein expression and secretion; U.S. Pat. NO.: 8,148,494 to Leonhartsberger et al., discloses a signal peptide with a cleavage site that can be fused with a recombinant protein; the contents of each of which are incorporated by reference in their entirety.

[0272] In some instances, the secreted signal sequences may be cytokine signal sequences such as, but not limited to, IL2 signal sequence or a p40 signal sequence.

[0273] In some instances, signal sequences directing the payload of interest to the surface membrane of the target cell may be used. Expression of the pay load on the surface of the target cell may be useful to limit the diffusion of the pay load to non-target in vivo environments, thereby potentially improving the safety profile of the payloads. Additionally, the membrane presentation of the payload may allow for physiologically and qualitative signaling as well as stabilization and recycling of the pay load for a longer half-life. Membrane sequences may be the endogenous signal sequence of the N terminal component of the payload of interest. Optionally, it may be desirable to exchange this sequence for a different signal sequence. Signal sequences may be selected based on their compatibility with the secretory pathway of the cell type of interest so that the pay load is presented on the surface of the T cell. In some embodiments, the signal sequence may be IgE signal sequence, CD8a signal sequence (also referred to as CD8a leader), or IL15Ra signal sequence (also referred to as IL15Ra leader) or Mldel CD8a signal sequence (also referred to as Mldel CD8 leader sequence).

[0274] Other signal sequence variants may be used in the present effector module may include those discussed in U.S. patent application publication NOs.: 2007 / 0141666; PCT patent application publication NOs.: 1993 / 018181; the contents of each of which are incorporated herein by reference in their entirety. 2026214111   10 Aug 2026

[0275] Other examples of signal sequences include, a variant may be a modified signal sequence discussed in U. S. Pat. 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.

[0276] In other examples, a signal sequence may be a heterogeneous signal sequence from other organisms such as vims, yeast and bacteria, which can direct an effector module to a particular cellular site, such as a nucleus (e.g., EP 1209450). Other examples may include Aspartic Protease (NSP24) signal sequences from Trichoderma that can increase secretion of fused protein such as enzymes (e.g., U. S. Pat. NO. 8,093,016 to Cervin and Kim), bacterial lipoprotein signal sequences (e.g., PCT application publication NO. WO 199109952 to Lau and Rioux), E.coli enterotoxin II signal peptides (e.g., U.S. Pat. NO. 6,605,697 to Kwon et al.), E.coli secretion signal sequence (e.g., U.S. patent publication NO. US2016090404 to Malley et al.), a lipase signal sequence from a methylotrophic yeast (e.g., U.S. Pat. NO. 8,975,041), and signal peptides for DNases derived from Coryneform bacteria (e.g., U.S. Pat. NO. 4,965,197); the contents of each of which are incorporated herein by reference in their entirety.

[0277] Signal sequences may also include nuclear localization signals (NLSs), nuclear export signals (NESs), polarized cell tubulo-vesicular structure localization signals (See, e.g., U.S. Pat. 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 to subcellular locations (e.g. lysosome, endoplasmic reticulum, golgi, mitochondria, plasma membrane and peroxisomes, etc.) (See, e.g., U.S. Pat. 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). Cleavage sites

[0278] In some embodiments, the effector module comprises a cleavage and / or processing feature.

[0279] The effector module of the present disclosure may include at least one protein cleavage signal / site. The protein cleavage signal / site may be located at the N-terminus, the C-terminus, at any space between the N-and the C-termini such as, but not limited to, half-way between the N-and C-termini, between the N-terminus and the half-way point, between the half-way point and the C-terminus, and combinations thereof.

[0280] The effector module may include one or more cleavage signal(s) / site(s) of any proteinases. The proteinases may be a serine proteinase, a cysteine proteinase, an endopeptidase, a dipeptidase, a metalloproteinase, a glutamic proteinase, a threonine proteinase and an aspartic proteinase. In some aspects, the cleavage site may be a signal sequence of furin, actinidain, calpam-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, formic acid, granzyme B, Matrix metallopeptidase-2, Matrix 2026214111   10 Aug 2026 metallopeptidase-3, pepsin, proteinase K, SUMO protease, subtilisin, TEV protease, thermolysin, thrombin, trypsin and TAGZyme. Tags

[0281] In some embodiments, the effector module comprises a protein tag.

[0282] The protein tag may be used for detecting and monitoring the process of the effector module. The effector module may include one or more tags such as an epitope tag (e.g., a FLAG or hemagglutinin (HA) tag). A large number of protein tags may be used for the present effector modules. They include, but are not limited to, self-labeling polypeptide tags (e.g., haloalkane dehalogenase (halotag2 or halotag?), ACP tag, clip tag, MCP tag, snap tag), 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 its variants), bioluminescent tags (e.g. luciferase and its variants), affinity tags (e.g., maltose-binding protein (MBP) tag, glutathione-S-transferase (GST) tag), 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 earner protein (BCCP), eXact, CBP, CYD, HPC, CBD intein-chitin binding domain, Trx, NorpA, and NusA.

[0283] In other embodiments, a tag may also be selected from those disclosed in U.S. Pat. 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.

[0284] In some aspects, a multiplicity of protein tags, either the same or different tags, may be used; each of the tags may be located at the same N or C terminus, whereas in other cases these tags may be located at each terminus. Linkers

[0285] In some embodiments, the effector module comprises a linker.

[0286] In some embodiments, the effector module of the disclosure may further comprise a linker sequence. The linker region serves primarily as a spacer between two or more polypeptides within the effector module. The "linker" or "spacer", as used herein, 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.

[0287] 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 from about 1 to 100 amino acids in length, which links together any of the domains / regions of the effector module (also called peptide linker). The peptide linker may 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 may also be optimized depending on the type of pay load utilized and based on the crystal structure of the pay load. In some instances, a shorter linker length may be preferably selected. In some aspects, the peptide linker is made up of amino acids linked together by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, 2026214111   10 Aug 2026 wherein the amino acids are 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), Aspartate (D), Glutamic acid (E), Asparagine (N), and Glutamine (Q). One or more of these amino acids may be glycosylated, as is understood by those in the art.

[0288] A linker sequence may be a natural linker derived from a multi-domain protein. A natural linker is a short peptide sequence that separates two different domains or motifs within a protein.

[0289] In some aspects, linkers may be flexible or rigid. In other aspects, linkers may be cleavable or non-cleavable. As used herein, the terms “cleavable linker domain or r...

Claims

1. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprising a H122Y mutation in the amino acid at position 122 (H122) of SEQ ID NO. 11717.

2. The composition of claim 1, wherein the DD further comprises:(i) a R27L mutation in the amino acid at position 27 (R27) of SEQ ID NO. 11717;(ii) a T87I mutation in the amino acid at position 87 (T87) of SEQ ID NO. 11717;(iii) a N252D mutation in the amino acid at position 252 (N252) of SEQ ID NO. 11717; or(iv) a combination of (i), (ii) and / or (iii).

3. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprising a E106D mutation in the amino acid at position 106 (E106) of SEQ ID NO. 11717.

4. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprising a W208S mutation in the amino acid at position 208 (W208) of SEQ ID NO. 11717.

5. The composition of claim 3 or 4, wherein the DD further comprises a C205S mutation in the amino acid atposition 205 (C205) of SEQ ID NO. 11717.

6. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprising a 159N mutation in the amino acid at position 59 (159) of SEQ ID NO. 11717.

7. The composition of claim 6, wherein the DD further comprises a G102R mutation in the amino acid at position 102 (G102) of SEQ ID NO. 11717.2026214111   10 Aug 20268. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprising a L156H mutation in the amino acid at position 156 (L156) of SEQ ID NO. 11717.

9. The composition of claim 8, wherein the DD further comprises:(i) a W4Y mutation in the amino acid at position 4 (W4) of SEQ ID NO. 11717;(ii) a F225L mutation in the amino acid at position 225 (F225) of SEQ ID NO. 11717;(iii) deletion of amino acids at positions 257-260 of SEQ ID NO. 11717;(iv) deletion of amino acids at positions 1-5 of SEQ ID NO. 11717; or(v) deletion of amino acids G234, E235 andP236 of SEQ ID NO. 11717.

10. The composition of claim 8, wherein the DD comprises four mutations relative to SEQ ID NO. 11717, said mutations corresponding to:(i) L156H, S172C, F178Y, andE186D; or(ii) D70N, D74N, D100N, and L156H.

11. A composition comprising an effector module, said effector module comprising a stimulus response element(SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole 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 a S73N mutation in the amino acid at position 73 (S73) of SEQ ID NO. 11717; and(ii) the second mutation is a substitution of F or Y at the amino acid position 89 (R89) of SEQ ID NO. 11717.

12. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprising a substitution of N or F at the ammo acid position 56 (S56) of SEQ ID NO. 11717.

13. The composition of claim 12, wherein the DD comprises two substitutions relative to SEQ ID NO. 11717 thatcorrespond to S56F andD71S.2026214111   10 Aug 202614. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprising one or more substitutions relative to SEQ ID NO. 11717, wherein at least one substitution is a substitution of D or N at the amino acid position 63 (G63) of SEQ ID NO. 11717, and wherein the one or more substitutions correspond to:G63D;G63D andM240L;G63D, E69VandN231I; orT55K, G63N and Q248N.

15. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole 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 the amino acid position 71 (D71) of SEQ ID NO. 11717, and wherein said two or more substitutions correspond to:D71LandT87N;D71L andL250R;D71L, T87N and L250R; or D71KandT192F.

16. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 11717), and further comprising two or more substitutions relative to SEQ ID NO. 11717, wherein at least one of the two or more substitutions is:(i) a substitution of F at the amino acid position 241 (V241) of SEQ ID NO. 11717; or(ii) a substitution of F or L at the ammo acid position 249 (P249) of SEQ ID NO. 11717; and wherein the two or more substitutions correspond to:D72F and V241F;D72FandP249L;D72FandP249F;2026214111   10 Aug 2026D72F, V241F andP249L;A77IandP249F; orV241F andP249L.

17. A composition comprising an effector module, said effector module comprising a stimulus response element (SRE) and at least one pay load which is operably linked to said SRE, wherein the SRE comprises a destabilizing domain (DD), said DD comprising a region of or the whole 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 the combination of V134F and L228F.

18. The composition of any one of claims 1-17, wherein the DD is derived from a region of CA2, said region comprising amino acids 2 to 260 of CA2 (SEQ ID NO. 210492).

19. The composition of any one of claims 1-18, wherein the SRE is responsive to one or more stimuli.

20. The composition of claim 19, wherein the stimulus is a small molecule, wherein the small molecule is selectedfrom Acetazolamide, Celecoxib, Valdecoxib, Rofecoxib, Methazolamide, Dorzolamide, Brinzolamide, Diclofenamide, Ethoxzolamide, Zonisamide, Dansylamide, or Dichlorphenamide.

21. The composition of any one of claims 1-20, wherein at least one mutation or substitution in the DD destabilizes the DD and the at least one pay load in the absence of a stimulus and wherein the DD and the pay load are stabilized in the presence of the stimulus.

22. The composition of any one of claims 1-21, wherein the at least one payload is a therapeutic agent, a natural protein, a fusion polypeptide, an antibody, or a variant or a fragment thereof.

23. The composition of any one of claims 1-22, wherein the composition is one or more polypeptides.

24. The composition of claim 22, wherein the therapeutic agent is a CD19 CAR.

25. A biocircuit system comprising the composition of any one of claims 1-24.2026214111   10 Aug 202626. A pharmaceutical composition comprising the composition of any one of claims 1-24, and a pharmaceutically acceptable excipient.

27. A polynucleotide encoding the composition of any one of claims 1-24.

28. A pharmaceutical composition comprising the polynucleotide of claim 27, and a pharmaceutically acceptableexcipient.

29. A vector comprising the polynucleotide of claim 27.

30. A cell comprising the polynucleotide of claim 27.

31. The cell of claim 30, wherein said cell is an immune cell for adoptive cell transfer (ACT).

32. A pharmaceutical composition comprising the cell of claim 30 or 31, and a pharmaceutically acceptable excipient.

33. A method of producing a modified cell, said method comprising introducing into a cell a nucleic acid molecule comprising the polynucleotide of claim 27.

34. A method of modulating expression, function, and / or level of a pay load in the cell of claim 30, said method comprising administering to the cell a stimulus, wherein the SRE is responsive to the stimulus and wherein the expression, function, and / or level of the payload is modulated in response to the stimulus.

35. A method of treating a disease in a subject in need thereof, said method comprising:(a) administering to the subject a therapeutically effective amount of the cell of claim 30, wherein the cell comprises a pay load that treats the disease; and(b) administering to the subject, a therapeutically effective amount of a stimulus, wherein the SRE is responsive to the stimulus and wherein expression of the pay load is modulated in response to the stimulus to thereby treat the disease.2026214111   10 Aug 202636. The method according to either of claims 34 or 35, wherein the stimulus is selected from Acetazolamide, Celecoxib, Valdecoxib, Rofecoxib, Methazolamide, Dorzolamide, Brinzolamide, Diclofenamide, Ethoxzolamide, Zonisamide, Dansylamide, or Dichlorphenamide.

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