Antibody-Based Fusion Proteins Allow Ca2+ Rewiring to Most

Dec 19, 2017 - Thus, the development of these antibody-based fusion proteins enables the rewiring of cell migration to most extracellular ligands when...
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Antibody-based fusion proteins allow Ca2+ rewiring to most extracellular ligands Anam Qudrat, and Kevin Truong ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00323 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

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Antibody-based fusion proteins allow Ca2+ rewiring to most extracellular ligands

Anam Qudrat1 and Kevin Truong1,2,*

1

Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario, M5S 3G9, Canada

2

Edward S. Rogers, Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King’s College Circle, Toronto, Ontario, M5S 3G4, Canada

Corresponding author (*): Kevin Truong [email protected], Tel: 416-978-7772, Fax: 416-978-4317 164 College Street Room 407, Rosebrugh Building, University of Toronto

Toronto, ON, M5S3G9, Canada

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Abstract The Ca2+ signaling toolkit is the set of proteins used by living systems to generate and respond to Ca2+ signals. The selective expression of these proteins in particular tissues, cell types and subcellular locations allows the Ca2+ signal to regulate a diverse set of cellular processes. Through synthetic biology, the Ca2+ signaling toolkit can be expanded beyond the natural repertoire to potentially allow a non-natural ligand to control downstream cellular processes. To realize this potential, we exploited the ability of an antibody to bind its antigen exclusively in combination with the ability of the cytoplasmic domain of vascular endothelial growth factor receptor 2 (VEGFR2) to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e. nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumour necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state but instead, only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively.

Keywords: monoclonal antibodies, nanobodies, Ca2+ signaling, migration, synthetic biology

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Background In nature, the Ca2+ signal can be rewired to regulate a diverse set of processes (e.g. motility, apoptosis, transcription and exocytosis) by selectively expressing proteins of the Ca2+ signaling toolkit (i.e. proteins that generate and respond to Ca2+ signals) in particular tissues, cell types and subcellular locations1. Following this principle, genetic circuits composed of natural or synthetic genes have likewise used the Ca2+ signal as an intermediate to rewire gene expression to light and magnetic fields2, 3. Advances in synthetic biology have expanded the Ca2+ signaling toolkit beyond the natural repertoire of extracellular ligands and intracellular processes that can directly generate and respond to Ca2+ signals. To rewire cell migration in direct response to a Ca2+ signal, the RhoA protein was engineered to be Ca2+-activated (named CaRQ)4. To expand the range of extracellular ligands that can generate a Ca2+ signal, our group developed a strategy for creating chimeric receptors that involved replacing the extracellular domain of VEGFR2 (vascular endothelial growth factor receptor 2) with the extracellular domain of the natural receptor that binds the extracellular ligand5. For this strategy to be effective, the extracellular ligand must oligomerize the extracellular domain (e.g. Ca2+ oligomerizes epithelial cadherin; tumour necrosis factor alpha oligomerizes its receptor) which in turn oligomerizes the cytoplasmic VEGFR2 domain, triggering downstream signaling to ultimately release Ca2+ from the endoplasmic reticulum (ER). As an example of re-wiring using the Ca2+ signal, a mammalian cell expressing both CaRQ and a particular chimeric receptor allowed direct migration to tumour necrosis factor alpha (TNFα)6. However, the strategy was limited by the existence of natural receptors that bind the particular target of interest at appropriate affinities. Furthermore, as natural receptors have differing folding tendencies, the success of one chimeric design may not directly predict the success of another design.

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Notably, the problem of producing a protein to binding almost any target is elegantly solved by the adaptive immune system7, 8. Through V(D)J recombination that randomly assembles particular gene segments, the adaptive immune system synthesizes a diverse set of antibodies to bind and remove a plethora of foreign antigens7, 8. The most studied class of antibodies is the monoclonal antibody (mAb) because it can tightly bind a single target. Monoclonal antibodies consist of two heavy chains and two light chain proteins that are covalently tethered by disulfide bonds between the heavy chains7. The variable domain of the heavy chain (VH) and the light chain (VL) together form an epitope where the target ligand specifically binds. Dozens of monoclonal antibodies have been approved to treat a wide range of diseases including the adalimumab antibody which binds TNFα to reduce inflammation associated with rheumatoid arthritis and Crohn’s disease8, 9. Since only the variable domains are required for target binding, single-chain antibodies (scFv) have been created by fusing VH and VL with a flexible linker of 10 to 25 amino acids10. In camelids (i.e. camels, llamas, dromedaries), there exists a class of antibodies that have an epitope formed from a single variable domain11. Thus, the smallest antibody-based protein required for binding a target is the single-domain antibody (also known as the nanobody), consisting of only the variable domain from the camelid antibody. Lastly, if needed, antibodies can be systematically tuned over a wide range of affinities12.

To expand Ca2+ rewiring to other extracellular ligands, even those without naturally-occurring binding partners, we investigated the design strategy of generating Ca2+ signals using fusion proteins consisting of antibody variants (e.g. mAbs, scFvs and nanobodies) with the cytoplasmic

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domain of VEGFR2. In theory, since scFv and nanobody bind a single target ligand, it cannot dimerize a chimeric receptor to generate a Ca2+ signal, unless the target of the scFv or nanobody is already oligomerized. If the target ligand happens to be a biological one, this does not pose a major limitation as most cellular proteins are oligomeric, either directly or indirectly through their binding partners13. Initially, it would appear that chimeric receptors made from the full mAb should not be effective because it would already be in a dimer due to the disulfide bond linking the heavy chains. However, if the target ligand is oligomeric, it can still stimulate higher order oligomerization of the chimeric receptor. Here, to demonstrate the feasibility of this antibody-based design strategy, we tested protein fusions of the cytoplasmic domain of VEGFR2 with four antibody variants: a green fluorescent protein (GFP) nanobody14, a red fluorescent protein (RFP) nanobody15, an anti-TNFα mAb (adalimumab)9 and an anti-CD14 scFv16. Each of these synthetic proteins responded to their respective extracellular stimuli by generating a Ca2+ signal. Further, when the anti-CD14 scFv chimeric protein was expressed with CaRQ4, it rewired cell migration towards sources of soluble CD14 (sCD14). Thus, the development of these antibody-based fusion proteins enables the rewiring of cell migration to most extracellular ligands when combined with CaRQ. Unlike fragments from natural receptors, the use of antibody fragments greatly simplifies future designs of chimeric receptor because protein folding is not an issue as all antibody-based chimeric receptors will essentially fold similarly. Since nonnatural signalling protein (e.g. GFP, RFP) acted as a signaling protein to cells expressing our antibody-based chimeric receptors, it suggests almost anything could have theoretically evolved to be a signaling protein.

Results and Discussion

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GFP nanobody-based fusion protein generates a Ca2+ signal in response to GFP stimuli A Ca2+ signal was generated by a GFP nanobody-based fusion protein (called GBPchi) when stimulated with the tandem fusion of monomeric GFP17 (tdGFP) (Figure 1A). In previous work5, to generate intracellular Ca2+ signals in response to high µM extracellular free Ca2+ (i.e. ~500 µM), we created a tandem fusion protein consisting of the leader sequence of immunoglobulin kappa (IgK) (i.e. 1METDTLLLWVLLLWVPGSTGD21), the first and second repeat of Ecadherin (Ecad12), and the transmembrane domain and the cytoplasmic domain of VEGFR2. High µM increases of extracellular free Ca2+ causes Ecad12 dimerization and subsequently an intracellular Ca2+ signal in cells5. The GBPchi instead has an additional GFP nanobody inserted between the leader sequence and Ecad12. We retained the Ecad12 component in GBPchi for two reasons: first, the Ecad12 component positions the GFP nanobody further away from the plasma membrane (PM); second, it allows testing whether the basal oligomeric state of the GBPchi, modulated by extracellular Ca2+, influences its ability to generate further Ca2+ signals when induced by another stimulus.

The stable HEK293 cell line (named GFP detector cells) was created by lentiviral infection with the GFP-detect transfer vector under zeocin selection (Figure S1). These cells expressed the GBPchi and the RCaMP1.07 Ca2+ sensor18 labelled to PM by the Lyn kinase peptide (1MGCIKSKGKDSA12) (Figure 1A). A dim red fluorescence outlined the cell periphery showing this PM localization (Video S1). Since the dissociation constant of the GFP nanobody to GFP is ~1 nM14, a bolus addition of [1 nM]f of monomeric GFP (mGFP) was added to the GFP detector cells in Ca2+-free PBS medium. As expected, there was no Ca2+ signal as binding mGFP should not oligomerize GBPchi (Figure 1E). A tandem fusion of mGFP (tdGFP) was used as a stimulus

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because it has two epitopes for the GBPchi. Bolus addition of [1 nM]f tdGFP to the extracellular medium of the GFP detector cells triggered a Ca2+ signal, lasting about 68.2 ± 11 s (Figure 1B,D). Another Ca2+ signal was triggered with another stimulation of these detector cells with tdGFP (Figure 1C) (Figure S3A). Acting through the Ecad12 component of GBPchi, a similar duration Ca2+ signal was induced by addition of [500 µM]f CaCl2 (Figure 1D). Interestingly, when the GFP detector cells in PBS medium containing [1 mM]f CaCl2, the bolus addition of [1 nM]f tdGFP also induced a Ca2+ signal (Figure 1D,E). Thus, stimulus that induces further oligomerization will trigger another Ca2+ signal. To abolish the Ca2+ signal, the GFP detector cells were pre-incubated with [50 nM]f staurosporine. Staurosporine (STS) is a broad spectrum kinase inhibitor that disrupts the Ca2+ signal by blocking auto-phosphorylation at the cytoplasmic domain of VEGFR219. A dose response curve showed percent cell response with respect to tdGFP concentration (Figure 1F). Using FRET (Fluorescence Resonance Energy Transfer) biosensors, downstream effector signaling of VEGFR2 was observed by measuring DAG activity (Figure S2A) and PLCγ activation (Figure S2B).

RFP nanobody-based fusion protein generates a Ca2+ signal in response to mCherry stimuli Likewise, a Ca2+ signal was generated by a RFP nanobody-based fusion protein (called RBPchi) when stimulated with the tandem fusion of monomeric Cherry20 (tdRFP) (Figure 2A). RBPchi is similar to GBPchi except the LaM-4 RFP nanobody15 replaced the GFP nanobody. The stable HEK293 cell line (named RFP detector cells) was created by lentiviral infection with the RFPdetect transfer vector under zeocin selection (Figure S1). Since the dissociation constant of the LaM-4 RFP nanobody to mCherry was reported in the subnanomolar range15, a bolus addition of [1 nM]f of monomeric Cherry (mCherry) was added to the RFP detector cells in Ca2+-free PBS

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medium, triggering no Ca2+ signal (Figure 2E). In contrast, when a tandem fusion of mCherry (tdRFP) was added at [1 nM]f concentration to the extracellular medium, it triggered a Ca2+ signal lasting about 56.1 ± 9.7 s (Figure 2B,D). Similarly, a second Ca2+ signal was triggered with more tdRFP stimulus (Figure 2C) (Figure S3B). Consistent with GBPchi, Ca2+ signals were triggered with Ca2+ addition or tdRFP in the presence of extracellular Ca2+ (Figure 2 D,E); Ca2+ signals were abolished by STS pre-incubation. Lastly, a dose response curve showed percent cell response with respect to tdRFP concentration (Figure 2F).

Adalimumab-based fusion protein generates Ca2+ signal in response to TNFα A Ca2+ signal was generated by an Adalimumab-based fusion protein (called Adachi) when stimulated with TNFα (Figure 3A). Adachi is composed of an Adachi light chain (Adachi-LC) and heavy chain subunit (Adachi-HC). The Adachi-LC is the same as the Adalimumab light chain, while the Adachi-HC is the Adalimumab heavy chain fused with the transmembrane and cytoplasmic domains of VEGFR2 (Figure 3A). While Adachi should be dimerized through the disulfide bonds between the heavy chains, the TNFα homotrimer21 could potentially further increase oligomeric state of Adachi to generate a Ca2+ signal. In fact, this scenario is akin to the cases where GBPchi and RBPchi were first dimerized by extracellular Ca2+ and then stimulated with their respective ligands to increase oligomeric state, generating a Ca2+ signal (Figure 1E, 2E). The stable HEK293 cell line (named TNFα detector cells) was created by lentiviral infection with the TNFα-detect transfer vector under zeocin selection (Figure S1). Bolus addition of 10 ng/mL of TNFα to the extracellular medium of the TNFα detector cells triggered a Ca2+ signal, lasting about 54.8 ± 9.9 s (Figure 3B) (Figure S3C). Ca2+ signals were triggered by the ATP positive control stimuli and Ca2+ signals were abolished by STS pre-incubation (Figure 3C).

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Lastly, a dose response curve showed percent cell response with respect to TNFα concentration (Figure 3D).

Anti-CD14 scFv-based fusion protein generates Ca2+ signal in response to soluble CD14 (sCD14) A Ca2+ signal was generated by an anti-CD14 scFv-based fusion protein (called scFVchi) when stimulated with sCD14 (Figure 4A). The scFVchi is similar to GBPchi except the anti-CD14 scFv16 (i.e. scFV2F9) replaced the GFP nanobody. CD14 is predominantly expressed on the plasma membrane (PM) of myeloid cells and serves as a co-receptor with Toll-like receptors for recognizing lipopolysaccharide released by bacteria during infections22. CD14 is shed from the PM as sCD1422 where it circulates at higher concentration during infection. The crystal structure of sCD14 revealed that it forms a horseshoe-shaped dimer23. The stable HEK293 cell line (named CD14 detector cells) was created by lentiviral infection with the CD14-detect transfer vector under zeocin selection (Figure S1).

Bolus addition of 10 ng/mL of sCD14 to the

extracellular medium of the CD14 detector cells triggered a Ca2+ signal, lasting about 52.7 ± 8.8 s (Figure 4B). Similarly, a second Ca2+ signal was triggered with more sCD14 stimulus (Figure 4C) (Figure S3D). Consistent with GBPchi and RBPchi, Ca2+ signals were triggered with Ca2+ addition or sCD14 in the presence of extracellular Ca2+ (Figure 4 D,E); Ca2+ signals were abolished by STS pre-incubation. Lastly, a dose response curve showed percent cell response with respect to sCD14 concentration (Figure 4F).

scFVchi and CaRQ rewires cell migration to sources of sCD14

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Cell migration was rewired to sources of sCD14 using a genetic circuit of scFVchi and CaRQ4 (an engineered Ca2+- activated RhoA protein) (Figure 5A). CaRQ transduces the Ca2+ signal generated by various natural receptors (e.g. channelrhodopsin-2 and VEGFR2)4 and synthetic receptors (e.g. CSF1Rchi24 and TNFR1chi6) into cellular blebbing that ultimately causes amoeboid-like cell migration to the respective target ligands of the receptors (e.g. blue light, VEGF, CSF1 and TNFα). The stable HEK293 cell line (named CD14 seeking cells) was created by lentiviral infection with the CD14-seek transfer vector, while the source cells expressing CD14 and PM-labelled mCherry was created by infection with CD14-source transfer vector (Figure S1). In CD14 seeking cells, CaRQ was visible by the yellow fluorescent outline of the cell periphery because it was fused to Venus25 and labelled to the PM (Figure 5B). A one-hour time lapse showed a CD14 seeking cell moving towards the CD14 source cell (Figure 5B, Video 2) at velocities averaging 43.3 ± 10.4 µm/hr (Figure 5C). The seeking cells appeared round because they were strained into single cells to increase motility since cells do not need to detach from the substrate or their neighbors before moving towards the source. In control experiments, when null cells (i.e. HEK293 cells expressing a fluorescent protein) were co-cultured with the source cluster, no physical displacement towards the source was seen (Figure 5C). To further the claim of chemotaxis, we conducted conventional transwell assays. When the CD14 seeking cells were plated in the apical chamber of the wells, they showed significant (n = 3, p < 0.01) migration towards the CD14 source cells, sCD14 or ATP in the basal chamber but not towards the null cells or a ROCK I/II inhibitor (Y-27632) (Figure 5D). This migration was not chemokinesis because the CD14 seeking cells did not significantly migrate across the membrane when sCD14 was added to both the apical and basal chambers (Figure 5D).

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Conclusion Using fusion proteins consisting of three types of antibody fragments (i.e. nanobodies, mAbs and scFvs), we triggered Ca2+ signals upon binding to their respective extracellular ligands – tdGFP, tdRFP, TNFα or sCD14. If the binding of the antibody to its extracellular ligand induced further oligomerization, a Ca2+ signal was triggered suggesting that the strict monomer to oligomer transition is not a prerequisite for signal transduction. The diversity of ligands that antibodies have and can be made to bind means that it is now possible to generate a Ca2+ signal in response to almost any extracellular ligand. The immediate benefit is Ca2+ rewiring. By rewiring these fusion proteins to Ca2+ indicators, it is possible to create cell-based screens for most extracellular ligands that have the specificity of an antibody and the optimizations of the Ca2+ indicators, now available in all fluorescent colours and boasting dynamic ranges exceeding 10-fold26, 27. For example, the scFVchi and RCaMP system offers a means to quickly screen for bacterial infection as sCD14 is elevated in the bloodstream during infection. By rewiring these fusion proteins to engineered Ca2+ activated small GTPases4, 28, it is also possible to program cells to migrate to almost any extracellular ligand. For example, the scFVchi and CaRQ system represents the first step in programming cells to migrate towards a site of infection that sheds sCD14 where the cell could then administer a local therapeutic intervention. As the Ca2+ signaling toolkit expands through synthetic biology, more biological processes can be controlled by extracellular ligands of our choice.

Methods

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Plasmid construction: The transfer vectors GBP-detector, RBP-detector, TNFα-detect, CD14detect, CD14-seek and CD14-source were synthesized by Genscript (Pescataway, NY) and subcloned into the pUC57-Simple vector using EcoRV. The sequences were synthesized for the cAbGFP4 GFP nanobody14, LaM-4 RFP nanobody15, adalimumab9, anti-CD14 single-chain antibody (scFV2F9)16 based on sequence information presented in their respective papers. Following our plasmid design structure29, cassettes were generated for the GFP nanobody, RFP nanobody, adalimumab and anti-CD14 scFv. Next, the chimeric proteins GBPchi, RBPchi, Adachi, scFVchi were modularly assembled using these new cassettes and cassettes from our other work5. All genes were highly expressed with the CMVp (cytomegalovirus promoter). All plasmid manipulations were performed by Genscript. All plasmids were transformed in E. coli DH5-α and were isolated using the Mini-prep kit (Invitrogen).

Cell Culture, Transfection and Stable Cell Line Generation: Human Embryonic Kidney (HEK293) (ATCC® CRL-1573TM) cells were used for ease of transfection/infection in the study. They were maintained in Dulbecco’s Modified Eagle’s Medium containing 25 mM D-glucose, 1 mM sodium pyruvate and 4 mM L-glutamine (Invitrogen, Carlsbad, CA) with 10% supplemented Fetal Bovine Serum (FBS) (Sigma Aldrich, St. Lois, MO) in T25 flasks (37 ºC and 5% CO2). Cells were passaged at 90% confluency using 0.05% TrypLE with Phenol Red (Invitrogen) and seeded onto 24-well Multiwell Plates (Falcon, Corning, NY) at a dilution of 1:20. Cells were transiently transfected using Lipofectamine 3000 according to manufacturer’s protocols (Invitrogen). Twenty-four hours post-transfection, cells were treated with 0.05% TrypLE with Phenol Red (Invitrogen) and plated in 6-well tissue culture plates (Falcon) at a serial dilution 1:2, 1:4, 1:8, 1:16 and 1:32. All stable HEK 293 cell lines were generated by

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lentiviral infection using the respective transfer vector and subsequent selection with blasticidin (10 ug/mL) or zeocin (200 µg/mL) for two weeks. Colonies were plated in 96-well tissue culture plates (Sarstedt, Numbrecht, Germany) for subsequent experiments and imaging. Illumination and Imaging: Imaging was performed using an inverted IX81 microscope with Lambda DG4 xenon lamp source and QuantEM 512SC CCD camera with a 10X or 40X objective (Olympus). Filter excitation (EX) and emission (EM) bandpass specifications were as follows (in nm): CFP (EX: 438/24, EM: 482/32), YFP (EX: 500/24, EM: 542/27), RFP (EX: 580/20, EM: 630/60) (Semrock). For FRET imaging, the CFP excitation and YFP emission filter was also used. Image acquisition and analysis was done with µManager and ImageJ software, respectively 30, 31. Cell Stimulation: Colonies plated in 96-well tissue culture plates (Sarstedt, Numbrecht, Germany) were washed with PBS and imaged in Serum-Free Media (SFM) (Thermo Fisher Scientific, Waltham, MA). TNFα (BioAspect), ATP (Sigma), soluble CD14 or CaCl2 was added as a bolus. Monomeric or tandem mGFP and mCherry were synthesized and purified from E.coli. To inhibit Ca2+ signals or blebbing, cells were pre-incubated for half an hour in PBS with [50 nM]f staurosporine (Sigma) or [1 µM]f Y-27632 (Sigma), respectively. Data Quantification: The Ca2+ signal was measured using the Live Intensity Plot pluggin for ImageJ 31. The duration of the signal was defined as the time between rising above 20% of the peak to falling below 20% of the peak. The signal intensity was normalized between 0 and 1, using the formula  = 

 

 

and reported as arbitrary units.

Statistical Analysis: All experiments for representative data showing Ca2+ traces were repeated 3 times independently with n = 12, unless otherwise indicated. All experiments were repeated at

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least 3 times and all data with normal distribution and similar variance were analyzed for statistical significance using two-tailed, unpaired Student’s t-test, unless otherwise indicated. Multiple group comparisons were made with one-factor ANOVA with Tukey-Kramer post-hoc test. For all tests, α was set at 0.05. p values smaller than 0.05 were considered significant and are indicated in text. Data expressed as mean ± s.d unless otherwise stated. Data were analyzed using Real Statistics Resource Pack for Excel (Microsoft, Redmond, WA). Supporting Information The Supporting Information includes 1 figure and 2 videos. Figure S1. (A) Schematic of transfer vectors. All of the detect transfer vector contains the selfinactivating promoter (SINp), a packaging signal (Ψ), a CMV promoter (CMVp) regulating PMlabelled RCaMP1.07, a CMVp regulating the respective fusion protein (i.e. GBPchi, RBPchi, Adachi or scFVchi), a SV40 promoter (SV40p) regulating zeocin resistance gene (zeor), and the 3’ long terminal repeat (LTR). The CD14-source transfer vector contains SINp, Ψ, CMVp regulating PM-labelled mCherry (pm-Cherry), CMVp regulating CD14, SV40p regulating blasticidin resistance gene (blastr), and the 3’ LTR. The CD14-seek transfer vector contains SINp, Ψ, CMVp regulating CaRQ, CMVp regulating scFVchi, a SV40p regulating zeor, and the 3’ LTR. Figure S2. GBPchi activates various downstream effectors in the VEGFR2 signaling when stimulated with 1 nM tdGFP. Post-stimulation, increases in FRET ratios of YFP:CFP were seen in (A) DAGR biosensor which measures diacylglycerol (DAG) activity and (B) CYPHR biosensor which measures PLC activation. Ca2+ transients from R-CaMP1.07 are observed along

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with a rise in FRET ratios. All experiments were repeated at least 3 with at least 4 cells per experiment. Figure S3. Average Ca2+ traces of two peaks from repeated respective stimuli for GBPchi (A), RBPchi (B), Adachi (C) and scFVchi (D). Error bars represent percent s.d. Experiments repeated 3 times. Video S1. A monolayer of GFP detector cell line responds to extracellular tdGFP. Upon addition of [1 nM]f tdGFP into the media, the GFP detector cells show an oscillation in fluorescence intensity, indicative of a Ca2+ signal. Scale bar is 100 µm. Experiment was repeated at least 3 times. n = 42. Video S2. CD14 seeking cell migrates towards a CD14 source cell. One-hour time-lapse of a CD14 seeking cell co-cultured with CD14 source cells. Scale bar is 20 µm. Experiment was repeated at least 3 times. n = 9.

Author Contributions Conceptualization, K.T.; Methodology, A.Q.; Formal Analysis, A.Q.; Investigation, A.Q.; Writing – Original Draft, A.Q.; Writing – Review & Editing, A.Q., K.T.; Visualization, A.Q.; Funding Acquisition, K.T.; Resources, K.T.; Supervision, K.T. Acknowledgments This work was funded by grants from the Canadian Cancer Society Research Institute (#701936) and NSERC (#05322-14). We thank Janice Wong and Abdullah Al Mosabbir for their helpful comments in reviewing this manuscript.

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Figure Legends Figure 1. GFP nanobody-based fusion protein (GBPchi) triggers a Ca2+ signal in response to extracellular tandem GFP (tdGFP). (A) Cartoon of the mechanism of activation of GBPchi. Gold oval represents the nanobody; light brown, Ecad12; dark brown, cytoplasmic domain of VEGFR2. (B) Representative Ca2+ trace observed with [1 nM]f tdGFP in GFP detector cell line expressing GBPchi and RCaMP1.07. (C) Representative multiple peaks after an additional stimulus of tdGFP. (D) Ca2+ signal durations with [500 µM]f CaCl2 and [1 nM]f tdGFP in extracellular media with and without Ca2+. Error bars show s.d. Experiments repeated 3 times. n = 26. (E) Percent cell response seen in GFP detector cells when stimulated with [500 µM]f CaCl2, [1 nM]f monomeric GFP (mGFP) or tdGFP in extracellular media with and without Ca2+ or [50 nM]f staurosporine (STS). Error bars show s.d. Samples compared with one-factor ANOVA followed by a Tukey-Kramer post-hoc test. Star indicates significance: p-value < 0.01. All experiments repeated 3 times. n = 33. (F) Percent response of GFP detector cells when stimulated with indicated tdGFP concentrations. Error bars show s.d. Experiments repeated 3 times. n = 18. Figure 2. RFP nanobody-based fusion protein (RBPchi) triggers a Ca2+ signal in response to extracellular tandem mCherry (tdRFP). (A) Cartoon of the mechanism of activation of RBPchi. Gold oval represents the nanobody; light brown, Ecad12; dark brown, cytoplasmic domain of VEGFR2. (B) Representative Ca2+ trace observed with [1 nM]f tdRFP RFP detector cell line expressing RBPchi and RCaMP1.07. (C) Representative multiple peaks after an additional stimulus of tdRFP. (D) Ca2+ signal durations with [500 µM]f CaCl2 and [1 nM]f tdRFP in extracellular media with and without Ca2+. Error bars show s.d. Experiments repeated 3 times. n = 27. (E) Percent cell response seen in RFP detector cells when stimulated with [500 µM]f

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CaCl2, [1 nM]f mCherry or tdRFP in extracellular media with and without Ca2+ or [50 nM]f STS. Error bars show s.d. Samples compared with one-factor ANOVA followed by a Tukey-Kramer post-hoc test. Star indicates significance: p-value < 0.01. All experiments were repeated at least 3 times. n = 26. (F) Percent response of RFP detector cells when stimulated with indicated tdRFP concentrations. Error bars show s.d. Experiments repeated 3 times. n = 17. Figure 3. Adalimumab-based fusion protein (Adachi) triggers a Ca2+ signal in response to extracellular TNFα. (A) Cartoon of the mechanism of activation of Adachi. Gold oval represents the Adalimumab; dark brown, cytoplasmic domain of VEGFR2. (B) Representative Ca2+ trace observed with 10 ng/mL of TNFα in TNFα detector cell line expressing Adachi and RCaMP1.07. (C) Percent cell response seen in TNFα detector cells when stimulated with [10 µM]f ATP, 10 ng/mL of TNFα or [50 nM]f staurosporine (STS). Error bars show s.d. Samples compared with one-factor ANOVA followed by a Tukey-Kramer post-hoc test. Star indicates significance: pvalue < 0.01. All experiments were repeated at least 3 times. n = 24. (D) Percent response of TNFα detector cells when stimulated with indicated TNFα concentrations. Error bars show s.d. Experiments repeated 3 times. n = 15. Figure 4. Anti-CD14 scFv-based fusion protein (scFVchi) triggers a Ca2+ signal in response to extracellular soluble CD14 (sCD14). (A) Cartoon of the mechanism of activation of scFVchi. Gold oval represents the scFV; light brown, Ecad12; dark brown, cytoplasmic domain of VEGFR2. (B) Representative Ca2+ trace observed with 10 ng/mL of sCD14 in CD14 detector cell line expressing scFVchi and RCaMP1.07. (C) Representative multiple peaks after an additional stimulus of sCD14. (D) Ca2+ signal durations with [500 µM]f CaCl2 and 10 ng/mL of sCD14 in extracellular media with and without Ca2+. Error bars show s.d. Experiments repeated 3 times. n = 28. (E) Percent cell response seen in CD14 detector cells when stimulated with [500

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µM]f CaCl2, 10 ng/mL of sCD14 in extracellular media with and without Ca2+ or [50 nM]f staurosporine (STS). Error bars show s.d. Samples compared with one-factor ANOVA followed by a Tukey-Kramer post-hoc test. Star indicates significance: p-value < 0.01. All experiments were repeated at least 3 times. n = 32. (F) Percent response of CD14 detector cells when stimulated with indicated sCD14 concentrations. Error bars show s.d. Experiments repeated 3 times. n = 21. Figure 5. scFVchi and CaRQ rewires cell migration to sources of sCD14. (A) Cartoon of activation mechanism of the scFVchi and CaRQ system. (B) Time-lapse images of a CD14 seeking cell co-cultured with a CD14 source cell showing displacement of the seeking cell towards the source cell at 12 minute intervals. Images are false colored: mCherry, red; YFP, green. Scale bar is 20 µm. n = 6. (C) Displacement of CD14 seeking cells or null cells to source cells over a time-lapse of 1 hour duration. Error bars show s.d. Samples compared with Student’s t-test. Star indicates significance; p-value < 0.01. Experiments were repeated 3 times. n = 9. (D) Transwell experiments showing cell migration in response to co-culturing with cell lines (i.e. CD14 source and null) or stimuli (i.e. [ATP]f = 10 µM, [soluble CD14]f = 10 ng/mL, or [Y27632]f = 1 µM (i.e. ROCK I and II inhibitor). A ++ indicates addition of sCD14. Error bars show s.d. Sample groups were compared with one-factor ANOVA followed by a Tukey-Kramer post-hoc test. Stars indicate significance; p-value < 0.01. n = 3.

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