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Tyrosinase-Mediated Synthesis of Nanobody–Cell Conjugates

  • Johnathan C. Maza
    Johnathan C. Maza
    Department of Chemistry, University of California, Berkeley, California 94720, United States
    More by Johnathan C. Maza
    Orcidhttps://orcid.org/0000-0003-2898-8770
  • , 
  • Derek M. García-Almedina
    Derek M. García-Almedina
    Department of Chemistry, University of California, Berkeley, California 94720, United States
    More by Derek M. García-Almedina
    Orcidhttps://orcid.org/0000-0002-0679-6930
  • , 
  • Lydia E. Boike
    Lydia E. Boike
    Department of Chemistry, University of California, Berkeley, California 94720, United States
    Novartis-Berkeley Center for Proteomics and Chemistry Technologies, Cambridge, Massachusetts 02139, United States
    More by Lydia E. Boike
  • , 
  • Noah X. Hamlish
    Noah X. Hamlish
    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, United States
    More by Noah X. Hamlish
  • , 
  • Daniel K. Nomura
    Daniel K. Nomura
    Department of Chemistry, University of California, Berkeley, California 94720, United States
    Novartis-Berkeley Center for Proteomics and Chemistry Technologies, Cambridge, Massachusetts 02139, United States
    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, United States
    Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, California 94720, United States
    Innovative Genomics Institute, Berkeley, California 94720, United States
    More by Daniel K. Nomura
    Orcidhttps://orcid.org/0000-0003-1614-8360
  • , and 
  • Matthew B. Francis*
    Matthew B. Francis
    Department of Chemistry, University of California, Berkeley, California 94720, United States
    Materials Sciences Division, Lawrence Berkeley National Laboratories, Berkeley, California 94720,United States
    *Email: [email protected]
    More by Matthew B. Francis
    Orcidhttps://orcid.org/0000-0003-2837-2538
Cite this: ACS Cent. Sci. 2022, 8, 7, 955–962
Publication Date (Web):June 22, 2022
https://doi.org/10.1021/acscentsci.1c01265

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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A convenient enzymatic strategy is reported for the modification of cell surfaces. Using a tyrosinase enzyme isolated from Agaricus bisporus, unique tyrosine residues introduced at the C-termini of nanobodies can be site-selectively oxidized to reactive o-quinones. These reactive intermediates undergo rapid modification with nucleophilic thiol, amine, and imidazole residues present on cell surfaces, producing novel nanobody–cell conjugates that display targeted antigen binding. We extend this approach toward the synthesis of nanobody–NK cell conjugates for targeted immunotherapy applications. The resulting NK cell conjugates exhibit targeted cell binding and elicit targeted cell death.

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Synopsis

A novel methodology is presented for using tyrosinase to synthesize protein−cell conjugates while retaining cell viability and function.

Introduction

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Cells are the basic units of life and are capable of dynamically sensing and responding to their environments through biomolecular interactions on their surfaces. As such, the ability to manipulate the cell surface has a broad range of applications in basic and applied science. For example, the attachment of DNA oligonucleotides to cells has enabled their controlled adhesion to glass surfaces and gold electrodes, (1−3) and the decoration of different cells with complementary DNA strands has allowed the formation of controlled cell–cell contacts. (4) Finally, the introduction of cancer-antigen-binding paratopes to the surfaces of immune cells has led to the development of cell-based immunotherapies against a variety of cancers. (5) While genetic engineering has been a key technology in many of these applications, new chemistries are emerging for the direct attachment of complex molecules to cell surfaces. (6) These methods could help overcome challenges associated with viral transduction vectors, like batch-to-batch heterogeneity and long-term safety concerns, while also representing a means for the scalable preparation of novel cell conjugates. (7−9)
Chemistries that modify cells must perform under a narrow set of conditions in order to maintain cell viability. They must proceed in buffered aqueous media at the optimal physiological pH─typically pH 7.4─and within a temperature range of 4–37 °C. Furthermore, these reactions must have sufficiently rapid kinetics to achieve high conversion even when confronted with the limits of surface diffusion characteristics. Due to these requirements, few chemistries exist that can attach molecules and proteins to live cells. A notable example includes metabolic engineering using unnatural oligosaccharides. This enables the decoration of cell surfaces with biorthogonal azide handles for subsequent modification with phosphine- or cyclooctyne-containing cargo. (10−12) This approach has also been used to engineer new interactions between immune cells and cancer. (13) Installation of DNA molecules onto cell surfaces has enabled the hybridization-directed installation of full-length IgG onto cell surfaces with applications in immunotherapy. (14,15) Finally, enzymes like sortase, (16,17) asparaginyl ligase, (18) and fucosyltransferase (19) have all been used to catalyze the attachment of small molecules and proteins to cells.
Recently, the enzyme tyrosinase has emerged as a useful tool for site-selective protein modification. (20−23) Canonically, the enzyme acts on free tyrosine amino acids to catalyze their oxidation to highly reactive o-quinone intermediates in the biosynthesis of melanin. Over the last two decades, our lab has used these o-quinones to modify protein N-termini (especially proline N-termini) as well as free thiols present on proteins (Figure 1a). (20,21) Recent advances have shown that the tyrosinase from Agaricus bisporus (abTYR) is also capable of oxidizing engineered tyrosine tags, like Ser–Gly4–Tyr tags at protein C-termini. This produces o-quinones in specific locations on protein surfaces, which can then be intercepted by a variety of small-molecule- or protein-based nucleophiles. (21−23) This strategy has been shown previously for the site-specific modification of both tyrosine-tagged single-chain variable fragments and full-length IgG. (21−23)

Figure 1

Figure 1. General strategy for modifying cell surfaces with nanobodies. (a) Tyrosinase catalyzes the oxidation of small-molecule phenols to highly reactive o-quinones, which can modify nucleophiles present on proteins. Engineered tyrosine tags at protein termini can also be oxidized by tyrosinase, producing a site-specific o-quinone on the protein that reacts with protein-based nucleophiles. (b) Tyrosine-tagged nanobodies can be site-specifically oxidized by tyrosinase for attachment of these proteins to cells. The resulting linkage produces a well-defined point of attachment for installing nanobodies on cell surfaces while imbuing the target cell with novel antigen-binding functionality. (c) Nanobodies are low-molecular-weight (∼10–15 kDa) antigen-binders derived from the variable region of the camelid antibody (PDB ID 3K1K).

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Herein, we adapt and expand this bioconjugation chemistry to achieve the one-step attachment of tyrosine-tagged proteins to cell surfaces. In this approach, a protein of interest is expressed with a C-terminal tyrosine, such as the sequence described above. The protein can be mixed with cells of interest and abTYR for site-selective activation of the introduced tyrosine to the corresponding o-quinone intermediate. These C-terminal o-quinones then react with endogenous nucleophiles present on cell surfaces, producing well-defined points of attachment (Figure 1b).
To demonstrate the applicability of this approach, we generated tyrosine-tagged nanobodies, which are small antigen-binders derived from the variable regions of camelid immunoglobulins (Figure 1c). (24) We show that abTYR site-specifically oxidizes introduced tyrosine tags and mediates the attachment of nanobodies to cell surfaces while retaining their antigen-binding abilities. We extend this strategy toward the synthesis of nanobody–natural killer (NK) cell conjugates, which exhibited targeted cell lysis. This strategy thus provides a simple, direct synthetic alternative to genetic engineering for cell-based immunotherapy applications.

Results and Discussion

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Tyrosinase-Mediated Synthesis of Nanobody–Cell Conjugates

To begin, a previously reported nanobody against GFP was expressed with a C-terminal Ser–Gly4–Tyr tag (nbGFPTyr). (25) To test the ability of abTYR to selectively oxidize the introduced tyrosine tag, 10 μM nbGFPTyr was exposed to 400 nM abTYR at 37 °C. A time-course experiment was performed, and analysis using ESI-TOF mass spectrometry revealed that the nbGFPTyr was fully converted to a singly oxidized product after 10 min, as indicated by a mass shift of ∼14 Da (Figure 2a and Supplementary Figure S1). Importantly, if the tyrosine residue in the tyrosine tag was swapped for a cysteine (nbGFPCys) no change in molecular weight was observed in the ESI-TOF MS trace, and no disulfide was formed. Taken together, these results indicate that it is the single phenol in the C-terminal tyrosine tag being oxidized by abTYR, providing a uniquely reactive o-quinone on the nanobody for attachment to cell surfaces (Supplementary Figure S1). This is in good agreement with previous work demonstrating the site-specific oxidation of tyrosine-tagged proteins. (21−23)

Figure 2

Figure 2. Modification of NK cell surfaces with nanobodies. (a) Tyrosinase enzyme produces a site-specific o-quinone at the C-terminal Ser–Gly4–Tyr tag installed on nanobodies, as evidenced by a 14 Da mass shift detected via ESI-TOF MS. (b) To verify that NK cell surfaces can be decorated with nanobodies using tyorsinase, a Tyr-tagged nanobody against GFP (nbGFPTyr) was designed. Using tyrosinase, nbGFPTyr can be attached to the cell surface, and 2° labeling with GFP can be used to analyze the reaction using flow cytometry. (c) Labeling experiments with nbGFPTyr validated attachment of the nanobody to the cell surface, as only cells treated with both nbGFPTyr and tyrosinase showed an increase in GFP fluorescence (red trace) over controls (blue and orange traces). (d) Using a Cys point mutant, a single FITC dye can be attached to each nbGFPTyr (nbFITC). After attachment of 10 μM nbFITC to cell surfaces, comparison against FITC-calibration beads determined that a median value of ∼120,000 copies of the nanobody were linked to the cells. Data are represented as box plots, with the top of the box representing the 75th percentile of the data, the middle line representing the median of the data, and the bottom of the box representing the 25th percentile of the data.

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Next we explored the ability of abTYR to catalyze the attachment of nbGFPTyr to cell surfaces. As a model cell system, we used NK cells, which have recently emerged as promising agents for targeted immunotherapies. Like T-cells, NK cells possess cytotoxic effector functions, and studies have shown that adoptively transferred NK cells are less prone to host rejection than their T-cell counterparts. (26,27) Additionally, methods for generating a robust supply of immortalized NK cells have been developed, enabling the scalable synthesis of engineered NK variants. NK-92MI is an immortalized NK cell line that constitutively expresses the gene for hIL-2, a cytokine required for NK cell proliferation and activation, facilitating cell culture and analysis of downstream effector functions. (28,29) However, these cells do not express Fc receptors, limiting their use in antibody-dependent cell cytotoxicity applications. Thus, the direct modification of this cell type with novel antigen-binding functionalities would provide exciting avenues for the synthesis of “off-the-shelf” NK-based immunotherapeutics.
As an initial proof of concept, ∼1 × 106 NK-92MI cells were exposed to 10 μM nbGFPTyr with 400 nM abTYR for 10 min at 37 °C in a cell incubator under 5% CO2. To validate successful attachment of the nbGFPTyr to the cell surfaces, cells were washed and subjected to a secondary labeling step with 1 μM sfGFP for 30 min (Figure 2b). After any unbound sfGFP was washed away, cells were analyzed for GFP fluorescence using flow cytometry. An increase in GFP fluorescence was only detected in the cells that had been treated with both nbGFPTyr and abTYR (Figure 2c, red trace). Cells exposed to only nbGFPTyr showed no change in fluorescence over no treatment controls (Figure 2c, blue and orange traces). These results indicate that the oxidation of nbGFPTyr by abTYR facilitates covalent attachment of the nanobody to the cell surface and that the attached nanobody retains antigen binding. Importantly, cell viability was unchanged after modification, as no increase in propidium iodide signal, a fluorescent indicator of cell death, was detected for those NK cells modified with nanobodies over controls (Supplementary Figure S2). In addition, half-life studies were also performed. It was determined that the attached nanobodies were retained on the cell surfaces with a half-life of ∼7.8 h (Supplementary Figure S3). This is comparable to other techniques that covalently attach proteins to the cell surface (14,19) and means NK cell surfaces should be returned to their unmodified state within 48 h.
To determine the number of nanobodies that were attached to the cell surface, we generated an nbGFPTyr bearing an alanine to cysteine mutation at position 76. We then site-specifically attached a maleimide–FITC dye to this cysteine (nbFITC, Supplementary Figure S4). Treatment of cells with this construct and abTYR resulted in attachment of nbFITC to the cell surface. Since a single FITC molecule is on every nanobody, comparison of the nbFITC-labeled cells to FITC calibrant beads, which contain a known number of FITC molecules, enabled the determination of the number of nanobodies on each cell surface. Using this approach, it was determined that a median of ∼120,000 copies of the nanobody were attached to each NK-92MI cell surface upon exposure to 10 μM nanobody and 400 nM abTYR for 10 min. The amount of nbFITC deposited on the cell surface can be tuned, as lower concentrations of the nanobody resulted in less modification. However, even nanobody concentrations as low as 1 μM resulted in fluorescence labeling above background levels (Figure 2d).
As a comparison, we also performed labeling reactions on Jurkat cells, an immortalized T-cell line. Approximately ∼1 × 106 Jurkat cells were incubated with varying concentrations of nbFITC in the presence or absence of 400 nM abTYR. After incubation for 10 min at 37 °C, cells were washed and analyzed for FITC fluorescence using flow cytometry. Jurkat cells exposed to both nbFITC and abTYR showed an increase in FITC signal over nbFITC only and untreated controls (Supplementary Figure S5). Once again, cell viability remained unchanged after modification, as no increase in propidium iodide signal was observed (Supplementary Figure S5). Comparison against FITC-calibration beads showed that using 10 μM nbFITC and 400 nM abTYR attached a median of ∼210,000 copies of the nanobodies to the cell surface (Supplementary Figure S5). Nanobody attachment could be tuned, with concentrations of nanobody as low as 1 μM resulting in fluorescence labeling over controls.
We also modified human PBMCs using this same approach. Gratifyingly, we see similar increases in fluorescence only when PBMCs are exposed to both 10 μM nbGFPTyr and 400 nM abTYR while retaining high cell viability (Supplementary Figure S6).
To validate that nanobody attachment was occurring at the cell surface, Jurkat cells were modified with 10 μM nbFITC in the presence or absence of 400 nM abTYR. After reaction, the cell membrane was then labeled with a cell-membrane-specific far-red dye. Imaging using a confocal microscope revealed distinct halos of FITC signal only in the cells treated with nbFITC and abTYR, which colocalized with the cell membrane dye (Supplementary Figure S7). Some nonspecific binding of nbFITC was observed in cells treated with nbFITC in the absence of abTYR, but no clear halo was observed.
Jurkat cells are a robust cell line and can be easily cultured to produce large quantities of cells. As a result, they are a useful cell line for proteomics experiments, which often require large amounts of cellular material. To understand the nature of the site of attachment between the cell surface and tyrosine-tagged nanobodies, we designed a proteomics experiment using an alkynyl-tyramide handle. This small-molecule probe has a phenol moiety for abTYR activation and attachment to cellular proteins as well as a terminal-alkyne moiety for reaction with azide-bearing molecules in a Cu(II)-mediated azido-alkyne cycloaddition. To ∼100 × 106 Jurkat cells was added 100 μM alkynyl-tyramide probe and 400 nM abTYR to a final volume of 10 mL. Cells were incubated at 37 °C for 20 min and then washed. After washing, cells were lysed, and modified proteins were labeled with an N3-TEV-biotin peptide tag. Proteins were then bound to a streptavidin–agarose resin and trypsin digested. Alkynyl-tyramide probe-modified peptides were released using TEV protease and then analyzed using mass spectrometry (Supplementary Figure S8).
Proteomic analysis identified 437 unique peptide sequences modified with the alkynyl-tyramide probe. Analysis of the amino acid residues modified with the probe identified lysine (50.6% of modified residues), histidine (30.2% of modified residues), and cysteine (19.2% of modified residues) as the nucleophilic sites responsible for modification (Supplementary Figure S8). While modification of thiol groups with abTYR-generated o-quinones has already been established, to the best of our knowledge, this is one of the first examples of o-quinones reacting with the amines and imidazoles of lysine and histidine. In purified protein labeling reactions, off-target modification with lysine and histidine residues has not been observed, even when the protein being modified contains a highly solvent-accessible His6 tag for purification. However, our previous work screening peptide N-termini for their ability to be modified by o-quinones found that both primary and secondary amines can be modified using this approach. (30) It is not unreasonable that reactions involving these residues are captured in our proteomics experiment. It is also possible that the modifications observed represent unique reactive contexts on the cell surface, where the heterogeneity of proteins, nucleophiles, and solvent-accessible sites combine to afford unanticipated modifications. While we are further exploring the nature of these reactions on cell surfaces, we stress that solution-based oxidative couplings using purified proteins proceed with excellent chemoselectivity. (20−22)
We also analyzed the uniquely identified proteins based on their annotated subcellular localization. We found that only ∼23% of the identified proteins represented known cell membrane or secreted proteins, with the rest of the annotations representing proteins inside the cell, suggesting that this small-molecule probe is capable of crossing the cell membrane before it quenches (Supplementary Figure S8). This is not surprising, as a paper published while this Article was under review also found that tyrosinase-generated small-molecule o-quinones can be used to label intracellular proteomes. (31)
To explore if our small-molecule proteomics data were representative of the modifications using tyrosine-tagged proteins, we performed a proteomics experiments using a biotinylated nbGFPTyr as bait (nbBAIT). After labeling the cell surface with this nanobody, we lysed the cells and captured any protein modified by the biotinylated nbBAIT protein using a streptavidin–agarose resin. The proteins were digested using trypsin, and the resulting peptides were submitted for proteomic analysis. Of the proteins captured in this experiment, only nine overlapped with the small-molecule proteomics experiment. Seven of these proteins have a known extracellular annotation and represent unique peptides in our small-molecule experiment that showed cysteine, lysine, and histidine modifications (Supplementary Figure S9).

Targeted Cell–Cell Interactions in abTYR-Synthesized Nanobody–NK Cell Conjugates

Once conditions for the attachment of nanobodies to cell surfaces had been established, we next explored the ability of nanobody–cell conjugates to engage in targeted cell–cell interactions. For this, we expressed a previously reported nanobody that binds the human epidermal growth factor receptor 2 (HER2) with the same C-terminal Ser–Gly4–Tyr tag (nbHER2Tyr). (24,32) The upregulation of HER2 is a hallmark of many breast cancers and makes targeting this receptor relevant for therapeutic purposes. Many HER2 targeting drugs are already available in the clinic where they show great efficacy against HER2+ cancers. (33)
To validate that the conjugation of nbHER2Tyr to proteins on the cell surface would not perturb HER2 binding, we synthesized a nbHER2Tyr–sfGFP conjugate using a sfGFP Y200C mutant and abTYR. When exposed to the HER2+ cell line SK-BR-3, this construct produced a distinct shift in fluorescence indicative of successful binding. In comparison, the HER2– cell line MDA-MB-468 exhibited no change in fluorescence, indicating that the o-quinoid linkage connecting the two proteins did not lead to off-target binding (Supplementary Figure S10).
Once again, 1 × 106 NK-92MI cells were treated with 10 μM nbHER2Tyr in the presence or absence of 400 nM abTYR for 10 min at 37 °C and with 5% CO2. After washing, the cells were assayed for their ability to bind FITC-labeled HER2 in solution. Only cells treated with the combination of nbHER2Tyr and abTYR were able to bind FITC-labeled HER2, as evidenced by a shift in the population of cells exhibiting fluorescence over an untreated control (Figure 3a, red trace). No shift in fluorescence was observed for cells treated with nbGFPTyr and abTYR (Figure 3a, blue trace). This indicates that antigen-binding of the conjugates is dependent on the target of the nanobody and is not influenced by the o-quionone-derived linkage between the nanobody and the cell surface.

Figure 3

Figure 3. Decoration of NK cells for nanobody-directed cell–cell interactions. (a) Using tyrosinase, a Tyr-tagged nanobody against HER2 (nbHER2Tyr) was attached to NK cells. Secondary labeling with a soluble FITC–HER2 showed that only cells exposed to nbHER2Tyr and tyrosinase exhibited a shift in FITC signal detected via flow cytometry (red trace) over controls. (b) To assess if tyrosinase-synthesized NK–nbHER2 conjugates can make targeted contacts with HER2+ cells, NK–nbHER2 cells were mixed with a HER2+ cell line (SK-BR-3) at a ratio of 2:1 (NK:target). Cells were allowed to bind and settle and then imaged using fluorscence microscopy. A nearest neighbor analysis was performed (CellProfiler), indicating that a statistically significant proportion of target cells (green) were bound to two or more NK–nbHER2 cells (red) only when the NK cells were pretreated with nbHER2Tyr and tyrosinase (orange bar). (c) Fluorescence microscopy images confirm rosette formation is only seen when NK cells are pretreated with both nbHER2Tyr and tyrosinase.

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An important component of NK engagement is the binding of these cells to cell targets. To validate that tyrosinase-synthesized NK–nbHER2Tyr could bind HER2+ cells, nanobody–NK conjugates were synthesized as described above. After synthesis, the conjugated NK cells were labeled with a MitoTracker Red dye. Simultaneously, the HER2+ cell line SK-BR-3 was labeled with a green CFSE dye, and the two cell types were mixed at a ratio of 2:1 (NK-conjugate:SK-BR-3). Cells were allowed to bind and settle for 2 h at 37 °C and 5% CO2, after which they were imaged using a high-throughput fluorescence microscope (ImageXpress Micro, Molecular Devices). A nearest neighbor analysis was performed using CellProfiler (Broad Institute) to determine the number of red-labeled NK cells touching each green-labeled target SK-BR-3 cell. A greater proportion of green target cells were bound by two or more red NK cells only if they had been pretreated with 10 μM nbHER2Tyr and 400 nM abTYR for 10 min (Figure 3b, orange bar). Absence of abTYR or conjugation with nbGFPTyr resulted in no statistically significant binding of target cells over untreated controls (Figure 3b, blue bars). Finally, a rosette pattern of binding between the green HER2+ cells and the red NK cells, commonly observed when cells are forming cell:cell contacts, was only observed for NK cells treated with nbHER2Tyr and abTYR (Figure 3c). Taken together, these results indicate that the tyrosinase-synthesized nanobody–NK conjugates form specific contacts with HER2+ cell types in a manner dependent on the antigen target of the nanobody.

Targeted Killing of nbHER2Tyr–NK Cell Conjugates

Finally, we explored the ability of tyrosinase-synthesized nanobody–NK conjugates to perform their effector functions and elicit targeted cell death. We used a fluorescence-based assay to measure NK-induced cell lysis. Briefly, HER2+ SK-BR-3 cells were preloaded with calcein AM dye, which becomes cell-impermeable after uptake. NK-induced cell death permeabilizes the cell membrane, and leakage of the dye into the supernatant allows fluorescent determination of NK cytotoxicity (Figure 4a).

Figure 4

Figure 4. Targeted cell killing elicited by tyrosinase-synthesized nanobody–NK cell conjugates. (a) Schematic representation of the fluorescence-based cell assay used to determine NK cytotoxicity. HER2+ cells (SK-BR-3) were preloaded with calcein AM dye, which is retained by the cell membrane after uptake. Lysis of the HER2+ cell releases dye into the supernatant, providing a measurement for cell lysis. Only NK cells pretreated with both nbHER2Tyr and tyrosinase (orange bar) show statistically significant specific cell lysis over control treatments. (b) To assess how the ratio of NK:target cell impacts specific cytotoxicity, NK–nbHER2 cells were synthesized using 10 μM nbHER2Tyr and 400 nM tyrosinase and mixed with calcein AM loaded HER2+ cells (SK-BR-3). Statistically significant cell death was observed at ratios even as low as 2:1 (effector:target). (c) To assess the required concentration of nbHER2Tyr needed to elict NK-mediated cell death, a variety of concentrations of nbHER2Tyr were used to label NK cells with tyrosinase. Increased lysis was observed when using 5 and 10 μM nbHER2, while a sharp reduction of NK lytic activity was observed at the higher concentration of 20 μM nbHER2Tyr.

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NK-92MI cells were treated with nbHER2Tyr in the presence or absence of abTYR. As an isotype control, NK cells were also treated with nbGFPTyr and abTYR. After treatment, cells were mixed with the calcein AM-labeled SK-BR-3 cells at a ratio of 5:1 (NK:SK-BR-3) and allowed to interact at 37 °C and 5% CO2. After 4 h, cells were spun down, and the supernatant was harvested for fluorescence determination. A statistically significant cytotoxic response was only observed in the NK cells pretreated with both nbHER2Tyr and abTYR (Figure 4a, orange bar). Exposure of NK cells to nbHER2Tyr alone did not elicit any significant response over no treatment controls, indicating that the cytotoxic response requires direct conjugation between the nanobody and the cell surface (Figure 4a). In addition, no significant cell death was observed in NK cells conjugated to nbGFPTyr via abTYR (Figure 4a). This indicates that it is not the o-quinone-derived linkage between the nanobody and the cell surface nor the exposure of the NK cells to abTYR itself that results in cell lysis. Upon varying the ratio of NK–nbHER2Tyr conjugates to SK-BR-3 cells, we found that the conjugates were capable of eliciting targeted cell death even at ratios as low as 2:1 (NK-conjugate:SK-BR-3, Figure 4b).
We also examined how different concentrations of nbHER2Tyr in the abTYR coupling step affected the lysis ability of the resulting NK cell conjugates. An increase in the ability of the NK-conjugates to elicit cell death was observed when they were exposed to nbHER2Tyr concentrations up to 10 μM. At the highest nbHERTyr concentration of 20 μM, however, the sharp decrease in cell lysis ability suggests that there is an optimal density of binding proteins on the cell surfaces, beyond which the NK cells lose their ability to engage their effector functions (Figure 4c). It is likely that very high levels of nanobody labeling preclude the binding of NK activating receptors to their cognate ligands.
One mechanism by which NK cells mediate their cytotoxic functionality is through NKG2D engagement of MICA/B on target cell surfaces. (34) As such, we explored the role of the NKG2D-MICA/B axis in the cell death mediated by our synthesized NK–nbHER2Tyr conjugates. We performed the same cell killing assays as above but blocked either NKG2D on the NK cell or MICA/B on the SK-BR-3 target cell using their respective antibodies. As expected, when MICA/B was blocked on the target cell, a statistically significant reduction in cell death was observed when compared to an isotype control (Supplementary Figure S11). Interestingly, blocking NKG2D had no effect compared to an isotype control (Supplementary Figure S11). Modification of the NKG2D receptor by our conjugated nanobody may occlude the antibody epitope. However, the marked decrease in cell killing observed after blocking MICA/B indicates that the NKG2D receptor–ligand axis can still engage and plays an important role in mediating the cell killing we have observed.

Conclusion

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Here, we have shown that the enzyme tyrosinase is capable of mediating the modification of nonengineered cell surfaces with full-size protein molecules. Using abTYR, unique tyrosine residues on the C-termini of nanobodies can be site-selectively oxidized. The resulting o-quinones undergo rapid reactions with amine- and thiol-based nucleophiles present on cell surfaces, as evidenced by proteomic analysis. Attachment of tyrosine-tagged proteins to the cell surface reached completion in ∼10 min and with no observed changes to cell viability. The resulting nanobody–cell conjugates are imbued with novel antigen-binding functionality, and we displayed the utility of this approach by synthesizing nbHER2-NK conjugates capable of eliciting targeted cell death in HER2+ model cell lines, with optimal cell killing occurring with the use of 10 μM of nbHER2Tyr. It is likely that different nanobody modification levels will be optimal for different types of immune cells and applications. A clear benefit of this approach is the ability to tune the level of modification by controlling the amount of protein used in the tyrosinase coupling reaction. Given the ease with which tyrosine tags can be introduced at protein C-termini during recombinant expression, we anticipate this approach will facilitate the attachment of a variety of different protein substrates to cell surfaces, including other tyrosine-tagged nanobodies, single-chain variable fragments, and even full-length IgG. Finally, the reliance of the method on low enzyme concentrations facilitates subsequent processing steps and offers excellent potential for the scalable synthesis of protein–cell conjugates in the future.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c01265.

  • Raw peptide lists for small-molecule proteomics (XLSX)

  • Unique peptides for small-molecule proteomics (XLSX)

  • Full experimental details and cloning procedures (PDF)

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Author Information

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  • Corresponding Author
    • Matthew B. Francis - Department of Chemistry, University of California, Berkeley, California 94720, United States;  Materials Sciences Division, Lawrence Berkeley National Laboratories, Berkeley, California 94720,United States;  Orcidhttps://orcid.org/0000-0003-2837-2538;  Email: [email protected]
  • Authors
    • Johnathan C. Maza - Department of Chemistry, University of California, Berkeley, California 94720, United States;  Orcidhttps://orcid.org/0000-0003-2898-8770
    • Derek M. García-Almedina - Department of Chemistry, University of California, Berkeley, California 94720, United States;  Orcidhttps://orcid.org/0000-0002-0679-6930
    • Lydia E. Boike - Department of Chemistry, University of California, Berkeley, California 94720, United States;  Novartis-Berkeley Center for Proteomics and Chemistry Technologies, Cambridge, Massachusetts 02139, United States
    • Noah X. Hamlish - Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, United States
    • Daniel K. Nomura - Department of Chemistry, University of California, Berkeley, California 94720, United States;  Novartis-Berkeley Center for Proteomics and Chemistry Technologies, Cambridge, Massachusetts 02139, United States;  Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, United States;  Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, California 94720, United States;  Innovative Genomics Institute, Berkeley, California 94720, United States;  Orcidhttps://orcid.org/0000-0003-1614-8360
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by the N.I.H. (R01GM138693) as well as the Chemical Biology Graduate Program at UC Berkeley (NIH T32-GM066698). J.C.M. was supported by a UC Berkeley Fellowship for Graduate Studies, and J.C.M. and L.B. were both supported by NSF Graduate Research Program Fellowships. D.M.G.A. was supported by a UC Berkeley Chancellor Fellowship. The authors thank Paul Huang for assistance in confocal imaging. The authors thank Dr. Mary West for flow cytometry and microscopy resources through the QB3 Cell and Tissue Analysis Facility.

Abbreviations

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abTYR

tyrosinase from Agaricus bisporus

sfGFP

superfolder green fluorescent protein

nbGFPTyr

GFP-binding nanobody with C-terminal Ser–Gly4–Tyr tag

nbGFPCys

GFP-binding nanobody with C-terminal Ser–Gly4–Cys tag

nbFITC

A76C mutant of nbGFPTyr, modified with FITC-maleimide

HER2

Human epidermal growth factor receptor 2

nbHER2Tyr

HER2-binding nanobody with C-terminal Ser–Gly4–Tyr tag

DPBS

Dulbecco’s phosphate buffered saline solution without Ca2+ or Mg2+

References

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    Li, Hao-Kang; Hsiao, Ching-Wen; Yang, Sen-Han; Yang, Hsiu-Ping; Wu, Tai-Sheng; Lee, Chia-Yun; Lin, Yan-Liang; Pan, Janet; Cheng, Zih-Fei; Lai, Yan-Da; Hsiao, Shih-Chia; Tang, Sai-Wen
    Cancers (2021), 13 (11), 2724CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
    Simple Summary: Chimeric antigen receptor T cell therapy has shown its potency against hematol. malignancies in autologous settings but also limited success against solid tumors with severe adverse events, including fatal cases of cytokine releasing syndrome. The aim of this research is to develop a novel off-the-shelf natural killer cell therapy against HER2-expressing cancers using Antibody-Cell Conjugation (ACC) technol. and the endogenous CD16-expressing oNK cell line. ACE1702, trastuzumab-armed oNK cells with γ irradn. and cryopreservation, present superior in vitro and in vivo potency against HER2-expressing cancer cells and shows no tumorigenic potential, indicating the clin. application fighting HER2-expressing solid tumors. These findings suggest that ACC technol. can be applied to allogeneic immune cells to provide off-the-shelf therapies for cancer patients. Natural killer (NK) cells harbor efficient cytotoxicity against tumor cells without causing life-threatening cytokine release syndrome (CRS) or graft-vs.-host disease (GvHD). When compared to chimeric antigen receptor (CAR) technol., Antibody-Cell Conjugation (ACC) technol. has been developed to provide an efficient platform to arm immune cells with cancer-targeting antibodies to recognize and attack cancer cells. Recently, we established an endogenous CD16-expressing oNK cell line (oNK) with a favorable expression pattern of NK activation/inhibitory receptors. In this study, we applied ACC platform to conjugate oNK with trastuzumab and an anti-human epidermal growth factor receptor 2 (HER2) antibody. Trastuzumab-conjugated oNK, ACE-oNK-HER2, executed in vitro and in vivo cytotoxicity against HER2-expressing cancer cells and secretion of IFNγ. The irradiated and cryopreserved ACE-oNK-HER2, designated as ACE1702, retained superior HER2-specific in vitro and in vivo potency with no tumorigenic potential. In conclusion, this study provides the evidence to support the potential clin. application of ACE1702 as a novel off-the-shelf NK cell therapy against HER2-expressing solid tumors.
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  16. 16
    Pishesha, N.; Bilate, A. M.; Wibowo, M. C.; Huang, N.-J.; Li, Z.; Deshycka, R.; Bousbaine, D.; Li, H.; Patterson, H. C.; Dougan, S. K.; Maruyama, T.; Lodish, H. F.; Ploegh, H. L. Engineered Erythrocytes Covalently Linked to Antigenic Peptides Can Protect against Autoimmune Disease. Proc. National Acad. Sci. 2017, 114, 3157– 3162,  DOI: 10.1073/pnas.1701746114
    [Crossref], [PubMed], [CAS], Google Scholar
    16
    Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease
    Pishesha, Novalia; Bilate, Angelina M.; Wibowo, Marsha C.; Huang, Nai-Jia; Li, Zeyang; Dhesycka, Rhogerry; Bousbaine, Djenet; Li, Hojun; Patterson, Heide C.; Dougan, Stephanie K.; Maruyama, Takeshi; Lodish, Harvey F.; Ploegh, Hidde L.
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (12), 3157-3162CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here the authors use the transpeptidase sortase to covalently attach disease-assocd. autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4+ and CD8+ T cells) in an antigen-specific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in exptl. autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes.
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  17. 17
    Shi, J.; Kundrat, L.; Pishesha, N.; Bilate, A.; Theile, C.; Maruyama, T.; Dougan, S. K.; Ploegh, H. L.; Lodish, H. F. Engineered Red Blood Cells as Carriers for Systemic Delivery of a Wide Array of Functional Probes. Proc. National Acad. Sci. 2014, 111, 10131– 10136,  DOI: 10.1073/pnas.1409861111
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    17
    Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes
    Shi, Jiahai; Kundrat, Lenka; Pishesha, Novalia; Bilate, Angelina; Theile, Chris; Maruyama, Takeshi; Dougan, Stephanie K.; Ploegh, Hidde L.; Lodish, Harvey F.
    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (28), 10131-10136CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. As proof of principle, we demonstrate site-specific conjugation of biotin to in vitro-differentiated mouse erythroblasts as well as to mature mouse RBCs. Thus modified, RBCs remain in the bloodstream for up to 28 d. A single domain antibody attached enzymically to RBCs enables them to bind specifically to target cells that express the antibody target. We extend these expts. to human RBCs and demonstrate efficient sortase-mediated labeling of in vitro-differentiated human reticulocytes.
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  18. 18
    Harmand, T. J.; Pishesha, N.; Rehm, F. B. H.; Ma, W.; Pinney, W. B.; Xie, Y. J.; Ploegh, H. L. Asparaginyl Ligase-Catalyzed One-Step Cell Surface Modification of Red Blood Cells. ACS Chem. Biol. 2021, 16, 1201– 1207,  DOI: 10.1021/acschembio.1c00216
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    18
    Asparaginyl Ligase-Catalyzed One-Step Cell Surface Modification of Red Blood Cells
    Harmand, Thibault J.; Pishesha, Novalia; Rehm, Fabian B. H.; Ma, Weiyi; Pinney, William B.; Xie, Yushu J.; Ploegh, Hidde L.
    ACS Chemical Biology (2021), 16 (7), 1201-1207CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Red blood cells (RBCs) can serve as vascular carriers for drugs, proteins, peptides, and nanoparticles. Human RBCs remain in the circulation for ∼120 days, are biocompatible, and are immunol. largely inert. RBCs are cleared by the reticuloendothelial system and can induce immune tolerance to foreign components attached to the RBC surface. RBC conjugates have been pursued in clin. trials to treat cancers and autoimmune diseases and to correct genetic disorders. Still, most methods used to modify RBCs require multiple steps, are resource-intensive and time-consuming, and increase the risk of inflicting damage to the RBCs. Here, we describe direct conjugation of peptides and proteins onto the surface of RBCs in a single step, catalyzed by a highly efficient, recombinant asparaginyl ligase under mild, physiol. conditions. In mice, the modified RBCs remain intact in the circulation, display a normal circulatory half-life, and retain their immune tolerance-inducing properties, as shown for protection against an accelerated model for type 1 diabetes. We conjugated different nanobodies to RBCs with retention of their binding properties, and these modified RBCs can target cancer cells in vitro. This approach provides an appealing alternative to current methods of RBC engineering. It provides ready access to more complex RBC constructs and highlights the general utility of asparaginyl ligases for the modification of native cell surfaces.
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  19. 19
    Li, J.; Chen, M.; Liu, Z.; Zhang, L.; Felding, B. H.; Moremen, K. W.; Lauvau, G.; Abadier, M.; Ley, K.; Wu, P. A Single-Step Chemoenzymatic Reaction for the Construction of Antibody–Cell Conjugates. ACS Central Sci. 2018, 4, 1633– 1641,  DOI: 10.1021/acscentsci.8b00552
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    19
    A Single-Step Chemoenzymatic Reaction for the Construction of Antibody-Cell Conjugates
    Li, Jie; Chen, Mingkuan; Liu, Zilei; Zhang, Linda; Felding, Brunie H.; Moremen, Kelley W.; Lauvau, Gregoire; Abadier, Michael; Ley, Klaus; Wu, Peng
    ACS Central Science (2018), 4 (12), 1633-1641CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    Employing live cells as therapeutics is a direction of future drug discovery. An easy and robust method to modify the surfaces of cells directly to incorporate novel functionalities is highly desirable. However, genetic methods for cell-surface engineering are laborious and limited by low efficiency for primary cell modification. Here we report a chemoenzymic approach that exploits a fucosyltransferase to transfer bio-macromols., such as an IgG antibody (MW∼ 150 kDa), to the glycocalyx on the surfaces of live cells when the antibody is conjugated to the enzyme's natural donor substrate GDP-Fucose. Requiring no genetic modification, this method is fast and biocompatible with little interference to cells' endogenous functions. We applied this method to construct two antibody-cell conjugates (ACCs) using both cell lines and primary cells, and the modified cells exhibited specific tumor targeting and resistance to inhibitory signals produced by tumor cells, resp. Remarkably, Herceptin-NK-92MI conjugates, a natural killer cell line modified with Herceptin, exhibit enhanced activities to induce the lysis of HER2+ cancer cells both ex vivo and in a human tumor xenograft model. Given the unprecedented substrate tolerance of the fucosyltransferase, this chemoenzymic method offers a general approach to engineer cells as research tools and for therapeutic applications.
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  20. 20
    Maza, J. C.; Bader, D. L. V.; Xiao, L.; Marmelstein, A. M.; Brauer, D. D.; ElSohly, A. M.; Smith, M. J.; Krska, S. W.; Parish, C. A.; Francis, M. B. Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives. J. Am. Chem. Soc. 2019, 141, 3885– 3892,  DOI: 10.1021/jacs.8b10845
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    20
    Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives
    Maza, Johnathan C.; Bader, Daniel L. V.; Xiao, Lifeng; Marmelstein, Alan M.; Brauer, Daniel D.; El Sohly, Adel M.; Smith, Matthew J.; Krska, Shane W.; Parish, Craig A.; Francis, Matthew B.
    Journal of the American Chemical Society (2019), 141 (9), 3885-3892CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A convenient enzymic strategy is reported for the modification of proline residues in the N-terminal positions of proteins. Using a tyrosinase enzyme isolated from Agaricus bisporus (abTYR), phenols and catechols are oxidized to highly reactive o-quinone intermediates that then couple to N-terminal proline residues in high yield. Key advantages of this bioconjugation method include (1) the use of air-stable precursors that can be prepd. on large scale if needed, (2) mild reaction conditions, including low temps., (3) the targeting of native functional groups that can be introduced readily on most proteins, and (4) the use of mol. oxygen as the sole oxidant. This coupling strategy was successfully demonstrated for the attachment of a variety of phenol-derivatized cargo mols. to a series of protein substrates, including self-assembled viral capsids, enzymes, and a chitin binding domain (CBD). The ability of the CBD to bind to the surfaces of yeast cells was found to be unperturbed by this modification reaction.
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  21. 21
    Lobba, M. J.; Fellmann, C.; Marmelstein, A. M.; Maza, J. C.; Kissman, E. N.; Robinson, S. A.; Staahl, B. T.; Urnes, C.; Lew, R. J.; Mogilevsky, C. S.; Doudna, J. A.; Francis, M. B. Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine–Cysteine Bond Formation. ACS Central Sci. 2020, 6, 1564– 1571,  DOI: 10.1021/acscentsci.0c00940
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    21
    Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine-Cysteine Bond Formation
    Lobba, Marco J.; Fellmann, Christof; Marmelstein, Alan M.; Maza, Johnathan C.; Kissman, Elijah N.; Robinson, Stephanie A.; Staahl, Brett T.; Urnes, Cole; Lew, Rachel J.; Mogilevsky, Casey S.; Doudna, Jennifer A.; Francis, Matthew B.
    ACS Central Science (2020), 6 (9), 1564-1571CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    The synthesis of protein-protein and protein-peptide conjugates is an important capability for producing vaccines, immunotherapeutics, and targeted delivery agents. Herein we show that the enzyme tyrosinase is capable of oxidizing exposed tyrosine residues into o-quinones that react rapidly with cysteine residues on target proteins. This coupling reaction occurs under mild aerobic conditions and has the rare ability to join full-size proteins in under 2 h. The utility of the approach is demonstrated for the attachment of cationic peptides to enhance the cellular delivery of CRISPR-Cas9 20-fold and for the coupling of reporter proteins to a cancer-targeting antibody fragment without loss of its cell-specific binding ability. The broad applicability of this technique provides a new building block approach for the synthesis of protein chimeras. Enzymic oxidn. of tyrosine residues followed by reaction with cysteine thiols allows the covalent coupling of proteins and peptides using only native amino acid side chains.
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  22. 22
    Marmelstein, A. M.; Lobba, M. J.; Mogilevsky, C. S.; Maza, J. C.; Brauer, D. D.; Francis, M. B. Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins. J. Am. Chem. Soc. 2020, 142, 5078– 5086,  DOI: 10.1021/jacs.9b12002
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    22
    Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins
    Marmelstein, Alan M.; Lobba, Marco J.; Mogilevsky, Casey S.; Maza, Johnathan C.; Brauer, Daniel D.; Francis, Matthew B.
    Journal of the American Chemical Society (2020), 142 (11), 5078-5086CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Oxidative coupling (OC) through o-quinone intermediates has been established as an efficient and site-selective way to modify protein N-termini and the unnatural amino acid p-aminophenylalanine (paF). Recently, we reported that the tyrosinase-mediated oxidn. of phenol-tagged cargo mols. is a particularly convenient method of generating o-quinones in situ. The coupling partners can be easily prepd. and stored, the reaction takes place under mild conditions (phosphate buffer, pH 6.5, 4 to 23 °C), and dissolved oxygen is the only oxidant required. Here, we show an important extension of this chem. for the activation of tyrosine residues that project into soln. from the N or C-termini of peptide and protein substrates. Generating the o-quinone electrophiles from tyrosine allows greater flexibility in choosing the nucleophilic coupling partner and expands the scope of the reaction to include C-terminal positions. We also introduce a new bacterial tyrosinase enzyme that shows improved activation for some tyrosine substrates. The efficacy of several secondary amines and aniline derivs. was evaluated in the coupling reactions, providing important information for coupling partner design. This strategy was used to modify the C-termini of an antibody scFv construct and of Protein L, a human IgG kappa light chain binding protein. The use of the modified proteins as immunolabeling agents was also demonstrated.
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  23. 23
    Bruins, J. J.; Westphal, A. H.; Albada, B.; Wagner, K.; Bartels, L.; Spits, H.; van Berkel, W. J. H.; van Delft, F. L. Inducible, Site-Specific Protein Labeling by Tyrosine Oxidation–Strain-Promoted (4 + 2) Cycloaddition. Bioconjugate Chem. 2017, 28, 1189– 1193,  DOI: 10.1021/acs.bioconjchem.7b00046
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    23
    Inducible, Site-Specific Protein Labeling by Tyrosine Oxidation-Strain-Promoted (4 + 2) Cycloaddition
    Bruins, Jorick J.; Westphal, Adrie H.; Albada, Bauke; Wagner, Koen; Bartels, Lina; Spits, Hergen; van Berkel, Willem J. H.; van Delft, Floris L.
    Bioconjugate Chemistry (2017), 28 (4), 1189-1193CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Genetically encoded tyrosine (Y-tag) can be utilized as a latent anchor for inducible and site-selective conjugation. Upon oxidn. of tyrosine with mushroom tyrosinase, strain-promoted cycloaddn. (SPOCQ) of the resulting 1,2-quinone with various bicyclo[6.1.0]nonyne (BCN) derivs. led to efficient conjugation. The method was applied for fluorophore labeling of laminarinase A and for the site-specific prepn. of an antibody-drug conjugate.
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  24. 24
    Wilton, E. E.; Opyr, M. P.; Kailasam, S.; Kothe, R. F.; Wieden, H.-J. SdAb-DB: The Single Domain Antibody Database. ACS Synth. Biol. 2018, 7, 2480– 2484,  DOI: 10.1021/acssynbio.8b00407
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    24
    sdAb-DB: The Single Domain Antibody Database
    Wilton, Emily E.; Opyr, Michael P.; Kailasam, Senthilkumar; Kothe, Ronja F.; Wieden, Hans-Joachim
    ACS Synthetic Biology (2018), 7 (11), 2480-2484CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    A review. The Single Domain Antibody Database, or sdAb-DB, (www.sdab-db.ca) is the first freely available repository for single domain antibodies and related classes of proteins. Due to their small size, modular structure, and ease of expression, single domain antibodies (sdAb) have a wide range of applications, including as a rational design tool, and are therefore of great interest for synthetic biologists and bioengineers. However, to enable effective use and sharing of existing sdAbs, including those with engineered functions (e.g., fusions with fluorescent proteins), as well as the rational design and engineering of new sdAbs, it is necessary to have access to sequences and exptl. data. We have therefore developed a publicly available, sdAb-focused database, providing access to manually curated sdAb data from protein databases, published scientific articles, and user submissions. The sdAb-DB is an open-source repository and sharing platform for the sdAb community, providing access to performance data and basic bioinformatic tools for use with previously described and validated sdAbs, as well as for the engineering of new sdAb-based designs and proteins.
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  25. 25
    Kirchhofer, A.; Helma, J.; Schmidthals, K.; Frauer, C.; Cui, S.; Karcher, A.; Pellis, M.; Muyldermans, S.; Casas-Delucchi, C. S.; Cardoso, M. C.; Leonhardt, H.; Hopfner, K.-P.; Rothbauer, U. Modulation of Protein Properties in Living Cells Using Nanobodies. Nat. Struct. Mol. Biol. 2010, 17, 133– 138,  DOI: 10.1038/nsmb.1727
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    25
    Modulation of protein properties in living cells using nanobodies
    Kirchhofer, Axel; Helma, Jonas; Schmidthals, Katrin; Frauer, Carina; Cui, Sheng; Karcher, Annette; Pellis, Mireille; Muyldermans, Serge; Casas-Delucchi, Corella S.; Cardoso, M. Cristina; Leonhardt, Heinrich; Hopfner, Karl-Peter; Rothbauer, Ulrich
    Nature Structural & Molecular Biology (2010), 17 (1), 133-138CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural anal. of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor (ER). This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.
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  26. 26
    Morvan, M. G.; Lanier, L. L. NK Cells and Cancer: You Can Teach Innate Cells New Tricks. Nat. Rev. Cancer. 2016, 16, 7– 19,  DOI: 10.1038/nrc.2015.5
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    26
    NK cells and cancer: you can teach innate cells new tricks
    Morvan, Maelig G.; Lanier, Lewis L.
    Nature Reviews Cancer (2016), 16 (1), 7-19CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
    Natural killer (NK) cells are the prototype innate lymphoid cells endowed with potent cytolytic function that provide host defense against microbial infection and tumors. Here, we review evidence for the role of NK cells in immune surveillance against cancer and highlight new therapeutic approaches for targeting NK cells in the treatment of cancer.
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  27. 27
    Bald, T.; Krummel, M. F.; Smyth, M. J.; Barry, K. C. The NK Cell–Cancer Cycle: Advances and New Challenges in NK Cell–Based Immunotherapies. Nat. Immunol. 2020, 21, 835– 847,  DOI: 10.1038/s41590-020-0728-z
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    27
    The NK cell-cancer cycle: advances and new challenges in NK cell-based immunotherapies
    Bald, Tobias; Krummel, Matthew F.; Smyth, Mark J.; Barry, Kevin C.
    Nature Immunology (2020), 21 (8), 835-847CODEN: NIAMCZ; ISSN:1529-2908. (Nature Research)
    A review. Abstr.: Natural killer (NK) cells belong to the innate immune system and contribute to protecting the host through killing of infected, foreign, stressed or transformed cells. Addnl., via cellular cross-talk, NK cells orchestrate antitumor immune responses. Hence, significant efforts have been undertaken to exploit the therapeutic properties of NK cells in cancer. Current strategies in preclin. and clin. development include adoptive transfer therapies, direct stimulation, recruitment of NK cells into the tumor microenvironment (TME), blockade of inhibitory receptors that limit NK cell functions, and therapeutic modulation of the TME to enhance antitumor NK cell function. In this Review, we introduce the NK cell-cancer cycle to highlight recent advances in NK cell biol. and to discuss the progress and problems of NK cell-based cancer immunotherapies.
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  28. 28
    Tam, Y. K.; Maki, G.; Miyagawa, B.; Hennemann, B.; Tonn, T.; Klingemann, H.-G. Characterization of Genetically Altered, Interleukin 2-Independent Natural Killer Cell Lines Suitable for Adoptive Cellular Immunotherapy. Hum. Gene Ther. 1999, 10, 1359– 1373,  DOI: 10.1089/10430349950018030
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    28
    Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy
    Tam, Y. K.; Maki, G.; Miyagawa, B.; Hennemann, B.; Tonn, T.; Klingemann, H.-G.
    Human Gene Therapy (1999), 10 (8), 1359-1373CODEN: HGTHE3; ISSN:1043-0342. (Mary Ann Liebert, Inc.)
    NK-92 is a highly cytotoxic natural killer (NK) tumor cell line that possesses properties that make it an excellent candidate for adoptive cellular immunotherapy. However, the cytotoxicity of NK cells is dependent on cytokines such as interleukin 2 (IL-2). Although NK-92 cells maintain cytotoxicity for a time after withdrawal of IL-2, clin. use will probably require prolonged treatment with fully activated cells to eliminate disease effectively. The ability to support cytotoxic cells with exogenously administered IL-2 is limited by assocd. toxicity. Therefore, the authors describe the transfection of the IL-2-dependent NK-92 cell line with human IL-2 (hIL-2) cDNA by particle-mediated gene transfer to create two IL-2-independent variants, NK-92MI and NK-92CI, and describe their characterization and comparison with parental cells. Both variants were shown to contain, express, and synthesize the hIL-2 cDNA. IL-2 synthesis was higher in NK-92MI cells compared with NK-92CI cells, with no expression in parental cells. Functionally, the cytotoxicity of all three cell lines was similar and coincubation with IL-2-independent variants did not affect hematopoietic progenitor cells. NK-92MI and NK-92CI cells were more radiosensitive than NK-92 cells, with proliferation inhibited at lower radiation doses and increased morality and decreased cytotoxicity compared with parental cells. Data presented here show that the authors have created by particle-mediated gene transfer two IL-2-independent variants of NK-92 that are identical to parental cells in virtually all respects, including high cytotoxic activity. The nonviral transfection of these cells makes them suitable for clin. applications. These IL-2-independent cells should allow prolonged treatment with fully active natural killer cells without the need for exogenous IL-2 support.
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  29. 29
    Suck, G.; Odendahl, M.; Nowakowska, P.; Seidl, C.; Wels, W. S.; Klingemann, H. G.; Tonn, T. NK-92: An ‘off-the-Shelf Therapeutic’ for Adoptive Natural Killer Cell-Based Cancer Immunotherapy. Cancer Immunol. Immunother. 2016, 65, 485– 492,  DOI: 10.1007/s00262-015-1761-x
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    29
    NK-92: an 'off-the-shelf therapeutic' for adoptive natural killer cell-based cancer immunotherapy
    Suck, Garnet; Odendahl, Marcus; Nowakowska, Paulina; Seidl, Christian; Wels, Winfried S.; Klingemann, Hans G.; Tonn, Torsten
    Cancer Immunology Immunotherapy (2016), 65 (4), 485-492CODEN: CIIMDN; ISSN:0340-7004. (Springer)
    Natural killer (NK) cells are increasingly considered as immunotherapeutic agents in particular in the fight against cancers. NK cell therapies are potentially broadly applicable and, different from their T cell counterparts, do not cause graft-vs.-host disease. Efficacy and clin. in vitro or in vivo expansion of primary NK cells will however always remain variable due to individual differences of donors or patients. Long-term storage of clin. NK cell lots to allow repeated clin. applications remains an addnl. challenge. In contrast, the established and well-characterized cell line NK-92 can be easily and reproducibly expanded from a good manufg. practice (GMP)-compliant cryopreserved master cell bank. Moreover, no cost-intensive cell purifn. methods are required. To date, NK-92 has been intensively studied. The cells displayed superior cytotoxicity against a no. of tumor types tested, which was confirmed in preclin. mouse studies. Subsequent clin. testing demonstrated safety of NK-92 infusions even at high doses. Despite the phase I nature of the trials conducted so far, some efficacy was noted, particularly against lung tumors. Furthermore, to overcome tumor resistance and for specific targeting, NK-92 has been engineered to express a no. of different chimeric antigen receptors (CARs), including targeting, for example, CD19 or CD20 (anti-B cell malignancies), CD38 (anti-myeloma) or human epidermal growth factor receptor 2 (HER2; ErbB2; anti-epithelial cancers). The concept of an NK cell line as an allogeneic cell therapeutic produced 'off-the-shelf' on demand holds great promise for the development of effective treatments.
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  30. 30
    Obermeyer, A. C.; Jarman, J. B.; Francis, M. B. N-Terminal Modification of Proteins with O-Aminophenols. J. Am. Chem. Soc. 2014, 136, 9572– 9579,  DOI: 10.1021/ja500728c
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    N-Terminal Modification of Proteins with o-Aminophenols
    Obermeyer, Allie C.; Jarman, John B.; Francis, Matthew B.
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    The synthetic modification of proteins plays an important role in chem. biol. and biomaterials science. These fields provide a const. need for chem. tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concns. of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.
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    Hurben, A. K.; Erber, L. N.; Tretyakova, N. Y.; Doran, T. M. Proteome-Wide Profiling of Cellular Targets Modified by Dopamine Metabolites Using a Bio-Orthogonally Functionalized Catecholamine. ACS Chem. Biol. 2021, 16, 2581– 2594,  DOI: 10.1021/acschembio.1c00629
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    Proteome-Wide Profiling of Cellular Targets Modified by Dopamine Metabolites Using a Bio-Orthogonally Functionalized Catecholamine
    Hurben, Alexander K.; Erber, Luke N.; Tretyakova, Natalia Y.; Doran, Todd M.
    ACS Chemical Biology (2021), 16 (11), 2581-2594CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Selective death of midbrain dopaminergic neurons is a hallmark pathol. of Parkinson's disease (PD), but the mol. mechanisms that initiate the cascade of events resulting in neurodegeneration in PD remain unclear. Compelling evidence suggests that dysregulation of dopamine (DA) induces neuronal stress and damage responses that are operative processes in striatal degeneration preceding PD-like symptoms. Improper DA sequestration to vesicles raises cytosolic DA levels, which is rapidly converted into electrophilic dopaquinone species (DQs) that react readily with protein nucleophiles forming covalent modifications that alter the native structure and function of proteins. These so-called DA-protein adducts (DPAs) have been reported to play a role in neurotoxicity, and their abundance with respect to neurodegeneration has been linked to clin. and pathol. features of PD that suggest that they play a causal role in PD pathogenesis. Therefore, characterizing DPAs is a crit. first step in understanding the susceptibility of midbrain dopaminergic neurons during PD. To help achieve this goal, we report here a novel DA-mimetic (DAyne) contg. a biorthogonal alkyne handle that exhibits a reactivity profile similar to DA in aq. buffers. By linking DPAs formed with DAyne to a fluorescent reporter mol., DPAs were visualized in fixed cells and within lysates. DAyne enabled global mapping of cellular proteins affected by DQ modification and their bioactive pathways through enrichment. Our proteomic profiling of DPAs in neuronal SH-SY5Y cells indicates that proteins susceptible to DPA formation are extant throughout the proteome, potentially influencing several diverse biol. pathways involved in PD such as endoplasmic reticulum (ER) stress, cytoskeletal instability, proteotoxicity, and clathrin function. We validated that a protein involved in the ER stress pathway, protein disulfide isomerase 3 (PDIA3), which was enriched in our chemoproteomic anal., is functionally inhibited by DA, providing evidence that dysregulated cellular DA may induce or exacerbate ER stress. Thus, DAyne provided new mechanistic insights into DA toxicity that may be obsd. during PD by enabling characterization of DPAs generated reproducibly at physiol. relevant quinone exposures. We anticipate our design and application of this reactivity-based probe will be generally applicable for clarifying mechanisms of metabolic quinone toxicity.
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  • Abstract

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    Figure 1

    Figure 1. General strategy for modifying cell surfaces with nanobodies. (a) Tyrosinase catalyzes the oxidation of small-molecule phenols to highly reactive o-quinones, which can modify nucleophiles present on proteins. Engineered tyrosine tags at protein termini can also be oxidized by tyrosinase, producing a site-specific o-quinone on the protein that reacts with protein-based nucleophiles. (b) Tyrosine-tagged nanobodies can be site-specifically oxidized by tyrosinase for attachment of these proteins to cells. The resulting linkage produces a well-defined point of attachment for installing nanobodies on cell surfaces while imbuing the target cell with novel antigen-binding functionality. (c) Nanobodies are low-molecular-weight (∼10–15 kDa) antigen-binders derived from the variable region of the camelid antibody (PDB ID 3K1K).

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    Figure 2

    Figure 2. Modification of NK cell surfaces with nanobodies. (a) Tyrosinase enzyme produces a site-specific o-quinone at the C-terminal Ser–Gly4–Tyr tag installed on nanobodies, as evidenced by a 14 Da mass shift detected via ESI-TOF MS. (b) To verify that NK cell surfaces can be decorated with nanobodies using tyorsinase, a Tyr-tagged nanobody against GFP (nbGFPTyr) was designed. Using tyrosinase, nbGFPTyr can be attached to the cell surface, and 2° labeling with GFP can be used to analyze the reaction using flow cytometry. (c) Labeling experiments with nbGFPTyr validated attachment of the nanobody to the cell surface, as only cells treated with both nbGFPTyr and tyrosinase showed an increase in GFP fluorescence (red trace) over controls (blue and orange traces). (d) Using a Cys point mutant, a single FITC dye can be attached to each nbGFPTyr (nbFITC). After attachment of 10 μM nbFITC to cell surfaces, comparison against FITC-calibration beads determined that a median value of ∼120,000 copies of the nanobody were linked to the cells. Data are represented as box plots, with the top of the box representing the 75th percentile of the data, the middle line representing the median of the data, and the bottom of the box representing the 25th percentile of the data.

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    Figure 3. Decoration of NK cells for nanobody-directed cell–cell interactions. (a) Using tyrosinase, a Tyr-tagged nanobody against HER2 (nbHER2Tyr) was attached to NK cells. Secondary labeling with a soluble FITC–HER2 showed that only cells exposed to nbHER2Tyr and tyrosinase exhibited a shift in FITC signal detected via flow cytometry (red trace) over controls. (b) To assess if tyrosinase-synthesized NK–nbHER2 conjugates can make targeted contacts with HER2+ cells, NK–nbHER2 cells were mixed with a HER2+ cell line (SK-BR-3) at a ratio of 2:1 (NK:target). Cells were allowed to bind and settle and then imaged using fluorscence microscopy. A nearest neighbor analysis was performed (CellProfiler), indicating that a statistically significant proportion of target cells (green) were bound to two or more NK–nbHER2 cells (red) only when the NK cells were pretreated with nbHER2Tyr and tyrosinase (orange bar). (c) Fluorescence microscopy images confirm rosette formation is only seen when NK cells are pretreated with both nbHER2Tyr and tyrosinase.

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    Figure 4

    Figure 4. Targeted cell killing elicited by tyrosinase-synthesized nanobody–NK cell conjugates. (a) Schematic representation of the fluorescence-based cell assay used to determine NK cytotoxicity. HER2+ cells (SK-BR-3) were preloaded with calcein AM dye, which is retained by the cell membrane after uptake. Lysis of the HER2+ cell releases dye into the supernatant, providing a measurement for cell lysis. Only NK cells pretreated with both nbHER2Tyr and tyrosinase (orange bar) show statistically significant specific cell lysis over control treatments. (b) To assess how the ratio of NK:target cell impacts specific cytotoxicity, NK–nbHER2 cells were synthesized using 10 μM nbHER2Tyr and 400 nM tyrosinase and mixed with calcein AM loaded HER2+ cells (SK-BR-3). Statistically significant cell death was observed at ratios even as low as 2:1 (effector:target). (c) To assess the required concentration of nbHER2Tyr needed to elict NK-mediated cell death, a variety of concentrations of nbHER2Tyr were used to label NK cells with tyrosinase. Increased lysis was observed when using 5 and 10 μM nbHER2, while a sharp reduction of NK lytic activity was observed at the higher concentration of 20 μM nbHER2Tyr.

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    This article references 34 other publications.

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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXpvVWqtbc%253D&md5=2a9955bfe0a5a1d915a7d58063464149
    8. 8
      Shearer, R. F.; Saunders, D. N. Experimental Design for Stable Genetic Manipulation in Mammalian Cell Lines: Lentivirus and Alternatives. Genes Cells. 2015, 20, 1– 10,  DOI: 10.1111/gtc.12183
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      8
      Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives
      Shearer, Robert F.; Saunders, Darren N.
      Genes to Cells (2015), 20 (1), 1-10CODEN: GECEFL; ISSN:1356-9597. (Wiley-Blackwell)
      A review. The use of third-generation lentiviral vectors is now commonplace in most areas of basic biol. These systems provide a fast, efficient means for modulating gene expression, but exptl. design needs to be carefully considered to minimize potential artifacts arising from off-target effects and other confounding factors. This review offers a starting point for those new to lentiviral-based vector systems, addressing the main issues involved with the use of lentiviral systems in vitro and outlines considerations which should be taken into account during exptl. design. Factors such as selecting an appropriate system and controls, and practical titrn. of viral transduction are important considerations for exptl. design. We also briefly describe some of the more recent advances in genome editing technol. TALENs and CRISPRs offer an alternative to lentivirus, providing endogenous gene editing with reduced off-target effects often at the expense of efficiency.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXislSgsA%253D%253D&md5=061a2b69ebbb5d5f10a0ad17ce935c07
    9. 9
      Kohn, D. B.; Sadelain, M.; Glorioso, J. C. Occurrence of Leukaemia Following Gene Therapy of X-Linked SCID. Nat. Rev. Cancer. 2003, 3, 477– 488,  DOI: 10.1038/nrc1122
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      9
      Occurrence of leukemia following gene therapy of X-linked SCID
      Kohn, Donald B.; Sadelain, Michel; Glorioso, Joseph C.
      Nature Reviews Cancer (2003), 3 (7), 477-488CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
      A review. Recombinant viral vectors have allowed gene transfer to be developed as a promising approach to the treatment of genetic diseases. Recently, gene therapy of children with X-linked severe combined immune deficiency resulted in impressive levels of immune reconstitution - a triumph that was later overshadowed by the development of leukemia in 2 patients. What were the causes of this cancer, and how can the therapeutic benefits of gene therapy be achieved while minimizing risk to the patient.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXltVSqtrw%253D&md5=a51e02e95ee60bd85e3a30a4767d8b98
    10. 10
      Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Chemical Remodelling of Cell Surfaces in Living Animals. Nature. 2004, 430, 873– 877,  DOI: 10.1038/nature02791
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      10
      Chemical remodelling of cell surfaces in living animals
      Prescher, Jennifer A.; Dube, Danielle H.; Bertozzi, Carolyn R.
      Nature (London, United Kingdom) (2004), 430 (7002), 873-877CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Cell surfaces are endowed with biol. functionality designed to mediate extracellular communication. The cell-surface repertoire can be expanded to include abiotic functionality through the biosynthetic introduction of unnatural sugars into cellular glycans, a process termed metabolic oligosaccharide engineering. This technique has been exploited in fundamental studies of glycan-dependent cell-cell and virus-cell interactions and also provides an avenue for the chem. remodelling of living cells. Unique chem. functional groups can be delivered to cell-surface glycans by metab. of the corresponding unnatural precursor sugars. These functional groups can then undergo covalent reaction with exogenous agents bearing complementary functionality. The exquisite chem. selectivity required of this process is supplied by the Staudinger ligation of azides and phosphines, a reaction that has been performed on cultured cells without detriment to their physiol. Here we demonstrate that the Staudinger ligation can be executed in living animals, enabling the chem. modification of cells within their native environment. The ability to tag cell-surface glycans in vivo may enable therapeutic targeting and non-invasive imaging of changes in glycosylation during disease progression.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmslCnu70%253D&md5=add5fdac7607d716a5564137ca6c06ba
    11. 11
      Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Copper-Free Click Chemistry for Dynamic in Vivo Imaging. Proc. National Acad. Sci. 2007, 104, 16793– 16797,  DOI: 10.1073/pnas.0707090104
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      11
      Copper-free click chemistry for dynamic in vivo imaging
      Baskin, Jeremy M.; Prescher, Jennifer A.; Laughlin, Scott T.; Agard, Nicholas J.; Chang, Pamela V.; Miller, Isaac A.; Lo, Anderson; Codelli, Julian A.; Bertozzi, Carolyn R.
      Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (43), 16793-16797CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomols. such as glycans and lipids. Here, the authors report a Cu-free variant of click chem. that can label these biomols. rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The crit. reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3+2] dipolar cycloaddn. with azides installed metabolically into biomols. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, the authors studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.
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    12. 12
      Saxon, E.; Bertozzi, C. R. Cell Surface Engineering by a Modified Staudinger Reaction. Science. 2000, 287, 2007– 2010,  DOI: 10.1126/science.287.5460.2007
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      12
      Cell surface engineering by a modified Staudinger reaction
      Saxon, Eliana; Bertozzi, Carolyn R.
      Science (Washington, D. C.) (2000), 287 (5460), 2007-2010CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Selective chem. reactions enacted within a cellular environment can be powerful tools for elucidating biol. processes or engineering novel interactions. A chem. transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chem. orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metab. of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.
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    13. 13
      Wang, X.; Lang, S.; Tian, Y.; Zhang, J.; Yan, X.; Fang, Z.; Weng, J.; Lu, N.; Wu, X.; Li, T.; Cao, H.; Li, Z.; Huang, X. Glycoengineering of Natural Killer Cells with CD22 Ligands for Enhanced Anticancer Immunotherapy. ACS Central Sci. 2020, 6, 382– 389,  DOI: 10.1021/acscentsci.9b00956
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      13
      Glycoengineering of Natural Killer Cells with CD22 Ligands for Enhanced Anticancer Immunotherapy
      Wang, Xianwu; Lang, Shuyao; Tian, Yunpeng; Zhang, Jianghong; Yan, Xu; Fang, Zhihong; Weng, Jian; Lu, Na; Wu, Xuanjun; Li, Tianlu; Cao, Hongzhi; Li, Zhu; Huang, Xuefei
      ACS Central Science (2020), 6 (3), 382-389CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Adoptive transfer of immune cells is being actively pursued for cancer treatment. Natural killer (NK) cells, a class of cytotoxic immune cells, generally lack inherent selectivities toward cancer. To bestow tumor-targeting abilities and enhance anticancer efficacy, a new strategy is established to glycoengineer NK cells. Carbohydrate-based ligands for CD22, a marker for B cell lymphoma, are introduced onto NK cells through either metabolic engineering or glyco-polymer insertion. Such NK cells exhibited greatly enhanced cytotoxicities toward CD22+ lymphoma cells in a CD22-dependent manner. Importantly, both CD22+ lymphoma cell lines and primary lymphoma cells from human cancer patients can be effectively killed by the engineered NK cells. Furthermore, glycoengineered NK cells provided significant protection to tumor-bearing mice. Thus, NK cell glycoengineering is an exciting new approach for cancer treatment complementing the current immune cell genetic engineering strategy. Sweetening natural killer cells enhanced their tumor-killing abilities in vitro and reduced tumor growth in vivo.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkt1yks70%253D&md5=5f6f36cf07a6f0428feb33487bfe6807
    14. 14
      Frank, M. J.; Olsson, N.; Huang, A.; Tang, S.-W.; Negrin, R. S.; Elias, J. E.; Meyer, E. H. A Novel Antibody-Cell Conjugation Method to Enhance and Characterize Cytokine-Induced Killer Cells. Cytotherapy. 2020, 22, 135– 143,  DOI: 10.1016/j.jcyt.2020.01.003
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      14
      A novel antibody-cell conjugation method to enhance and characterize cytokine-induced killer cells
      Frank, Matthew J.; Olsson, Niclas; Huang, Andy; Tang, Sai-Wen; Negrin, Robert S.; Elias, Joshua E.; Meyer, Everett H.
      Cytotherapy (2020), 22 (3), 135-143CODEN: CYTRF3; ISSN:1465-3249. (Elsevier Inc.)
      Cytokine-induced killer (CIK) cells are an ex vivo-expanded cellular therapy product with potent anti-tumor activity in a subset of patients with solid and hematol. malignancies. We hypothesize that directing CIK cells to a specific tumor antigen will enhance CIK cell anti-tumor cytotoxicity. We present a newly developed method for affixing antibodies directly to cell surface proteins. First, we evaluated the anti-tumor potential of CIK cells after affixing tumor-antigen targeting monoclonal antibodies. Second, we evaluated whether this antibody-conjugation method can profile the surface proteome of CIK cells. We demonstrated that affixing rituximab or daratumumab to CIK cells enhances cytotoxic killing of multiple lymphoma cell lines in vitro. These armed CIK cells exhibited enhanced intracellular signaling after engaging tumor targets. Cell surface proteome profiling suggested mechanisms by which antibody-armed CIK cells concurrently activated multiple surface proteins, leading to enhanced cytolytic activity. Our surface proteome anal. indicated that CIK cells display enhanced protein signatures indicative of immune responses, cellular activation and leukocyte migration. Here, we characterize the cell surface proteome of CIK cells using a novel methodol. that can be rapidly applied to other cell types. Without genetic modification CIK cells can be rapidly armed with monoclonal antibodies, which endows them with high specificity to kill tumor targets.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXltFykt7w%253D&md5=c61c8f677e77dd3c8edd1a4c3e24f9d4
    15. 15
      Li, H.-K.; Hsiao, C.-W.; Yang, S.-H.; Yang, H.-P.; Wu, T.-S.; Lee, C.-Y.; Lin, Y.-L.; Pan, J.; Cheng, Z.-F.; Lai, Y.-D.; Hsiao, S.-C.; Tang, S.-W. A Novel Off-the-Shelf Trastuzumab-Armed NK Cell Therapy (ACE1702) Using Antibody-Cell-Conjugation Technology. Cancers. 2021, 13 (11), 2724,  DOI: 10.3390/cancers13112724
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      15
      A Novel off-the-Shelf Trastuzumab-Armed NK Cell Therapy (ACE1702) Using Antibody-Cell-Conjugation Technology
      Li, Hao-Kang; Hsiao, Ching-Wen; Yang, Sen-Han; Yang, Hsiu-Ping; Wu, Tai-Sheng; Lee, Chia-Yun; Lin, Yan-Liang; Pan, Janet; Cheng, Zih-Fei; Lai, Yan-Da; Hsiao, Shih-Chia; Tang, Sai-Wen
      Cancers (2021), 13 (11), 2724CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
      Simple Summary: Chimeric antigen receptor T cell therapy has shown its potency against hematol. malignancies in autologous settings but also limited success against solid tumors with severe adverse events, including fatal cases of cytokine releasing syndrome. The aim of this research is to develop a novel off-the-shelf natural killer cell therapy against HER2-expressing cancers using Antibody-Cell Conjugation (ACC) technol. and the endogenous CD16-expressing oNK cell line. ACE1702, trastuzumab-armed oNK cells with γ irradn. and cryopreservation, present superior in vitro and in vivo potency against HER2-expressing cancer cells and shows no tumorigenic potential, indicating the clin. application fighting HER2-expressing solid tumors. These findings suggest that ACC technol. can be applied to allogeneic immune cells to provide off-the-shelf therapies for cancer patients. Natural killer (NK) cells harbor efficient cytotoxicity against tumor cells without causing life-threatening cytokine release syndrome (CRS) or graft-vs.-host disease (GvHD). When compared to chimeric antigen receptor (CAR) technol., Antibody-Cell Conjugation (ACC) technol. has been developed to provide an efficient platform to arm immune cells with cancer-targeting antibodies to recognize and attack cancer cells. Recently, we established an endogenous CD16-expressing oNK cell line (oNK) with a favorable expression pattern of NK activation/inhibitory receptors. In this study, we applied ACC platform to conjugate oNK with trastuzumab and an anti-human epidermal growth factor receptor 2 (HER2) antibody. Trastuzumab-conjugated oNK, ACE-oNK-HER2, executed in vitro and in vivo cytotoxicity against HER2-expressing cancer cells and secretion of IFNγ. The irradiated and cryopreserved ACE-oNK-HER2, designated as ACE1702, retained superior HER2-specific in vitro and in vivo potency with no tumorigenic potential. In conclusion, this study provides the evidence to support the potential clin. application of ACE1702 as a novel off-the-shelf NK cell therapy against HER2-expressing solid tumors.
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    16. 16
      Pishesha, N.; Bilate, A. M.; Wibowo, M. C.; Huang, N.-J.; Li, Z.; Deshycka, R.; Bousbaine, D.; Li, H.; Patterson, H. C.; Dougan, S. K.; Maruyama, T.; Lodish, H. F.; Ploegh, H. L. Engineered Erythrocytes Covalently Linked to Antigenic Peptides Can Protect against Autoimmune Disease. Proc. National Acad. Sci. 2017, 114, 3157– 3162,  DOI: 10.1073/pnas.1701746114
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      16
      Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease
      Pishesha, Novalia; Bilate, Angelina M.; Wibowo, Marsha C.; Huang, Nai-Jia; Li, Zeyang; Dhesycka, Rhogerry; Bousbaine, Djenet; Li, Hojun; Patterson, Heide C.; Dougan, Stephanie K.; Maruyama, Takeshi; Lodish, Harvey F.; Ploegh, Hidde L.
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (12), 3157-3162CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here the authors use the transpeptidase sortase to covalently attach disease-assocd. autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4+ and CD8+ T cells) in an antigen-specific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in exptl. autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjvVyjs7w%253D&md5=6a129adca06524e386953054bc3dd62e
    17. 17
      Shi, J.; Kundrat, L.; Pishesha, N.; Bilate, A.; Theile, C.; Maruyama, T.; Dougan, S. K.; Ploegh, H. L.; Lodish, H. F. Engineered Red Blood Cells as Carriers for Systemic Delivery of a Wide Array of Functional Probes. Proc. National Acad. Sci. 2014, 111, 10131– 10136,  DOI: 10.1073/pnas.1409861111
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      17
      Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes
      Shi, Jiahai; Kundrat, Lenka; Pishesha, Novalia; Bilate, Angelina; Theile, Chris; Maruyama, Takeshi; Dougan, Stephanie K.; Ploegh, Hidde L.; Lodish, Harvey F.
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (28), 10131-10136CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. As proof of principle, we demonstrate site-specific conjugation of biotin to in vitro-differentiated mouse erythroblasts as well as to mature mouse RBCs. Thus modified, RBCs remain in the bloodstream for up to 28 d. A single domain antibody attached enzymically to RBCs enables them to bind specifically to target cells that express the antibody target. We extend these expts. to human RBCs and demonstrate efficient sortase-mediated labeling of in vitro-differentiated human reticulocytes.
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    18. 18
      Harmand, T. J.; Pishesha, N.; Rehm, F. B. H.; Ma, W.; Pinney, W. B.; Xie, Y. J.; Ploegh, H. L. Asparaginyl Ligase-Catalyzed One-Step Cell Surface Modification of Red Blood Cells. ACS Chem. Biol. 2021, 16, 1201– 1207,  DOI: 10.1021/acschembio.1c00216
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      18
      Asparaginyl Ligase-Catalyzed One-Step Cell Surface Modification of Red Blood Cells
      Harmand, Thibault J.; Pishesha, Novalia; Rehm, Fabian B. H.; Ma, Weiyi; Pinney, William B.; Xie, Yushu J.; Ploegh, Hidde L.
      ACS Chemical Biology (2021), 16 (7), 1201-1207CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Red blood cells (RBCs) can serve as vascular carriers for drugs, proteins, peptides, and nanoparticles. Human RBCs remain in the circulation for ∼120 days, are biocompatible, and are immunol. largely inert. RBCs are cleared by the reticuloendothelial system and can induce immune tolerance to foreign components attached to the RBC surface. RBC conjugates have been pursued in clin. trials to treat cancers and autoimmune diseases and to correct genetic disorders. Still, most methods used to modify RBCs require multiple steps, are resource-intensive and time-consuming, and increase the risk of inflicting damage to the RBCs. Here, we describe direct conjugation of peptides and proteins onto the surface of RBCs in a single step, catalyzed by a highly efficient, recombinant asparaginyl ligase under mild, physiol. conditions. In mice, the modified RBCs remain intact in the circulation, display a normal circulatory half-life, and retain their immune tolerance-inducing properties, as shown for protection against an accelerated model for type 1 diabetes. We conjugated different nanobodies to RBCs with retention of their binding properties, and these modified RBCs can target cancer cells in vitro. This approach provides an appealing alternative to current methods of RBC engineering. It provides ready access to more complex RBC constructs and highlights the general utility of asparaginyl ligases for the modification of native cell surfaces.
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    19. 19
      Li, J.; Chen, M.; Liu, Z.; Zhang, L.; Felding, B. H.; Moremen, K. W.; Lauvau, G.; Abadier, M.; Ley, K.; Wu, P. A Single-Step Chemoenzymatic Reaction for the Construction of Antibody–Cell Conjugates. ACS Central Sci. 2018, 4, 1633– 1641,  DOI: 10.1021/acscentsci.8b00552
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      19
      A Single-Step Chemoenzymatic Reaction for the Construction of Antibody-Cell Conjugates
      Li, Jie; Chen, Mingkuan; Liu, Zilei; Zhang, Linda; Felding, Brunie H.; Moremen, Kelley W.; Lauvau, Gregoire; Abadier, Michael; Ley, Klaus; Wu, Peng
      ACS Central Science (2018), 4 (12), 1633-1641CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Employing live cells as therapeutics is a direction of future drug discovery. An easy and robust method to modify the surfaces of cells directly to incorporate novel functionalities is highly desirable. However, genetic methods for cell-surface engineering are laborious and limited by low efficiency for primary cell modification. Here we report a chemoenzymic approach that exploits a fucosyltransferase to transfer bio-macromols., such as an IgG antibody (MW∼ 150 kDa), to the glycocalyx on the surfaces of live cells when the antibody is conjugated to the enzyme's natural donor substrate GDP-Fucose. Requiring no genetic modification, this method is fast and biocompatible with little interference to cells' endogenous functions. We applied this method to construct two antibody-cell conjugates (ACCs) using both cell lines and primary cells, and the modified cells exhibited specific tumor targeting and resistance to inhibitory signals produced by tumor cells, resp. Remarkably, Herceptin-NK-92MI conjugates, a natural killer cell line modified with Herceptin, exhibit enhanced activities to induce the lysis of HER2+ cancer cells both ex vivo and in a human tumor xenograft model. Given the unprecedented substrate tolerance of the fucosyltransferase, this chemoenzymic method offers a general approach to engineer cells as research tools and for therapeutic applications.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisVaqsrbP&md5=59cb733e3db9f5b9ffb711451afd68b4
    20. 20
      Maza, J. C.; Bader, D. L. V.; Xiao, L.; Marmelstein, A. M.; Brauer, D. D.; ElSohly, A. M.; Smith, M. J.; Krska, S. W.; Parish, C. A.; Francis, M. B. Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives. J. Am. Chem. Soc. 2019, 141, 3885– 3892,  DOI: 10.1021/jacs.8b10845
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      20
      Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives
      Maza, Johnathan C.; Bader, Daniel L. V.; Xiao, Lifeng; Marmelstein, Alan M.; Brauer, Daniel D.; El Sohly, Adel M.; Smith, Matthew J.; Krska, Shane W.; Parish, Craig A.; Francis, Matthew B.
      Journal of the American Chemical Society (2019), 141 (9), 3885-3892CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A convenient enzymic strategy is reported for the modification of proline residues in the N-terminal positions of proteins. Using a tyrosinase enzyme isolated from Agaricus bisporus (abTYR), phenols and catechols are oxidized to highly reactive o-quinone intermediates that then couple to N-terminal proline residues in high yield. Key advantages of this bioconjugation method include (1) the use of air-stable precursors that can be prepd. on large scale if needed, (2) mild reaction conditions, including low temps., (3) the targeting of native functional groups that can be introduced readily on most proteins, and (4) the use of mol. oxygen as the sole oxidant. This coupling strategy was successfully demonstrated for the attachment of a variety of phenol-derivatized cargo mols. to a series of protein substrates, including self-assembled viral capsids, enzymes, and a chitin binding domain (CBD). The ability of the CBD to bind to the surfaces of yeast cells was found to be unperturbed by this modification reaction.
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    21. 21
      Lobba, M. J.; Fellmann, C.; Marmelstein, A. M.; Maza, J. C.; Kissman, E. N.; Robinson, S. A.; Staahl, B. T.; Urnes, C.; Lew, R. J.; Mogilevsky, C. S.; Doudna, J. A.; Francis, M. B. Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine–Cysteine Bond Formation. ACS Central Sci. 2020, 6, 1564– 1571,  DOI: 10.1021/acscentsci.0c00940
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      21
      Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine-Cysteine Bond Formation
      Lobba, Marco J.; Fellmann, Christof; Marmelstein, Alan M.; Maza, Johnathan C.; Kissman, Elijah N.; Robinson, Stephanie A.; Staahl, Brett T.; Urnes, Cole; Lew, Rachel J.; Mogilevsky, Casey S.; Doudna, Jennifer A.; Francis, Matthew B.
      ACS Central Science (2020), 6 (9), 1564-1571CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      The synthesis of protein-protein and protein-peptide conjugates is an important capability for producing vaccines, immunotherapeutics, and targeted delivery agents. Herein we show that the enzyme tyrosinase is capable of oxidizing exposed tyrosine residues into o-quinones that react rapidly with cysteine residues on target proteins. This coupling reaction occurs under mild aerobic conditions and has the rare ability to join full-size proteins in under 2 h. The utility of the approach is demonstrated for the attachment of cationic peptides to enhance the cellular delivery of CRISPR-Cas9 20-fold and for the coupling of reporter proteins to a cancer-targeting antibody fragment without loss of its cell-specific binding ability. The broad applicability of this technique provides a new building block approach for the synthesis of protein chimeras. Enzymic oxidn. of tyrosine residues followed by reaction with cysteine thiols allows the covalent coupling of proteins and peptides using only native amino acid side chains.
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    22. 22
      Marmelstein, A. M.; Lobba, M. J.; Mogilevsky, C. S.; Maza, J. C.; Brauer, D. D.; Francis, M. B. Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins. J. Am. Chem. Soc. 2020, 142, 5078– 5086,  DOI: 10.1021/jacs.9b12002
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      22
      Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins
      Marmelstein, Alan M.; Lobba, Marco J.; Mogilevsky, Casey S.; Maza, Johnathan C.; Brauer, Daniel D.; Francis, Matthew B.
      Journal of the American Chemical Society (2020), 142 (11), 5078-5086CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Oxidative coupling (OC) through o-quinone intermediates has been established as an efficient and site-selective way to modify protein N-termini and the unnatural amino acid p-aminophenylalanine (paF). Recently, we reported that the tyrosinase-mediated oxidn. of phenol-tagged cargo mols. is a particularly convenient method of generating o-quinones in situ. The coupling partners can be easily prepd. and stored, the reaction takes place under mild conditions (phosphate buffer, pH 6.5, 4 to 23 °C), and dissolved oxygen is the only oxidant required. Here, we show an important extension of this chem. for the activation of tyrosine residues that project into soln. from the N or C-termini of peptide and protein substrates. Generating the o-quinone electrophiles from tyrosine allows greater flexibility in choosing the nucleophilic coupling partner and expands the scope of the reaction to include C-terminal positions. We also introduce a new bacterial tyrosinase enzyme that shows improved activation for some tyrosine substrates. The efficacy of several secondary amines and aniline derivs. was evaluated in the coupling reactions, providing important information for coupling partner design. This strategy was used to modify the C-termini of an antibody scFv construct and of Protein L, a human IgG kappa light chain binding protein. The use of the modified proteins as immunolabeling agents was also demonstrated.
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    23. 23
      Bruins, J. J.; Westphal, A. H.; Albada, B.; Wagner, K.; Bartels, L.; Spits, H.; van Berkel, W. J. H.; van Delft, F. L. Inducible, Site-Specific Protein Labeling by Tyrosine Oxidation–Strain-Promoted (4 + 2) Cycloaddition. Bioconjugate Chem. 2017, 28, 1189– 1193,  DOI: 10.1021/acs.bioconjchem.7b00046
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      23
      Inducible, Site-Specific Protein Labeling by Tyrosine Oxidation-Strain-Promoted (4 + 2) Cycloaddition
      Bruins, Jorick J.; Westphal, Adrie H.; Albada, Bauke; Wagner, Koen; Bartels, Lina; Spits, Hergen; van Berkel, Willem J. H.; van Delft, Floris L.
      Bioconjugate Chemistry (2017), 28 (4), 1189-1193CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Genetically encoded tyrosine (Y-tag) can be utilized as a latent anchor for inducible and site-selective conjugation. Upon oxidn. of tyrosine with mushroom tyrosinase, strain-promoted cycloaddn. (SPOCQ) of the resulting 1,2-quinone with various bicyclo[6.1.0]nonyne (BCN) derivs. led to efficient conjugation. The method was applied for fluorophore labeling of laminarinase A and for the site-specific prepn. of an antibody-drug conjugate.
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    24. 24
      Wilton, E. E.; Opyr, M. P.; Kailasam, S.; Kothe, R. F.; Wieden, H.-J. SdAb-DB: The Single Domain Antibody Database. ACS Synth. Biol. 2018, 7, 2480– 2484,  DOI: 10.1021/acssynbio.8b00407
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      24
      sdAb-DB: The Single Domain Antibody Database
      Wilton, Emily E.; Opyr, Michael P.; Kailasam, Senthilkumar; Kothe, Ronja F.; Wieden, Hans-Joachim
      ACS Synthetic Biology (2018), 7 (11), 2480-2484CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      A review. The Single Domain Antibody Database, or sdAb-DB, (www.sdab-db.ca) is the first freely available repository for single domain antibodies and related classes of proteins. Due to their small size, modular structure, and ease of expression, single domain antibodies (sdAb) have a wide range of applications, including as a rational design tool, and are therefore of great interest for synthetic biologists and bioengineers. However, to enable effective use and sharing of existing sdAbs, including those with engineered functions (e.g., fusions with fluorescent proteins), as well as the rational design and engineering of new sdAbs, it is necessary to have access to sequences and exptl. data. We have therefore developed a publicly available, sdAb-focused database, providing access to manually curated sdAb data from protein databases, published scientific articles, and user submissions. The sdAb-DB is an open-source repository and sharing platform for the sdAb community, providing access to performance data and basic bioinformatic tools for use with previously described and validated sdAbs, as well as for the engineering of new sdAb-based designs and proteins.
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    25. 25
      Kirchhofer, A.; Helma, J.; Schmidthals, K.; Frauer, C.; Cui, S.; Karcher, A.; Pellis, M.; Muyldermans, S.; Casas-Delucchi, C. S.; Cardoso, M. C.; Leonhardt, H.; Hopfner, K.-P.; Rothbauer, U. Modulation of Protein Properties in Living Cells Using Nanobodies. Nat. Struct. Mol. Biol. 2010, 17, 133– 138,  DOI: 10.1038/nsmb.1727
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      25
      Modulation of protein properties in living cells using nanobodies
      Kirchhofer, Axel; Helma, Jonas; Schmidthals, Katrin; Frauer, Carina; Cui, Sheng; Karcher, Annette; Pellis, Mireille; Muyldermans, Serge; Casas-Delucchi, Corella S.; Cardoso, M. Cristina; Leonhardt, Heinrich; Hopfner, Karl-Peter; Rothbauer, Ulrich
      Nature Structural & Molecular Biology (2010), 17 (1), 133-138CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural anal. of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor (ER). This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.
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    26. 26
      Morvan, M. G.; Lanier, L. L. NK Cells and Cancer: You Can Teach Innate Cells New Tricks. Nat. Rev. Cancer. 2016, 16, 7– 19,  DOI: 10.1038/nrc.2015.5
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      26
      NK cells and cancer: you can teach innate cells new tricks
      Morvan, Maelig G.; Lanier, Lewis L.
      Nature Reviews Cancer (2016), 16 (1), 7-19CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
      Natural killer (NK) cells are the prototype innate lymphoid cells endowed with potent cytolytic function that provide host defense against microbial infection and tumors. Here, we review evidence for the role of NK cells in immune surveillance against cancer and highlight new therapeutic approaches for targeting NK cells in the treatment of cancer.
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    27. 27
      Bald, T.; Krummel, M. F.; Smyth, M. J.; Barry, K. C. The NK Cell–Cancer Cycle: Advances and New Challenges in NK Cell–Based Immunotherapies. Nat. Immunol. 2020, 21, 835– 847,  DOI: 10.1038/s41590-020-0728-z
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      27
      The NK cell-cancer cycle: advances and new challenges in NK cell-based immunotherapies
      Bald, Tobias; Krummel, Matthew F.; Smyth, Mark J.; Barry, Kevin C.
      Nature Immunology (2020), 21 (8), 835-847CODEN: NIAMCZ; ISSN:1529-2908. (Nature Research)
      A review. Abstr.: Natural killer (NK) cells belong to the innate immune system and contribute to protecting the host through killing of infected, foreign, stressed or transformed cells. Addnl., via cellular cross-talk, NK cells orchestrate antitumor immune responses. Hence, significant efforts have been undertaken to exploit the therapeutic properties of NK cells in cancer. Current strategies in preclin. and clin. development include adoptive transfer therapies, direct stimulation, recruitment of NK cells into the tumor microenvironment (TME), blockade of inhibitory receptors that limit NK cell functions, and therapeutic modulation of the TME to enhance antitumor NK cell function. In this Review, we introduce the NK cell-cancer cycle to highlight recent advances in NK cell biol. and to discuss the progress and problems of NK cell-based cancer immunotherapies.
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    28. 28
      Tam, Y. K.; Maki, G.; Miyagawa, B.; Hennemann, B.; Tonn, T.; Klingemann, H.-G. Characterization of Genetically Altered, Interleukin 2-Independent Natural Killer Cell Lines Suitable for Adoptive Cellular Immunotherapy. Hum. Gene Ther. 1999, 10, 1359– 1373,  DOI: 10.1089/10430349950018030
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      28
      Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy
      Tam, Y. K.; Maki, G.; Miyagawa, B.; Hennemann, B.; Tonn, T.; Klingemann, H.-G.
      Human Gene Therapy (1999), 10 (8), 1359-1373CODEN: HGTHE3; ISSN:1043-0342. (Mary Ann Liebert, Inc.)
      NK-92 is a highly cytotoxic natural killer (NK) tumor cell line that possesses properties that make it an excellent candidate for adoptive cellular immunotherapy. However, the cytotoxicity of NK cells is dependent on cytokines such as interleukin 2 (IL-2). Although NK-92 cells maintain cytotoxicity for a time after withdrawal of IL-2, clin. use will probably require prolonged treatment with fully activated cells to eliminate disease effectively. The ability to support cytotoxic cells with exogenously administered IL-2 is limited by assocd. toxicity. Therefore, the authors describe the transfection of the IL-2-dependent NK-92 cell line with human IL-2 (hIL-2) cDNA by particle-mediated gene transfer to create two IL-2-independent variants, NK-92MI and NK-92CI, and describe their characterization and comparison with parental cells. Both variants were shown to contain, express, and synthesize the hIL-2 cDNA. IL-2 synthesis was higher in NK-92MI cells compared with NK-92CI cells, with no expression in parental cells. Functionally, the cytotoxicity of all three cell lines was similar and coincubation with IL-2-independent variants did not affect hematopoietic progenitor cells. NK-92MI and NK-92CI cells were more radiosensitive than NK-92 cells, with proliferation inhibited at lower radiation doses and increased morality and decreased cytotoxicity compared with parental cells. Data presented here show that the authors have created by particle-mediated gene transfer two IL-2-independent variants of NK-92 that are identical to parental cells in virtually all respects, including high cytotoxic activity. The nonviral transfection of these cells makes them suitable for clin. applications. These IL-2-independent cells should allow prolonged treatment with fully active natural killer cells without the need for exogenous IL-2 support.
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    29. 29
      Suck, G.; Odendahl, M.; Nowakowska, P.; Seidl, C.; Wels, W. S.; Klingemann, H. G.; Tonn, T. NK-92: An ‘off-the-Shelf Therapeutic’ for Adoptive Natural Killer Cell-Based Cancer Immunotherapy. Cancer Immunol. Immunother. 2016, 65, 485– 492,  DOI: 10.1007/s00262-015-1761-x
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      29
      NK-92: an 'off-the-shelf therapeutic' for adoptive natural killer cell-based cancer immunotherapy
      Suck, Garnet; Odendahl, Marcus; Nowakowska, Paulina; Seidl, Christian; Wels, Winfried S.; Klingemann, Hans G.; Tonn, Torsten
      Cancer Immunology Immunotherapy (2016), 65 (4), 485-492CODEN: CIIMDN; ISSN:0340-7004. (Springer)
      Natural killer (NK) cells are increasingly considered as immunotherapeutic agents in particular in the fight against cancers. NK cell therapies are potentially broadly applicable and, different from their T cell counterparts, do not cause graft-vs.-host disease. Efficacy and clin. in vitro or in vivo expansion of primary NK cells will however always remain variable due to individual differences of donors or patients. Long-term storage of clin. NK cell lots to allow repeated clin. applications remains an addnl. challenge. In contrast, the established and well-characterized cell line NK-92 can be easily and reproducibly expanded from a good manufg. practice (GMP)-compliant cryopreserved master cell bank. Moreover, no cost-intensive cell purifn. methods are required. To date, NK-92 has been intensively studied. The cells displayed superior cytotoxicity against a no. of tumor types tested, which was confirmed in preclin. mouse studies. Subsequent clin. testing demonstrated safety of NK-92 infusions even at high doses. Despite the phase I nature of the trials conducted so far, some efficacy was noted, particularly against lung tumors. Furthermore, to overcome tumor resistance and for specific targeting, NK-92 has been engineered to express a no. of different chimeric antigen receptors (CARs), including targeting, for example, CD19 or CD20 (anti-B cell malignancies), CD38 (anti-myeloma) or human epidermal growth factor receptor 2 (HER2; ErbB2; anti-epithelial cancers). The concept of an NK cell line as an allogeneic cell therapeutic produced 'off-the-shelf' on demand holds great promise for the development of effective treatments.
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    30. 30
      Obermeyer, A. C.; Jarman, J. B.; Francis, M. B. N-Terminal Modification of Proteins with O-Aminophenols. J. Am. Chem. Soc. 2014, 136, 9572– 9579,  DOI: 10.1021/ja500728c
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      30
      N-Terminal Modification of Proteins with o-Aminophenols
      Obermeyer, Allie C.; Jarman, John B.; Francis, Matthew B.
      Journal of the American Chemical Society (2014), 136 (27), 9572-9579CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The synthetic modification of proteins plays an important role in chem. biol. and biomaterials science. These fields provide a const. need for chem. tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concns. of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.
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    31. 31
      Hurben, A. K.; Erber, L. N.; Tretyakova, N. Y.; Doran, T. M. Proteome-Wide Profiling of Cellular Targets Modified by Dopamine Metabolites Using a Bio-Orthogonally Functionalized Catecholamine. ACS Chem. Biol. 2021, 16, 2581– 2594,  DOI: 10.1021/acschembio.1c00629
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      31
      Proteome-Wide Profiling of Cellular Targets Modified by Dopamine Metabolites Using a Bio-Orthogonally Functionalized Catecholamine
      Hurben, Alexander K.; Erber, Luke N.; Tretyakova, Natalia Y.; Doran, Todd M.
      ACS Chemical Biology (2021), 16 (11), 2581-2594CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Selective death of midbrain dopaminergic neurons is a hallmark pathol. of Parkinson's disease (PD), but the mol. mechanisms that initiate the cascade of events resulting in neurodegeneration in PD remain unclear. Compelling evidence suggests that dysregulation of dopamine (DA) induces neuronal stress and damage responses that are operative processes in striatal degeneration preceding PD-like symptoms. Improper DA sequestration to vesicles raises cytosolic DA levels, which is rapidly converted into electrophilic dopaquinone species (DQs) that react readily with protein nucleophiles forming covalent modifications that alter the native structure and function of proteins. These so-called DA-protein adducts (DPAs) have been reported to play a role in neurotoxicity, and their abundance with respect to neurodegeneration has been linked to clin. and pathol. features of PD that suggest that they play a causal role in PD pathogenesis. Therefore, characterizing DPAs is a crit. first step in understanding the susceptibility of midbrain dopaminergic neurons during PD. To help achieve this goal, we report here a novel DA-mimetic (DAyne) contg. a biorthogonal alkyne handle that exhibits a reactivity profile similar to DA in aq. buffers. By linking DPAs formed with DAyne to a fluorescent reporter mol., DPAs were visualized in fixed cells and within lysates. DAyne enabled global mapping of cellular proteins affected by DQ modification and their bioactive pathways through enrichment. Our proteomic profiling of DPAs in neuronal SH-SY5Y cells indicates that proteins susceptible to DPA formation are extant throughout the proteome, potentially influencing several diverse biol. pathways involved in PD such as endoplasmic reticulum (ER) stress, cytoskeletal instability, proteotoxicity, and clathrin function. We validated that a protein involved in the ER stress pathway, protein disulfide isomerase 3 (PDIA3), which was enriched in our chemoproteomic anal., is functionally inhibited by DA, providing evidence that dysregulated cellular DA may induce or exacerbate ER stress. Thus, DAyne provided new mechanistic insights into DA toxicity that may be obsd. during PD by enabling characterization of DPAs generated reproducibly at physiol. relevant quinone exposures. We anticipate our design and application of this reactivity-based probe will be generally applicable for clarifying mechanisms of metabolic quinone toxicity.
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    32. 32
      Bruce, V. J.; Lopez-Islas, M.; McNaughton, B. R. Resurfaced Cell-Penetrating Nanobodies: A Potentially General Scaffold for Intracellularly Targeted Protein Discovery. Protein Sci. 2016, 25, 1129– 1137,  DOI: 10.1002/pro.2926
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      32
      Resurfaced cell-penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery
      Bruce, Virginia J.; Lopez-Islas, Monica; McNaughton, Brian R.
      Protein Science (2016), 25 (6), 1129-1137CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease-relevant macromols. that present a challenge to small-mol. reagents. Addnl., high-throughput screening and evolution-based methods often make the discovery of new protein binders simpler than the analogous small-mol. discovery process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease-relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, in our experience, many proteins are not amenable to such extensive mutagenesis. Here, we report that nanobodies-a small and stable protein that can be evolved to recognize virtually any disease-relevant receptor-are amenable to cationic resurfacing, which results in cell internalization. Once internalized, these nanobodies access the cytosol. Polycationic resurfacing does not appreciably alter the structure, expression, and function (target recognition) of a previously reported GFP-binding nanobody, and multiple nanobody scaffolds are amenable to polycationic resurfacing. Given this, we propose that polycationic resurfaced cell-penetrating nanobodies might represent a general scaffold for intracellularly targeted protein drug discovery.
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    33. 33
      Lyon, R. Drawing Lessons from the Clinical Development of Antibody-Drug Conjugates. Drug Discovery Today Technologies. 2018, 30, 105– 109,  DOI: 10.1016/j.ddtec.2018.10.001
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      33
      Drawing lessons from the clinical development of antibody-drug conjugates
      Lyon Robert
      Drug discovery today. Technologies (2018), 30 (), 105-109 ISSN:.
      The antibody-drug conjugate (ADC) field has seen a remarkable expansion in the number of entrants in clinical studies. Many of these agents employ newer conjugation technologies that have been developed over the last decade that confer various attributes to the ADCs prepared with them, including stability, potency, and homogeneity. In many cases, these new ADCs appear demonstrably superior to earlier technologies in preclinical models of activity and toxicology, but the degree to which these improvements will translate to the clinic is only starting to be seen. Many of these technologies are now competing head-to-head by targeting the same antigen in similar patient populations, allowing for a direct comparison of their clinical performance properties. As lessons from these experiences feed back into discovery research, future iterations of ADC design may be expected to bring improved therapeutics into the clinic.
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    34. 34
      Bauer, S.; Groh, V.; Wu, J.; Steinle, A.; Phillips, J. H.; Lanier, L. L.; Spies, T. Activation of NK Cells and T Cells by NKG2D, a Receptor for Stress-Inducible MICA. Science. 1999, 285, 727– 729,  DOI: 10.1126/science.285.5428.727
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      34
      Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA
      Bauer, Stefan; Groh, Veronika; Phillips, Joseph H.; Lainer, Lewis L.; Spies, Thomas
      Science (Washington, D. C.) (1999), 285 (5428), 727-729CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Stress-inducible MICA, a distant homolog of major histocompatibility complex (MHC) class I, functions as an antigen for γδ T cells and is frequently expressed in epithelial tumors. A receptor for MICA was detected on most γδ T cells, CD8+ αβ T cells, and natural killer (NK) cells and was identified as NKG2D. Effector cells from all these subsets could be a stimulated by ligation of NKG2D. Engagement of NKG2D activated cytolytic responses of γδ T cells and NK cells against transfectants and epithelial tumor cells expressing MICA. These results define an activating immunoreceptor-MHC ligand interaction that may promote antitumor NK and T cell responses.
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