Mutant aslov2 domains and uses thereof

Abstract

Provided herein are Avena sativa light, oxygen, voltage 2 (asLOV2) protein domains comprising one or more amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain.

Description

MUTANT ASLOV2 DOMAINS AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 568,263, filed March 21, 2024 and U.S. Provisional Application No. 63 / 758,104, filed February 13, 2025, both which are hereby incorporated in their entirety by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is hereby incorporated by reference in its entirety. Said XML copy, created on March 11, 2025, is named CLS- 040WO_SL, and is 13,703 bytes in size.BACKGROUND

[0003] Protein based therapeutic molecules, such as antibodies, are powerful tools in treating diseases. However, many antibodies have undesirable off target effects. For example, the antibody herceptin is used to treat some breast cancers. However, herceptin has been reported to cause off target heart damage. Thus, additional methods of controlling off target effects of therapeutic drugs are required.

[0004] The LOV domain is a photosensory module that confers light response to a variety of proteins in many organisms like plants, fungi and bacteria. Depending on the type of LOV domain, it can undergo a light-induced conformational change (like the LOV2 domain from Avena sativa) or dimerize in response to light (like VVD, the LOV domain from Neurospora crassa). This light induced change in conformation or oligomeric state has been used to build many optogenetic tools and control processes like transcription, enzymatic activity and localization of proteins. However, such optogenetic tools require the application of light to the tools, which is not feasible in all circumstances, such as internal therapeutic applications. Thus, additional LOV domains that are responsive to non-light stimuli are required.SUMMARY

[0005] In one aspect, provided herein are mutant Avena sativa light, oxygen, voltage 2 (asLOV2) protein domains comprising one or more amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain.

[0006] In some embodiments, the one or more amino acid substitution(s) comprises one or more amino acid substitution(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acidsubstitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the wild type asLOV2 domain of the full length phototropin 1 protein (SEQ ID NO: 11).

[0007] In some embodiments, the mutant asLOV2 domain comprises one or more of a C48P, C48A, R46W, F92L, L94V, QI 11 A, Q111K, E123G, G126R, G126K, and / or DI 38M amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or C450P, C450A, L496V, Q513A, Q513K, R448W, E525G, F494L, G528R, G528K, and / or D540M amino acid substitution(s) as compared to the full length phototropin 1 protein (SEQ ID NO: 11).

[0008] In some embodiments, the mutant asLOV2 domain comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0009] In some embodiments, the mutant asLOV2 domain does not comprise an N-terminal methionine residue as compared to the sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0010] In some embodiments, the increased magnetic response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or greater as compared to a wild type asLOV2 domain.

[0011] In some embodiments, the increased magnetic response is a decrease in fluorescence as compared to a wild type asLOV2 domain.

[0012] In one aspect, provided herein are chimeric polypeptides comprising a mutant Avena sativa light, oxygen, voltage 2 (asLOV2) domain, wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain, and at least a first protein.

[0013] In some embodiments, the mutant asLOV2 domain comprises one or more amino acid substitution(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the full length phototropin 1 protein (SEQ ID NO: 11).

[0014] In some embodiments, the mutant asLOV2 domain comprises one or more of a C48P, C48A, R46W, F92L, L94V, Q111A, Q111K, E123G, G126R, and / or D138M amino acid substitution(s) as compared to wild type asLOV2 (SEQ ID NO: 1).

[0015] In some embodiments, the mutant asLOV2 domain comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0016] In some embodiments, the mutant asLOV2 domain does not comprise an N-terminal methionine residue as compared to the sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0017] In some embodiments, the increased magnetic response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or greater as compared to a wild type asLOV2 domain.

[0018] In some embodiments, the increased magnetic response is a decrease in fluorescence as compared to a wild type asLOV2 domain.

[0019] In some embodiments, the first protein is an antibody, an antibody fragment, a nanobody, a monobody, an scFv, an sdAb, a Fab, an antibody-drug conjugate (ADC), a cytokine, a chemokine, a peptide, an esterase, a chimeric antigen receptor (CAR), a synthetic receptor, or a synthetic transcriptional regulator.

[0020] In some embodiments, the mutant asLOV2 domain is fused to the N terminus of the first protein.

[0021] In some embodiments, the mutant asLOV2 domain is fused to the C terminus of the first protein.

[0022] In some embodiments, the mutant asLOV2 domain is inserted in a loop or domain of the first protein.

[0023] In some embodiments, the first protein is an antibody or an antibody fragment and the mutant asLOV2 domain is inserted in a loop of the variable light (VL) domain of the light chain, inserted in a loop of the variable heavy (VH) domain of the heavy chain fused to the N terminus of the light chain or light chain variable (VL) region, the N terminus of the heavy chain or heavy chain variable (VH) region, the C terminus of the light chain or light chain variable (VL) region, or the C terminus of the heavy chain or heavy chain variable (VH) region.

[0024] In some embodiments, the first protein is a bioluminescent protein.

[0025] In some embodiments, the bioluminescent protein is a luciferase, optionally wherein the luciferase is selected from the group consisting of NanoLuc, firefly luciferase, Renilla reniformis luciferase, copepod luciferase, bacterial luciferase, or Dinoflagellate luciferase.

[0026] In some embodiments, the first protein is a fluorescent protein.

[0027] In some embodiments, comprising a second protein fused to the first protein of the mutant asLOV2 domain.

[0028] In some embodiments, the second protein is a fluorescent protein.

[0029] In one aspect, provided herein are magneto-responsive gene expression systems, comprising: i. a magneto-responsive synthetic transcriptional regulator comprising a magneto-responsive domain comprising a mutant Avena sativa light, oxygen, voltage 2 (asLOV2) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain, a DNA-binding domain, and a trans activation domain; and ii. a target transcription unit, comprising a DNA motif capable of being bound by the DNA-binding domain, a transcriptional control element, and a nucleic acid sequence encoding a target protein.

[0030] In some embodiments, the one or more amino acid substitution(s) comprises one or more mutation(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the full length phototropin 1 protein (SEQ ID NO: 11)

[0031] In some embodiments, the mutant asLOV2 domain comprises one or more of a C48P, C48A, R46W, F92L, L94V, QI HA, Q111K, E123G, G126R, G126K, and / or DI 38M amino acid substitution(s) as compared to wild type asLOV2 (SEQ ID NO: 1).

[0032] In some embodiments, the mutant asLOV2 domain comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0033] In some embodiments, the mutant asLOV2 domain does not comprise an N-terminal methionine residue as compared to the sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0034] In some embodiments, the mutant asLOV2 domain has an increased magnetic response as compared to a wild type asLOV domain (SEQ ID NO: 1).

[0035] In some embodiments, the increased magnetic response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or greater as compared to a wild type asLOV2 domain.

[0036] In some embodiments, the target protein is an antibody, an antibody fragment, a nanobody, a monobody, an scFv, an sdAb, a Fab, an antibody-drug conjugate (ADC), a cytokine, a chemokine, a peptide, an esterase, a chimeric antigen receptor (CAR), or a synthetic receptor.

[0037] In some embodiments, the DNA binding domain is a helix-turn-helix DNA-binding domain, zinc finger motif or zinc cluster DNA-binding domain, leucine zipper DNA-binding domain, winged helix DNA-binding domain, winged helix-tum-helix DNA-binding domain, helix-loop-helix DNA-binding domain, high mobility family DNA-binding domain, or a B3 DNA-binding domain.

[0038] In some embodiments, the transactivation domain is a transcriptional activation domain rich in acidic amino acids, transcriptional activation domains rich in proline, transcriptional activation domains rich in serine / threonine and transcriptional activation domains rich in glutamine, and Kruppel-related box transcriptional repression domain.

[0039] In some embodiments, the transactivation domain comprises a herpes simplex virus VP 16 particle protein transcriptional activation domain, a yeast Gal4 protein transcriptional activation domain, an NF-KB p65 subunit transcriptional activation domain, a yeast general control protein 4 transcriptional activation domain, or a Kruppel-related box transcriptional repression domain of the zinc finger 354A protein, a YAP transcriptional activation domain, a WWTR1 (TAZ) transcriptional activation domain, and a CREB3 (LZIP) transcriptional activation domain.

[0040] In some embodiments, the transcriptional control element is an inducible promoter.

[0041] In some embodiments, the inducible promoter is operably linked to the nucleic acid sequence encoding a target protein.

[0042] In some embodiments, the transcriptional control element is selected from the adenovirus late promoter, cytomegalovirus (CMV) minimal promoter, yeast Gall gene promoter and SV40 promoter.

[0043] In one aspect, provided herein are methods of inducing protein expression in one or more cells comprising obtaining a cell comprising the magneto-responsive gene expression system of any one of claims 25-38 and applying a magnetic field to the one or more cells.

[0044] In some embodiments, the one or more cells are a bacterial cell, a eukaryotic cell, a mammalian cell, or a human cell.

[0045] In some embodiments, the method is performed in vitro, in vivo, or ex vivo.

[0046] In some embodiments, the method is performed in vitro on a human subject’s one or more cells.

[0047] In one aspect, provided herein are methods of assessing binding of a mutant Avena sativa light, oxygen, voltage 2 (asLOV2) domain as compared to the wild type asLOV2 domain (SEQ ID NO: 1), to a target protein comprising: i. providing a cell or solution comprising the mutant asLOV2 domain and a first target protein, wherein the mutant asLOV2 domain and the first target protein are operably linked; and ii. applying a magnetic force to the cell or solution and assessing binding of the asLOV2 domain to the first target protein.

[0048] In some embodiments, the magnetic force induces binding of the asLOV2 domain to the first target protein or induces unbinding of the asLOV2 domain to the first target protein.

[0049] In some embodiments, assessing binding of the asLOV2 domain to the first target protein comprises assessing localization of the first target protein in the cell or solution.

[0050] In some embodiments, assessing binding of the asLOV2 domain to the first target protein comprises assessing fluorescence in the cell or solution, optionally wherein the fluorescence is determined by fluorescence microscopy or a Fluorescence Resonance Energy Transfer (FRET) assay.

[0051] In some embodiments, assessing binding of the asLOV2 domain to the first target protein comprises assessing binding of the first target protein to a second target protein, optionally wherein the binding of the first target protein and the target protein is determined by surface plasmon resonance.

[0052] In some embodiments, the mutant asLOV2 domain and the target protein are covalently linked, optionally wherein the mutant asLOV2 domain and the target protein are a chimeric polypeptide.

[0053] In some embodiments, the mutant asLOV2 domain and the target protein are non- covalently linked.

[0054] In some embodiments, the mutant asLOV2 domain comprises one or more amino acid substitution(s) at positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the full length phototropin 1 protein (SEQ ID NO: 11).

[0055] In one aspect, provided herein are methods of treating a subject in need comprising administering a chimeric polypeptide comprising a protein and a mutant Avena sativa light, oxygen, voltage (asLOV) 2 domain comprising one or more amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain; and applying a magnetic field to the subject.

[0056] In some embodiments, the one or more amino acid substitution(s) comprises one or more amino acid substitution(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the full length photo tropin 1 protein (SEQ ID NO: 11).

[0057] In some embodiments, the mutant asLOV2 domain comprises one or more of a R46W, C48P, F92L, L94V, Q111K, E123G, G126R, and / or D138M amino acid substitution(s) as compared to wild type asLOV2 (SEQ ID NO: 1).

[0058] In some embodiments, the mutant asLOV2 domain comprises one or more of a R46W, C48P, C48A, F92L, L94V, Q111A, Q111K, E123G, G126R, G126K, and / or D138M amino acid substitution(s) as compared to wild type asLOV2 (SEQ ID NO: 1).

[0059] In some embodiments, the mutant asLOV2 domain comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0060] In some embodiments, the mutant asLOV2 domain does not comprise an N-terminal methionine residue as compared to the sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0061] In some embodiments, the increased magnetic response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or greater as compared to a wild type asLOV2 domain.

[0062] In some embodiments, the protein is an antibody, an antibody fragment, a nanobody, a monobody, an scFv, an sdAb, a Fab, an antibody-drug conjugate (ADC), a cytokine, a chemokine, a peptide, an esterase, a chimeric antigen receptor (CAR), a synthetic receptor, or a synthetic transcriptional regulator.

[0063] In some embodiments, the mutant asLOV2 domain is fused to the N terminus of the protein or target protein.

[0064] In some embodiments, the mutant asLOV2 domain is fused to the C terminus of the protein or target protein.

[0065] In some embodiments, the protein or target protein is an antibody or an antibody fragment and the mutant asLOV2 domain is inserted in a loop of the variable light (VL) domain of the light chain, inserted in a loop of the variable heavy (VH) domain of the heavy chain fused to the N terminus of the light chain or light chain variable (VL) region, the N terminus of the heavy chain or heavy chain variable (VH) region, the C terminus of the light chain or light chain variable (VL) region, or the C terminus of the heavy chain or heavy chain variable (VH) region.

[0066] In some embodiments, the magnetic field is generated by a handheld magnet.

[0067] In some embodiments, the handheld magnet is a rare earth, ferrite, or alnico magnet.

[0068] In some embodiments, the rare earth is a neodymium or samarium-cobalt magnet.

[0069] In some embodiments, the magnetic field is between about 0.5 milli Tesla (mlT) to 50 mlT.

[0070] In some embodiments, the magnetic field is applied to the subject or cell for about 1, 5, 10, 20, 30, 40, 50, or 60 seconds or more.

[0071] In some embodiments, the magnetic field is applied to the subject or cell for about 1, 5, 10, 20, 30, 40, 50, or 60 minutes or more.

[0072] In some embodiments, the magnetic field is applied to the subject or cell for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more.

[0073] In some embodiments, the magnetic field is applied to the subject or cell for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days or more.

[0074] In some embodiments, the subject in need has cancer, an autoimmune disorder, or an infectious disease.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0075] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0076] FIG. 1 provides a diagram of an exemplary method of identifying mutations in the LOV domain that result in increased magnetic responsiveness.

[0077] FIG. 2A shows the magnetic response of a mutant with a C48A mutation (SEQ ID NO: 2) as compared to the wild type asLOV2 domain provided in SEQ ID NO: 1 (which corresponds to a C450A mutation in the full length Avena saliva phototropin 1 protein, SEQ ID NO; 11). FIG. 2B shows the magnetic response of a mutant with a C48A D138M (C450A D540M as compared to SEQ ID NO: 11) mutation (SEQ ID NO: 3). FIG. 2C shows the magnetic response of a mutant with a C48A QI HA D138M (C450A Q513A D540M as compared to SEQ ID NO: 11) mutation (SEQ ID NO: 4). FIG. 2D shows the magnetic response of a mutant with a C48A L94V Q111A D138M (C450A L496V Q513A D540M as compared to SEQ ID NO: 11) mutation (SEQ ID NO: 5). FIG. 2E shows the magnetic response of a mutant with a C48P L94V Q111A D138M (C450P L496V Q513A D540M as compared to SEQ ID NO: 11) mutation (SEQ ID NO: 6). FIG. 2F shows the magnetic response of a mutant with a C48P L94V Q111K D138M (C450P L496V Q513K D540M as compared to SEQ ID NO: 11) mutation (SEQ ID NO: 7). FIG. 2G shows the magnetic response of a mutant with a C48P L94V Qll IK D138M and a C terminus truncation at residue 157 (C450P L496V Q513A D540M and 544 truncation as compared to SEQ ID NO: 11) mutation (SEQ ID NO: 8).

[0078] FIG. 3 provide an exemplary diagram of a magnetic field-induced conformational change in mutant asLOV2 domain that results in modulation of the conformation of a target protein of interest and binding of the target protein to its cognate ligand.

[0079] FIG. 4 shows the shows the magnetic response of an asLOV2 protein mutant with a C48P L94V Q111K G126K D138M (C450P L496V Q513K G528K D540M as compared to SEQ ID NO: 11) mutation (SEQ ID NO: 9)

[0080] FIG. 5 shows the magnetic response of an asLOV2 protein mutant with R46W, C48P, F92L, L94V, QI U K, E123G, G126R, D138M (R448W C450P F494L L496V Q513K E252G G528R D540M as compared to SEQ ID NO: 11) mutations (SEQ ID NO: 10).DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0081] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0082] The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0083] The term “in situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.

[0084] The term “in vivo” refers to processes that occur in a living organism.

[0085] The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0086] The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0087] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0088] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0089] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0090] The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[0091] The term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

[0092] The terms “protein,” “polypeptide,” and “peptide” are used herein interchangeably.

[0093] “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Operably linked nucleic acid sequences may but need not necessarily be adjacent. For example, in some instances a coding sequence operably linked to a promoter may be adjacent to the promoter. In some instances, a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences. Also, in some instances, more than two sequences may be operably linked including but not limited to e.g., where two or more coding sequences are operably linked to a single promoter.

[0094] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.Mutant LOY domain proteins

[0095] The impact of magnetic fields on biological systems is currently poorly understood. Conflicting reports of evidence for and against magnet field effects in Drosophila (see e.g., Nature 454, 1014-1018 (2008) compared to Nature 620, 595-599(2023)), monarch butterflies (see e.g., Nature Communications 5: 4164 (2014) compared to Ecol. Evol 12(l l):e9498(2022), and Saccharomyces cerevisiae (see e.g., PLoS ONE 14(1): e0209843, Rendiconti Lincei. Scienze Fisishe e Natural! 30, 775-784 (2019) compared toBioelectromagnetics, 31(1) :28-38 (2010) and Plant Physiology, 53(2) (974)), among others, confound the general understanding of the effect of magnetic fields on biologic processes.

[0096] Some fluorescent proteins have shown magneto responsive behavior in living systems, but the purified fluorescent proteins exhibit no magnetic responsiveness in vitro. All such fluorescent proteins rely on cofactors, such as riboflavin, for the magnetic response, and are not themselves magneto-responsive.

[0097] The light, oxygen, voltage (LOV2) domain of phytochrome 1 (phototropin 1) from Avena sativa is a light sensitive phototropin with fluorescent properties when expressed in conjunction with a flavin mononucleotide (FMN) cofactor. The Avena sativa phototropin 1 protein is also known as non-specific serine / threonine protein kinase, encoded by the NPH1-1 gene (UniProt: 049003). The LOV2 domain is a fluorescent domain that absorbs light in the cyan spectrum and emits light in the green spectrum. LOV domains have an FMN cofactor that is required for their fluorescent function. The conformational change has been reported to depend on a cysteine in the active site, which forms a temporary covalent bond to the flavin. In addition, the LOV2 domain also undergoes a conformational change when it absorbs cyan light, resulting changing the shape of the protein structure. Some fluorescent proteins exhibit magneto-responsive behavior, including the LOV domain, GFP, and mScarlet. However, other fluorescent proteins such as GFP and mScarlet require an exogenous FMN cofactor for their magnetic response. Neither GFP nor FMN are individually magneto-responsive. Unlike GFP and mScarlet, the LOV domain includes an endogenous FMN cofactor and thus does not require any additional exogenous cofactor for its magneto-responsive behavior. However, the magnetic responsiveness of wild type asLOV2 is weak, and thus additional mutations are required in order to increase the magnetic responsiveness of the asLOV2 domain.

[0098] Provided herein are Avena sativa light, oxygen, voltage 2 (asLOV2) protein domains comprising one or more amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain.

[0099] The sequences of wild type and mutant asLOV2 domains and the full length phototropin 1 protein are provided in Table 1.

[0100] In some embodiments, mutant Avena saliva light, oxygen, voltage 2 (asLOV2) protein domain comprises one, two, three, four, or five or more amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain

[0101] In some embodiments, the one or more amino acid substitution(s)comprises one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the wild type asLOV2 domain of the full phototropin 1 protein (SEQ ID NO: 11). In some embodiments, the one or more amino acid substitution(s)comprises one, two, three, four, five, six, seven, eight, or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the wild type asLOV2 domain of the full phototropin 1 protein (SEQ ID NO: 11).

[0102] In some embodiments, the one or more amino acid substitution(s)comprises one or more amino acid substitution(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the one or more amino acid substitution(s)comprises one, two, three, four, five, six, seven, eight, or more amino acid substitution(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1).

[0103] In some embodiments, the mutant asLOV2 domain does not comprise an N terminal methionine residue shown in the sequences provided in SEQ ID NOs: 1-10.

[0104] In some embodiments, the mutant asLOV2 domain comprises one or more of a C450P, C450A, L496V, Q513A, Q513K, R448W, E525G, F494L, G528R, G528K, and / or D540M amino acid substitution(s) as compared to the wild type asLOV2 domain of the phototropin 1 protein (SEQ ID NO: 11). In some embodiments, the mutant asLOV2 domain comprises a C450P amino acid substitution as compared to the wild type asLOV2 domain of the phototropin 1 protein (SEQ ID NO: 11). In some embodiments, the mutant asLOV2 domain comprises a C450A amino acid substitution as compared to the wild type asLOV2 domain of the phototropin 1 protein (SEQ ID NO: 11). In some embodiments, the mutant asLOV2 domain comprises a C450A D540M amino acid substitution as compared to the wild type asLOV2 domain of the phototropin 1 protein (SEQ ID NO: 11). In some embodiments, the mutant asLOV2 domain comprises a C450A D540M Q513A amino acid substitution as compared to the wild type asLOV2 domain of the phototropin 1 protein (SEQ ID NO: 11). In some embodiments, the mutant asLOV2 domain comprises a C450A D540M Q513A L496V amino acid substitution as compared to the wild type asLOV2 domain of the phototropin 1 protein (SEQ ID NO: 11). In some embodiments, the mutant asLOV2 domain comprises a C450P L496V Q513A D540M amino acid substitution as compared to the wild type asLOV2 domain of the phototropin 1 protein (SEQ ID NO: 11). In some embodiments, the mutant asLOV2 domain comprises a C450P L496V Q513K D540M amino acid substitution as compared to the wild type asLOV2 domain of the phototropin 1 protein (SEQ ID NO: 11).

[0105] In some embodiments, the mutant asLOV2 domain comprises one or more of a R46W, C48P, C48A, F92L, L94V, Q111A, Q111K, E123G, G126R, and / or D138M amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a C48P amino acid substitution as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a C48A amino acid substitution as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a C48A D138M amino acid substitution as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a C48A D138M Q111A amino acid substitution as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a C48A D138M Q111A L94V amino acid substitution as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a C48P L94VQI HA D138M amino acid substitution as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a C48P L94V QI UK D138M amino acid substitution as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a C48P L94V QI UK G126K D138M amino acid substitution as compared to the wild type asLOV2 domain (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a R46W, C48P, F92L, L94V, Q111K, E123G, G126R, D138M amino acid substitution as compared to the wild type asLOV2 domain (SEQ ID NO: 1).

[0106] In some embodiments, the mutant asLOV2 domain comprises a C48P L94V QI UK D138M amino acid substitution and a deletion of the three C-terminus amino acids as compared to the wild type asLOV2 domain (SEQ ID NO: 1), wherein the three C-terminus amino acids are replaced with the sequence RSCNSRSAAGTMEFEA (SEQ ID NO: 12).

[0107] In some embodiments, the mutant asLOV2 domain comprises a sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0108] In some embodiments, the increased magnetic response is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or greater as compared to a wild type asLOV2 domain. In some embodiments, the increased magnetic response is at least about between l%-25%, 2%-25%, 3%-25%, 4%-25%, 5%-25%, 6%-25%, 7%-25%, 8%-25%, 9%-25%, 10%-25%, ll%-25% 12%-25%, 13%-25%, 14%-25%, 15%- 25%, 16%-25%, 17%-25% 18%-25%, 19%-25%, 20%-25%, 21%-25%, 22%-25%, 23%- 25%, 24%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%- 60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 8O%-85%, 85%-90%, 90%-95%, 95%- 100%, 100%-125%, 125%-150%, 150%-175%, 175%-200%, 200%-225%, 225%-250%, 250%-275%, 275%-300%, or greater as compared to a wild type asLOV2 domain.

[0109] In some embodiments, the mutant asLOV2 domain further comprises a deletion of the three C-terminus amino acids as compared to wild type asLOV2 domain (SEQ ID NO: 1), wherein the three C-terminus amino acids are replaced with the sequence RSCNSRSAAGTMEFEA (SEQ ID NO: 12).

[0110] In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some aspects, the amino acid substitutions are not conservative amino acidsubstitutions. In some embodiments, the mutant asLOV2 domain proteins described in this paragraph are referred to herein as “variants” or “analogues”. In some embodiments, such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining mutant asLOV2 domain proteins.Chimeric proteins

[0111] The mutant asLOV2 domain proteins provided herein can be used to generate reversible magnetic-switchable control proteins by inserting or fusing the mutant asLOV2 domain to a protein of interest (e.g., a target protein), thus forming a chimeric protein. Without wishing to be bound by theory, magnetic-induced changes in the shape of the mutant asLOV2 protein can be used to drive additional conformational changes in the target protein of interest, e.g., an antibody, ligand, or enzyme. For example, without wishing to be bound by theory, if the protein of interest to be controlled by the mutant asLOV2 domain is an antibody, the application of a magnetic field to the chimeric asLOV2-antibody polypeptide can induce conformational changes in the antibody that result in increased or decreased binding of the antibody to its cognate ligand. A schematic of the magnetic field induced conformational change in mutant asLOV2 domain that results in modulation of the conformation of a target protein of interest is provided in FIG. 3. In the exemplary diagram of FIG. 3, the application of the magnetic field induced changes in the asLOV2 domain that induce conformational change in a binding protein that results in binding of the binding protein to its cognate ligand. As another example, without wishing to be bound by theory, if the protein of interest to be controlled by the mutant asLOV2 domain is an enzyme such as an esterase, the application of a magnetic field to the chimeric asLOV2- esterase polypeptide can induce conformational changes in the esterase that result in increased or decreased enzymatic activity. The asLOV domain and the first and / or second protein can be linked directly or operatively via a linker peptide. The amino acid number of the linker peptide can be variable, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids or more.Antibodies and antigen binding fragments

[0112] The mutant asLOV2 domain can be expressed as a chimeric protein (e.g., a fusion protein) with any antibody or antigen binding fragment thereof. The mutant asLOV2 domain can be fused or inserted at any suitable spot to the antibody or antigen bindingfragment thereof. For example, the mutant asL0V2 domain can be inserted in a loop of the variable light (VL) domain of the light chain, inserted in a loop of the variable heavy (VH) domain of the heavy chain fused to the N terminus of the light chain or light chain variable (VL) region, the N terminus of the heavy chain or heavy chain variable (VH) region, the C terminus of the light chain or light chain variable (VL) region, or the C terminus of the heavy chain or heavy chain variable (VH) region. Suitable insertion sites in antibodies or antigen binding fragment thereof include the conserved solvent exposed sites in the variable domains, such as loop 1, loop 2, loop 3, loop 4, lop 5, loop 6, loop 7, or loop 8. Suitable residues pair to target for insertion include, but are not limited to KE43, PG41, SV62, GR55, SL84, GG15, and AK74. Insert of wild type LOV proteins into antibodies to form opto-nanobodies is described in Gil, A.A., et al., Nat Commun 11, 4044 (2020) which is hereby incorporated by reference in its entirety.

[0113] When the mutant asLOV2 domain is inserted into or fused to an antibody or antigen binding fragment thereof, the application of a magnetic field can induce a change in the binding affinity of the antibody to its cognate target ligand. The magnet-controlled binding is a reversible effect that can either increase or decrease the binding affinity of the antibody to it ligand. Without wishing to be bound by theory, this magnet-controlled change in binding may be due to magnetic-induced changes in the shape of the mutant asLOV2 protein that then induced conformational changes in the fusion protein, e.g., an antibody. In some embodiments, the application of a magnetic field to the chimeric asLOV2-antibody polypeptide increases the binding affinity of the antibody to its ligand. In some embodiments, the application of a magnetic field to the chimeric asLOV2-antibody polypeptide decreases the binding affinity of the antibody to its ligand. Thus, the mutant asLOV2 domain proteins provided herein can be used to generate reversible magnetic-switchable binding proteins.

[0114] The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific antibodies.

[0115] In some embodiments, the antigen-binding moiety includes one or more antigen-binding determinants of an antibody or a functional antigen-binding fragment or equivalent thereof. In some embodiments, the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a monobody, a diabody, a triabody, or a minibody, a F(ab')2 fragment, a Fab fragment, a single chain variable fragment (scFv), a VHH, and a single domain antibody (sdAb), or a functional fragment thereof.

[0116] As used herein, the term “single-chain” refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the singlechain Fab molecule. As described in more detail herein, an scFv has a variable domain of light chain (VL) connected from its C-terminus to the N-terminal end of a variable domain of heavy chain (VH) by a polypeptide chain. Alternately the scFv comprises of polypeptide chain where in the C-terminal end of the VH is connected to the N-terminal end of VL by a polypeptide chain.

[0117] The “Fab fragment” (also referred to as fragment antigen-binding) contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains respectively. The variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are involved in antigen-binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.

[0118] “F(ab’)2” fragments contain two Fab’ fragments joined, near the hinge region, by disulfide bonds. F(ab’)2 fragments may be generated, for example, by recombinant or synthetic methods or by pepsin digestion of an intact antibody. The F(ab’) fragments can be dissociated, for example, by treatment with B-mercaptoethanol.

[0119] “ Fv” fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

[0120] The “Single-chain Fv” or “scFv” includes the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen-binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in WO93 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

[0121] The term “single domain antibody” or “sdAb” refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain. Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi el al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies. Sdabs are fairly stable andeasy to express as fusion partner with the Fc chain of an antibody (Harmsen MM, De Haard HJ (2007). “Properties, production, and applications of camelid single-domain antibody fragments”. Appl. Microbiol Biotechnol. 77(1): 13-22). The terms “single domain antibody” and “sdAb” are used interchangeably herein to refer to an antibody comprising at least one monomeric domain, such as a VHH domain, or a VNAR domain (from a shark antibody) without a light chain, and an Fc region.

[0122] The term “VHH” or “VHH domain” or “VHH antigen-binding domain” as used herein refers to the antigen-binding portion of a single-domain antibody (sdAb), such as a camelid antibody. In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0123] Exemplary antibodies that can be used in the chimeric protein with the mutant asLOV domain include, but are not limited to, Abagovomab, Abciximab, Abituzumab, Abrezekimab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afasevikumab, Afelimomab, Alacizumabpegol, Alemtuzumab, Alirocumab, Altumomabpentetate, Amatuximab, Amivantamab, Anatumomabmafenatox, Andecaliximab, Anetumabravtansine, Anifrolumab, Ansuvimab, Anrukinzumab,(=IMA-638), Apolizumab, Aprutumabixadotin, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atidortoxumab, Atinumab, Atoltivimab, Atoltivimab / maftivimab / odesivimab, Atorolimumab, Avelumab, Azintuxizumabvedotin, Bamlanivimab, Bapineuzumab, Basiliximab, Bavituximab, BCD-100, Bebtelovimab, Bectumomab, Bedinvetmab, Begelomab, Belantamabmafodotin, Belimumab, Bemarituzumab, Benralizumab, Berlimatoxumab, Bermekimab, Bersanlimab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Birtamimab,Bivatuzumab, Bleselumab, Blinatumomab, Blontuvetmab, Blosozumab, Bococizumab, Brazikumab, Brentuximabvedotin, Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Burosumab, Cabiralizumab, Camidanlumabtesirine, Camrelizumab, Canakinumab, Cantuzumabmertansine, Cantuzumabravtansine, Caplacizumab, Casirivimab, Capromab, Carlumab, Carotuximab, Catumaxomab, cBR96-doxorubicinimmunoconjugate, Cedelizumab, Cemiplimab, Cergutuzumabamunaleukin, Certolizumabpegol, Cetrelimab, Cetuximab, Cibisatamab, Cilgavimab, Cirmtuzumab, Citatuzumabbogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumabtetraxetan, Codrituzumab, Cofetuzumabpelidotin, Coltuximabravtansine, Conatumumab, Concizumab, Cosfroviximab, Crenezumab, Crizanlizumab, Crotedumab, CR6261, Cusatuzumab, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumabpegol, Daratumumab, Dectrekumab,Demcizumab, Denintuzumabmafodotin, Denosumab, Depatuxizumabmafodotin, Derlotuximabbiotin, Detumomab, Dezamizumab, Dinutuximab, Dinutuximabbeta, Diridavumab, Divozilimab, Domagrozumab, Donanemab, Dorlimomabaritox, Dostarlimab, Drozitumab, DS-8201, Duligotuzumab, Dupilumab, Durvalumab, Dusigitumab, Duvortuxizumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elezanumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emapalumab, Emibetuzumab, Emicizumab, Enapotamabvedotin, Enavatuzumab, Enfortumabvedotin, Enlimomabpegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epcoritamab, Epitumomabcituxetan, Epratuzumab, Eptinezumab, Erenumab, Erlizumab, Ertumaxomab, Etaracizumab, Etesevimab, Etigilimab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Faricimab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Flotetuzumab, Fontolizumab, Foralumab, Foravirumab, Fremanezumab, Fresolimumab, Frovocimab, Frunevetmab, Fulranumab, Futuximab, Galcanezumab, Galiximab, Gancotamab,Ganitumab, Gantenerumab, Gatipotuzumab, Gavilimomab, Gedivumab, Gemtuzumabozogamicin, Gevokizumab, Gilvetmab, Gimsilumab, Girentuximab, Glembatumumabvedotin, Glofitamab, Golimumab, Gomiliximab, Gosuranemab,Guselkumab, lanalumab, Ibalizumab, Sintilimab, Ibritumomabtiuxetan, Icrucumab, Idarucizumab, Ifabotuzumab, Igovomab, Iladatuzumabvedotin, Imalumab, Imaprelimab, Imciromab, Imdevimab, Imgatuzumab, Inclacumab, Indatuximabravtansine, Indusatumabvedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin, Ipilimumab, lomab-B, Iratumumab, Isatuximab, Iscalimab, Istiratumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lacnotuzumab, Ladiratuzumabvedotin, Lampalizumab, Lanadelumab, Landogrozumab, Laprituximabemtansine, Larcaviximab, Lebrikizumab, Lecanemab, Lemalesomab, Lendalizumab, Lenvervimab, Lenzilumab, Lerdelimumab, Leronlimab, Lesofavumab, Letolizumab, Lexatumumab, Libivirumab, Lifastuzumabvedotin, Ligelizumab, Loncastuximabtesirine, Losatuxizumabvedotin, Lilotomabsatetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumabmertansine, Lucatumumab, Lulizumabpegol, Lumiliximab, Lumretuzumab, Lupartumab, Lupartumabamadotin, Lutikizumab, Maftivimab, Mapatumumab, Margetuximab, Marstacimab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minrelumomab, Mirikizumab, Mirvetuximabsoravtansine, Mitumomab, Modotuximab, Mogamulizumab, Monalizumab, Morolimumab, Mosunetuzumab, Motavizumab, Moxetumomabpasudotox,Muromonab-CD3, Nacolomabtafenatox, Namilumab, Naptumomabestafenatox, Naratuximabemtansine, Namatumab, Natalizumab, Navicixizumab, Navivumab, Naxitamab, Nebacumab, Necitumumab, Nemolizumab, NEODOOl, Nerelimomab, Nesvacumab, Netakimab, Nimotuzumab, Nirsevimab, Nivolumab, Nofetumomabmerpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odesivimab, Odulimomab, Ofatumumab, Olaratumab, Oleclumab, Olendalizumab, Olokizumab,Omalizumab,Omburtamab, OMS721, Onartuzumab, Ontuxizumab, Onvatilimab, Opicinumab, Oportuzumabmonatox, Oregovomab, Orticumab, Otelixizumab, Otilimab,Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, PDR001, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumabvedotin, Pintumomab, Placulumab, Pozelimab, Prezalumab, Plozalizumab, Pogalizumab, Polatuzumabvedotin, Ponezumab, Porgaviximab, Prasinezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PROMO, Quilizumab, Racotumomab,Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranevetmab,Ranibizumab, Raxibacumab, Ravagalimab, Ravulizumab, Refanezumab, Regavirumab, Regdanvimab, Relatlimab, Remtolumab, Reslizumab, Retifanlimab, Rilotumumab, Rinucumab, Risankizumab, Rituximab, Rivabazumabpegol, Robatumumab, Rmab, Roledumab, Romilkimab, Romosozumab, Rontalizumab, Rosmantuzumab, Rovalpituzumabtesirine, Rovelizumab, Rozanolixizumab, Ruplizumab, SA237, Sacituzumabgovitecan, Samalizumab, Samrotamabvedotin, Sarilumab, Satralizumab, Satumomabpendetide, Secukinumab, Selicrelumab, Seribantumab, Setoxaximab, Setrusumab, Sevirumab, Sibrotuzumab, SGN-CD19A, SHP647, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirtratumabvedotin, Sirukumab, Sofituzumabvedotin, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Sotrovimab, Spartalizumab, Spesolimab, Stamulumab, Sulesomab, Suptavumab, Sutimlimab, Suvizumab, Suvratoxumab, Tabalumab, Tacatuzumabtetraxetan, Tadocizumab, Tafasitamab, Talacotuzumab, Talizumab, Talquetamab, Tamtuvetmab, Tanezumab, Taplitumomabpaptox, Tarextumab, Tavolimab,Teclistamab, Tefibazumab, Telimomabaritox, Telisotuzumab, Telisotuzumabvedotin, Tenatumomab, Teneliximab, Teplizumab, Tepoditamab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Tibulizumab, Tildrakizumab, Tigatuzumab, Timigutuzumab, Timolumab, tiragolumab, Tiragotumab, Tislelizumab, Tisotumabvedotin, Tixagevimab, TNX-650, Tocilizumab, Tomuzotuximab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab,Trastuzumabduocarmazine, Trastuzumabemtansine, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumabcelmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab, Vadastuximabtalirine, Vanalimab, Vandortuzumabvedotin, Vantictumab, Vanucizumab, Vapaliximab, Varisacumab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Vilobelimab, Visilizumab, Vobarilizumab, Volociximab, Vonlerolizumab, Vopratelimab, Vorsetuzumabmafodotin, Votumumab, Vunakizumab, Xentuzumab, XMAB-5574, Zalutumumab, Zanolimumab, Zatuximab, Zenocutuzumab, Ziralimumab, Zolbetuximab, or Zolimomabaritox. Sequences of the exemplary antibodies can found online at drugbank.com.

[0124] The antibody or antigen binding domain of the chimeric asLOV2 protein may further be incorporated into an antibody drug conjugate (ADC). Any appropriate linker and drug moiety can be used in the ADC. Exemplary drug moieties include, but are not limited to, microtubulin inhibitors monomethyl auristatin A MMAE, monomethyl auristatin F MMA and mertansine, DNA binder calicheamicin, and topoisomerase 1 inhibitors SN-38 and exatecan. Exemplary linkers include, but are not limited to, disulfides, hydrazones or peptides (cleavable), or thioethers (non-cleavable). Cleavable linkers include, but are not limited to, Acid-cleavable linkers, Glutathione (GSH)-cleavable linkers, Fe(II) cleavable linkers, P- glucuronidase-cleavable linkers, P-galactosidase-cleavable linkers, photo-responsive cleavable linkers, and biorthogonal cleavable linkers. Additional linkers for ADCS are provided in Su Zet al., Acta Pharm Sin B. 2021 Dec; 11(12):3889-3907, hereby incorporated by reference in its entirety.Chemokines and Cytokines

[0125] The mutant asLOV2 domain can be expressed as a chimeric protein (e.g., a fusion protein) with a chemokine or cytokine. Suitable chemokines include, but are not limited to, IL-1, TNF-alpha, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10. Suitable cytokines include but are not limited to, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, the IL-2 subfamily, the IL-10 subfamily, interferons alpha, beta, and gamma, TGF- L TGF- 2, TGF- 3, or the IL- 17 subfamily.Esterases

[0126] The mutant asLOV2 domain can be expressed as a chimeric protein (e.g., a fusion protein) with an esterase. Esterases hydrolyze esters into acids and alcohol. Suitable esterases include, but are not limited to, Acetylesterase, Cholinesterase, Pectinesterase,Thioesterase, Phosphatase, Alkaline phosphatase, Phosphodiesterase, Fructose bisphosphatase, and Phosphoric diester hydrolases.Synthetic receptors

[0127] In some aspects, the mutant asLOV domains provided herein are expressed as a chimeric polypeptide with a synthetic receptor. Exemplary synthetic receptors include chimeric antigen receptors (CARs) and synthetic transcriptional regulators. Synthetic transcriptional regulators comprise a transcriptional activator domain comprising a DNA- binding domain that can bind to a DNA reaction element and a trans activation domain that can interact with a transcriptional control element.Chimeric Antigen Receptors

[0128] In some aspects, the synthetic immune receptor is a chimeric antigen receptor (CAR). The CAR may be a human CAR, comprising fully human sequences, e.g., natural human sequences.

[0129] In some embodiments, the chimeric antigen receptor includes an extracellular portion comprising an antigen binding domain. The antigen recognition domain of a receptor such as a CAR can be linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and / or signal via another cell surface receptor. Thus, in some embodiments, the extracellular binding component e.g., ligand-binding or antigenbinding domain) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0130] In some aspects, the chimeric antigen receptor includes an extracellular portion comprising an antigen binding domain and an intracellular signaling domain. In some embodiments, the antigen binding domain (e.g., an antibody or antigen binding fragment thereof) is referred to as a “binder.” In some embodiments, the antigen-binding domain is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, or a minibody, a F(ab')2 fragment, a Fab fragment, a single chain variable fragment (scFv), and a single domain antibody (sdAb), or a functional fragment thereof. In some embodiments, theantigen-binding moiety comprises an scFv. The antigen-binding moiety can include naturally -occurring amino acid sequences or can be engineered, designed, or modified so as to provide desired and / or improved properties, e.g. , increased binding affinity. In some embodiments, an antibody or fragment includes an scFv, a VH, or a single-domain VH antibody and the intracellular domain contains an IT AM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain.

[0131] In some aspects, the transmembrane domain contains a transmembrane portion of CD8a or CD28. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0132] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, and / or CD154.Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and / or transmembrane domain(s).

[0133] In some embodiments, the transmembrane domain of the receptor, e.g., the CAR, is a transmembrane domain of human CD28 or variant thereof, e.g., a 27- amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1). In some embodiments, the CAR comprises a CD 8a transmembrane domain.

[0134] In some embodiments, the CAR further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., a CD8a hinge, a CD4 hinge, a CD28 hinge, an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some embodiments, the constantregion or portion is of a human IgG, such as IgG4 or IgGl. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include CD8a hinge, IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or international patent application publication number W02014031687.

[0135] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine- serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the receptor.

[0136] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the receptor. In some embodiments, the CAR comprises means for activating at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the receptor. For example, in some contexts, the receptor induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cellreceptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and / or any derivative or variant of such molecules, and / or any synthetic sequence that has the same functional capability. In some embodiments, the means for at least one of the normal effector functions or responses of the immune cell comprises an CAR intracellular activation domain, e.g., an intracellular activation domain provided herein or an equivalent thereof. In some embodiments, the means for at least one of the normal effector functions or responses of the immune cell comprises an CAR intracellular activation domain and a CAR co-stimulatory domain, e.g., a co-stimulatory domain provided herein or an equivalent thereof.

[0137] In some aspects, the receptor includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

[0138] In some embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as a 112 AA cytoplasmic domain of isoform 3 of human CD3.zeta. (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993.

[0139] The receptor, e.g., the CAR, can include at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the extracellular domain is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor-gamma, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta or Fc receptor-gamma and CD8, CD4, CD25 or CD16.

[0140] In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4- IBB or functional variant or portion thereof, such as a 42-amino acid cytoplasmic domain of a human 4-1BB (Accession No. Q07011.1) or functional variant or portion thereof.

[0141] In some embodiments, the receptor encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary receptors include intracellular components of CD3-zeta, CD28, and 4-1BB. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is 4- IBB.

[0142] In some embodiments, the receptor includes a signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40, DAP10, and ICOS. In some aspects, the same receptor includes both the activating and costimulatory components. In some embodiments, the same receptor includes multiple costimulatory components.

[0143] In certain embodiments, the intracellular signaling domain comprises a CD8a transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a 4-1BB (CD137, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain. In some embodiments, the CAR comprises a 4- IBB co-stimulatory domain.Synthetic transcriptional regulators

[0144] A synthetic transcriptional regulator can comprise a transcriptional activator and optional addition protein domains. A transcriptional activator can comprise a DNA- binding domain operably linked to a transactivation domain. In some cases the transcriptional activator is an engineered protein, such as a zinc finger or TALE based DNA binding domain fused to an effector domain such as VP64.

[0145] The expression system nucleic acid can be incorporated into an expression vector. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., Hum Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al.,PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In some cases, the vector is a lentivirus vector. Also suitable are transposon-mediated vectors, such as piggyback and sleeping beauty vectors.

[0146] The expression system nucleic acid can be delivered to a cell via viral or non- viral methods. Non-viral delivery methods include electroporation or chemical treatment. Viral methods include the use of any one of the viral vectors described herein.

[0147] Any suitable protein can be expressed as the target protein by the expression system. Suitable proteins include, but are not limited to, an antibody, an antibody fragment, a nanobody, a monobody, an scFv, an sdAb, a Fab, an antibody-drug conjugate (ADC), a cytokine, a chemokine, a peptide, an esterase, or a chimeric antigen receptor (CAR).DNA-binding domains

[0148] The DNA binding domain assists in targeting the transactivation domain to the regulatory element (e.g., the inducible promoter) of the target gene. Any suitable DNA binding domain can be used, including but not limited to, a helix-tum-helix DNA-binding domain, a zinc finger (ZF) motif or zinc cluster DNA-binding domain, a transcription activator-like effectors (TALEs), a Cas (CRISPR-Cas) (including the sgRNA), a leucine zipper DNA-binding domain, a winged helix DNA-binding domain, a winged helix-turn- helix DNA-binding domain, a helix-loop-helix DNA-binding domain, a high mobility family DNA-binding domain, B3 DNA-binding domain.

[0149] In some embodiments, the synthetic transcriptional regulator comprises one or more intracellular “DNA-binding domains” (or “DB domains”). Such “DNA-binding domains” refer to sequence-specific DNA binding domains that bind a particular DNA sequence element. Accordingly, as used herein, a “sequence-specific DNA-binding domain”refers to a protein domain portion that has the ability to selectively bind DNA having a specific, predetermined sequence. A sequence-specific DNA binding domain can comprise a wild-type or naturally occurring sequence, or it can be a modified, mutant, or derivative version of the original domain that has the desired ability to bind to a desired sequence. In some embodiments, the sequence-specific DNA binding domain is engineered to bind a desired sequence. Non-limiting examples of proteins having sequence-specific DNA binding domains that can be used in synthetic proteins described herein include HNFla, Gal4, GCN4, reverse tetracycline receptor, THY1, SYN1, NSE / RU5', AGRP, CALB2, CAMK2A, CCK, CHAT, DLX6A, EMX1, zinc finger proteins or domains thereof, CRISPR / Cas proteins, such as Cas9, Cas3, Cas4, Cas5, Cas5e (or CasD), Cash, Casbe, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul96, and TALEN.

[0150] In those embodiments where a CRISPR / Cas-like protein is used, the CRISPR / Cas-like protein can be a wild type CRISPR / Cas protein, a modified CRISPR / Cas protein, or a fragment of a wild type or modified CRISPR / Cas protein. The CRISPR / Cas-like protein can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. For example, nuclease (i.e. , DNase, RNase) domains of the CRISPR / Cas-like protein can be modified, deleted, or inactivated. Alternatively, the CRISPR / Cas-like protein can be truncated to remove domains that are not essential for the functions of the systems described herein. For example, a CRISPR enzyme that is used as a DNA binding protein or domain thereof can be mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR or domain thereof lacks the ability to cleave a nucleic acid sequence containing a DNA binding domain target site. For example, a D10A mutation can be combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity.Transactivation domains

[0151] Any suitable trans activation domain may find use in the transcriptional activator. Within a cell or system, a transcriptional activator may be paired with a transcriptional control element that is responsive to the transcriptional activator, e.g., a promoter to drive expression of a nucleic acid encoding a polypeptide of interest that isoperably linked to the transcriptional control element. Useful transcriptional activators, transcriptional control elements, activator / control element pairs, and components of such systems may include but are not limited to e.g., those used in inducible expression systems including but not limited to e.g., those described in Goverdhana et al. Mol Ther. (2005) 12(2): 189-211; U.S. Patent Application Pub. Nos. 20160152701, 20150376627, 20130212722, 20070077642, 20050164237, 20050066376, 20040235169, 20040038249, 20030220286, 20030199022, 20020106720; the disclosures of which are incorporated herein by reference in their entirety.

[0152] In some instances, useful transcriptional activators may include mammalian transcription factors or engineered or mutated forms thereof. In some instances, useful transcriptional activators may include human transcription factors or engineered or mutated forms thereof. In some instances, useful transcriptional activators may include mouse transcription factors or engineered or mutated forms thereof. In some instances, useful transcriptional activators may include rat transcription factors or engineered or mutated forms thereof. In some instances, useful transcriptional activators may include cow transcription factors or engineered or mutated forms thereof. In some instances, useful transcriptional activators may include pig transcription factors or engineered or mutated forms thereof.

[0153] In some instances, use of a mammalian transcription factor may reduce the chance that the transcription factor induces an immune response in a mammal. In some instances, use of an engineered or mutated transcription factor, including e.g., mutated or engineered mammalian transcription factors, may reduce the chance that the transcription factor induces an immune response in a mammal.

[0154] In some embodiments, the transcriptional activator is GAL4-VP16. In some cases, the transcriptional activator is VP64 Zip(+). In some embodiments, the transcriptional activator is an engineered protein, such as a zinc finger or TALE based DNA binding domain fused to an effector domain such as VP64. A variety of other transcriptional trans activators known in the art are suitable for use.

[0155] In some embodiments, the transactivation domain comprises a herpes simplex virus VP 16 particle protein transcriptional activation domain, a yeast Gal4 protein transcriptional activation domain, an NF-KB p65 subunit transcriptional activation domain, a yeast general control protein 4 transcriptional activation domain, or a Kruppel-related box transcriptional repression domain of the zinc finger 354A protein, a YAP transcriptional activation domain, a WWTR1 (TAZ) transcriptional activation domain, and a CREB3 (LZIP) transcriptional activation domain.Transcriptional control elements

[0156] In some embodiments, the target transcription unit comprises a Transcriptional control elements. Such transcriptional control elements can be promoters. In some embodiments, the target transcription unit comprises an inducible promoter. A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / “ON” state) or an inducible promoter (i.e., a promoter whose state, active / “ON” or inactive / “OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.

[0157] Suitable promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. In some embodiments, the transcriptional control element is selected from the adenovirus late promoter, cytomegalovirus (CMV) minimal promoter, yeast Gall gene promoter and SV40 promoter.Expression systems

[0158] In one aspect, provided herein are magneto-responsive gene expression system, comprising: a magneto-responsive transcription factor comprising a magneto- responsive domain comprising a mutant Avena sativa light, oxygen, voltage 2 (asLOV2) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain, a DNA-binding domain, and a transactivation domain; and a target transcription unit, comprising a DNA motif capable of being bound by the DNA-binding domain, a transcriptional control element, and a nucleic acid sequence encoding a target protein. Any synthetic transcriptional regulator described herein can be used in the expression system. Any suitable target gene can be controlled by the expression system described herein, such as, but not limited to an antibody, an antibody fragment, a nanobody, a monobody, an scFv, an sdAb, a Fab, a cytokine, a chemokine, a peptide, an esterase, or a chimeric antigen receptor (CAR).

[0159] In one aspect, provided herein are methods of inducing protein expression in one or more cells comprising obtaining a cell comprising the magneto-responsive gene expression system and applying a magnetic field to the one or more cells.

[0160] In some embodiments, the one or more cells are a bacterial cell, a eukaryotic cell, a mammalian cell, or a human cell.

[0161] In some embodiments, the method is performed in vitro, in vivo, or ex vivo.

[0162] In some embodiments, the method is performed in vitro on a human subject’s one or more cells.Bioluminescence proteins

[0163] The mutant asLOV domain can be fused to a bioluminescent enzyme such as a luciferase or NanoLuc luciferase (available from Promega). Any suitable luciferase can be used, including but not limited to, NanoLuc, firefly (e.g., Pholinus py rails) luciferase, Renilla reniformis luciferase, copepod (e.g., Metridia longa) luciferase, bacterial (e.g., Vibrio fischeri, Vibrio haweyi, or Vibrio harveyi) luciferase, or Dinoflagellate luciferase.Fluorescent proteins

[0164] In some aspects, provided herein are fusion proteins of the mutant asLOV domain and one or more fluorescent proteins (e.g., fluorophores) that can be used in screening assays, such as FRET. A mutant asLOV2 domain protein described herein can be expressed as a fusion protein in between an energy donor (e.g., photon donor), such as CFP, and an energy acceptor (e.g., photon acceptor), such as YFP, in a cell. The energy acceptordonor pair can be fluorescent proteins (FPs), and quantum dots (QDs). Additional FRET pairs are described in Bajar BT et al., A Guide to Fluorescent Protein FRET Pairs. Sensors (Basel). 2016 Sep 14; 16(9): 1488, hereby incorporated by reference in its entirety. Exemplary FRET donors and acceptors include, but are not limited to, ECFP-EYFP, mTurquoise2-sEYFP, mTurquoise2-mVenus, EGFP-mCherry, Clover-mRuby2, mClover3-mRuby3, mNeonGreen- mRuby3, eqFP650-iRFP, mAmetrine-tdTomato, LSSmOrange-mKate2, EGFP-sREACh, EGFP-ShadowG, EGFP-activated PA-GFP, EGFP-Phanta, mTagBFP-sfGFP, mVenus- DIKOK, and CyOFPl-mCardinal.Cells

[0165] The mutant asLOV2 domains can be expressed in any suitable host cell. Both eukaryotic and bacterial cells can be used to express the mutant asLOV2 domain proteins described here. Exemplary cells include unicellular eukaryotic cells such as cultured BY4741 and AH109 Saccharomyces cerevisiae yeast, fission yeast, P. pastoris yeast, klebsiella lactate,H. polymorpha yeast cells, and fungal cells. Immortalized mammalian cell lines comprising human embryonic kidney epithelial cells (HEK293), African green monkey kidney fibroblast COS-7, human cervix carcinoma cells (Hela), mouse fibroblasts (NIH3T3), etc., can also be used. The suitable cells also include non-immortalized cells such as ex vivo transgenic or homologous recombinant animal embryo cells for the purpose of gene therapy. Cells isolated form a human patient can also be used to express a mutant asLOV2 domain protein described herein. Suitable cells include, but are not limited to, immune cells such as T cells (CD4 or CD8), NK cells, hematopoietic stem cells, osteoplasts, hepatocytes, leukocytes, neuronal cells, skin epithelium and airway epithelium cells; ex vivo transgenic or homologous recombinant animal embryo stem cells and fertilized egg cells. Suitable cells also comprising non-mammalian eukaryotic cells such as bacterial, fungal, yeast, plant, drosophila, inset, zebra fish, or nematode. Exemplary bacterial cells include, but are not limited to, E.coli, Bacillus, Lactoccocus lactis, Erwinia chrysanthemi and Clostridium histolyticum.Magnets

[0166] The magnetic field used to induce the conformational change of the asLOV2 mutant proteins described herein can be generated by a handheld magnet. Handheld magnets generate a magnetic field stronger than the earths magnetic field, but weaker than the strong magnetic force generated by an MRI. Suitable handheld magnets include rare earth, ferrite, or alnico magnet. Rare earth magnets include neodymium or samarium-cobalt magnet.

[0167] In some embodiments, the handheld magnet is a rare earth, ferrite, or alnico magnet.

[0168] In some embodiments, the rare earth is a neodymium or samarium-cobalt magnet.

[0169] In some embodiments, the handheld magnet generates a magnetic field between about 0.5 milli Tesla (mT) to 50 mT. In some embodiments, the handheld magnet generates a magnetic field of between about 0.5-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 21-22, 22-23, 23-24 ,24-25, 25-26, 26-27, 27-28, 28-29, 29-30, 31-32, 32-33, 33-34 ,34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 41-42, 42-43, 43-44 ,44-45, 45-46, 46-47, 47-48, 48-49, 49-50 milli Tesla (mT). In some embodiments, the handheld magnet generates a magnetic field of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, or 50 milli Tesla (mT).Methods of Treating Dieases

[0170] A method of treating a subject in need comprising administering a chimeric polypeptide comprising a protein and a mutant Avena sativa light, oxygen, voltage (asLOV) 2 domain comprising one or more amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain; and applying a magnetic field to the subject. In such a method, the application of the magnetic field induces a magnet- controllable effect on the mutant asLOV2 domain, e.g., a conformational change, as described herein. Without wishing to be bound by theory, such magnet-controllable effects can be increasing or decreasing antigen binding affinity of an antibody or antigen binding fragment, or increasing or decreasing enzymatic activity. The benefit of the method is that magnetic field can penetrate the optically dense tissue of the subject and exert the modulatory effect on the mutant asLOV2 domain in the absence of light. In this way, a therapeutic molecule, e.g., an antibody, can be administered systemically to a subject, but the effect of the antibody can be spatially controlled by only applying a magnetic field to the therapeutic region of interest (e.g., the breast area for breast cancer).

[0171] In some embodiments, the mutant asLOV2 domain comprises one or more of a C450A, C450P, L496V, Q513A, Q513K, or D540M amino acid substitution(s) as compared to the phototropin 1 protein (SEQ ID NO: 11). In some embodiments, the mutant asLOV2 domain comprises one or more of a C48A, C48P, L94V, QI 11A, QI 1 IK, or D138M amino acid substitution(s) as compared to wild type asLOV2 (SEQ ID NO: 1). In some embodiments, the mutant asLOV2 domain comprises a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the mutant asLOV2 domain has an increased magnetic response as compared to a wild type asLOV2 domain (SEQ ID NO: 1).

[0172] In some embodiments, increased magnetic response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% or greater as compared to a wild type asLOV2 domain.

[0173] In some embodiments, the protein is an antibody, an antibody fragment, a nanobody, a monobody, an scFv, an sdAb, a Fab, an antibody-drug conjugate (ADC), a cytokine, a chemokine, a peptide, an esterase, a chimeric antigen receptor (CAR), a synthetic receptor, or a transcription factor as described herein.

[0174] In some embodiments, the magnetic field is applied to the subject or cell for about 1, 5, 10, 20, 30, 40, 50, or 60 seconds or more. In some embodiments, the magnetic field is applied to the subject or cell for about 1, 5, 10, 20, 30, 40, 50, or 60 minutes or more.In some embodiments, the magnetic field is applied to the subject or cell for about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more. In some embodiments, the magnetic field is applied to the subject or cell for about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days or more.

[0175] In some embodiments, the disease is a cancer, an autoimmune disorder, or an infectious disease.Methods of Assessing Binding of Proteins

[0176] In one aspect, provided herein are methods of assessing binding of a mutant Avena sativa light, oxygen, voltage 2 (asLOV2) domain as compared to the wild type asLOV2 domain (SEQ ID NO: 1), to a target protein comprising: providing a cell or solution comprising the mutant asLOV2 domain and a first target protein, wherein the mutant asLOV2 domain and the first target protein are operably linked; and applying a magnetic force to the cell or solution and assessing binding of the asLOV2 domain to the first target protein.

[0177] In some embodiments, the magnetic force induces binding of the asLOV2 domain to the first target protein or induces unbinding of the asLOV2 domain to the first target protein.

[0178] In some embodiments, assessing binding of the asLOV2 domain to the first target protein comprises assessing localization of the first target protein in the cell or solution. An exemplary method of assessing cellular localization of proteins using a wild type LOV protein is described in Wang H, et al., Nat Methods. 2016 September ; 13(9): 755-758, hereby incorporated by reference in its entirety.

[0179] In some embodiments, assessing binding of the asLOV2 domain to the first target protein comprises assessing fluorescence in the cell or solution, optionally wherein the fluorescence is determined by fluorescence microscopy or a Fluorescence Resonance Energy Transfer (FRET) assay. FRET assays in live cell imagining are described in Sekar RB, Periasamy A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol. 2003 Mar 3;160(5):629-33, hereby incorporated by reference in its entirety.

[0180] In some embodiments, assessing binding of the asLOV2 domain to the first target protein comprises assessing binding of the first target protein to a second target protein, optionally wherein the binding of the first target protein and the target protein is determined by surface plasmon resonance. Methods of assessing protein binding via surface plasmonresonance are generally known in the art and are reviewed in “Protein-Protein Interactions: Surface Plasmon Resonance” by B. Douzi Bacterial Protein Secretion Systems pp 257-275, 2017, hereby incorporated by reference in its entirety.

[0181] In some embodiments, the mutant asLOV2 domain and the target protein are covalently linked, optionally wherein the mutant asLOV2 domain and the target protein are a chimeric polypeptide. In some embodiments, the mutant asLOV2 domain and the target protein are non-covalently linked.Methods of Modulating Protein Activity

[0182] In one aspect, provided herein are methods of modulating the activity of a protein, such as enzymatic activity or protein (e.g., antigen or ligand binding), comprising providing a chimeric protein comprising at least one mutant asLOV2 domain and a first protein; and applying a magnetic force to the chimeric protein and assessing the activity of the first protein.

[0183] Control of enzymatic activity by photosensitive proteins is described in Zhou XX, et al., Optical control of protein activity by fluorescent protein domains. Science. 2012 Nov 9;338(6108):810-4. doi: 10.1126 / science.1226854. In such a method, the chimeric protein of the mutant asLOV2 domain and protein of interest is inactive when there is no magnetic field. Application of the magnetic field can alter the shape of the mutant asLOV2 domain, thereby activating the protein of interest enzymatic activity. In some embodiments, the chimeric protein comprises one mutant asLOV2 domain fused to the N-terminus or the C- terminus of the protein of interest. In some embodiments, the chimeric protein comprises a mutant asLOV2 domain fused to both the N-terminus and the C-terminus of the protein of interest.

[0184] Reversible control of protein binding by light sensitive proteins is described in Gil, A.A., et al., Nat Commun 11, 4044 (2020) which is hereby incorporated by reference in its entirety. In such a method, the chimeric protein of the mutant asLOV2 domain and protein of interest (e.g., a therapeutic protein such as an antibody) cannot bind the cognate ligand of the protein of interest in the absence of a magnetic field due to the conformation of the asLOV2 domain mutant protein that affects the binding site of the protein of interest, by steric or allosteric inhibitory effects. Without wishing to be bound by theory, application of the magnetic field can alter the shape of the mutant asLOV2 domain, thereby resulting in a conformational change that releases the steric or allosteric inhibitory effect on the protein of interest. Once the magnetic field is applied, the protein of interest can bind to its cognateligand. In another method, the chimeric protein of the mutant asL0V2 domain and protein of interest (e.g., a therapeutic protein such as an antibody) binds the cognate ligand of the protein of interest in the absence of a magnetic field, but the binding affinity can be modulated by application of a magnetic field. Without wishing to be bound by theory, application of the magnetic field can alter the shape of the mutant asL0V2 domain, thereby resulting in a conformational change that modulates the binding affinity of the protein of interest, either increasing it or decreasing. In such a method, the application of a magnetic field can result in decreased binding of the protein of interest to its cognate ligand.Methods of modulating fluorescence with mutant asLOV2 domains

[0185] In one aspect, provided herein are methods of modulating fluorescence with an asLOV2 mutant describe herein. Such methods comprise expressing an asLOV2 mutant describe herein operably linked to a second fluorescent protein, such as Scarlet, and applying a light source and a magnetic field. When the magnetic field is applied, the fluorescence of the second fluorescent protein is altered and the light emission alteration is measured. In some embodiments, an array of two or more different asLOV2 mutants describe herein operably linked to a second fluorescent protein is constructed. Such an array allows for multiplexing of the fluorescent protein light emission alteration.Pharmaceutical compositions

[0186] Methods for treatment of diseases are also encompassed by the present disclosure. Said methods include administering a therapeutically effective amount of a mutant asLOV2 domain comprising one or more amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain. The mutant asLOV2 domain of the invention can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the mutant asLOV2 domain, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0187] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

[0188] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives can be included, as required.

[0189] Administration is preferably in a “therapeutically effective amount” or “prophylactically effective amount”(as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0190] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.EXAMPLES

[0191] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0192] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., currentaddition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rdEd. (Plenum Press) Vols A and B(1992).Example 1: Generation and Characterization of Mutant asLOV2 Domain Proteins

[0193] Materials and Methods

[0194] The asLOV2 domain sequence was cloned into the bacterial vector pRSET, which contains a T7 promoter, an ampicillin selection marker, and an N-terminal HIS tag. Libraries of all possible single mutants were generated by combining 4 ul PCR reactions per amino acid using NNK semi-random complementary primers, 0.2 ng / ul template and Phusion Enzyme 2x mastermix in GC buffer. The melting temperature decreased from 65 °C to 45 °C in the course of 30 cycles, with a 3 minute extension time. After pooling, the template was digested with Dpnl, the DNA was transformed into BL21(DE3) E. coli cells, and transformed cells were selected on ampicillin-LB plates incubated at 37 °C.

[0195] The magnetic field response of the LOV domain was screened by imaging the petri dishes on a custom-built microscope setup capable of delivering 10 mW / cm2 cyan light (470 / 40 nm). Images were acquired continuously, while switching the electromagnet (KK- P80-10) underneath the plates every 5 seconds for a minute. The electromagnet generated an electromagnetic field of 5 milli Tesla (mT). The colony that displayed the largest modulation amplitude of fluorescence was picked, and the mutated plasmid was as a template for the following round of library generation as described above. A diagram of the mutant selection process is provided in FIG. 1.

[0196] Results

[0197] As show in in FIGs. 2A-2G, the directed evolution of the asLOV2 protein via random mutagenesis increased the magnetic response of the asLOV2 protein. FIG. 2A shows the magnetic response of a mutant with a C48A mutation (SEQ ID NO: 2) as compared to the wild type asLOV2 domain provided in SEQ ID NO: 1 (which corresponds to a C450A mutation in the full length Avena sativa phototropin 1 protein). FIG. 2B shows the magnetic response of a mutant with a C48A D138M (C450A D540M) mutation (SEQ ID NO: 3). FIG. 2C shows the magnetic response of a mutant with a C48A QI HA D138M (C450A Q513A D540M) mutation (SEQ ID NO: 4). FIG. 2D shows the magnetic response of a mutant with a C48A L94V QI 11A D138M (C450A L496V Q513A D540M) mutation (SEQ ID NO: 5).FIG. 2E shows the magnetic response of a mutant with a C48P L94V Q111A D138M (C450P L496V Q513A D540M) mutation (SEQ ID NO: 6). FIG. 2F shows the magnetic response of a mutant with a C48P L94V QI 1 IK D138M (C450P L496V Q513K D540M) mutation (SEQ ID NO: 7). FIG. 2G shows the magnetic response of a mutant with a C48P L94V QI 1 IK D138M and a C terminus truncation at residue 157 (C450P L496V Q513A D540M and 544 truncation) mutation (SEQ ID NO: 8). In each figure, the fluorescence decreases when the electromagnetic field is activated, and recovers when the electromagnetic field is deactivated. With each round of mutagenesis, the asLOV2 fluorescent response to the magnetic field increased. The results show that directed evolution was successful in amplify the asLOV2 fluorescence response to magnetic fields by more than 25%, as compared to wild type asLOV2.

[0198] Enhancing the fluorescence response to magnetic fields, can be used in applications such as lock-in detection, optically detected imaging (if there is resonance with IR or RF), or improving imaging in scattering media, or direct control of protein function. Furthermore, without wishing to be bound by theory, asLOV2’s fluorescence response to magnetic fields is a result of the structural shape of the asLOV2 domain, and thus applying a magnetic field to the asLOV2 mutant proteins resulted in a conformational change in the asLOV2 protein, as shown by the reduction in fluorescence of the asLOV2 protein during application of the magnetic field.Example 2; Generation and Characterization of Chimeric asLOV2 Domain Proteins

[0199] Luminescent chimeric mutant asLOV2 domain polypeptides

[0200] A chimeric polypeptide comprising a mutant asLOV2 domain protein described herein fused to a bioluminescent enzyme, such as NanoLuc, a luciferase, is constructed. The luminescence of the bioluminescent protein in combination with application of a magnetic field induced changes in the conformation of the asLOV2 domain.

[0201] Antibody chimeric mutant asLOV2 domain polypeptides

[0202] A chimeric polypeptide comprising a mutant asLOV2 domain protein described herein and an antibody or antigen binding fragment thereof is constructed. The mutant asLOV2 domain protein can be fused to the C terminus or N terminus of the antibody or antigen binding fragment, or incorporated into a loop region of the variable domain. In one method, the antibody chimera is unable to bind to its cognate ligand in the absence of a magnetic field, while the antibody chimera is able to bind to its cognate ligand when a magnetic field is applied. In another method, the antibody chimera is able to bind to itscognate ligand in the absence of a magnetic field, while the antibody chimera is unable to bind to its cognate ligand when a magnetic field is applied. The antibody-LOV domain chimera may also include a luminescent protein as described herein.Example 3: Use of Mutant asLOV2 Domain Proteins in Screening Assays

[0203] Monitoring the ability of LOV to interact with binding partners can be used as a screening platform via an applicable method, such as microscopy, FRET, or cell fractionation.

[0204] A mutant asLOV2 domain protein described herein is either bound or fused to one of a pair of binding proteins. The mutant asLOV2 domain protein and bound protein or chimeric asLOV2 domain protein fused to the binding protein is expressed in a cell. A magnetic field is applied to the cell. The binding state of the asLOV2 chimeric binding protein is then assessed by determining changes in localization of one of the binding partners if the other one is confined to a structurally defined area like the mitochondria surface or the inner side of the plasma membrane. For example, the mutant asLOV2 chimeric protein can be the protein confined to a structurally defined area or the free protein. The application of the magnetic field results in changes in the binding of the asLOV2 domain to the bound protein or the accessibility of a binding site on the binding protein. After application of the magnetic field, changes in the cell localization of the one of the binding partners is assessed via live cell microscopy or cell fractionation. Additional details on an exemplary binding assay can be found in Wang et al, Nat Methods. 2016 Sep; 13(9): 755-758, incorporated by reference in its entirety.

[0205] A Fluorescence Resonance Energy Transfer (FRET) assay can also be used to assess conformational changes of the asLOV2 domain. FRET assays also have the advantage of being used in E. coli for faster and easier screening selection. A mutant asLOV2 domain protein described herein is expressed as a fusion protein in between an energy donor (e.g., photon donor), such as CFP, and an energy acceptor (e.g., photon acceptor), such as YFP, in a cell. The energy acceptor-donor pair can be fluorescent proteins (FPs), and quantum dots (QDs). Additional FRET pairs are described in Bajar BT et al., A Guide to Fluorescent Protein FRET Pairs. Sensors (Basel). 2016 Sep 14; 16(9): 1488. An energy source such as a photon stream from a laser is applied to the cell. When a magnetic field is not applied to the fusion protein, the asLOV domain conformation is not altered and the energy donor and acceptor are not in proximity to each other to transfer the energy between the donor and acceptor, resulting in no photon transfer or fluorescence of the photon acceptor. When themagnetic field is applied to the fusion protein, the conformational change in the asLOV2 domain brings the energy donor and acceptor into proximity and the photon is transferred between the acceptor and donor, resulting in fluorescence of the donor protein.

[0206] Additional details on an exemplary FRET assay can be found in Murakoshi H et al, Sci Rep. 2015 Oct 15;5: 15334, incorporated by reference in its entirety.

[0207] Antigen binding screening assay

[0208] A magnetic nanobody is constructed by inserting a mutant asLOV2 domain described herein into a variable region of an antibody or antigen binding fragment (e.g., a loop of the variable region), or at the C or N terminus of the antibody or antigen binding fragment. The chimeric protein is bound to the surface of a bead, and binding to the target can be monitored on a fluorescence microscope by following the accumulation of a red- protein labeled target. The advantage of this assay is that it would allow selection of positive or negative nanobodies, as binding can be induced or inhibited by light depending on the insertion site of the LOV domain on the nanobody.Example 4; Treatment of disease with a chimeric asLOV2 domain protein

[0209] A therapeutic chimeric polypeptide comprising a mutant asLOV2 domain protein described herein and therapeutic molecule, such as an antibody or antigen binding fragment thereof is constructed. The therapeutic chimeric polypeptide is administered to a patient in need of the therapeutic molecule. In one therapeutic method, the therapeutic molecule is unable to bind to its cognate ligand (if it is an antibody) or exhibit enzymatic activity (if is it an enzyme) in the absence of a magnetic field, but is able to bind to its cognate ligand or exhibit enzymatic activity in the presence of a magnetic field. In another therapeutic method, the therapeutic molecule binds to its cognate ligand (if it is an antibody) or exhibits enzymatic activity (if is it an enzyme) in the absence of a magnetic field, but is unable to unable to bind to its cognate ligand or exhibit enzymatic activity in the presence of a magnetic field. A targeted or localized magnetic field is applied to the patient at the site of the desired therapeutic effect (e.g., at the breast if the antibody is an antibody that targets a breast cancer antigen) for a suitable time to allow the asLOV2 domain to undergo a conformational change. The therapeutic molecule exerts its therapeutic effect at the desired location, thereby treating the disease. A reduction in the patient’ s disease or symptoms is observed.Example 5; Generation and Characterization of Additional Mutant asLOV2 Domain Proteins

[0210] Additional random mutagenesis mutant asLOV2 domain proteins were generated and characterized as described in Example 1.

[0211] The magnetic response of the asLOV2 mutants was assessed by blotting the E. coli colonies expressing the mutant proteins onto nitrocellulose paper and imaging the paper as described in Example 1 while delivering 10 mW / cm2 cyan light (470 / 40 nm) and applying an electromagnet that generated an electromagnetic field of 10 milli Tesla (mT) every 5 seconds.

[0212] A further asLOV2 domain mutant incorporating a G126K mutation in the C48P L94V Qll IK D138M background was generated and characterized (C48P L94V QI 1 IK D138M G126K, SEQ ID NO: 9.) The top panel of FIG. 4 shows the fluorescence of the asLOV2 domain mutant as a 10 mT electromagnetic field was periodically applied over time (seconds). The bottom panel shows the percent relative increase in magnetic response of the mutant asLOV2 protein relative to the lowest fluorescence of the mutant asLOV2 protein using the algorithm (F-Fpit) / (FFit) where F is the fluorescence (counts / pixel) and Ffltis the smoothed lower bound fit of the fluorescence curve in the upper panel. In particular, the C48P L94V Q111K D138M G126K mutant exhibited a 75% increase in fluorescence response to the magnetic field as compared to wild type asLOV2 domain proteins (FIG. 4).

[0213] Further mutagenesis resulted in an asLOV2 mutant comprising R46W, C48P, F92L, L94V, Ql l IK, E123G, G126R, D138M mutations (SEQ ID NO: 10) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) that exhibited an approximately 300% increase in fluorescence amplitude when a magnetic field was applied. FIG. 5 shows fluorescence as a function of time, while a 10 mT magnetic field was applied to the mutant asLOV2 protein. The top panel of FIG. 5 shows the fluorescence of the asLOV2 domain mutant as a 10 mT electromagnetic field was periodically applied over time (seconds). The bottom panel shows the percent relative increase in magnetic response of the mutant asLOV2 protein relative to the lowest fluorescence of the mutant asLOV2 protein using the algorithm (F-Fmin) / (Fmin) where F is the fluorescence (counts / pixel) and Fminis the smoothed lower bound fit of the fluorescence curve in the upper panel. The fluorescence of the asLOV2 mutant decreased when the magnet was activated, and recovered when the magnet was deactivated, indicating that the application of the magnetic field altered the structure of the mutant asLOV2 protein, thus affected the fluorescence of the protein.

[0214] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0215] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

CLAIMS1. A mutant Avena sativa light, oxygen, voltage 2 (asLOV2) protein domain comprising one or more amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain.

2. The mutant asLOV2 domain of claim 1, wherein the one or more amino acid substitution(s) comprises one or more amino acid substitution(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the wild type asLOV2 domain of the full length phototropin1 protein (SEQ ID NO: 11).

3. The mutant asLOV2 domain of claim 1 or 2, wherein the mutant asLOV2 domain comprises one or more of a C48P, C48A, R46W, F92L, L94V, Q111A, Q111K, E123G, G126R, G126K, and / or D138M amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or C450P, C450A, L496V, Q513A, Q513K, R448W, E525G, F494L, G528R, G528K, and / or D540M amino acid substitution(s) as compared to the full length photo tropin 1 protein (SEQ ID NO: 11).

4. The mutant asLOV2 domain of any one of claims 1-3, wherein the mutant asLOV2 domain comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

5. The mutant asLOV2 domain of any one of claims 1-4, wherein the mutant asLOV2 domain does not comprise an N-terminal methionine residue as compared to the sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

6. The mutant asLOV2 domain of any one of claims 1-5, wherein the increased magnetic response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or greater as compared to a wild type asLOV2 domain.

7. The mutant asLOV2 domain of claim 6, wherein the increased magnetic response is a decrease in fluorescence as compared to a wild type asLOV2 domain.

8. A chimeric polypeptide comprising a mutant Avena sativa light, oxygen, voltage 2 (asLOV2) domain, wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain, and at least a first protein.

9. The chimeric polypeptide of claim 8, wherein the mutant asLOV2 domain comprises one or more amino acid substitution(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the full length phototropin 1 protein (SEQ ID NO: 11).

10. The chimeric polypeptide of claim 8 or 9, wherein the mutant asLOV2 domain comprises one or more of a C48P, C48A, R46W, F92L, L94V, Q111A, Q111K, E123G, G126R, G126K and / or D138M amino acid substitution(s) as compared to wild type asLOV2 (SEQ ID NO: 1).

11. The chimeric polypeptide of any one of claims 8-10, wherein the mutant asLOV2 domain comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

12. The chimeric polypeptide of any one of claims 8-11, wherein the mutant asLOV2 domain does not comprise an N-terminal methionine residue as compared to the sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

13. The chimeric polypeptide of any one of claims 8-12, wherein the increased magnetic response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or greater as compared to a wild type asLOV2 domain.

14. The chimeric polypeptide of claim 13, wherein the increased magnetic response is a decrease in fluorescence as compared to a wild type asLOV2 domain.

15. The chimeric polypeptide of any one of claims 8-14, wherein the first protein is an antibody, an antibody fragment, a nanobody, a monobody, an scFv, an sdAb, a Fab, anantibody-drug conjugate (ADC), a cytokine, a chemokine, a peptide, an esterase, a chimeric antigen receptor (CAR), a synthetic receptor, or a synthetic transcriptional regulator.

16. The chimeric polypeptide of any one of claims 8-15, wherein the mutant asL0V2 domain is fused to the N terminus of the first protein.

17. The chimeric polypeptide of any one of claims 8-15, wherein the mutant asL0V2 domain is fused to the C terminus of the first protein.

18. The chimeric polypeptide of any one of claims 8-15, wherein the mutant asL0V2 domain is inserted in a loop or domain of the first protein.

19. The chimeric polypeptide of any one of claims 8-16, wherein the first protein is an antibody or an antibody fragment and the mutant asLOV2 domain is inserted in a loop of the variable light (VL) domain of the light chain, inserted in a loop of the variable heavy (VH) domain of the heavy chain fused to the N terminus of the light chain or light chain variable (VL) region, the N terminus of the heavy chain or heavy chain variable (VH) region, the C terminus of the light chain or light chain variable (VL) region, or the C terminus of the heavy chain or heavy chain variable (VH) region.

20. The chimeric polypeptide of any one of claims 8-14, wherein the first protein is a bioluminescent protein.

21. The chimeric polypeptide of claim 20, wherein the bioluminescent protein is a luciferase, optionally wherein the luciferase is selected from the group consisting of NanoLuc, firefly luciferase, Renilla reniformis luciferase, copepod luciferase, bacterial luciferase, or Dinoflagellate luciferase.

22. The chimeric polypeptide of any one of claims 8-14, wherein the first protein is a fluorescent protein.

23. The chimeric polypeptide of any one of claims 8-14 or 22, further comprising a second protein fused to the first protein of the mutant asLOV2 domain.

24. The chimeric polypeptide of claim 23, wherein the second protein is a fluorescent protein.

25. A magneto-responsive gene expression system, comprising: a. a magneto-responsive synthetic transcriptional regulator comprising a magneto-responsive domain comprising a mutant Avena saliva light, oxygen,voltage 2 (asLOV2) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain, a DNA- binding domain, and a transactivation domain; and b. a target transcription unit, comprising a DNA motif capable of being bound by the DNA-binding domain, a transcriptional control element, and a nucleic acid sequence encoding a target protein.

26. The magneto-responsive gene expression system of claim 25, wherein the one or more amino acid substitution(s) comprises one or more mutation(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the full length phototropin 1 protein (SEQ ID NO: 11)27. The magneto-responsive gene expression system of claim 25 or 26, wherein the mutant asLOV2 domain comprises one or more of a C48P, C48A, R46W, F92L, L94V, QI HA, Q111K, E123G, G126R, G126K, and / or D138M amino acid substitution(s) as compared to wild type asLOV2 (SEQ ID NO: 1).

28. The magneto-responsive gene expression system of any one of claims 25-27, wherein the mutant asLOV2 domain comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

29. The magneto-responsive gene expression system of any one of claims 25-28, wherein the mutant asLOV2 domain does not comprise an N-terminal methionine residue as compared to the sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

30. The magneto-responsive gene expression system of any one of claims 25-29, wherein the mutant asLOV2 domain has an increased magnetic response as compared to a wild type asLOV domain (SEQ ID NO: 1).

31. The magneto-responsive gene expression system of claim 30, wherein the increased magnetic response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or greater as compared to a wild type asLOV2 domain.

32. The magneto-responsive gene expression system of any one of claims 25-31, wherein the target protein is an antibody, an antibody fragment, a nanobody, a monobody, an scFv, an sdAb, a Fab, an antibody-drug conjugate (ADC), a cytokine, a chemokine, a peptide, an esterase, a chimeric antigen receptor (CAR), or a synthetic receptor.

33. The magneto-responsive gene expression system of any one of claims 25-32, wherein the DNA binding domain is a helix-tum-helix DNA-binding domain, zinc finger motif or zinc cluster DNA-binding domain, leucine zipper DNA-binding domain, winged helix DNA- binding domain, winged helix-turn-helix DNA-binding domain, helix-loop-helix DNA- binding domain, high mobility family DNA-binding domain, or a B3 DNA-binding domain.

34. The magneto-responsive gene expression system of any one of claims 25-33, wherein the transactivation domain is a transcriptional activation domain rich in acidic amino acids, transcriptional activation domains rich in proline, transcriptional activation domains rich in serine / threonine and transcriptional activation domains rich in glutamine, and Kruppel-related box transcriptional repression domain.

35. The magneto-responsive gene expression system of any one of claims 25-34, wherein the transactivation domain comprises a herpes simplex virus VP16 particle protein transcriptional activation domain, a yeast Gal4 protein transcriptional activation domain, an NF-KB p65 subunit transcriptional activation domain, a yeast general control protein 4 transcriptional activation domain, or a Kruppel-related box transcriptional repression domain of the zinc finger 354A protein, a YAP transcriptional activation domain, a WWTR1 (TAZ) transcriptional activation domain, and a CREB3 (LZ1P) transcriptional activation domain.

36. The magneto-responsive gene expression system of any one of claims 25-35, wherein the transcriptional control element is an inducible promoter.

37. The magneto-responsive gene expression system of claim 36, wherein the inducible promoter is operably linked to the nucleic acid sequence encoding a target protein.

38. The magneto-responsive gene expression system of any one of claims 25-37, wherein the transcriptional control element is selected from the adenovirus late promoter, cytomegalovirus (CMV) minimal promoter, yeast Gall gene promoter and SV40 promoter.

39. A method of inducing protein expression in one or more cells comprising obtaining a cell comprising the magneto-responsive gene expression system of any one of claims 25-38 and applying a magnetic field to the one or more cells.

40. The method of claim 39, wherein the one or more cells are a bacterial cell, a eukaryotic cell, a mammalian cell, or a human cell.

41. The method of claim 39 or 40, wherein the method is performed in vitro, in vivo, or ex vivo.

42. The method of claim 39-41, wherein the method is performed in vitro on a human subject’s one or more cells.

43. A method of assessing binding of a mutant Avena sativa light, oxygen, voltage 2 (asLOV2) domain as compared to the wild type asLOV2 domain (SEQ ID NO: 1), to a target protein comprising: a. providing a cell or solution comprising the mutant asLOV2 domain and a first target protein, wherein the mutant asLOV2 domain and the first target protein are operably linked; and b. applying a magnetic force to the cell or solution and assessing binding of the asLOV2 domain to the first target protein.

44. The method of claim 43, wherein the magnetic force induces binding of the asLOV2 domain to the first target protein or induces unbinding of the asLOV2 domain to the first target protein.

45. The method of claim 43 or 44, wherein assessing binding of the asLOV2 domain to the first target protein comprises assessing localization of the first target protein in the cell or solution.

46. The method of claim 43-45, wherein assessing binding of the asLOV2 domain to the first target protein comprises assessing fluorescence in the cell or solution, optionally wherein the fluorescence is determined by fluorescence microscopy or a Fluorescence Resonance Energy Transfer (FRET) assay.

47. The method of claim 43 or 44, wherein assessing binding of the asLOV2 domain to the first target protein comprises assessing binding of the first target protein to a second target protein, optionally wherein the binding of the first target protein and the target protein is determined by surface plasmon resonance.

48. The method of any one of claim 43-47, wherein the mutant asLOV2 domain and the target protein are covalently linked, optionally wherein the mutant asLOV2 domain and the target protein are a chimeric polypeptide.

49. The method of any one of claim 43-47, wherein the mutant asLOV2 domain and the target protein are non-covalently linked.

50. The method of any one of claim 43-49, wherein the mutant asLOV2 domain comprises one or more amino acid substitution(s) at positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the full length phototropin 1 protein (SEQ ID NO: 11).

51. A method of treating a subject in need comprising administering a chimeric polypeptide comprising a protein and a mutant Avena sativa light, oxygen, voltage (asLOV) 2 domain comprising one or more amino acid substitution(s) as compared to the wild type asLOV2 domain (SEQ ID NO: 1) wherein the mutant asLOV2 domain has an increased magnetic response as compared to the wild type asLOV domain; and applying a magnetic field to the subject.

52. The method of claim 51 , wherein the one or more amino acid substitution(s) comprises one or more amino acid substitution(s) at residue positions 46, 48, 92, 94, 111, 123, 126, and / or 138, as compared to the wild type asLOV2 domain (SEQ ID NO: 1) or one or more amino acid substitution(s) at residue positions 448, 450, 494, 496, 513, 525, 528 or 540, as compared to the full length phototropin 1 protein (SEQ ID NO: 11).

53. The method of any one of claim 50-52, wherein the mutant asLOV2 domain comprises one or more of a C48P, C48A, R46W, F92L, L94V, Q111A, Q111K, E123G, G126R, G126K, and / or D138M amino acid substitution(s) as compared to wild type asLOV2 (SEQ ID NO: 1).

54. The method of any one of claims 43-53, wherein the mutant asLOV2 domain comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

55. The method of any one of claims 43-54, wherein the mutant asLOV2 domain does not comprise an N-terminal methionine residue as compared to the sequence as set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

56. The method of any one of claims 43-55, wherein the increased magnetic response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or greater as compared to a wild type asLOV2 domain.

57. The method of any one of claims 43-56, wherein the protein is an antibody, an antibody fragment, a nanobody, a monobody, an scFv, an sdAb, a Fab, an antibody-drug conjugate (ADC), a cytokine, a chemokine, a peptide, an esterase, a chimeric antigen receptor (CAR), a synthetic receptor, or a synthetic transcriptional regulator.

58. The method of any one of claims 43-57, wherein the mutant asLOV2 domain is fused to the N terminus of the protein or target protein.

59. The method of any one of claims 43-58, wherein the mutant asLOV2 domain is fused to the C terminus of the protein or target protein.

60. The method of any one of claims 43-59, wherein the protein or target protein is an antibody or an antibody fragment and the mutant asLOV2 domain is inserted in a loop of the variable light (VL) domain of the light chain, inserted in a loop of the variable heavy (VH) domain of the heavy chain fused to the N terminus of the light chain or light chain variable (VL) region, the N terminus of the heavy chain or heavy chain variable (VH) region, the C terminus of the light chain or light chain variable (VL) region, or the C terminus of the heavy chain or heavy chain variable (VH) region.

61. The method of any one of claims 39-60, wherein the magnetic field is generated by a handheld magnet.

62. The method of claim 61, wherein the handheld magnet is a rare earth, ferrite, or alnico magnet.

63. The method of claim 62, wherein the rare earth is a neodymium or samarium-cobalt magnet.

64. The method of any one of claims 39-63, wherein the magnetic field is between about 0.5 milli Tesla (mlT) to 50 mlT.

65. The method of any one of claims 39-64, wherein the magnetic field is applied to the subject or cell for about 1, 5, 10, 20, 30, 40, 50, or 60 seconds or more.

66. The method of any one of claims 39-64, wherein the magnetic field is applied to the subject or cell for about 1, 5, 10, 20, 30, 40, 50, or 60 minutes or more.

67. The method of any one of claims 39-64, wherein the magnetic field is applied to the subject or cell for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more.

68. The method of any one of claims 39-64, wherein the magnetic field is applied to the subject or cell for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days or more.

69. The method of any one of claims 51-68, wherein the subject in need has cancer, an autoimmune disorder, or an infectious disease.

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