METHODS AND SYSTEMS FOR CONTROLLING THE AGONISTIC PROPERTIES OF ANTIBODY VARIABLE DOMAINS BY LIGHT

Abstract

The inventors has developed a recombinant molecular system, named OptoFab, allowing the accurate control of the agonistic properties of an antibody-derived Fab fragment in time and in space using specific wavelengths of light. It consists in a Fab fragment derived from an agonistic antibody of interest, linked to optogenetic modules that confer a light response capacity. Indeed, antibody derived Fab fragments generally keep the specificity of the antibody for its epitope, but not its agonistic properties. However, when Fab fragments are oligomerized, they recover the agonistic properties of the whole antibody. These characteristics, are at the basis of the OptoFab concept as its objective is to manipulate the oligomerization/immobilization statue of a Fab fragment using optogenetics to control its agonistic property. The present invention relates to methods and systems for controlling the agonistic properties of antibody variable domains by light.

Claims

1. A recombinant protein comprising a variable domain of an antibody that is fused at its c-terminal end to a factor that can interact with a photoreceptor protein in a light-dependent manner.

2. The recombinant protein of claim 1 wherein the variable domain is selected from the group consisting of VH domains, VL domains, Of and single domain antibodies (sdAbs).

3. The recombinant of claim 1 which comprises a Fab fragment wherein the VH domain of the Fab fragment is fused at its c-terminal end to the factor that can interact with a photoreceptor protein in a light-dependent manner.

4. The recombinant protein of claim 3 wherein the Fab fragment derives from an agonistic antibody.

5. The recombinant protein of claim 4 wherein the agonistic antibody is specific for a receptor of an immune cell.

6. The recombinant protein of claim 5 wherein the agonistic antibody is specific for a costimulatory receptor selected from the group consisting of CD134 (OX40), CD137 (4-1BB), CD28, GITR, CD27, CD70, ICOS, RANKL, TNFRSF25 (DR3), CD258 (LIGHT), CD40 and HVEM.

7. The recombinant protein of claim 1 wherein the factor is selected from the group consisting of Phytochrome Interacting Factors (PIFs), FHY1/FHL, Phytochrome kinase substrate 1 (PKS1), nucleoside diphosphate kinase 2 (NDPK2), cryptochromes, Aux/IAA proteins, phosphatases, E3 ubiquitin ligases, and ARR4.

8. The recombinant protein of claim 1 wherein the factor is selected from the group consisting of PIF1, PIF2, PIF3, PIF4, PIF5, PIF6, and PIF7.

9. The recombinant protein of claim 1 wherein the factor comprises an amino acid sequence that has at least 90% identity with the amino acid sequence as set forth in SEQ ID NO:1.

10. A nucleic acid encoding for the recombinant protein of claim 1.

11. A host cell transformed with the nucleic acid of claim 10.

12. An optogenetic system comprising at least one recombinant protein of claim 1 and at least one photoreceptor protein.

13. The optogenetic system of claim 2 wherein the at least one photoreceptor protein is a phytochrome selected from the group consisting of Phytochrome A (PhyA), Phytochrome B (PhyB), Phytochrome C (PhyC), Phytochrome D (PhyD), and Phytochrome E (PhyE).

14. The optogenetic system of claim 13 wherein the at least one photoreceptor protein comprises an amino acid sequence that has at least 90% identity with the amino acid sequence as set forth in SEQ ID NO:2.

15. The optogenetic system of claim 12 wherein the at least one photoreceptor protein is immobilized on a solid surface.

16. A method of activating on demand a cell or a plurality of cells comprising i) contacting the cell or the plurality of cells with the optogenetic system of claim 12 and ii) exposing the cell or the plurality of cells to a wavelength of light sufficient to oligomerize the at least one recombinant protein and activate the cell or the plurality of cells.

17. The method of claim 16 wherein the cell or the plurality of cells is embedded in a tissue, organ or organism.

18. The method of claim 16 wherein the cell or the plurality of cells are lymphocytes; natural killer cells; or myeloid cells.

19. A method of modulating an immune response in a tissue, organ or organism comprising, i) contacting a cell or a plurality of cells in the tissue, organ or organism with the optogenetic system of claim 12, and ii) exposing the cell or the plurality of cells to a wavelength of light sufficient to oligomerize the at least one recombinant protein and modulate the immune response.

20. A method of treating cancer or an inflammatory auto-immune disease in a subject in need thereof, comprising i) contacting a cell or a plurality of cells in a tissue, organ or organism of the subject with a therapeutically effective amount of the optogenetic system of claim 12, and ii) exposing the cell or the plurality of cells to a wavelength of light sufficient to oligomerize the at least one recombinant protein, modulate the immune response of the subject and treat the cancer or the inflammatory auto-immune disease.

21. The recombinant protein of claim 7 wherein the cryptochrome is CRY1 or CRY2, the phosphatase is FyPP or PAPP5, and the E3 ubiquitin ligase is COP1.

22. The method of claim 16 wherein the recombinant protein comprises a Fab fragment.

23. The method of claim 18, wherein the lymphocytes are B cells and/or T cells; and the myeloid cells are monocytes, macrophages, eosinophils, mast cells, basophils, or granulocytes.

Description

FIGURES

[0071] FIG. 1: Representation of the H57OptoFab system involving PHYB/PIF Optogenetic module. A. PIF6 peptide is cloned at C terminus of the H57 Fab Heavy chain. 650 nm wavelength light triggers the binding of the OptoFab to PHYB. This process is reversed following a 730 nm wavelength light. B. The H57 OptoFab system allows the light dependent immobilization of the TCR allowing an accurate control of TCR signaling.

[0072] FIG. 2: Western blot analysis of OptoFab production. Affinity purified proteins from the supernatant of OptoFab transfected HEK cells analyzed by western blot under reducing condition A. Or non-reducing conditions B. (FT: column flow-through, Elution: eluted proteins).

[0073] FIG. 3: Analysis of the H57 OptoFab specificity for TCR. A. Murine primary CD4 T cells were incubated or not with H57 OptoFab, then labelled with anti-His Alexa647 and analyzed by cytometry. B. Murine primary CD4 T cells were incubated with the indicated dose of the H57 OptoFab, then labelled with anti-His Alexa647 and analyzed by cytometry. The graph shows the quantity of protein versus the MFI. C. Competition experiment between a recombinant H57 Fab Alexa 488 and the H57 OptoFab.

[0074] FIG. 4: Analysis of HoloPhyB purity and functionality. A. SDS-PAGE analysis of affinity purified HoloPhyB: MW: Molecular Weight, FT: Flow Through, Elution: Eluted HoloPhyB. B. Absorption spectrum of purified HoloPhyB kept in dark (dotted black curve), illuminated with a 656 nm wavelength light (curve 1) and illuminated with a 656 nm wavelength light followed by a 740 nm wavelength light (curve 2).

[0075] FIG. 5: H57 OptoFab bind to HoloPhyB in a light dependent manner. Pull-Down experiment of H57-OptoFab binding to HoloPhyB-coated beads. Upper panel: western blot analysis of the pulled-down proteins following different illumination patterns. Lower panel: signal quantification using Fiji software.

[0076] FIG. 6: H57 OptoFab allows the light-controlled stimulation of murine primary T lymphocytes. Primary T lymphocytes loaded with PBDX Calcium indicator and incubated with H57 OptoFab were dropped on a HoloPhyB coated cover slip and exposed to the indicated light wavelengths. A. Microscope field containing primary T cells exposed to a 740 nm wavelength light (left panel), then to a 656 nm wavelength light (right panel). B. Quantification of calcium indicator fluorescence in live primary T cells during specific light exposure. Cell fluorescence has been quantified over time using FIJI software. Each symbol correspond to an individual cell.

EXAMPLE 1: METHODS

[0077] Generation of the H57-PIF6 OptoFab:

[0078] pYD7-HC-PIF (HC=fragment of the Heavy Chain derived from the monoclonal antibody H57) plasmid and pTT22-LC (LC=Light Chain derived from the monoclonal antibody H57) were cloned using In-Fusion HD cloning kit (Clonetech) in a standard reaction mixture. Oligonucleotide were synthesized by Sigma Aldrich. PCR templates are listed in table 3. PCR-amplified fragments were purified following the manufacturer's instructions. Host vector backbone was linearized using restriction enzyme. All cloning products were confirmed by sequencing (Eurofins Genomics).

[0079] H57 F(ab)-Pif was produced by cotransfecting HEK293T ebna cells with 10 μg of pYD7-HC-PIF plasmid and 30 μg of pTT22-LC plasmid (ratio 1:3) using Polyethylenimine. Cells were then maintained in DMEM high glucose (GIBCO) 2% FBS+0.5% Tryptone TN1+1.25 mM valproic acid supplemented with G418 at 37° C. with 5% CO2 in a humidified incubator. Supernatants were collected 7 days later and the recombinant OptoFab was purified by Ni-NTA affinity chromatography. Production of F(ab)-PIF was verified by western blotting.

[0080] Production of HoloPhyB:

[0081] HoloPhyB-StrepTag-pet28a was obtained by site-directed mutagenesis. StrepTag was added inside HoloPhyB-pet28a plasmid using QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer's instructions. Oligonucleotides were synthesized by Sigma Aldrich.

[0082] To produce HoloPhyB protein, E. coli cells BL21 were transformed with a combination of one plasmid encoding for PΦB synthase lacking transit peptide (AHY2), and a second plasmid driving heme oxygenase-1 expression and a third plasmid for HoloPhyB synthesis (HoloPhyB-strepTag expression) (Leung et al., 2008).

[0083] Single colonies were selected and grown in synthetic complete medium (LB broth with kanamycin) overnight at 37° C. Production was induced using IPTG at OD600 of 0.4-0.6 and kept overnight at 17° C. Proteins was extracted using a lysis buffer (Tris 50 mM+NaCl 300 mM+Imidazole 10 mM+lysozyme 0.25 mg/ml-pH 8) with DNase and MgSO4. Finally, proteins were purified by Ni-NTA affinity chromatography. Production of HoloPhyB was verified by western blotting.

TABLE-US-00003 TABLE 1 PCR templates Inserts Forward (5′-3′) Reverse (5′-3′) LC gatctctagcgaattcatgaaatacctattgcctacggc ggccgctagcaagctttcagcactcgccc agccgctggattgttattactcgcggcccagccggccat ctgttgaa ggcctatgagctgatacagccttcc HC gcgaattccctctagaatggagtttgggctgagctgggt cggcctcgagcggccgcttaatgatggtg tttcctcgttgctctttttagaggtgtccagtgtgaggt atgatgatagaaccggagccggtcttgtc gtacttggtggaatctgg acagctcttggg PIF tgacaagaccggcgccggtagcggcagtggtagtggta ggcctcgagcggcgccggggatccttaat gatggtgat StrepTag cgacggcgccggatcctggagccacccgcaatttgaaa tgaaaatacaggttttctttttcaaattg aagaaaacctgtattttca cgggtggctccaggatccggcgccgtcg

[0084] Photoconversion and Absorption Spectrum Analysis:

[0085] The photoconversions of HoloPhyB were assessed using BioLED light source at 656 nm or 740 nm for the indicated times. The absorption spectrum of holoPhyB was determined between 260 nm and 700 nm using a spectrophotometer (NanoDrop).

[0086] Measurement of the OptoFab Binding to TCR Using Flow Cytometry

[0087] Murine CD4+ T lymphocytes were incubated with H57 OptoFab diluted in PBS containing 2% SVF 1h at 10° C. Then after 2 washes, cells were incubated with an Alexa647 conjugated anti-his antibody (BD-Pharmingen) 30 minutes at 4° C. The binding of the H57 OptoFab was measured using a flow cytometer (BD FACSCanto-BD Biosciences)

[0088] Pull-Down Assay

[0089] HoloPhyB has been attached to strep-tactin coated beads (ferrimagnetic agarose beads coupled to the Strep-Tactin® IBA) in PBS containing 0.1% Triton X-100, 5 mM β-mercaptoethanol, and 1 mm PMSF). Then, soluble OptoFab has been added to the beads and exposed to the different pattern of light. Samples were then washed three times and D-Biotin was added to elute PhyB. The amount of OptoFab interacting with PhyB in each condition was assessed by western blot. The measurement and quantification of the western blot has been performed using a CCD camera (Azure system).

[0090] T Cell Stimulations and Ca2+ Influx Analyses:

[0091] Murine primary CD4 T cells has been loaded with PBDX Calcium dye (Sigma) 1 h at 37° C., then incubated with H57 OptoFab 1 h at 10° C. After 3 washes, T cells are then dropped into HoloPhyB-coated glass bottom LabTek chambers at 37° C. and imaged using a videomicroscope (Zeiss). BioLED illumination system has been plugged in the white-light path and exposure to 646 nm or 740 nm lights are set using BioLED light source control Module from Mightex. Quantifications has been performed using Fiji software.

EXAMPLE 2: THE OPTOFAB SYSTEM

[0092] OptoFab is a recombinant molecular system allowing the accurate control of the agonistic properties of an Antibody-derived Fab fragment in time and in space using specific wavelengths of light. It consists in a Fab fragment derived from an antibody of interest, linked to optogenetic modules that confer a light response capacity. Indeed, antibody derived Fab fragments generally keep the specificity of the antibody for its epitope, but not its agonistic properties. However, when Fab fragments are immobilized or oligomerized, they recover the agonistic properties of the whole antibody. These characteristics that are shared by numerous Fab fragments derived from agonistic antibodies, are at the basis of the OptoFab concept as its objective is to manipulate the oligomerization/immobilization statue of a Fab fragment using optogenetics to control its agonistic property. Optogenetics is a rising technology that consist in using light sensitive domains from plant or prokaryote proteins to control with light biological processes such as protein-protein interactions (Repina et al., 2017). Optogenetics provide the control of biological process with a temporal resolution in the millisecond range and a spatial resolution above the micrometer scale (restricted by the light diffraction limit).

[0093] We first generated an OptoFab system allowing to reversibly control T lymphocyte activation using specific wavelengths of light. Thus, we generated a recombinant protein composed of i) a Fab fragment derived from the H57 monoclonal antibody that recognizes an epitope in the β-chain of the TCR and has agonistic properties that drive T cell activation, coupled to ii) the Phytochrome Interacting Factor 6 (PIF6). The Phytochrome B of Arabidopsis Thaliana, when exposed to a light at 650 nm, experience a conformational change leading to the opening of a binding site for PIF6. An exposure to light at 730 nm reverses this process and free PIF6 (Leung et al., 2008; Toettcher et al., 2013).

[0094] The idea here is to use light to accurately control in time and space the agonistic property of the H57 Fab fragment by inducing is aggregation/immobilization on surfaces coated with recombinant Phy-B derived molecule (FIG. 1A). By this way, we aim to drive the capture of TCR and thus, T cell stimulation (FIG. 1B).

[0095] Phytochrome Interacting Factor 6 Peptide does not Alter Fab Fragment Derived from H57 Specificity:

[0096] We wanted to generate a Fab fragment derived from H57 monoclonal antibody that can be immobilized or clustered using light. We thus decided to clone a sequence encoding for the Phytochrome Interacting Factor 6 peptide (PIF6) at the 3′ end of the fragment of the H57 heavy chain forming the Fab fragment. The goal is to generate a Fab fragment whose immobilization or clustering can be controlled with light through its interaction with Phytochrom B (FIG. 1). We co-transfected this construct with a plasmid encoding for H57 Fab light chain in HEK cells to produce a PIF6 modified H57 Fab (optoFab) (FIG. 1). The optoFab has been affinity purified on Ni column thanks to a 6×His tag added at the carboxy-terminal end of PIF6. Western blot analysis of the purified OptoFab in reducing condition showed that, as expected, the modified heavy chain migrate at 41 kDa (FIG. 2A). Under non-reducing condition, the OptoFab migrates at 67 kDa (FIG. 2B). This shift in the migration is due to the interaction of the heavy chain with the light chain that is preserved under non-reducing condition. This data suggested that the purified OptoFab is correctly folded and is composed of one fragment of H57 heavy chain-PIF6, and one light chain fragment. PIF6 peptide doesn't seem to alter H57 Fab folding and secretion.

[0097] To go further in optoFab functionality analyses, we tested if the OptoFab derived from H57 antibody kept its specificity for the TCRβ chain. To test if the H57 OptoFab maintain its specificity for TCR, T cells have been incubated with a solution containing 2 ug/mL of OptoFab, 1 h at 10° C., then labelled with an anti-6×His antibody coupled to Alexa647 and analyzed by flow cytometry (FIG. 3). We observed a labelling of T cell similar to the one obtained with a fluorescent H57 Fab fragment. We then performed competition experiments to verify that the H57 OptoFab has the same specificity than the original H57 Fab. A 10-fold excess of OptoFab have been added on cells incubated with H57 Fab coupled to Alexa 488. This treatment fully abrogates the fluorescent H57 Fab labelling, showing that OptoFab competed with the native Fab for TCR binding. These experiments show that the H57-derived OptoFab is specific for TCR β chain.

[0098] Production of Functional Recombinant HoloPhyB:

[0099] To capture the OptoFab, we decided to produce a truncated form of PhyB containing the 1-650 aminoacids named Holo-PHYB (Leung et al., 2008). We cloned a single StrepTag followed by a 6×His Tag at the carboxy-terminal end of Holo-PHYB. This protein has been co-transfected in BL21 bacteria with a plasmid encoding Heme Oxygenase 1 and a plasmid encoding PΦB synthase lacking the transit peptide (Leung et al., 2008). These two constructs generate a PΦB adduct in the bacteria that favour the production of soluble HoloPhyB complexed with PΦB chromophore, which is required for its functionality. HoloPhyB was then affinity purified on Ni column, followed by a second step of purification using ion exchange column. As expected, a SDS-PAGE followed by a Coomassie staining analysis showed that the purified protein migrated at 75 kDa (FIG. 4A). A characteristic of the complex HoloPhyB-PΦB is its absorption spectrum. A spectral analysis showed that the protein we purified had two absorption peaks, one at 280 nm, and another at 660 nm (FIG. 4B), indicating that we succeed in producing the HoloPhyB-PΦB complex. When functional, this complex can display two distinct spectrums. In its Pr form, it absorbs red-light (at 665 nm) as shown in FIG. 4B. But following red light exposure, the complex switch in its Pfr form and can absorb Far-red light. As shown in FIG. 4B, a 5 minutes exposure of the purified HoloPhyB-PΦB complex to a 656 nm red light modified its absorption spectrum with the appearance of a peak around 730 nm (far-red light). Interestingly, a 740 nm far-red light reverse this phenomenon inducing a return to the Pr form. Altogether, these data show that we produced a photo-convertible functional HoloPhyB-PΦB complex.

[0100] The H57 Derived OptoFab Interacts Specifically with HoloPhyB-PΦB Complex in a Light Dependent Manner:

[0101] Soluble H57 Fab binds to the TCR without triggering any signalling. However, when immobilized on a planar surface or on beads, it triggers TCR signalling (data not shown). To verify if H57 OptoFab can be used as a molecular switch to control T cell activation, we next tested if the OptoFab can be specifically and reversibly immobilized with appropriate light wavelength. HoloPhyB has been coated on streptactin-coated beads, then incubated with OptoFab and exposed to different pattern of lights before a pull-down experiment (FIG. 5). As shown by western blot, when the system has been kept in the dark, the OptoFab is not pulled-down with HoloPhyB-coated beads. However, when the system is exposed to a 656 nm wave length light during 5 minutes, we observed that the OptoFab is pulled-down with HoloPhyB-coated beads. This data shows that an exposure to a red light triggers the binding of the OptoFab to HoloPhyB. Furthermore, when the system was exposed to a 740 nm wavelength light or kept 1 hour in the dark following the exposure to the red light, the interaction between the OptoFab and HoloPhyB is disrupted. All together, these data show that the light responding module of the recombinant proteins we produced is functional. The H57-derived OptoFab can be reversibly immobilized “on demand” using different wavelength of light.

[0102] H57 Derived OptoFab Acts as a Cellular On-Off Switch for T Cells:

[0103] Considering that the H57-derived OptoFab is specific for TCR and can be immobilized with light, we then wanted to test if it could allow a time gated control of TCR stimulations. We first verified that as the unmodified H57 Fab, the H57-derived OptoFab does not triggers TCR signalling per se. A very sensitive way to detect TCR signalling under a microscope is the analysis of Ca.sup.t+ influx in live cells using specific Ca.sup.t+ probes. Naïve primary T cells loaded with the calcium indicator PBDX have been incubated in the presence of 1 ug/mL of OptoFab, were dropped on a glass surface coated with holoPhyB and analysed under the microscope (FIG. 6A). When the system was exposed to a 740 nm wavelength light, no Ca.sup.t+ influxes were detected in the T cells, indicating that they remained unstimulated. However, a 656 nm wavelength light exposure triggered calcium influxes a few seconds later, indicating that the light dependent immobilization of the OptoFab driven by HoloPhyB triggered TCR stimulation. A consecutive exposure to a 730 nm wavelength light induced a rapid decrease of Ca.sup.t+ concentration in T cells, showing that TCR stimulation has been interrupted (FIG. 6B). Altogether, these data showed that the H57 OptoFab module allows a time gated control of TCR triggering. It acts as a molecular on/off switch for T lymphocytes.

EXAMPLE 3: OPTOFAB AND HOLOPHYB SEQUENCES

[0104] H57 OptoFab

TABLE-US-00004 H57 Heavy Chain-PIF: SEQ ID NO: 3 MEFGLSWVFLVALFRGVQCEVYLVESGGDLVQPGSSLKVSCAASGFTFSDF WMYWVRQAPGKGLEWVGRIKNIPNNYATEYADSVRGRFTISRDDSRNSIYL QMNRLRVDDTAIYYCTRAGRFDHFDYWGQGTMVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGAGSGSGSGS GSMMFLPTDYCCRLSDQEYMELVFENGQILAKGQRSNVSLHNQRTKSIMDL YEAEYNEDFMKSIIHGGGGAITNLGDTQVVPQSHVAAAHETNMLESNKHVD GSGSGSGSGSENLYFQGHHHHHH* H57 Light Chain : SEQ ID NO: 4 MKYLLPTAAAGLLLLAAQPAMAYELIQPSSASVTVGETVKITCSGDQLPKN FAYWFQQKSDKNILLLIYMDNKRPSGIPERFSGSTSGTTATLTISGAQPED EAAYYCLSSYGDNNDLVFGSGTQLTVLRGRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* HoloPhyB: SEQ ID NO: 2 MVSGVGGSGGGRGGGRGGEEEPSSSHTPNNRRGGEQAQSSGTKSLRPRSNT ESMSKAIQQYTVDARLHAVFEQSGESGKSFDYSQSLKTTTYGSSVPEQQIT AYLSRIQRGGYIQPFGCMIAVDESSFRIIGYSENAREMLGIMPQSVPTLEK PEILAMGTDVRSLFTSSSSILLERAFVAREITLLNPVWIHSKNTGKPFYAI LHRIDVGVVIDLEPARTEDPALSIAGAVQSQKLAVRAISQLQALPGGDIKL LCDTVVESVRDLTGYDRVMVYKFHEDEHGEVVAESKRDDLEPYIGLHYPAT DIPQASRFLFKQNRVRMIVDCNATPVLVVQDDRLTQSMCLVGSTLRAPHGC HSQYMANMGSIASLAMAVIINGNEDDGSNVASGRSSMRLWGLVVCHHTSSR CIPFPLRYACEFLMQAFGLQLNMELQLALQMSEKRVLRTQTLLCDMLLRDS PAGIVTQSPSIMDLVKCDGAAFLYHGKYYPLGVAPSEVQIKDVVEWLLANH ADSTGLSTDSLGDAGYPGAAALGDAVCGMAVAYITKRDFLFWFRSHTAKEI KWGGAKHHPEDKDDGQRMHPRSSFQAFLEVVKSRSQPWETAEMDAIHSLQL ILRDSFKESEAAMNSKVVDGVVQPCRDMAGEQGIDELGAGTLEKLVDGAGS WSHPQFEKENLYFQGLEHHHHHH*

REFERENCES

[0105] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0106] Leung, D. W., Otomo, C., Chory, J., and Rosen, M. K. (2008). Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway. Proc Natl Acad Sci USA 105, 12797-12802. [0107] Repina, N. A., Rosenbloom, A., Mukherjee, A., Schaffer, D. V., and Kane, R. S. (2017). At Light Speed: Advances in Optogenetic Systems for Regulating Cell Signaling and Behavior. Annu Rev Chem Biomol Eng 8, 13-39. [0108] Toettcher, J. E., Weiner, O. D., and Lim, W. A. (2013). Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155, 1422-1434.