Patent Application
20240238451
Optical imaging with NIR-FPs provides increased tissue penetration depths and better signal-to-noise ratio due to reduced light-scattering, tissue absorption and autofluorescence in NIR region (650-900 nm). NIR FPs allow labeling of whole organisms, specific cell populations, organelles, or individual proteins, and enable spectral multiplexing with FPs, biosensors and optogenetic tools active in visible range. While direct tagging of proteins with FPs ensures specificity and allows studying of protein dynamics in live cells, in some cases, FP-fusion constructs behave differently from their endogenous analogs. This might be due to altered expression level, turnover, or blocked by FP-tag functional domains.
Endogenous, not modified, proteins may be visualized by nanobodies (Nbs), which are single-domain 15 kDa antigen-binding fragments derived from camelid heavy-chain-only antibodies. Despite their small size, Nbs bind antigens with high affinity and specificity. The advantage of Nbs is their ability to recognize and bind a cognate (specific) antigen intracellularly. It has been shown that genetic fusions of Nbs with FPs of GFP-like family enable visualization of protein dynamics in live cells. However, nanobodies are usually stable irrespective of the antigen level. Accordingly, a large pool of nanobodies remains unbound to antigen. This large excess of free unbound nanobodies complicates, and in some cases, makes nearly impossible the nanobodies' use for visualization or manipulation/modification of intracellular targets.
The expression level of Nb-FP fusions may be decreased by weak or inducible promoters, or by the development of stable cell lines with low Nb-FP fusions expression. These approaches allow moderation of the impact of background signal but do not allow studies of cell populations heterogeneous by antigen expression.
To control the level of intracellular Nb-FP fusions, the use of conditionally stable Nbs containing six-point mutations in the Nb framework regions has been suggested. The diversity of Nbs is mainly determined by three complementarity determining regions (CDRs), whereas four framework regions are relatively conserved. However, the introduction of mutations at the frameworks may affect the Nb antigen-binding properties. Analysis of structures of Nb-antigen complexes shows that the considerable number of contacts with antigen is mediated by the residues in the framework regions, which limits this mutation approach to reduce intracellular level of Nbs.
Recently, an autorepression system based on the Krüppel associated box (KRAB) transcriptional repressor fused with antibody-like fibronectin-derived intrabodies that, in turn, fused to an FP, has been suggested. In these fusions, the intrabodies' expression depends on the antigen level because the unbound intrabodies fusions are translocated to the nucleus where the KRAB domain represses their transcription. However, the high accumulation of intrabodies in the nuclei (where they produce strong fluorescent signal, even if the cognate antigen is not expressed) limits this approach to non-nuclear antigens and to homogeneously antigen-expressing cell populations.
Accordingly, novel approaches for using nanobody to visualize or manipulate/modify intracellular targets are urgently needed.
The present disclosure provides fusion proteins (termed NIR-Fbs) comprising antigen-dependent near-infrared (NIR) fluorescent nanobodies that are allosterically unstable and, consequently, degrade in mammalian cells. These NIR-Fbs become highly stabilized and, consequently, brightly fluorescent, when bound to the cognate intracellular antigen. Also disclosed are methods of making and using the fusion proteins disclosed herein.
Provided herein are fusion proteins comprising a single domain antibody (sdAb) (including, but not limited to, a nanobody) that binds selectively to a specific antigen, wherein a second polypeptide is inserted into the single domain antibody, generating an internal fusion. Also provided are methods of making and methods of using the fusion proteins disclosed herein.
In one aspect, provided is an internal fusion protein comprising a nanobody, wherein a polypeptide is inserted into the nanobody. In some embodiments, the internal fusion protein comprises:
In some embodiments, the internal fusion protein comprises:
In some embodiments the first polypeptide forming an alpha helix located comprises a sequence that is at least 90% identical to SEQ ID NO:33 and the second polypeptide forming an alpha helix comprises a sequence that is at least 90% identical to SEQ ID NO:34. In some embodiment, the first polypeptide forming an alpha helix located comprises a sequence that is at least 90% identical to SEQ ID NO:34 and the second polypeptide forming an alpha helix comprises a sequence that is at least 90% identical to SEQ ID NO:33.
In some embodiments, the first polypeptide forming an alpha helix located comprises SEQ ID NO:33 and the second polypeptide forming an alpha helix comprises SEQ ID NO:34. In some embodiments, the first polypeptide forming an alpha helix located comprises SEQ ID NO:34 and the second polypeptide forming an alpha helix comprises SEQ ID NO:33.
In some embodiments, the polypeptide is inserted into the nanobody at a position corresponding to the i) G44/K45, (ii) S65/V66, or (iii) P90/E91 insertion site in a nanobody of SEQ ID NO:8. In some embodiments, the polypeptide inserted into the nanobody is a fluorescent protein, a drug, a toxin.
In some embodiments, the polypeptide inserted into the nanobody is a non-fluorescent protein.
In some embodiments, the polypeptide inserted into the nanobody is a fluorescent protein, wherein the fluorescent protein is a near-infrared fluorescent protein. In some embodiments, the near-infrared fluorescent protein inserted into the nanobody comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1-6. In some embodiments, the near-infrared fluorescent protein inserted into the nanobody comprises any one of SEQ ID NOs: 1-6.
In some embodiments, the polypeptide inserted into the nanobody is a toxin, wherein the toxin is caspase-3 mutant V266E, diphtheria toxin A subunit, Pseudomonas exotoxin, ricin, gelonin, or cucurmosin.
In some embodiments, the nanobody specifically binds to a fluorescent protein, a tumor antigen, or an intracellular protein.
In some embodiments the internal fusion protein is fused to a transcription factor.
In some embodiments, the polypeptide inserted into the nanobody is a kinase inhibitor. In some embodiments, the kinase inhibitor comprises the sequence GRTGRRNAI (SEQ ID NO:39) or RPKRPTTLNLF (SEQ ID NO:40).
In one aspect, provided is a multi-modular fusion protein comprising:
In some embodiments, the nanobody of the first internal fusion protein binds to a first antigen and the nanobody of the second internal fusion protein binds to a second antigen, and wherein the first and the second antigen are different. In some embodiments, the second antigen is targeted for degradation.
Provided is an internal fusion protein or a multi-modular fusion protein, wherein the internal fusion protein or the multi-modular fusion protein comprises a signal peptide.
Provided herein are nucleic acids encoding the internal fusion proteins or the multi-modular fusion proteins disclosed herein. Provided herein are vectors comprising the nucleic acids disclosed herein. In some embodiments, the vector is a viral vector. Provided herein is a cell comprising a nucleic acid disclosed herein or a vector disclosed herein. In some embodiments, the cell is an isolated cell.
In one aspect, provided is a method of recoloring a cell expressing a first fluorescent protein, the method comprising:
In one aspect, provided is a method of detecting the simultaneous presence of a first and a second antigen in a cell, the method comprising:
In one aspect, provided is a method of promoting degradation of a target antigen in a cell, the method comprising expressing in the cell an internal fusion protein disclosed herein comprising a first nanobody directed against a first antigen, wherein the internal fusion protein is fused to second nanobody directed against a second antigen that is to be targeted for degradation and wherein (a) in the presence of the first antigen, degradation of the second antigen is not promoted: and (b) in the absence of the first antigen, degradation of the second antigen is promoted. In some embodiments, a fluorescent protein is inserted into the first nanobody, and wherein (a) in the presence of the first antigen, the fluorescent protein emits fluorescence: and (b) in the absence of the first antigen, the fluorescent protein does not emit fluorescence.
In one aspect, provided is a method of regulating the expression of a protein of interest in a cell, the method comprising expressing in the cell an internal fusion protein disclosed herein comprising a nanobody directed against an antigen, wherein the internal fusion protein is fused to transcription factor that controls the expressing of the protein of interest, wherein (a) in the presence of the antigen, expression of the protein of interest is increased: and (b) in the absence of the antigen, expression of the protein of interest is decreased. In some embodiments, a fluorescent protein is inserted into the first nanobody, and wherein (a) in the presence of the antigen, the fluorescent protein emits fluorescence: and (b) in the absence of the antigen, the fluorescent protein does not emit fluorescence.
In one aspect, provided is a method of regulating kinase activity in a cell, the method comprising expressing in the cell an internal fusion protein disclosed herein comprising a nanobody directed against an antigen, wherein the internal fusion protein is fused to a peptide that reduces the activity of a kinase and wherein (a) in the presence of the antigen, the activity of the kinase is reduced; and (b) in the absence of the antigen, the activity of the kinase is not reduced. In some embodiments, a fluorescent protein is inserted into the first nanobody, and wherein (a) in the presence of the antigen, the fluorescent protein emits fluorescence; and (b) in the absence of the antigen, the fluorescent protein does not emit fluorescence.
Provided herein are fusion proteins comprising a single domain antibody (sdAb) (including, but not limited to, a nanobody) that binds selectively to a specific antigen, wherein a second polypeptide is inserted into the single domain antibody, generating an internal fusion. Also provided are methods of making and methods of using the fusion proteins disclosed herein.
As a non-limiting example, the fusion proteins disclosed herein may be used for imaging. Fusion proteins disclosed herein enable re-coloring of cellular structures labeled with visible range fluorescent proteins (FPs), e.g., EGFP or mCherry, using fusion proteins comprising Nbs against these FPs.
Additionally, NIR-Fb fusion proteins comprising Nbs against intracellular proteins are capable of efficiently binding and visualizing endogenous proteins, and background-free images are observed.
Moreover, fusion proteins disclosed herein enable manipulation of endogenous molecules by serving as a destabilizing fusion-partner for various effector proteins and peptides. To this end, molecular constructs were developed for directed degradation of targeted proteins, controllable protein expression, and modulation of the activity of enzymes in an antigen-dependent manner.
Additionally, fusion proteins disclosed herein can be used for the detection of rare endogenous proteins (i.e., can be used as biosensors) and for the manipulation of various endogenous proteins, signaling pathways, and even cell fate. For example, NIR-Fbs specific to active conformations of signaling molecules can enable monitoring their activity and depending on intracellular localization. Moreover, fusions of fusion proteins disclosed herein with Cre, Cas9 and Flp recombinases or transcriptional factors can control gene expression and genome editing selectively in cells, expressing specific intracellular epitopes.
Notably, because NIR-Fbs exhibit bright fluorescence in near-infrared tissue transparency window, they can be used in living mammals, including humans, in various deep-tissue applications. For example, NIR-Fbs specific for cancer biomarker or pathogen (viruses or bacteria) antigens, fused with cellular toxin and delivered by AAV vehicle, can enable initially visualization and then elimination of the tumor or pathogen-infected cells in a subject.
Due to the high homology and similar structural organization among different NBs, it has been discovered that the Nb fusion approach described herein is generalizable and works well for a large variety of Nbs. Accordingly, the fusion proteins disclosed herein open completely new ways for development and screening for high-affinity Nbs and their uses in cells and in vivo. Nbs can be screened as a library of NIR-Fb fusions expressed in mammalian cells using flow cell sorters, thus enormously speeding up screening, allowing screening of significantly larger Nb synthetic and naïve libraries, and yielding Nbs with higher affinity that are well-folded in mammalian cells, as compared to current phage, cell-surface and ribosome displays, which mainly allow enriching Nb repertoires.
Fluorescent Fusion Proteins that Specifically Bind to Targets of Interest
Provided herein are fusion proteins comprising a single domain antibody (sdAb) that binds selectively to a specific antigen, wherein a second polypeptide is inserted into the single domain antibody, generating an internal fusion.
As used herein, the term “single domain antibody” or “sdAb” refers to an antibody fragment consisting of a single monomeric variable antibody domain. In one embodiment, the sdAb is derived from the antigen-binding portion of a camelid heavy-chain-only antibody. Such an sdAb is also referred to as a VHH fragment or a nanobody. In one embodiment, the sdAb is derived from the antigen-binding portion of a cartilaginous fish heavy-chain-only antibodies (IgNAR, ‘immunoglobulin new antigen receptor’). Such an sdAb is called a VNAR fragment. Alternatively, an sdAb can be generated from conventional IgGs by obtaining or engineering monomeric, stable VH or VL domains.
A single-domain antibody comprises a variable region primarily responsible for antigen recognition and binding and a framework region. The “variable region,” also called the “complementarity determining region” (CDR), comprises loops which differ extensively in size and sequence based on antigen recognition. CDRs are generally responsible for the binding specificity of the single-domain antibody. Distinct from the CDRs is the framework region. The framework region is relatively conserved and assists in overall protein structure. The framework region may comprise a large solvent-exposed surface consisting of a ß-sheet and loop structure.
The sdAbs disclosed herein can be directed against any antigen, including, but not limited to, a fluorescent protein, a tumor antigen, or an intracellular antigen.
Examples of fluorescent proteins that the sdAb may bind to include, but are not limited to, a red fluorescent protein, green fluorescent protein, (3-F)Tyr-EGFP, A44-KR, aacuGFP1, aacuGFP2, aceGFP, aceGFP-G222E-Y220L, aceGFP-h, AcGFP1, AdRed, AdRed-C148S, aeurGFP, afraGFP, alajGFP1, alajGFP2, alajGFP3, amCyan1, amFP486, amFP495, amFP506, amFP515, amilFP484, amilFP490, amilFP497, amilFP504, amilFP512, amilFP513, amilFP593, amilFP597, anm1GFP1, anm1GFP2, anm2CP, anobCFP1, anobCFP2, anobGFP, apulFP483, AQ14, AQ143, Aquamarine, asCP562, asFP499, AsRed2, asulCP, atenFP, avGFP, avGFP454, avGFP480, avGFP509, avGFP510, avGFP514, avGFP523, AzamiGreen, Azurite, BDFP1.6, bfloGFPal, bfloGFPcl, BFP. BFP.A5, BFP5, bsDronpa (On), ccalGFP1, ccalGFP3, ccalOFP1, ccalRFP1, ccalYFP1, cEGFP, cerFP505, Cerulean, CFP, cFP484, cfSGFP2, cgfmKate2, CGFP, cgfTagRFP, cgigGFP, cgreGFP, CheGFP1, CheGFP2, CheGFP4, Citrine, Citrine2, Clomeleon, Clover, cp-mKate, cpCitrine, cpT-Sapphire174-173, CyOFP1, CyPet, CyRFP1 (CyRFP1), d-RFP618, D10, d1EosFP (Green), d1EosFP (Red), d2EosFP (Green), d2EosFP (Red), deGFP1, deGFP2, deGFP3, deGFP4, dendFP (Green), dendFP (Red), Dendra (Green), Dendra (Red), Dendra2 (Green), Dendra2 (Red), Dendra2-M159A (Green), Dendra2-M159A (Orange), Dendra2-T69A (Green), Dendra2-T69A (Orange), dfGFP, dimer1, dimer2, dis2RFP, dis3GFP, dKeima, dKeima570, dLanYFP, DrCBD, Dreiklang (On), Dronpa (On), Dronpa-2 (On), Dronpa-3 (On), dsFP483, DspR1, DsRed, DsRed-Express, DsRed-Express2, DsRed-Max, DsRed.M1, DsRed.T3, DsRed, T4, DsRed2, DstC1, dTFP0.1, dTFP0.2, dTG, dTomato, dVFP, E2-Crimson, E2-Orange, E2-Red/Green, EaGFP, EBFP, EBFP1.2, EBFP1.5, EBFP2, ECFP, ECFPH148D, ECGFP, eechGFP1, eechGFP2, eechGFP3, eechRFP, efasCFP, efasGFP, eforCP, EGFP, eGFP203C, eGFP205C, Emerald, Enhanced Cyan-Emitting GFP, EosFP (Green), EosFP (Red), eqFP578, eqFP611, eqFP611V124T, eqFP650, eqFP670, EYFP, EYFP-Q69K, fabdGFP, ffDronpa (On), FoldingReporterGFP, FP586, FPrf12.3, FR-1, FusionRed, FusionRed-M, G1, G2, G3, Gamillus (On), Gamillus0.1, Gamillus0.2, Gamillus0.3, Gamillus0.4, GCaMP2, gfasGFP, GFP(S65T), GFP-151pyTyrCu, GFP-Tyr151pyz, GFPmut2, GFPmut3, GFPxm16, GFPxm161, GFPxm162, GFPxm163, GFPxm18, GFPxm181uv, GFPxm18uv, GFPxm19, GFPxm191uv, GFPxm19uv, H9, HcRed, HcRed-Tandem, HcRed7, hcriGFP, hmGFP, HriCFP, HriGFP, iFP1.4, iFP2.0, iLov, iq-EBFP2, iq-mApple, iq-mCerulean3, iq-mEmerald, iq-mKate2, iq-mVenus, iRFP670, iRFP682, iRFP702, iRFP713, iRFP720, IrisFP (Green), IrisFP (Orange), IrisFP-M159A (Green), Jred, Kaede (Green), Kaede (Red), Katushka, Katushka-9-5, Katushka2S, KCY, KCY-G4219, KCY-G4219-38L, KCY-R1, KCY-R1-158A, KCY-R1-38H, KCY-R1-38L, KFP1 (On), KikGR1 (Green), KikGR1 (Red), KillerOrange, KillerRed, KO, Kohinoor (On), laesGFP, laGFP, LanFP1, LanFP2, lanRFP-ΔS831, LanYFP, laRFP, LSS-mKate1, LSS-mKate2, LSSmOrange, M355NA, mAmetrine, mApple, Maroon0.1, mAzamiGreen, mBanana, mBeRFP, mBlueberry 1, mBlueberry2, mc1, mc2, mc3, mc4, mc5, mc6, McaG1, McaG1ea, McaG2, mCardinal, mCarmine, mcavFP, mcavGFP, mcavRFP, mcCFP, mCerulean, mCerulean.B, mCerulean.B2, mCerulean.B24, mCerulean2, mCerulean2.D3, mCerulean2.N, mCerulean2.N(T65S), mCerulean3, mCherry, mCherry2, mCitrine, mClavGR2 (Green), mClavGR2 (Red), mClover3, mCyRFP1, mECFP, meffCFP, meffGFP, meffRFP, mEGFP, meleCFP, meleRFP, mEmerald, mEos2 (Green), mEos2 (Red), mEos2-A69T (Green), mEos2-A69T (Orange), mEos3.1 (Green), mEos3.1 (Red), mEos3.2 (Green), mEos3.2 (Red), mEos4a (Green), mEos4a (Red), mEos4b (Green), mEos4b (Red), mEosFP (Green), mEosFP (Red), mEosFP-F173S (Green), mEosFP-F173S (Red), mEosFP-M159A (Green), mEYFP, MfaG1, mGarnet, mGarnet2, mGeos-C(On), mGeos-E (On), mGeos-F (On), mGeos-L (On), mGeos-M (On), mGeos-S(On), mGinger1, mGinger2, mGrape1, mGrape2, mGrape3, mHoneydew, MiCy, mIFP, miniSOG, miniSOGQ103V, miniSOG2, miRFP, miRFP670, miRFP670nano, miRFP670v1, miRFP703, miRFP709, miRFP720, mIrisFP (Green), mIrisFP (Red), mK-GO (Early), mK-GO (Late), mKalama1, mKate, mKateM41GS158C, mKateS158A, mKateS158C, mKate2, mKeima, mKelly1, mKelly2, mKG, mKikGR (Green), mKikGR (Red), mKillerOrange, mKO, mKO2, mKOK, mLumin, mMaple (Green), mMaple (Red), mMaple2 (Green), mMaple2 (Red), mMaple3 (Green), mMaple3 (Red), mMaroon1, mmGFP, mMiCy, mmilCFP, mNectarine, mNeonGreen, mNeptune, mNeptune2, mNeptune2.5, mNeptune681, mNeptune684, Montiporasp, #20-9115, mOrange, mOrange2, moxBFP, moxCerulean3, moxDendra2 (Green), moxDendra2 (Red), moxGFP, moxMaple3 (Green), moxMaple3 (Red), mox NeonGreen, mox Venus, mPapaya, mPapaya0.7, mPlum, mPlum-E16P, mRaspberry, mRed7, mRed7Q1, mRed7Q1S1, mRed7Q1S1BM, mRFP1, mRFP1-Q66C, mRFP1-Q66S, mRFP1-Q66T, mRFP1.1, mRFP1.2, mRojoA, mRojoB, mRouge, mRtms5, mRuby, mRuby2, mRuby 3, mScarlet, mScarlet-H, mScarlet-I, mStable, mStrawberry, mT-Sapphire, mTagBFP2, mTangerine, mTFP0.3, mTFP0.7 (On), mTFP1, mTFP1-Y67W, mTurquoise, mTurquoise2, muGFP, mUkG, mVenus, mVenus-Q69M, mVFP, mVFP1, mWasabi, Neptune, NijiFP (Green), NijiFP (Orange), NowGFP, obeCFP, obeGFP, obeYFP, OFP, OFPxm, oxBFP, oxCerulean, oxGFP, ox Venus, P11, P4, P4-1, P4-3E, P9, PA-GFP (On), Padron (On), Padron(star) (On). Padron0.9 (On), PAmCherry1 (On), PAmCherry2 (On), PAmCherry3 (On), PAmKate (On), PATagRFP (On), PATagRFP1297 (On), PATagRFP1314 (On), pcDronpa (Green), pcDronpa (Red), pcDronpa2 (Green), pcDronpa2 (Red), PdaC1, pdae1GFP, phiYFP, phiYFPv, pHluorin, ecliptic, pHluorin, ecliptic (acidic), pHluorin, ratiometric (acidic), pHluorin, ratiometric (alkaline), pHluorin2 (acidic), pHluorin2 (alkaline), pHuji, PlamGFP, pmeaGFP1, pmeaGFP2, pmimGFP1, pmimGFP2, Pp2FbFP, Pp2FbFPL30M, ppluGFP1, ppluGFP2, pporGFP, pporRFP, PS-CFP (Cyan), PS-CFP (Green), PS-CFP2 (Cyan), PS-CFP2 (Green), psamCFP, PSmOrange (Far-red), PSmOrange (Orange), PSmOrange2 (Far-red), PSmOrange2 (Orange), ptilGFP, R3-2+PCB, RCaMP, RDSmCherry0.1, RDSmCherry0.2, RDSmCherry0.5, RDSmCherry 1, rfloGFP, rfloRFP, RFP611, RFP618, RFP630, RFP637, RFP639, roGFP1, roGFP1-R1, roGFP1-R8, roGFP2, rrenGFP, RRYT, rsCherry (On), rsCherry Rev (On), rsCherryRev1.4 (On), rsEGFP (On), rsEGFP2 (On), rsFastLime (On), rsFolder (Green), rsFolder2 (Green), rsFusionRed1 (On), rsFusionRed2 (On), rsFusionRed3 (On), rsTagRFP (ON), Sandercyanin, Sapphire, sarcGFP, SBFP1, SBFP2, SCFP1, SCFP2, SCFP3A, SCFP3B, scubGFP1, scubGFP2, scubRFP, secBFP2, SEYFP, sg11, sg12, sg25, sg42, sg50, SGFP1, SGFP2, SGFP2(206A), SGFP2(E222Q). SGFP2(T65G), SHardonnay, shBFP, shBFP-N158S/L173I, ShG24, Sirius, SiriusGFP. Skylan-NS (On), Skylan-S(On), smURFP, SNIFP, SOPP, SOPP2, SOPP3, SPOON (on), stylGFP, SuperfolderGFP, SuperfoldermTurquoise2, SuperfoldermTurquoise2ox, SuperNovaGreen, SuperNovaRed, SYFP2, T-Sapphire, TagBFP, TagCFP, TagGFP, TagGFP2, TagRFP, TagRFP-T, TagRFP657, TagRFP675, TagYFP, td-RFP611, td-RFP639, tdimer2(12), tdKatushka2, TDsmURFP, tdTomato, tKeima, Topaz, TurboGFP, TurboGFP-V197L, TurboRFP, Turquoise-GL, Ultramarine, UnaG, usGFP, Venus, VFP, vsfGFP-0, vsfGFP-9, W1C, W2, W7, WasCFP, Wi-Phy, YPet, zFP538, zoan2RFP, ZsGreen, ZsYellow1, αGFP, 10B, 22G, 5B, 6C, Ala, aacuCP, acanFP, ahyaCP, amilCP, amilCP580, amilCP586, amilCP604, apulCP584, BFPsol, Blue102, CFP4, cgigCP, CheGFP3, Clover1.5, cpasCP, Cy11.5, dClavGR1.6, dClover2, dClover2A206K, dhorGFP, dhorRFP, dPapaya0.1, Dronpa-C62S, DsRed-Timer, echFP, echiFP, EYFP-F46L, fcFP, fcomFP, Fpaagar, Fpag_frag, Fpcondchrom, FPmann, FPmcavgr7.7, Gamillus0.5, gdjiCP, gfasCP, GFPhal, gtenCP, hcriCP, hfriFP, KikG, LEA, mcFP497, mcFP503, mcFP506, mCherry 1.5, mClavGR1, mClavGR1.1, mClavGR1.8, mClover1.5, mcRFP, meffCP, mEos2-NA, meruFP, mKate2.5, mOFP.T.12, mOFP.T.8, montFP, moxEos3.2, mPA-GFP, mPapaya0.3, mPapaya0.6, mRFP1.3, mRFP1.4, mRFP1.5, mTFP0.4, mTFP0.5, mTFP0.6, mTFP0.8, mTFP0.9, mTFP1-Y67H, mTurquoise-146G, mTurquoise-146S, mTurquoise-DR, mTurquoise-GL, mTurquoise-GV, mTurquoise-RA, mTurquoise2-G, NpR3784g, PDM1-4, psupFP, Q80R, rfloGFP2, RpBphP1, RpBphP2, RpBphP6, rrGFP, RSGFP1, RSGFP2, RSGFP3, RSGFP4, RSGFP6, RSGFP7, Rtms5, scleFP1, scleFP2, spisCP, stylCP, sympFP, TeAPCα, tPapaya0.01, Trp-lessGFP, vsGFP, Xpa, yEGFP, YFP3, zGFP, and zRFP.
The term “tumor antigen” as used herein includes both tumor associated antigens (TAAs) and tumor specific antigens (TSAs). A tumor associated antigen means an antigen that is expressed by a tumor cell in higher amounts than is expressed by normal cells or an antigen that is expressed by normal cells during fetal development. A tumor specific antigen is an antigen that is unique to tumor cells and is not expressed by normal cells. The term tumor antigen includes TAAs or TSAs that have been already identified and those that have yet to be identified and includes fragments, epitopes and any and all modifications to the tumor antigens. Not-limiting examples of tumor antigens that the sdAbs may bind to include, but are not limited to, CD19, CD20, CD30, CD33, CD38, CD133, BCMA, TEM8, EpCAM, ROR1, Folate Receptor, CD70, MAGE-1, MAGE-2, MAGE-3, CEA, tyrosinase, midkin, BAGE, CASP-8, β-catenin, CA-125, CDK-1, ESO-1, gp75, gp100, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL 13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orf112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orf153, NKTR, NSEP1, U2AFIL, CYNL2, TPR GOLGA, BMII, COX-2, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, Nestin, OLIG2, ART1, ART4, B-cycline, Gli1, Cav-1, Cathepsin B, CD74, E-Cadherin, EphA2/Eck, Fra-1/Fosl 1, GAGE-1, Ganglioside/GD2, GnT-V, β1, 6-N, Ki67, Ku70/80, PROX1, PSCA, SOX10, SOX11, Survivin, βhCG, WT1, mesothelin, melan-A, NY-BR-1, NY-CO-58, MN (gp250)), telomerase, SSX-2, PRAME, PLK1, VEGF-A, VEGFR2, and Tie-2.
Intracellular proteins that the sdAb may bind to include, but are not limited to, antigens that are found, for example, in the cytoplasm and/or nucleus of a cell. Examples of an intracellular antigen include, but are not limited to, a receptor (e.g., cytoplasmic receptors such as peroxisome proliferator-activated receptors and nuclear receptors such as steroid hormone receptor, aryl hydrocarbon receptor), a transcription factor (e.g., SPI, AP-1, C/EBP, Heat shock factor, ATF/CREB, c-Myc, 1-Oct, NF-1, STAT3), a cytokine (e.g., interleukins, interferons, erythropoietin, thrombopoietin, colony stimulating factors), a growth factor (EGF, HGF, BMP, VEGF), an enzyme (e.g., protease, kinase, phosphatase), messengers (e.g., hormones such as vasopressin, follicle stimulating hormone, luteinizing hormone or neurotransmitters such as somatostatin or substance P), a member of a signaling pathway (e.g., MAPK pathway, Wnt pathway, Hedgehog pathway, Retinoic acid pathway, TGF beta pathway, JAK-STAT pathway, cAMP-dependent pathway), a carrier protein (e.g., electron carriers, such as oxidoreductases, NADPH oxidases), or a structural protein (e.g., actin, tubulin).
In one aspect, provided is an internal fusion protein comprising a nanobody, wherein a polypeptide is inserted into the nanobody.
In some embodiments, the inserted polypeptide is a fluorescent protein, a drug, a toxin, an enzyme, and/or a polypeptide with inhibitory function. In some embodiments, the polypeptide is a non-fluorescent protein. A non-limiting example of a non-fluorescent protein is the miRFP670nano3 variant Tyr58Cys (SEQ ID NO:7).
Non limiting examples of fluorescent proteins that may be inserted into the sdAbs include, but are not limited to, any of the fluorescent proteins disclosed herein. In some embodiments, the inserted protein is a near-infrared (NIR) fluorescent protein (FP). In one embodiment, provided is an internal fusion protein comprising a nanobody, wherein a polypeptide is inserted into the nanobody and wherein the polypeptide inserted into the nanobody is a miRFPnano. The resulting fusion protein is referred to as a near-infrared fluorescent nanobody (NIR-Fb). In some embodiments, the inserted protein is a miRPFnano variant. In some embodiments, the inserted protein comprises miRFP670nano, miRFP670nano R57C C86, miRFP670nano R57C C86S L114F V115S, miRFP670nano3, miRFP704nano, miRFP718nano, or a non-fluorescent miRFP670nano variant. In some embodiments, the miRFPnano is covalently linked to a chromophore. In some embodiments, the chromophore is biliverdin IVα (BV). In some embodiments, the chromophore is phycocyanobilin (PCB) tetrapyrrole.
Non limiting examples of drugs that may be inserted into the sdAbs include, but are not limited to, human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons, colony stimulating factors, interleukins, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoproteins, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha-1 antitrypsin, albumin and fragment polypeptides thereof, apolipoprotein-E, erythropoietin, factor VII, factor VIII, factor IX, plasminogen activating factor, urokinase, streptokinase, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, platelet-derived growth factor, epidermal growth factor, osteogenic growth factor, bone stimulating protein, calcitonin, insulin, atriopeptin, cartilage inducing factor, connective tissue activating factor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, monoclonal or polyclonal antibodies against various viruses, bacteria, toxins, etc., and virus-derived vaccine antigens, human serum albumin, human growth hormone, interferon alpha, erythropoietin, colony stimulating factors, immunoglobulins, angiogenin, bone morphogenic protein, chemokines, leptin, inhibitory factor, stem cell factor, transforming growth factor, and tumor necrosis factor.
Non limiting examples of toxins that may be inserted into the sdAbs include, but are not limited to, cholesterol dependent cytolysins, ADP-ribosylating toxins, plant toxins, bacterial toxins, viral toxins, pore forming toxins, and cell penetrating peptides. Non-limiting examples of toxins include caspase-3 mutant V266E, Pseudomonas exotoxin, ricin, gelonin, cucurmosin, diphtheria toxin fragment A, diphtheria toxin fragment A/B, tetanus toxin, E. coli heat labile toxin (LTI and/or LTII), cholera toxin, C. perfringes iota toxin, shiga toxin, anthrax toxin, MTX (B. sphaericus mosquilicidal toxin), perfringolysin 0, streptolysin, barley toxin, mellitin, anthrax toxins LF and EF, adenylate cyclase toxin, botulinolysin B, botulinolysin E3, botulinolysin C, botulinum toxin A, cholera toxin, clostridium toxins A, B, and alpha, shiga A toxin, shiga-like A toxin, cholera A toxin, pertussis Si toxin, E, coli heat labile toxin (LTB), pH stable variants of listeriolysin 0 (pH-independent; amino acid substitution L461T), thermostable variants of listeriolysin 0 (amino acid substitutions E247M, D320K), pH and thermostable variants of listeriolysin 0 (amino acid substitutions E247M, D320K, and L46IT), streptolysin 0), streptolysin c, streptolysin 0 e, sphaericolysin, anthrolysin 0, cereolysin, thuringiensilysin 0, 41 3275813vl weihenstephanensilysin, alveolysin, brevilysin, butyriculysin, tetanolysin 0, novyilysin, lectinolysin, pneumolysin, mitilysin, pseudopneumolysin, suilysin, intermedilysin, ivanolysin, seeligeriolysin 0, vaginolysin, and pyolysin.
Non limiting examples of enzymes that may be inserted into the sdAbs include, but are not limited to, lysozyme, pronase, serrapeptase, streptokinase, streptodolase, urokinase, hyaluronidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, iduronidase, and iduronate 2-Sulfatase (iduronate-2-sulfatase), galactose-6-sulfatase, alpha-glucosidase, acid ceramidase, acid sphingomyelina Acid sphingomyelinsase, galactocerebrosidsase, arylsulfatase A, B, beta-hexosaminidase A, B, heparin-N-sulfatase-sulfatase), alpha-D-mannosidase, beta-glucuronidase, N-acetylgalactosamine-6-sulfatase (N-acetylgalactosamine-6 sulfatase), Lysosomal acid lipase, alpha-N-acetyl-glucosaminidase, glucocere Brocidase (glucocerebrosidase), butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, Platelet activating factor Acetylhydrolase, neutral endopeptidase, myeloperoxidase, iduronate sulfatase, Agalsidase, Taliglucerase, Velaglucerase, Alglucerase, Sebelipase, Laronidase, Idursulfase, Galsulfase, and Elosulfase.
In some embodiments, a polypeptide with inhibitory function that may be inserted into the sdAbs include, wherein the polypeptide reduces the biological activity of another biological molecule. In some embodiments, the polypeptide with inhibitory function is an enzyme inhibitor. In some embodiments, the polypeptide with inhibitory function is an antibody. In some embodiments, the polypeptide with inhibitory function binds to a regulatory sequence element and inhibits or activates the transcription of a sequence operatively linked to a regulatory sequence element, respectively.
In some embodiments, the polypeptide inserted into the nanobody is connected to the N-terminal and the C-terminal portion of the nanobody with a flexible linker. In some embodiments, the flexible linker is between 3 and 30 amino acids long. In some embodiments, the flexible linker predominantly comprises glycine and serine residues. In some embodiments, the flexible linker comprises a sequence selected from the group consisting of GGS, GGGGS (SEQ ID NO:30), GGSGGGS (SEQ ID NO:31), or GGGGSGGGGS (SEQ ID NO:32) or repeats of these sequences. In some embodiments, the polypeptide inserted into the nanobody is connected to the N-terminal and the C-terminal portion of the nanobody with rigid linkers.
In some embodiments, the fusion proteins disclosed herein comprise the following sequences from N- to C-terminus: N-terminal portion of the nanobody-linker 1-first polypeptide forming an alpha helix-inserted polypeptide-second polypeptide forming an alpha helix-linker 2-C-terminal portion of the nanobody.
In some embodiments, the fusion proteins disclosed herein comprise the following sequences from N- to C-terminus: N-terminal portion of the nanobody-linker 1-first polypeptide forming an alpha helix-linker 2-inserted polypeptide-linker 3-second polypeptide forming an alpha helix-linker 4-C-terminal portion of the nanobody.
The linkers may or may not have the same sequence/length. In some embodiments, one of the two polypeptides forming an alpha helix comprises the sequence MANLDKMLNTTVTEVRKF (SEQ ID NO:33) or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.
In some embodiments, one of the two polypeptides forming an alpha helix comprises the sequence TWEIDFLKQQAVVMGIAIQQS (SEQ ID NO:34) or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.
In some embodiments, the insertion site in the nanobody for the polypeptide (and any linkers and/or alpha helices forming sequences if applicable) corresponds to the G44/K45 site of the nanobody to GFP shown in
In some embodiments, provided herein is a multi-modular fusion protein comprising an internal fusion protein disclosed herein fused to one more internal fusion proteins disclosed herein.
In some embodiments, the multi-modular fusion protein comprises two fusion proteins disclosed herein. In some embodiments, the nanobody of the first fusion protein binds to a different antigen than the nanobody of the second fusion protein, generating a bispecific multi-modular fusion protein. In some embodiments, the nanobody of the first fusion protein binds to the same antigen as the nanobody of the second fusion protein.
In some embodiments, the multi-modular fusion protein is fused to one or more additional proteins.
In embodiments, the multi-modular fusion protein a trispecific multi-modular fusion protein.
In some embodiments, the internal fusion proteins or the multi-modular fusion proteins disclosed herein comprise a signal sequence (also referred to as a signal peptide). Signal sequences are well known in the art. See, e.g., Owji et al., comprehensive review of signal peptides: Structure, roles, and applications. Eur J Cell Biol 2018 August; 97(6): 422-441, incorporated herein in its entirety. In one embodiment, the signal sequence comprises (MGVKVLFALICIAVAE, SEQ ID NO:35).
In some embodiments, provided is an internal fusion protein comprising a first nanobody directed against a first antigen, optionally wherein a fluorescent protein is inserted into the first nanobody, wherein the internal fusion protein is fused to second nanobody directed against a second antigen that is to be targeted for degradation.
In some embodiments, provided is an internal fusion protein comprising a nanobody directed against a first protein, wherein the internal fusion protein is fused to a transcription factor that controls expression of a second protein. In some embodiments, the transcription factor is GAL, which is used in the GAL4/UAS expression system.
In some embodiments, provided is an internal fusion protein comprising a nanobody directed against a protein of interest, wherein the internal fusion protein is fused to a peptide that inhibits the activity of a kinase. In some embodiments, the peptide inhibits the activity of protein Kinase A (PKA) or c-Jun N-terminal kinase (JNK). In some embodiments, the peptide comprises the sequence GRTGRRNAI (SEQ ID NO:36). In some embodiments, the peptide comprises the sequence RPKRPTTLNLF (SEQ ID NO:37).
Also provided herein are nucleic acids encoding the fusion proteins disclosed herein, as well as vectors, host cells, and expression systems.
The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
The nucleic acids encoding the fusion proteins disclosed may be, e.g., DNA, cDNA, RNA, synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination. For example, provided is an expression vector comprising a polynucleotide sequence encoding a fusion protein described herein operably linked to expression control sequences suitable for expression in a eukaryotic and/or prokaryotic host cell.
The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A “vector” includes, but is not limited to, a viral vector, a plasmid, an RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. In some embodiments, the employed vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available. Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno associated viruses, AAV), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, and spumavirus.
A variety of expression vectors have been developed for the efficient synthesis of fusion proteins in prokaryotic cells such as bacteria and in eukaryotic systems, including but not limited to yeast and mammalian cell culture systems have been developed. The vectors can comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences. Also provided are cells comprising expression vectors for the expression of fusion proteins disclosed herein.
Provided herein is a method of recoloring a cell expressing a first fluorescent protein, the method comprising:
Provided herein is a method of detecting the simultaneous presence of a first and a second antigen in a cell, the method comprising
Provided herein is a method of promoting degradation of a target antigen in a cell, the method comprising expressing in the cell an internal fusion protein comprising a first nanobody directed against a first antigen, wherein the fusion protein is fused to second nanobody directed against an antigen that is to be targeted for degradation (i.e., the second antigen): and wherein:
In one embodiment, a fluorescent protein is inserted into the first nanobody, and wherein:
Provided herein is a method of regulating the expression of a protein of interest in a cell, the method comprising expressing in the cell an internal fusion protein comprising a nanobody directed against an antigen, wherein the fusion protein is fused to transcription factor that controls the expressing of the protein of interest, wherein:
In one embodiment, a fluorescent protein is inserted into the first nanobody, and wherein:
Provided herein is a method of regulating kinase activity in a cell, the method comprising expressing an internal fusion protein comprising a nanobody directed against an antigen, wherein the internal fusion protein is fused to a peptide that inhibits the activity of a kinase and wherein:
In one embodiment, a fluorescent protein is inserted into the first nanobody, and wherein:
Provided herein is a method of regulating kinase activity in a cell, the method comprising expressing an internal fusion protein comprising a nanobody directed against an antigen, wherein the internal fusion protein is fused to a peptide that reduces the activity of a kinase and wherein:
In one embodiment, a fluorescent protein is inserted into the first nanobody, and wherein:
It is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the disclosure. It is further to be understood that this disclosure includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment, or a particular claim, that feature can also be used, to the extent possible, in combination with and/or in the context of other particular aspects and embodiments.
Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).
All other referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
To facilitate a better understanding of the disclosure, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the disclosure.
The genes encoding miRFPnano proteins and their mutants were amplified by polymerase chain reaction (PCR) and inserted into the pBAD/His-B vector (Invitrogen/Thermo-Fisher Scientific) at KpnI/EcoRI sites. All oligonucleotide PCR primers were purchased from Biomers.
For BV synthesis, E, coli host cells were cotransformed with a pWA23 h plasmid encoding heme oxygenase from Bradyrhizobium ORS278 under rhamnose promoter.
Random mutagenesis of genes encoding miRFPnanos was performed with a GeneMorph II random mutagenesis kit (Agilent Technologies). Site-specific mutagenesis and saturated mutagenesis were performed by overlap-extension PCR. For library construction, a mixture of mutated genes was cloned into pBAD/His-B vector and electroporated into LMG194 host cells (Invitrogen/Thermo-Fisher Scientific), containing a pWA23 h plasmid. Typical mutant libraries contained 107-108 clones.
Flow cytometry screening of mutant libraries was performed using an Influx cell sorter (BD Biosciences). 640 nm laser for excitation and 670/30 nm or 725/40 nm emission filters were used for the selection of positive clones. Before sorting, cells were grown in LB/ampicillin/kanamycin medium supplemented with 0.02% rhamnose and 0.005% arabinose for 5 h at 37° C., and then for 20 h at 22° C. The next day bacterial cells were pelleted, washed and diluted with phosphate-buffered saline (PBS) to an optical density of 0.03 at 600 nm. Cells collected after sorting were incubated in SOC medium for 1 h at 37° C., and then plated on LB/ampicillin/kanamycin Petri dishes supplemented with 0.005% arabinose and 0.02% rhamnose overnight at 37° C. Screening of brightest clones was performed with Leica M205 fluorescence stereomicroscope equipped with CCD camera
(Tucsen), using two filter sets: 650/45 nm excitation and 710/50 nm emission, and 700/20 nm excitation and 730 nm LP emission. About 30 selected clones were subcloned into a pcDNA3.1 plasmid (Invitrogen/Thermo-Fisher Scientific) and evaluated in transiently transfected HeLa cells.
Proteins were expressed in LMG194 bacterial cells, cotransformed with pWA23 h plasmid, encoding heme oxygenase. Bacterial cells were grown to an optical density of 0.5-0.7 at 600 nm in LB/ampicillin/kanamycin medium supplemented with 0.02% rhamnose and, then, to induce miRFPnanos or NIR-Fb expression, 0.005% arabinose was added. Bacteria were cultured for 5 h at 37° C., and, then, at 22° C., for 20 h.
Protein purification was performed with Ni-NTA agarose (Qiagen). Proteins were eluted with PBS containing 100 mM EDTA.
Fluorescence spectra were recorded with a Cary Eclipse fluorimeter (Agilent Technologies). Absorbance measurements were performed with a Hitachi U-2000 spectrophotometer. The extinction coefficients of miRFPnanos were determined as a ratio between the absorbance value of the main peak at the Q-band and the value of the peak at the Soret band, assuming the latter to have the extinction coefficient of free BV of 39,900 M−1 cm−1. The fluorescence quantum yields of miRFPnanos were determined using a Nile blue dye and miRFP709 as standards.
The pH stability was studied using a series of buffers (100 mM sodium acetate, 300 mM NaCl for pH 2.5-5.0, and 100 mM NaH2PO4, 300 mM NaCl for pH 4.5-9.0).
Distance between the adjacent B-strands of an Nb after insertion of miRFP670nano3 was estimated with a Coot software (Crystallographic Object-Oriented Toolkit) using an Nb structure from the crystallized GFP:antiGFP-Nb complex (PDB ID: 3OGO).
For crystallization, miRFP670nano3 and miRFP718nano were equilibrated in 20 mM Tris-HCl, 300 mM NaCl at pH 8.0 buffer and concentrated to 27.3 and 27.8 mg ml−1, respectively. Initial crystallization conditions were found with the NT8 crystallization robot (Formulatrix) using Hampton Research, Jena Bioscience, and Molecular Dimensions screens. The conditions were further optimized with additive screens. The best crystals could be obtained from (8.4% PEG 4000, 3.6% MPD, 0.06 M sodium/potassium phosphate buffer pH 6.3) and (12.6% PEG 6000, 0.1 M lithium sulfate, 0.07 M citric acid buffer pH 3.5, 2.1% D-sorbitol) for miRFP670nano3 and miRFP718nano, respectively. The crystals suitable for X-ray data collection were grown by the hanging-drop vapor diffusion method. In the large-scale crystallization experiment, 2 μl of the protein solution was mixed with 2 μl of the reservoir solution and incubated against 500 ml of the same reservoir solution at 20° C. for a week.
X-ray data were acquired on SER-CAT 22-ID and 22-BM beamline stations (Advanced Photon Source, Argonne National Laboratory, Argonne, IL). Before data collection, the crystals were flash-frozen in a 100 K nitrogen gas stream. Diffraction images were processed with HKL200049. The structures were solved by the molecular replacement method with MOLREP50 using the structure of miRFP670nano (PDB ID: 6MGH) as a search model. To remove model bias, the structures were rebuilt with ARP/wARP model building and density improvement software. The structure refinement was carried out with REFMAC5 (CCP4 suite) and PHENIX.REFINE (PHENIX suite) programs. Realspace model correction and structure validation were performed with COOT.
2 μl of EGFP or NIR-FbGFP and control antigen bovine serum albumin were spotted in PBS (proteins concentration 1 μ/ml) to the nitrocellulose membranes. The membranes were air-dried and incubated with 10% non-fat skimmed milk in PBS at 37° C., for 1 h. Then membranes were washed with PBS and incubated with EGFP or NIR-FbGFP (1:100 dilution, PBS, 0.05% Tween-20), respectively, at 37º C for 1 h. After incubation membraned were washed and imaged using the Leica M205 fluorescence stereomicroscope, using two filter sets: 650/45 nm excitation and 710/50 nm emission, for NIR-FbGFP and 480/40 nm excitation and 535/50 nm emission for EGFP.
To construct plasmids encoding miRFPnanos or their mutants, the respective genes were inserted into the pcDNA3.1 plasmid (Invitrogen/Thermo Fisher Scientific) at KpnI/EcoRI sites.
To engineer plasmids for protein tagging and labeling of intracellular structures, the miRFP670nano3 (GenBank miRFP670nano3), miRFP704nano (GenBank MW627295), or miRFP718nano (GenBank MW627296) genes were swapped with miRFP703 either as C- (for LAMP1 and H2B) or N-terminal fusions (for α-tubulin, β-actin, myosin and clathrin).
Nanobodies internally fused with miRFPnanos were generated by overlap PCR, and the resulted constructs were inserted into the pcDNA plasmid at the KpnI/EcoRI sites. Nb to GFP was amplified from a pcDNA3.1-NSImb-vhhGFP4 plasmid (Addgene #35579). Nb to actin was amplified from a plasmid commercially available from ChromoTek. Nb to mCherry was amplified from a pGEX6P1-mCherry-Nanobody plasmid (Addgene #70696). Nb to ALFA-tag was amplified from a pET51b(+)-EGFP-NbALFA plasmid (Addgene #136626). Genes of Nb 2E7 to HIV gp41, Nb21 and Nbm6 to SARS-COV-2 to spike protein were synthesized by GenScript. A NIR-Fbactin/Y58C-EGFP fusion was generated by overlap PCR and then a Tyr58Cys mutation was introduced to miRFP670nano3 by site-directed mutagenesis.
The gene encoding RBD (333-529) of SARS-COV-2 spike protein was amplified from a pDONR223-SARS-COV-2 S plasmid (Addgene #149329). The gene encoding human β-catenin was amplified from a pCI-neo-β-catenin plasmid (Addgene #16518). The gene encoding HIV p24 was amplified from a pET51b(+)-SNAP-p24 plasmid (Addgene #130718). The gene encoding DHFR was amplified from a pET22b-ecDHFR plasmid (Addgene #109055). The gene encoding HIV gp41 was amplified from a pLAI-Env plasmid (Addgene #133996). The PCR-amplified genes were then fused with msfGFP by SOE-PCR and inserted into a pcDNA3.1 plasmid. ALFA-fused α-tubulin, β-actin, myosin, and clathrin were generated by PCR with primer encoding ALFA-tag.
To generate NIR-Fb fusions, a 20 amino acid linker (Gly4Ser)4 (SEQ ID NO:41) was inserted into the pcDNA plasmid by the BamHI/NotI sites. For the construction of bispecific NIR-Fbs, NIR-FbGFP was inserted into the pcDNA3.1 plasmid, containing the (Gly4Ser)4 (SEQ ID NO:41) linker at the KpnI/BamHI sites, and NIR-FbmCherry was inserted at the NotI/XbaI sites. To generate a NIR-FbmCherry-EGFP fusion, EGFP was inserted into the pcDNA3.1 plasmid, containing a linker (Gly4Ser)4 and NIR-FbmCherry at NotI/XbaI site.
To engineer a NIR-FbGFP-GAL4 fusion, the GAL4-VP16 sequence was PCR-amplified from a pGV-ER plasmid (Systasy) and inserted at the NotI/XbaI sites into the pcDNA3.1 plasmid containing NIR-FbGFP and linker (Gly4Ser)4. To generate a NIR-FbGFP-NbALFA, the NIR-FbGFP was inserted into the pcDNA-3.1 plasmid, containing the (Gly4Ser)4 linker at the KpnI/BamHI sites, and NbALFA was inserted at the NotI/XbaI sites. To generate biNIR-FbGFP-GAL4 and biNIR-FbGFP-NbALFA fusions, two NIR-FbGFP genes were joined by SOE-PCR and swapped with NIR-FbGFP. The ALFA-tag was added to the N-terminus of GAL4-VP16 with oligonucleotide primer and inserted into the pcDNA3.1 plasmid, at the KpnI/XbaI site.
To generate fusions with inhibitory peptides, the peptide sequences were introduced to the NIR-FbGFP with the (Gly4Ser)4 linker by PCR, and a Y58C mutation was introduced to miRFP670nano3 by overlap PCR.
To construct Gluc-biNIR-FbGFP fusion Gluc gene was amplified from pUAS-Gluc plasmid and inserted to plasmid encoding biNIR-FbGFP at the HindIII/KpnI sites. To generate a biNIR-FbGFP-caspase/V266E and biNIR-FbGFP-DTA plasmids the biNIR-FbGFP was inserted into the pcDNA-3.1 plasmid, containing the (Gly4Ser)4 linker at the KpnI/BamHI sites, and caspase/V266E or DTA, respectively, were inserted at the NotI/XbaI sites. The gene encoding caspase/V266E was amplified from a pET21b-Caspase-3(V266E) plasmid (Addgene #90089). The gene encoding DTA was amplified from a pAAV-mCherry-flex-dtA plasmid (Addgene #58536).
HeLa (CCL-2), N2A (CCL-131), U-2 OS (HTB-96), HEK293T (CRL-3216), and NIH3T3 (CRL-1658) cells were obtained from the ATCC.
Cells were cultured in a DMEM medium supplemented with 10% FBS, 0.5% penicillin-streptomycin and 2 mM glutamine (Invitrogen/Thermo-Fisher Scientific) at 37° C.
For live-cell fluorescence microscopy, cells were plated in 35 mm glass-bottom Petri dishes (Greiner Bio-One International). Transient transfections were performed using polyethyleneimine or Effectene Transfection Reagent (Qiagen).
Primary rat cortical neurons were prepared in the Neuronal Cell Culture Unit, University of Helsinki. All animal work was performed under the ethical guidelines of the European convention and regulations of the Ethics Committee for Animal Research of the University of Helsinki. Cells were plated at a density of 500,000-700,000 per 35 mm glass-bottom dish, coated with Poly-L-Lysine (0.01 mg/ml) (Merck). Neurons were grown at 37° C. and 5% CO2 in neurobasal medium (Gibco) supplemented with B27 (Invitrogen/Thermo-Fisher Scientific), L-glutamine (Invitrogen/Thermo-Fisher Scientific), and penicillin-streptomycin (Lonza). Cultured neurons were transfected with pcDNA plasmids encoding respective miRFPnano at 4-5 days in vitro (DIV) using Effectene Transfection Reagent (Qiagen) and imaged 48-72 h after transfection.
Live cells were imaged with an Olympus IX81 inverted epifluorescence microscope, equipped with a Xenon lamp (Lambda LS, Sutter). An ORCA-Flash4.0 V3 camera (Hamamatsu) was used for image acquisition. Cells were imaged using either a 20×0.75 NA air or a 60×1.35 NA oil objective lens (UPlanSApo, Olympus). During imaging, HeLa cells were incubated in a cell imaging solution (Life Technologies-Invitrogen) and kept at 37° C. The microscope was operated with a SlideBook v.6.0.8 software (Intelligent Imaging Innovations). To separately image miRFP670nano3 and miRFP18nano, two filter sets (605/30 nm exciter with 667/30 nm emitter, and 685/20 nm exciter with 725/40 nm emitter) (Chroma) were used. The data were analyzed using a SlideBook v. 6.0.8 (Intelligent Imaging Innovations) and a Fiji v. 1.50b software.
Photobleaching measurements were performed in live HeLa cells 48 h after the transfection using 60×1.35NA oil objective lens (UPlanSApo, Olympus). Obtained raw data were normalized to corresponding absorbance spectra and extinction coefficients of the miRFPnanos, the spectrum of Xenon lamp and the transmission of the 665/45 nm photobleaching filter.
Flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD Biosciences). Before analysis, live cells were washed and diluted in cold PBS to a density of 500,000 cells per ml. At least 50,000 cells per sample were recorded. The fluorescence intensity of miRFP670nano, miRFP670nano3, miRFP704nano and miRFP718nano expressing cells was analyzed using the 640 nm excitation laser and 675/25 nm or 670 nm LP emission filters. The fluorescence intensity of EGFP was analyzed using 488 nm laser for excitation, and its fluorescence was detected with a 510/15 nm emission filter. The data were analyzed using FlowJo v.7.6.2 software.
For an antigen-dependent GAL4 stabilization, Hela cells were co-transfected with plasmids encoding NIR-FbGFP-GAL4 or biNIR-FbGFP-GAL4, reporter plasmid pUAS-Gluc (5×UAS), and with either EGFP or mTagBFP in a 1:10:89 ratio. HeLa cells were co-transfected with plasmids encoding ALFA-tagged GAL4-VP16, reporter plasmid pUAS-Gluc (5×UAS), NIR-FbGFP-NbALFA or biNIR-FbGFP-NbALFA, and with either EGFP or mTagBFP2 in the 1:10:44.5:44.5 ratio. To measure Gluc activity, the co-transfected Hela cells were grown in 24-well plates. 10 h after the transfection mixture was removed, a fresh growth medium was added, and cells were incubated for 48 h. Then, 5 μl of culture media was mixed with 100 μl of 2 μM coelenterazine (Invitrogen/Thermo Fisher Scientific) in PBS in wells of a 96-well half-area white plate (Costar). Bioluminescence was measured using a Victor X3 multilabel plate reader (PerkinElmer).
The viability of cells transfected with caspase/V266E and biNIR-FbGFP-caspase/V266E fusion was monitored by Annexin V staining (Annexin V-AlexaFluor555 conjugate: Invitrogen/Thermo Fisher Scientific). The viability of cells transfected with DTA and biNIR-FbGFP-DTA fusion was quantified by MTT test. Transfection efficiency was measured by flow cytometry and signal of non-transfected cells was subtracted.
The miRFP670nano3 gene was PCR amplified and subcloned into an AAV transfer vector downstream of the human synapsin promoter and upstream of WPRE and hGHpA sequences. This vector was co-transfected into HEK293-AAV cells (Vector Biolabs) along with a pAdeno-helper vector and a pRC-AAV9 rep-cap plasmid. Recombinant AAV9 production was then carried out using a protocol developed by the Byungkook Lim laboratory at the University of California at San-Diego. The recombinant AAV9-hSYN-miRFP670nano3-WPRE-hGHpA was titered by qPCR using primers designed to the hGHpA sequence. The titer of the virus was 1.4E+12 GC ml−1. The vector (volume: 0.4 l:μ undiluted) was injected into the cortex (coordinates: AP −0.8-(−1.75) mm, ML 1.45-1.65 mm, DV 0.25-1.1 mm) or dorsal horn of the L3-L4 spinal cord (ML 0.3, DV 0.3-0.5 mm). For surgical procedures, thin-wall glass pipettes were pulled on a Sutter Flaming/Brown micropipette puller (model P-97). Pipette tips were cut at an acute angle under 10× magnification using sterile techniques. Tip diameters were typically 15-20 μm. Pipettes that did not result in sharp bevels nor had larger tip diameters were discarded. Millimeter tick marks were made on each pulled needle to measure the virus volume injected into the brain or spinal cord.
Mice were anesthetized with isoflurane (4% for induction: 1%-1.5% for maintenance) and positioned in a computer-assisted stereotactic system with digital coordinate readout and atlas targeting (Leica Angle Two). Body temperature was maintained at 36-37° C., with a direct current (DC) temperature controller and ophthalmic ointment was used to prevent eyes from drying. A small amount of depilator cream (Nair) was used to remove hair over the dorsal areas of the injection site thoroughly. The skin was cleaned and sterilized with a two-stage scrub of betadine and 70% ethanol.
For brain injections, a midline incision was made beginning just posterior to the eyes and ending just passed the lambda suture. The scalp was pulled open and periosteum cleaned using scalpel and forceps to expose the desired hemisphere for calibrating the digital atlas and coordinate marking. Once reference points (bregma and lambda) were positioned using the pipette needle and entered into the program, the desired target was set on the digital atlas. The injection pipette was carefully moved to the target site (using AP and ML coordinates). Next, the craniotomy site was marked and an electrical micro-drill with a fluted bit (0.5 mm tip diameter) was used to thin a 0.5-1 mm diameter part of the bone over the target injection site. Once the bone was thin enough to flex gently, a 30G needle with an attached syringe was used to carefully cut and lift a small (0.3-0.4 mm) segment of bone.
For spinal cord injections, surgical scissors were used to make a small (around 10 mm) incision along the midline. Fascia connecting the skin to the underlying muscle was removed with forceps. The skin was held back by retractors. Using blunt dissection, lateral edges of the spinal column were isolated from connective tissue and muscle. Tissue from the vertebra of interest and one vertebra rostral and caudal to the site of spinal cord exposure was removed with forceps. The spine was then stabilized using Cunningham vertebral clamps and any remaining connective tissue on top of the exposed vertebrae removed with a spatula. Using a small sterile needle, an approximately 0).3 mm opening was made in the tissue overlying the designated injection site.
For injection, a drop of the virus was carefully pipetted onto parafilm (1-2 □l) for filling the pulled injection needle with the desired volume. Once loaded with sufficient volume, the injection needle was slowly lowered into the brain or spinal cord until the target depth was reached. Manual pressure was applied using a 30-ml syringe connected by shrink tubing and 0.4 μl of the virus was slowly injected over 5-10 min. Once the virus was injected, the syringe's pressure valve was locked. The position was maintained for approximately 10 min to allow the virus to spread and to avoid backflow upon needle retraction. Following the injection, head or spinal cord clamps were removed, muscle approximated, and the skin sutured along the incision. Mice were given subcutaneous Buprenex SR (0).5 mg per kg) and allowed to recover before placement in their cage.
All live animal procedures were performed following the guidelines of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at the Salk Institute. Male Cx3cr1GFP/+ mice (stock #005582, Jackson Laboratories) with an age of 8-14 weeks at the time of imaging (6-9 weeks at the time of stereotactic injection) were used.
For surgical procedures, mice were anesthetized with isoflurane (4-5% for induction: 1%-1.5% for maintenance) and implanted with a head or spinal plate on a custom surgical bed (Thorlabs). Body temperature was maintained at 36-37° C. with a DC temperature control system and ophthalmic ointment was used to prevent eyes from drying. Depilator cream (Nair) was used to remove hair above the imaging site. The skin was thoroughly cleansed and disinfected with a two-stage scrub of betadine and 70% ethanol.
For brain imaging, a scalp portion was surgically removed to expose frontal, parietal, and interparietal skull segments. Scalp edges were attached to the lateral sides of the skull using a tissue-compatible adhesive (3M Vetbond). A custom-machined metal plate was affixed to the skull with dental cement (Coltene Whaledent, cat, no. H00335), allowing the head to be stabilized with a custom holder. An approximately 3 mm diameter craniotomy was made over the AAV injection site. A 1.5% agarose solution and coverslip were applied to the exposed tissue, and a coverslip affixed to the skull with dental cement to control tissue motion. Imaging commenced immediately after optical window preparation.
For spinal cord imaging, a laminectomy was performed over the AAV injection site. The dura mater overlying the spinal cord was kept intact. A custom-cut #0 coverslip was placed over the exposed tissue and fixed to the spine with dental cement to control tissue motion. The depth of anesthesia was monitored throughout the experiment and adjusted as needed to maintain a breath rate of approximately 55-65 breaths per minute. Saline was supplemented as needed to compensate for fluid loss.
Live animal imaging was performed 2-5 weeks after AAV injection. Briefly, a Sutter Movable Objective Microscope equipped with a pulsed femtosecond Ti:Sapphire laser (Chameleon Ultra II, Coherent) with two fluorescence detection channels was used for imaging (green emission filter: ET-525/70M (Chroma); near-infrared emission filters: ET645/75M (Chroma) in conjunction with FF01-720/SP (Semrock); dichroic beamsplitter: 565DCXR (Chroma); photomultiplier tubes: H7422-40 GaAsP (Hamamatsu)). The laser excitation wavelength was set between 880-930 nm. The average laser power was <10-15 mW at the tissue surface and adjusted with depth as needed to compensate for signal loss due to scattering and absorption. An Olympus 20×1.0 NA water immersion objective was used for light delivery and collection. Z-stacks included up to 900 images, acquired at 1-2 μm axial step size, used a two- to four-frame average, 256 or 512×512-pixel resolution, and 1.0×-2.5× zoom (corresponding to 701-282 μm fields of view).
Data were processed in ImageJ or Fiji software (SciJava, https://fiji.sc) using the ‘Remove Outliers’ (radius: 1 pixel: threshold: 75) and ‘Subtract Background’ (rolling ball radius: 256 pixels) functions, xz projections were created from xy fluorescence image stacks using the ‘Reslice’ and ‘3D Project’ functions on 15 μm-sized sub-volumes. Motion artifacts in spinal cord images were reduced using the ‘Image Stabilizer’ plugin (transformation: affine).
To generate fluorescent protein (FP) variants with improved properties, miRFP670nano (see Table 1) was used as a starting point. miRFP670nano is a single-domain fluorescent protein that uses biliverdin IVa as a chromophore. In miRFP670nano, the chromophore forms a covalent thioether bond between the C31 atom of ring A of BV and Cys86 residue. The formation of the covalent bond is accompanied by a reduction of ring A, shortening of the conjugated system of the chromophore and, hence, resulting in a hypsochromic shift of its fluorescence. In the cystral structure of miRFP670nano, the Cβ and Cγ atoms of Arg57 are positioned close to the C32 atom of ring A of BV.
To enable a covalent binding of BV via the C32 atom, resulting in spectral red-shift, a R57C C86S double mutant of miRFP670nano was generated (see Table 1). Indeed, the fluorescence spectra for miRFP670nano R57C C86S double mutant were red-shifted by 35 nm as compared to parental miRFP670nano (
To red-shift the spectra further, saturated mutagenesis of the miRFP670nano R57C C86S double mutant was performed at residues 113-117, which surround chromophore ring D. An even more red-shifted variant with two extra mutations L114F and V115S was identified (miRFP670nano R57C C86S L114F V115S). (
Single-domain 17 kDa miRFP704nano and miRFP718nano red-shifted NIR FPs exhibited excitation/emission maxima at 680/704 nm and 690/718 nm, respectively. Enhanced miRFP670nano3 protein with excitation/emission at 645/670 nm exhibited spectral properties similar to those of parental miRFP670nano (
In addition to BV, miRFP670nano3 is also capable of binding a phycocyanobilin (PCB) tetrapyrrole, which is a natural chromophore for wild-type CBCRs27. The PCB chromophore increased miRFP670nano3 quantum yield to 22.4% (
References listed in Table 2: (1) Shcherbakova, D. M., Stepanenko, O. V., Turoverov, K. K. & Verkhusha, V. V. Near-Infrared Fluorescent Proteins: Multiplexing and Optogenetics across Scales. Trends Biotechnol 36, 1230-1243 (2018); (2) Chernov, K. G., Redchuk, T. A., Omelina, E. S. & Verkhusha, V. V. Near-Infrared Fluorescent Proteins, Biosensors, and Optogenetic Tools Engineered from Phytochromes. Chem Rev 117, 6423-6446 (2017); (3) Snapp, E. Design and use of fluorescent fusion proteins in cell biology. Curr Protoc Cell Biol Chapter 21, 21 24 21-21 24 13 (2005); (4) Bathula, N. V., Bommadevara, H. & Hayes, J. M. Nanobodies: The Future of Antibody-Based Immune Therapeutics. Cancer Biother Radiopharm (2020); (5) Akiba, H, et al. Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody. Sci Rep 9, 15481 (2019); (6) Deschaght, P, et al. Large Diversity of Functional Nanobodies from a Camelid Immune Library Revealed by an Alternative Analysis of Next-Generation Sequencing Data. Front Immunol 8, 420 (2017); (7) Gonzalez-Sapienza, G., Sofia, M. A. R. & Rosa, T.-d. Single-Domain Antibodies As versatile Affinity Reagents for Analytical and Diagnostic Applications. Frontiers in Immunology 8 (2017); (8) Cheloha, R. W., Harmand, T. J., Wijne, C., Schwartz, T. U. & Ploegh, H. L. Exploring cellular biochemistry with nanobodies. J Biol Chem 295, 15307-15327 (2020).
Engineered miRFPnanos variants were brightly fluorescent in mammalian cells without adding exogenous BV. The cellular (a.k.a, effective) brightness of miRFP670nano3 was >4-fold higher than that of miRFP670nano in all tested mammalian cell lines (
miRFP670nano3 exhibited photostability close to that of miRFP670nano (which already has increased photostability as compared to BphP-derived NIR FPs) while the photostability of miRFP704nano and miRFP718nano were even 8.8- and 1.8-fold higher than that for parental miRFP670nano, respectively (
Further, the engineered miRFPnanos exhibited high protein stability in mammalian cells. After a 4 h incubation with cycloheximide, a protein synthesis inhibitor, cells expressing engineered miRFPnanos retained 85-90% of their fluorescence (
Due to their high brightness, pH-stability, and compact single-domain fold, the engineered miRFPnanos are well suited for their use in N- or C-terminal fusion constructs and for labeling intracellular structures.
In two-color NIR imaging, different co-expressed fusions of miRFP670nano3 or miRFP718nano had proper localization and clear separation of miRFP670nano3 and miRFP718 fluorescence signals (
Primary rat cortical neurons expressing engineered miRFPnanos miRFP670nano3, miRFP704nano, or miRFP718nano exhibited bright homogenous fluorescence without the supply of exogenous BV (
To investigate the structural basis for the changes in the biophysical and biochemical properties of the new miRFPnanos, the crystal structures of miRFP670nano3 and miRFP718nano were determined at 1.8 Å and 1.7 Å resolution, respectively. The crystal structures were compared with the structures of parental miRFP670nano, with which miRFP670nano3 and miRFP718nano share ˜ 80% of the sequence identity (
miRIP670nano3
In miRFP670nano3, the chromophore BV is covalently attached by a thioether bond between the Cys86 residue and the C31 atom of the ring A, similar to the orientation in the parent miRFP670nano. The chromophore of miRFP670nano3 is stabilized by eight hydrogen bonds with the nearby residues Asp56, Tyr67, Arg71, Val84, and His117, a face-to-face stacking with Tyr87, and an edge-to-face stacking with Phe59.
The substantially increased quantum yield and brightness of miRFP670nano3 are likely provided by three mutations Val38Ser, Val39His, and Val140Met. The side chain of Ser38 forms a strong H-bond with the side chain of His39 locking it in the conformation favoring H-bonding with the side chain of Asp5. This H-bond stabilizes the spatial position of the N-terminal a-helix, tethering it to the second β-strand of the protein. The long side-chain of Met140 is the central element of the dense hydrophobic cluster formed by residues Leu8, Thr11, Val26, Ile104, Leu114, and Met140. This cluster effectively interconnects N- and C-terminal α-helices to the central β-sheet of the protein, making the fold of miRFP670nano3 more rigid than in parental miRFP670nano. The enhanced rigidity of the protein restricts BV mobility, makes the non-radiative transition of the chromophore less favorable, and hence provides for increased quantum yield and brightness of miRFP670nano3.
miRFP718nano
In miRFP718nano, the chromophore that forms a covalent thioether bond between the C32 atom of BV ring A and the rationally introduced Cys57 (
An internal fusion of an engineered miRFPnano variant, here miRFP670nano3, with a well-characterized nanobody (Nb) directed against green fluorescent protein (NbGFP) was generated. Three insertion sites for miRFP670nano3 in NbGFP were tested (
In cells coexpressing enhanced (EGFP), all internal fusions exhibited NIR fluorescence, which varied depending on the insertion site and linkers length. In the absence of EGFP, fluorescence of internal fusions was not detected (
NIR-FbGFP was expressed in E, coli. In contrast to mammalian cells, bacterial cells expressing NIR-Fb without antigen exhibited NIR fluorescence (
To determine whether a specificity to antigen affected NIR-Fb behavior, an EGFP variant bearing an Asn146Ile mutation was used. This mutation dramatically reduces binding to NbGFP. Coexpression of NIR-FbGFP and EGFP/Asn146Ile caused a >10-fold decrease in NIR fluorescence of HeLa cells as compared to the cells co-expressed NIR-FbGFP and non-mutated EGFP (
To assess whether an antigen concentration affected the protein level of NIR-Fb, the same amount of NIR-FbGFP plasmid was co-transfected with different amounts of EGFP plasmid. Fluorescence of NIR-FbGFP strongly depended on EGFP expression level (
The data indicated that a possible mechanism of NIR-FbGFP behavior in mammalian cells is the stabilization by antigen binding and degradation in the unbound state by the ubiquitin-proteasome system. To further evaluate this hypothesis, cells transfected with NIR-FbGFP were treated with bortezomib, an inhibitor of proteasome-dependent protein degradation. Whereas the non-treated cells were non-fluorescent, cells treated with bortezomib demonstrated strong NIR fluorescence (
Similarly, NIR-FbGFP constructs comprising miRFP704nano (
To study whether the NIR-Fb approach can be extended to other Nbs, the approach was applied to nine more Nbs, such as Nb specific to actin, BC2 Nb to β-catenin, LaM4 Nb to mCherry, Nb against ALFA-tag, Nb ca1698 to E, coli DHFR (dihydrofolate reductase), Nb 59H10 and 2E7 to HIV (human immunodeficiency virus) antigens p2436 and gp41, and Nb 21 and m6 specific to spike SARS-Cov-2 antigen (
Although these Nbs have similar structural architecture, their mode of binding to antigen is different. NbGFP employs for antigen binding all three CDR regions (PDB ID: 3OGO). In contrast, in the LaM4 interaction with mCherry, the binding occurs via the CDR3 and only partly via CDR1 (PDB ID: 6IR1). Nb m6 binding to the spike SARS-COV-2 RBD (receptor binding domain) is mediated by the CDR1 and CDR2. The interactions in the BC2 Nb are formed by the CDR3 and framework regions 2 and 3 (PDB ID: 5IVN). NbALFA binding of the ALFA-peptide is mainly mediated by CDRs 2 and 3 but also involves framework regions 2 (PDB ID: 6I2G). Nb ca1698 binds to DHFR using residues from all three CDRs and framework 2 (PDB ID: 4EIG). To interact with the C-terminal domain of the HIV p24 antigen, Nb 59H10 uses its framework region 2 and all three CDRs (PDB ID: 5O2U)36. The major contacts of the Nb 2E7 and HIV gp41 antigen are formed by residues in CDR1, CDR2 and framework region 2 (PDB ID:5HM1). Nb 21 binds to spike SARS-COV-2 RBD via all three CDRs and framework regions 2 and 339.
Despite the differences in the structural basis of antigen binding, all engineered NIR-Fbs exhibited antigen-dependent stabilization in mammalian cells, similar to NIR-FbGFP. The eight engineered NIR-Fbs exhibited strong fluorescence in cells co-expressing their cognate antigens and degraded in the absence of specific antigens (
To date, many transgenic mice and cell lines expressing proteins of interest labeled with EGFP or mCherry are available. Accordingly, the NIR-Fb technology was next used for re-coloring of EGFP- and mCherry-labeled proteins. HeLa cells were co-transfected with EGFP- or mCherry-tagged β-actin and α-tubulin and a 10-fold excess of NIR-FbGFP and NIR-FbmCherry encoding plasmids. Despite this, both NIR-FbGFB and NIR-FbmCherry strongly colocalized with the EGFP- or mCherry-labeled cytoskeletal proteins, and no background signal was observed (
The NIR re-coloring with NIR-FbGFP and NIR-FbmCherry can be applied to numerous available transgenic mice and cell lines expressing proteins of interest tagged with EGFP or mCherry. NIR-Fbs exhibit antigen-dependence stabilization in the dose-dependent manner (
Notably, NIR-FbALFA engineered with Nb specific to ALFA tag performed well in the labeling of various ALFA-tagged cellular proteins (
Next, it was demonstrated that NIR-Fbs can be used for the indirect labeling of a protein of interest (e.g., actin), by fusion a non-fluorescent miRFPnano variant with another fluorescent protein (e.g., EGFP). To avoid double-labeling, miRFP670nano3/Tyr58Cys was generated to serve as a non-fluorescent miRFPnano variant (
Next, a bispecific fusion construct containing NIR-Fbs to two different cognate antigens, EGFP and mCherry, was generated (
Co-expression of both EGFP and mCherry resulted in miRFP670nano3 fluorescence, indicating that the bispecific NIR-FbGFP-NIR-FbmCherry fusion was stabilized by binding with antigens. In contrast, when the bispecific NIR-FbGFP-NIR-FbmCherry fusion was coexpressed with either only EGFP or only mCherry, the NIR fluorescence dropped >10-fold, indicating that the bispecific fusion had been degraded (
Thus, bispecific NIR-Fbs permit specific labeling of double-positive cell populations expressing both targeted antigens, efficiently acting as Boolean “and” element. This property makes these constructs very useful for the design of new signaling pathways and other synthetic biology applications in mammalian cells.
Next, the suitability of NIR-Fb as destabilizing fusion-partner for regulatory proteins as determined.
First, to confirm that NIR-Fb mediates antigen-dependent stability of its fusion-partner, a NIR-FbmCherry-EGFP fusion was generated and its behavior in live cells cotransfected with mCherry or mTagBFP2, respectively, was evaluated (
Then, this approach was applied to antigen-dependent gene expression. For this, a GAL4/UAS system was used in which the GAL4 transcription factor drives expression of the reporter gene downstream of its UAS (upstream activating sequence)'s DNA-binding site. GAL4 was fused to NIR-FbGFP (
In a related experiment, GAL4 was fused with NIR-FbGFP (
In sum, these data showed that cellular proteins can be targeted for the directed degradation in an antigen-dependent manner by replacing the Nb directed against GFP in the NIR-FbmCherry-NbGFP fusion with a Nb directed against the protein of interest.
Next, it was demonstrated that NIR-Fbs can be used as a destabilizing component for proteolysis of a targeted protein, such as EGFP. To demonstrate this, a fusion of NIR-FbmCherry and NbGFP was constructed (
In a related experiment, NbALFA and its small antigen ALFA peptide (SRLEEELRRRLTE, SEQ ID NO:38) fused to a protein were used. The degradation of the GAL4 transcription factor was tested by fusing NIR-FbGFP and biNIR-FbGFP with NbALFA and GAL4 with ALFA peptide (
These data show that intracellular proteins can be targeted for the directed antigen-dependent degradation mediated by binding with NIR-Fb fusions.
Next, NIR-Fb was generated for the modulation of protein kinase activity.
Protein Kinase A (PKA) and c-Jun N-terminal kinase (JNK) were selected. PKA is one of the key effectors of the CAMP-dependent pathway, while JNK regulates cellular responses to stress signals. The activity of PKA could be specifically inhibited by a peptide comprising amino acids 14-22 (GRTGRRNAI, SEQ ID NO:36) of PKA inhibitor protein PKI. This peptide binds to the catalytic subunit of PKA and acts as pseudosubstrate. For inhibition of JNK activity, the widely used inhibitory peptide composed of the amino acids 153-163 (RPKRPTTLNLF, SEQ ID NO:40) of JNK-interacting protein-1 JIP-1 was chosen. The binding of this peptide induces rearrangement between the N- and C-terminal domains of JNK and distorts the ATP-binding pocket, inhibiting the catalytic activity.
To develop antigen-dependent inhibitors for PKA and JNK kinases, constructs were engineered that consisted of corresponding inhibitory peptides and the non-fluorescent NIR-FbGFP/Tyr58Cys variant (
Co-transfection with NIR-FbGFP/Y58C-PKI (
These results demonstrate the suitability of NIR-Fb inhibitory fusions to modulate kinases signaling events in an antigen-dependent manner.
To evaluate the utility of miRFP670nano3 for in vivo imaging, the miRFPnano variant was subcloned into an adeno-associated viral (AAV) vector carrying a synapsin promoter. The vector was then injected into the somatosensory cortex or spinal dorsal horn of Cx3cr1GFP/+ mice with labeled microglia. Two to five weeks after injection, animals were prepared for in vivo two-photon imaging to determine protein expression and attainable imaging depth. NIR FPs in addition to their absorption peaks in the so-called Q band (i.e., 30-70 GM at 1200-1280 nm), exhibit high cross-sectional values in the so-called Soret band (180-450 GM at 890-950 nm). This feature permits multiplex, subcellular resolution imaging of miRFP670nano3 and EGFP with single-wavelength two-photon excitation using a standard Ti:Sapphire laser. The experiments used excitation light powers that, do not activate microglia, which are highly sensitive to perturbations in tissue physiology.
miRFP670nano3 brightly fluoresced in transduced cells without the need for external BV. At the 920 nm wavelength optimally exciting EGFP, miRFP670nano3-positive cells were routinely visualized through the entire depth of the cortex, down to the entorhinal cortex level (˜850 μm) that exceeded those with EGFP in Cx3cr1GFP/+ mice with brightly EGFP-labeled microglia. No photobleaching or cytotoxicity was observed.
It has been demonstrated that microglia respond to neuronal tissue perturbations with morphological changes and functional alterations. Such changes were not observed in response to the long-term (3-5 weeks) miRFP670nano3 expression (data not shown), demonstrating that miRFPnanos do not cause cytotoxicity and are well tolerated in vivo.
Immunotoxins are chimeric proteins containing an antibody or other antigen binding molecules attached to a toxin. Typically, antigen-binding components are specific to surface proteins of cancer cells and mediated delivery of toxic agents, which killed targeted cells. Therefore, targeted molecules of classical immunotoxins are limited to those expressed on the cell surface. In contrast, the antigen-dependent properties of NIR-Fbs' fusions allow their use as intracellular immunotoxins, specific to targeted molecules present inside cells.
To test the utility of NIR-Fb for targeted intracellular antigen dependent killing of cells, a fusion of biNIR-FbGFP and constitutively active caspase-3 mutant V266E, which induces apoptosis, was constructed. The biNIR-FbGFP caspase/V266E fusion was expressed in the presence and absence of EGFP and cell viability was monitored by Annexin V staining. Co-transfection with biNIR-FbGFP-caspase/V266E and with EGFP resulted in cell death, similar to transfection with caspase/V266E. When mTagBFP2 was co-expressed with biNIR-FbGFP-caspase/V266E instead of EGFP, observed apoptosis level was close to that observed for control cells (
Next, a fusion of biNIR-FbGFP and diphtheria toxin A subunit (DTA), one of the most frequently used toxic component for immunotoxin development, was constructed. Co-transfection with biNIR-FbGFP-DTA and with EGFP resulted in robust cell death, similar to observed under cell transfection with DTA. Instead, the viability of cells transfected with biNIR-FbGFP-DTA, but without EGFP was close to control cells (
Next, it was determined whether NIR-Fbs can mediate specific transport of intracellular molecules for their further detection or presentation on cell membrane. For this, biNIR-FbGFP was fused to Gluc, containing natural secretion signal peptide (MGVKVLFALICIAVAE, SEQ ID NO:35). Then, cells were co-transfected with Gluc-biNIR-FbGFP and with either EGFP or mTagBFP2. Both, EGFP and mTagBFP2 did not contain any secretion signals.
Co-expression of Gluc-biNIR-FbGFP fusion with EGFP resulted in robust Gluc signal (
In bold are sequences of N- and C-terminal helices of miRFP670nano3. The sequence of miRFP670nano3 is underlined.
MANLDKMLNTTVTEVRKF
LQADRVCVFKFEEDYSGTVSHEAVDDRWISILKTQVQDRYFMETRGEEY
VHGRYQAIADIYTANLVECYRDLLIEFQVRAILAVPILQGKKLWGLLVA
HQLAGPREWQ
TWEIDFLKQQAVVMGIAIQQS
MANLDKMLNTTVTEVRKF
LQADRVCVFKFEEDYSGTVSHEAVDDRWISILKTQVQDRYFMETRGEEYV
HGRYQAIADIYTANLVECYRDLLIEFQVRAILAVPILQGKKLWGLLVAHQ
LAGPREWQ
TWEIDFLKQQAVVMGIAIQQS
MANLDKMLNTTVTEVRKF
LQADRVCVFKFEEDYSGTVSHEAVDDRWISILKTQVQDRYFMETRGEEYV
HGRYQAIADIYTANLVECYRDLLIEFQVRAILAVPILQGKKLWGLLVAHQ
LAGPREWQ
TWEIDFLKQQAVVMGIAIQQS
While the foregoing written description enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The disclosure should therefore not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the disclosure.
This invention was made with U.S. government support under Federal Grant No. GM122567 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/31015 | 5/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63194624 | May 2021 | US | |
| 63271439 | Oct 2021 | US |
