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Cytosolic delivery of inhibitory antibodieswith cationic
lipidsHejia Henry Wanga and Andrew Tsourkasb,1
aGraduate Group in Biochemistry and Molecular Biophysics,
University of Pennsylvania, Philadelphia, PA 19104; and bDepartment
of Bioengineering,University of Pennsylvania, Philadelphia, PA
19104
Edited by Richard A. Lerner, Scripps Research Institute, La
Jolla, CA, and approved September 26, 2019 (received for review
August 12, 2019)
Antibodies can be developed to directly inhibit almost any
protein,but their inability to enter the cytosol limits inhibitory
antibodiesto membrane-associated or extracellular targets.
Developing acytosolic antibody delivery systemwould offer unique
opportunitiesto directly inhibit and study intracellular protein
function. Here wedemonstrate that IgG antibodies that are
conjugated with anionicpolypeptides (ApPs) can be complexed with
cationic lipids originallydesigned for nucleic acid delivery
through electrostatic interactions,enabling close to 90% cytosolic
delivery efficiencywith only 500 nM IgG.The ApP is fused to a small
photoreactive antibody-binding domain(pAbBD) that can be
site-specifically photocrosslinked to nearly alloff-the-shelf IgGs,
enabling easy exchange of cargo IgGs. We showthat cytosolically
delivered IgGs can inhibit the drug efflux pumpmultidrug
resistance-associated protein 1 (MRP1) and the tran-scription
factor NFκB. This work establishes an approach forusing existing
antibody collections to modulate intracellular proteinfunction.
antibody | protein delivery | cytosolic | intracellular |
penetrating
Antibodies have become important research tools becausethey can
be developed to bind nearly any exposed proteinepitope with high
affinity and specificity through either tradi-tional immunization
or in vitro display approaches (1). By bindingto an appropriate
epitope, antibodies can also directly inhibit theirantigen’s
biological activity by either sterically blocking the antigenfrom
binding to interaction partners or locking the antigen inan
inactive conformation (1, 2). Indeed, microinjected antibodieshave
not only shown that antibody-dependent inhibition of intra-cellular
proteins is possible, but have also been used to uncoverthe
biological roles of oncogenes (3), stress response proteins (4),and
regulatory proteins (5, 6). Although physical delivery tech-niques
such as microinjection (3–7) or electroporation (7, 8) arevery
effective at cytosolic antibody delivery, they are low through-put
and can result in significant toxicity, significantly limiting
theutility of cytosolic antibodies as research
tools.Unsurprisingly, many alternative approaches have been ex-
plored for cytosolic antibody delivery (9, 10). For example,
anti-body fragments or antibody-like binding proteins can be
engineeredfor cytosolic stability and expressed intracellularly as
intrabodies(9, 10). Antibodies or antibody fragments can also be
fused to orincubated with cell-penetrating peptides (CPPs) to
induce theirendocytic uptake into cells followed by endosomal
escape into thecytosol (11, 12). Finally, antibodies with an innate
ability to enterthe cytosol have recently been developed in which
the deliverymoiety lies within the light chain variable region
(13).Carrier-mediated approaches for cytosolic protein delivery,
in
which cargo proteins are encapsulated by delivery lipids
orpolymers, have also been explored and capitalize on advances
innonviral nucleic acid delivery formulations that have alreadybeen
proven effective (14, 15). Although carrier-mediated cyto-solic
antibody delivery has been reported multiple times (9, 10),those
claims must be evaluated cautiously. A stringent assess-ment of
commercially available carrier-mediated antibody de-livery
platforms revealed that none were capable of cytosolicdelivery to
>6% of cells (8). However, recent progress in delivering
the Cas9 protein, which is similar in size to IgG antibodies,
withlipid nanoparticles for genome editing (16) suggests that
carrier-mediated approaches are still viable strategies for
cytosolicantibody delivery.Inspired by strategies for complexing
proteins with cationic
lipids (16), polymers (17–19), and nanoparticles (20), we
hypoth-esized that IgGs functionalized with anionic polypeptides
(ApPs)could mimic the polyanionic nature of nucleic acids and
becomplexed with cationic lipids designed for nucleic acid
de-livery. Rather than engineering IgGs directly, we fused ApPs toa
photoreactive antibody-binding domain (pAbBD) that couldbe
photocrosslinked to each heavy chain of an IgG to createhighly
negatively charged IgG-(pAbBD-ApP)2 conjugates withoutperturbing
binding affinity (Fig. 1) (21). Because functionality isbuilt into
the pAbBD rather than the IgG, cargo IgGs can beeasily exchanged
without genetic reengineering, allowing most off-the-shelf IgGs to
be easily functionalized.Here we report that our cytosolic antibody
delivery approach
enables close to 90% delivery efficiency at a concentration of
only500 nM IgG. Our modular antibody functionalization strategy
iscompatible with IgGs from many different species and isotypesand
our complexation approach is compatible with a diverse set
ofcationic lipids. Finally, we demonstrate that cytosolically
deliveredIgGs are functional and can inhibit not only the drug
efflux pumpMRP1 to sensitize cancer cells to chemotherapeutic
drugs, butalso the transcription factor NFκB. These findings
establish thatour method enables easy and efficient cytosolic
delivery of almost
Significance
Antibodies are important research tools because they canbe
developed to bind to as well as directly inhibit almost anyprotein.
Unfortunately, antibodies cannot cross the plasmamembrane and are
therefore limited to perturbing membraneor secreted protein
activity. We found that by appending anionicpolypeptides (ApPs) to
immunoglobulin G (IgG) antibodies, theycould be complexed with
cationic lipids, originally designed fornucleic acid delivery,
through electrostatic interactions to enableefficient cytosolic
antibody delivery. By fusing ApPs to photo-reactive
antibody-binding domains, we can rapidly functionalizealmost any
off-the-shelf IgGwithout genetic reengineering of thecargo IgG. Our
cytosolically delivered antibodies are capable ofinhibiting
intracellular proteins, establishing a new approach forstudying
intracellular protein function with inhibitory antibodies.
Author contributions: H.H.W. and A.T. designed research; H.H.W.
performed research;H.H.W. and A.T. analyzed data; and H.H.W. and
A.T. wrote the paper.
Competing interest statement: H.H.W. and A.T. have a pending
patent on this technol-ogy. A.T. is a founder and owns equity in
AlphaThera, a biotechnology company that sellspAbBD-based
products.
This article is a PNAS Direct Submission.
Published under the PNAS license.1To whom correspondence may be
addressed. Email: [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1913973116/-/DCSupplemental.
First published October 14, 2019.
22132–22139 | PNAS | October 29, 2019 | vol. 116 | no. 44
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any off-the-shelf IgG antibody and can extend the potential
ap-plications of existing IgG antibody collections.
ResultsStringent Detection of Cytosolic Protein Delivery. When
developingintracellular protein delivery technologies, it is
imperative to usean assay that only detects cargo proteins that
have been success-fully delivered to the cytosol. Endosome escape
is the majorbottleneck for cytosolic delivery, resulting in large
false-positiverates for assays that measure total cellular uptake
of cargo pro-teins (22, 23). Accordingly, we used a stringent
self-assemblingsplitGFP (24) reporter system in which one half of
the splitGFP,the S11 peptide, is fused to pAbBD-ApP, whereas the
other half,splitGFP(1–10), is expressed in reporter cells (25–28).
Only onceIgG-(pAbBD-ApP-S11)2 is successfully delivered into the
cytosoldoes splitGFP complementation and turn-on fluorescence
occur(Fig. 1). Furthermore, fluorescence intensity is directly
correlatedwith the amount of protein that is cytosolically
delivered.We prepared pAbBD-ApP-S11 with 10 to 30 residues-long
polyaspartate or polyglutamate ApPs and confirmed that theycould
photocrosslink to rituximab (Ritux) (SI Appendix, Fig. S1 Aand B).
We chose rituximab because its antigen, CD20, is notexpressed in
our reporter cells, removing a potential confound-ing factor for
delivery. To validate that our protein cargos arecompatible with
the splitGFP reporter system, we confirmed thatsplitGFP
complementation occurred when either pAbBD-S11 orRitux-(pAbBD-S11)2
were incubated with purified splitGFP(1–10)or physically delivered
into HEK293T splitGFP(1–10) reportercells by electroporation (SI
Appendix, Fig. S1 C–F). Due to thetime required for chromophore
maturation (SI Appendix, Fig. S1C andD), reporter cells were
assessed for splitGFP fluorescence 6 hfollowing electroporation
with either flow cytometry or live-cellfluorescence microscopy. As
expected, fluorescence microscopyrevealed diffuse splitGFP
fluorescence with both pAbBD-S11 andRitux-(pAbBD-S11)2, but
fluorescence was depleted from thenucleus only with
Ritux-(pAbBD-S11)2 (SI Appendix, Fig. S1F).Because pAbBD-S11 is
only ∼11 kDa, it is capable of passivelytranslocating across the
nuclear pore complex, whereas Ritux-(pAbBD-S11)2 is ∼170 kDa and is
very inefficient at passivenuclear translocation (29).
Cytosolic pAbBD Delivery. Initially, we wondered whether
pAbBD-ApP-S11 alone, without IgG, could be cytosolically delivered
intoHEK293T splitGFP(1–10) cells once complexed with a
commercially
available cationic transfection lipid, Lipofectamine (Lipo)
2000.We tested simple polyaspartate or polyglutamate ApPs thatwere
10 to 30 residues long. Live-cell fluorescence micros-copy showed
robust splitGFP fluorescence, indicating substantialcytosolic
delivery, once polyaspartate ApPs were at least 15residues long
(D15), with a peak at 20 aspartate residues (D20)(Fig. 2A). With
polyglutamate ApPs, splitGFP fluorescenceincreased with length up
to 30 glutamate residues (E30) (Fig.2B). Even though the base
pAbBD-S11 has a net charge of −7,no cytosolic delivery could be
detected without ApPs (Fig. 2 Aand B).Using flow cytometry, we
quantified splitGFP fluorescence as
either the percentage of splitGFP-positive cells, which
reflectsdelivery efficiency, or the fold increase in splitGFP
fluorescence,which reflects the amount of protein delivered (Fig. 2
C and Dand SI Appendix, Fig. S2). Flow cytometry confirmed the
trendsseen between ApP length and delivery efficiency identified
viamicroscopy. Increasing the ratio of cationic lipid to
proteingenerally improved delivery efficiency, but at the cost of
in-creased toxicity (SI Appendix, Fig. S2). We were able to
identifyregimes, though, where viability remained greater than 90%
withexcellent delivery efficiency (Fig. 2 C and D and SI Appendix,
Fig.S2). The best polyglutamate and polyaspartate ApPs were D20and
E30 with delivery efficiencies of 53.9 ± 2.6% and 50.5 ±5.4%,
respectively, when 500 nM pAbBD-ApP-S11 was complexedwith 2 μL Lipo
2000 (Fig. 2 C and D).Together, these results demonstrate that a
small protein,
pAbBD-S11 (∼11 kDa), can be efficiently delivered into
thecytosol simply by fusing it to polyaspartate or
polyglutamateApPs via complexation with cationic lipids. We believe
that thisstrategy can be easily adopted for cytosolic delivery of
smallantibody-like binding proteins such as affibodies (∼6 kDa),
mono-bodies (∼10 kDa), or nanobodies (∼15 kDa), but ApP lengthmay
need to be reoptimized for maximal delivery.
Cytosolic IgG Delivery. Next, we tested whether
Ritux-(pAbBD-ApP-S11)2 could also be cytosolically delivered when
complexedwith Lipo 2000. As expected, no splitGFP fluorescence
could bedetected by microscopy with Ritux-(pAbBD-S11)2, which has
abase net charge of +4 (Fig. 3 A and B). Once both polyaspartateand
polyglutamate ApPs reached at least 20 residues long,however,
microscopy revealed diffuse splitGFP fluorescence withnuclear
depletion (Fig. 3 A and B). Because nuclear depletionindicates that
the S11 reporter peptide remained linked to Ritux-(pAbBD-ApP-S11)2
following endosomal escape, we are confi-dent that the splitGFP
fluorescence is reflective of significantcytosolic delivery of
Ritux-(pAbBD-ApP-S11)2.Flow cytometry of splitGFP fluorescence
corresponded well to
microscopy, but additionally revealed that delivery peaked
with25 aspartates (D25), but plateaued with 20 glutamates
(E20)(Fig. 3 C and D and SI Appendix, Fig. S3). Lipo RNAiMax,
acationic lipid designed for siRNA delivery, was more effectivethan
Lipo 2000 at delivering Ritux-(pAbBD-ApP-S11)2 withshort ApPs, but
worse with longer ApPs (SI Appendix, Fig. S4).Similarly to
pAbBD-ApP-S11 delivery, increasing the ratio of ei-ther lipids to
Ritux-(pAbBD-ApP-S11)2 generally improved de-livery, but also
increased toxicity (SI Appendix, Figs. S3 and S4).After significant
optimization, we achieved a maximal deliveryefficiency of 65.7 ±
3.6% in HEK293T splitGFP(1–10) cellswith >90% viability when 500
nM Ritux-(pAbBD-D25-S11)2 wascomplexed with 2 μL Lipo 2000 (Fig.
3C). Interestingly, deliveryefficiencies for Ritux-(pAbBD-ApP-S11)2
were higher than that ofpAbBD-ApP-S11 alone, likely due to having 2
ApPs linked toeach rituximab molecule.To demonstrate the
generalizability of our IgG delivery approach,
we tested several other commercially available cationic lipids
andreporter cell lines. All conditions resulted in efficient
cytosolic de-livery of Ritux-(pAbBD-D25-S11)2 and
Ritux-(pAbBD-E25-S11)2,
Fig. 1. Schematic of antibody delivery approach. (1) pAbBD is
purified as afusion to an ApP as well as the splitGFP S11 reporter
peptide and thenphotocrosslinked to a cargo IgG. (2)
IgG-(pAbBD-ApP-S11)2 conjugates arecomplexed with cationic delivery
lipids. (3) Lipid-IgG complexes are takenup by cells via
endocytosis. (4) Delivery lipids promote endosome escapeand
cytosolic IgG-(pAbBD-ApP-S11)2 delivery. (5) Cytosolic
IgG-(pAbBD-ApP-S11)2 delivery can be detected by splitGFP
complementation and turn-onfluorescence.
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albeit to varying degrees (Fig. 4 A–C and SI Appendix, Figs. S5
A–C,S6, and S7). Delivery efficiency was significantly higher in
HT1080and A549 splitGFP(1–10) cells, reaching up to 72.7 ± 3.9%and
86.4 ± 1.1%, respectively, with 500 nM Ritux-(pAbBD-D25-S11)2 (Fig.
4B and SI Appendix, Fig. S7). Other than rituximab(human IgG,
hIgG1), pAbBD-D25-S11 could also photocrosslinkand cytosolically
deliver hIgG2, mouse IgG2a (mIgG2a), mIgG2b,mIgG3, rat IgG2c
(rIgG2c), and rabbit IgG (rabIgG) into A549splitGFP(1–10) cells
(Fig. 4 D and E). Delivery efficiencyremained high with all tested
IgG species and isotypes,except for rIgG2c, which had a moderate
delivery efficiency(Fig. 4D).Finally, we measured how delivery
efficiency varied with Ritux-
(pAbBD-D25-S11)2 and Ritux-(pAbBD-E25-S11)2 dose (Fig. 4F–H and
SI Appendix, Fig. S5 D–F). At 100 nM Ritux-(pAbBD-D25-S11)2, a
5-fold decrease in concentration, delivery efficiencywas only
slightly reduced to 73.5 ± 3.0% in A549 splitGFP(1–10)cells with
Lipo 2000 (Fig. 4G). Even at 1 nM Ritux-(pAbBD-D25-S11)2 or
Ritux-(pAbBD-E25-S11)2, cytosolic delivery was stilldetectable,
albeit low (Fig. 4 F–H and SI Appendix, Fig. S5 D–F).Collectively,
these results demonstrate the versatility of our
IgG delivery approach with regards to the following factors:
1)We can deliver off-the-shelf IgGs from a variety of species
andisotypes. 2) It is compatible with all tested cationic lipids
and celllines thus far. 3) Delivery efficiency is maintained at low
IgG-(pAbBD-ApP-S11)2 concentrations. We observe that although
increasing the net negative charge of cargo proteins is critical
forcomplexation and delivery, it eventually becomes unproductiveor
even deleterious for delivery efficiency.
Cytosolically Delivered QCRL3 Can Inhibit MRP1, a Drug Efflux
Pump.Having shown robust delivery, we next sought to demonstrate
theutility of cytosolic IgGs by inhibiting MRP1, a drug-export
pumpassociated with chemotherapy resistance (30). Although it isa
transmembrane protein, we chose MRP1 because it can beinhibited by
a well-characterized monoclonal IgG, QCRL3, whichinhibits via
binding to one of MRP1’s cytosolic nucleotide bindingdomains
(30–33).Initially, we used the calcein-efflux assay to assess MRP1
ac-
tivity in which MRP1 inhibition results in calcein
fluorescenceretention (Fig. 5A). A total of 500
nMQCRL3-(pAbBD-D25-S11)2delivery resulted in calcein retention in
HEK293T, HT1080, andA549 cells (Fig. 5 B and C), indicating
inhibition of endogenousMRP1 activity. No inhibition was observed
following delivery ofthe mIgG2a-(pAbBD-D25-S11)2 isotype control or
simply incu-bating cells with QCRL3-(pAbBD-D25-S11)2.
QCRL3-(pAbBD-D25-S11)2 delivery performed as well as MK571, a
nonselectivesmall-molecule MRP1 inhibitor (30), in all except for
HEK293Tcells (Fig. 5C). We attribute this to the high expression of
otherefflux pumps that are also inhibited by MK571 in HEK293Tcells.
Finally, photocrosslinking is necessary for IgG delivery,
asdelivery of QCRL3 mixed with pAbBD-D25-S11 did not result in
Fig. 2. Optimizing ApPs for cytosolic pAbBD delivery. A total of
500 nM pAbBD-S11 (negative control) or pAbBD-ApP-S11 with either
polyaspartate orpolyglutamate ApPs 10 15, 20, 25, or 30 residues
long were complexed with 2 μL Lipo 2000 and added to HEK293T
splitGFP(1–10) cells for 6 h. (A and B)Representative live-cell
fluorescence microscopy images following delivery with
polyaspartate (A) and polyglutamate (B) ApPs shows diffuse splitGFP
fluo-rescence indicating significant cytosolic delivery. (C and D)
Flow cytometry of splitGFP fluorescence following delivery with
polyaspartate (C) and poly-glutamate (D) ApPs. (Left)
Representative flow cytometry histograms. (Center) Percent of cells
splitGFP-positive. (Right) Fold increase in median
splitGFPfluorescence over negative control. The dotted line
indicates either 90% of the cell population (Center) or no increase
in fluorescence (Right). Viability wasdetermined with the LDH
assay. Data are mean ± SEM, n = 4; *P < 0.05, **P < 0.01
(1-sided 1-sample t test of log ratios).
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calcein retention (Fig. 5D). This suggests that during
complexa-tion, noncovalent interactions between pAbBD and IgGs
aredisrupted.Next, we attempted to sensitize A549 cells to
doxorubicin and
vincristine, which are chemotherapeutic drugs known to be
MRP1substrates (30, 34). Cytosolic delivery of 500
nMQCRL3-(pAbBD-D25-S11)2 was able to sensitize A549 cells to
doxorubicin by 3.7 ±0.45 fold and vincristine by 9.0 ± 2.0 fold
(Fig. 5 E and F). Incomparison, MK571 treatment resulted in only
moderate sensiti-zation, and delivery of the mIgG2a isotype control
resulted in nosensitization (Fig. 5 E and F). Thus, cytosolically
delivered IgGsremain functional following delivery and can inhibit
biologicallyinteresting proteins.
Cytosolically Delivered IgGs Can Inhibit NFκB. Finally, we
investi-gated whether cytosolically delivered IgGs could inhibit
protein–protein interactions, which are particularly difficult to
perturbwith small molecules. We targeted the transcription factor
NFκB,which is a heterodimer between p50 and RelA (p65) whose
nu-clear localization signals (NLSs) are normally masked by
IκBα,sequestering NFκB in the cytosol. With TNFα stimulation, IκBα
isdegraded, allowing NFκB to enter the nucleus to stimulate
tran-scription (35). We hypothesized that an anti-RelA NLS IgG
couldsterically block RelA from engaging with its cognate nuclear
im-port factor (NIF) to prevent RelA nuclear translocation and
NFκB-mediated transcription (Fig. 6A). We also tested an
anti-RelA C terminus IgG that bound to an epitope distinct fromthe
NLS.Initially, we delivered 150 nM anti-RelA NLS-(pAbBD-D25-
S11)2, anti-RelA C-term-(pAbBD-D25-S11)2, or their
isotypecontrols (mIgG3 for anti-RelA NLS, rabIgG for
anti-RelAC-term) into A549 cells and assessed for RelA nuclear
translocationfollowing TNFα stimulation. Immunofluorescence
revealed thatboth anti-RelA NLS and anti-RelA C-term delivery
reduced RelAnuclear translocation to 48.0 ± 0.8% and 60.1 ± 5.9% of
that ofnormal cells, respectively (Fig. 6 B and C and SI Appendix,
Fig. S8).Next, by using a NFκB-driven luciferase reporter plasmid,
weshowed that delivery of both anti-RelA NLS and anti-RelA
C-termreduced NFκB transcriptional activity to 52.4 ± 1.1% and 68.3
±2.6% of that of normal cells, respectively (100 ng/mL TNFα)
(Fig.6D). This degree of inhibition is excellent considering the
deliveryefficiencies with 150 nM anti-RelA NLS and anti-RelA
C-termwere 70.9 ± 0.8% and 29.7 ± 4.5%, respectively (SI
Appendix,Fig. S9). Because anti-RelA C-term IgG delivery was
capable ofinhibiting NFκB, antibody binding to non-NLS epitopes may
besufficient to sterically block nuclear translocation of many
targetproteins. We anticipate that cytosolic IgG-dependent
cytoplas-mic sequestration can be easily adopted to inhibit other
tran-scription factors or nuclear proteins.
Fig. 3. Optimizing ApPs for cytosolic IgG delivery. A total of
500 nM Ritux-(pAbBD-S11)2 (negative control) or
Ritux-(pAbBD-ApP-S11)2 with eitherpolyaspartate or polyglutamate
ApPs 10, 15, 20, 25, or 30 residues long were complexed with 2 μL
Lipo 2000 and added to HEK293T splitGFP(1–10) cellsfor 6 h. (A and
B) Representative live-cell fluorescence microscopy images
following delivery with polyaspartate (A) and polyglutamate (B)
ApPs showsdiffuse splitGFP fluorescence with nuclear depletion
indicating significant cytosolic delivery. (C and D) Flow cytometry
of splitGFP fluorescence followingdelivery with polyaspartate (C )
and polyglutamate (D) ApPs. (Left) Representative flow cytometry
histograms. (Center) Percent of cells splitGFP-positive.(Right)
Fold increase in median splitGFP fluorescence over negative
control. The dotted line indicates either 90% of the cell
population (Center) or no increasein fluorescence (Right).
Viability was determined with the LDH assay. Data are mean ± SEM, n
= 4; *P < 0.05, **P < 0.01, ***P < 0.001 (1-sided 1-sample
t test oflog ratios).
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DiscussionEfficient cytosolic delivery of proteins, particularly
antibodies,has long been sought for expanding the toolbox that one
can usefor perturbing biological systems. By leveraging a modular
anti-body functionalization technology (21), we were able to
easilyappend ApPs onto off-the-shelf IgGs to enable their
complexa-tion with a variety of commercially available cationic
lipids andefficiently deliver them into the cytosol of cells.
Cytosolicallydelivered IgGs remain functional and are capable of
inhibitingdiverse proteins such as MRP1 and NFκB.When compared to
CPP-mediated delivery of small peptides
or proteins, our approach enables cytosolic delivery of a
muchlarger IgG cargo with similar or greater efficiencies at an
∼100-fold lower concentration (25–28). It is difficult to directly
com-pare our technology to previous carrier-mediated approachesdue
to the use of different reporter systems. We note, however,
that previous studies have relied on either a reporter that
onlydetects total cellular uptake (17) or by delivering proteins
ca-pable of greatly amplifying their signal, such as
Cre-recombinaseor enzymes (16, 18–20). In contrast, our functional
assays—MRP1 and NFκB inhibition—are more stringent and
requiredelivery of close to stoichiometric amounts of IgG
relativeto their target, which is more representative of most
proteininhibition assays.Intrabodies have long been used to
modulate cell biology,
ranging from simply inhibiting target proteins (8–10, 36, 37)
tomarking target proteins for degradation (38). However,
thoseapproaches require either expertise in in vitro display
technolo-gies or antibody engineering to create fragments that can
foldproperly in the cytoplasmic environment, which can be
majorbarriers for easy adoption by researchers. Because IgG
anti-bodies have been invaluable research tools for decades,
there
Fig. 4. IgG delivery scope. (A–C) A total of 500 nM
Ritux-(pAbBD-S11)2 (negative control) or Ritux-(pAbBD-D25-S11)2 was
complexed with 2 μL of the in-dicated cationic lipid and added to
the indicated splitGFP(1–10) reporter cells for 6 h. Representative
flow cytometry histograms of splitGFP fluorescence areshown in A.
Flow cytometry data were quantified as the percent of cells
splitGFP-positive (B) and the fold increase in median splitGFP
fluorescence overnegative control (C). (D and E) Same as for B and
C but 500 nM IgG-(pAbBD-D25-S11)2 of the indicated species and
isotype was complexed with 2 μL Lipo 2000and added to A549
splitGFP(1–10) cells. (F–H) Same as for A–C, but indicated
concentrations of Ritux-(pAbBD-D25-S11)2 were complexed with 2 μL
Lipo 2000and added to A549 splitGFP(1–10) cells. The dotted line
indicates either no increase in fluorescence (C, E, and H) or 90%
of the cell population (G). Viabilitywas determined with the LDH
assay. Data are mean ± SEM, n = 4; **P < 0.01, ***P < 0.001
(1-sided 1-sample t test of log ratios).
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exists large and well-validated antibody collections (39, 40)
that,using our modular IgG functionalization and cytosolic
deliveryapproach, can now be repurposed for perturbing the activity
ofintracellular proteins. Although IgG binding is not guaranteed
toinhibit all target proteins, we believe that the large size of
IgGs issufficient to sterically block many biological
interactions.Cytosolic delivery of inhibitory antibodies also
offers unique
advantages over traditional genetic or small-molecule
approachesfor modulating protein function. Because genetic
approaches forprotein knockdown or knockout do not directly act on
targetproteins, they perform poorly against proteins with long
half-lives(41) and can induce significant cellular compensatory
responses(42). Small-molecule inhibitors and modulators can avoid
theselimitations, but many proteins are not druggable by small
mole-cules and for those that are, identifying potent compounds
andthen validating their selectivity can be challenging (43). In
con-trast, inhibitory antibodies directly bind to target proteins,
act onfast time scales, and can be generated far more easily than
smallmolecules. Finally, cytosolic inhibitory antibodies also offer
theopportunity to discriminate between and modulate the activity
of
proteins with specific posttranslational modifications or
certainisoforms of a protein, which are generally not possible
withtraditional approaches.Cytosolic IgGs have previously been
reported to be capable of
engaging the TRIM21 E3 ubiquitin ligase to degrade their
targetproteins (7), but pAbBD photocrosslinking sterically blocks
theTRIM21 binding site (21, 44, 45) and prevents our
cytosolicallydelivered IgGs from harnessing any endogenous protein
degra-dation machinery. Future studies could address this by
engineeringalternative antibody-binding domains (46) to
photocrosslink toIgGs outside of the Fc region. In this study we
complexed IgGswith commercially available cationic lipids. Although
the resultingcomplexes perform efficiently in cultured cells, their
poor phar-macokinetics render them unsuitable for in vivo studies.
Futurestudies should test alternative delivery formulations for in
vivodelivery, such as those containing ionizable and
poly(ethyleneglycol)-conjugated lipids (14), which have shown
promise for thein vivo delivery of siRNA. However, it is not clear
whether theseformulations, when prepared with antibodies, will have
adequatepharmacokinetics, be capable of extravasating from the
vasculature
Fig. 5. Cytosolic QCRL3 delivery inhibits MRP1. (A) Schematic of
assays that assess MRP1 inhibition. In the calcein efflux assay,
cells are first loaded withcalcein, a fluorescent
membrane-impermeable MRP1 substrate. Cells with high MRP1 activity
will rapidly export calcein, whereas MRP1 inhibition results
incalcein fluorescence retention. In the chemotherapeutic
sensitization assay, MRP1 inhibition results in greater
intracellular accumulation of doxorubicin orvincristine, resulting
in sensitization to both compounds. (B) Representative flow
cytometry histograms of calcein fluorescence after 16 h of export
in calcein-loaded HT1080 cells treated with 20 μM MK571, 500 nM
QCRL3-(pAbBD-D25-S11)2, 500 nM cytosolically delivered
mIgG2a-(pAbBD-D25-S11)2, or 500 nMcytosolically delivered
QCRL3-(pAbBD-D25-S11)2. (C) Calcein-efflux assay quantification
across HEK293T, HT1080, and A549 cell lines. Only QCRL3 delivery
andMK571 treatment resulted in calcein fluorescence retention. Data
are mean ± SEM, n = 4, ***P < 0.001 (1-sided 1-sample t test of
log ratios). (D) Same as for B,but in calcein-loaded A549 cells
treated with cytosolic delivery of 500 nM QCRL3 with or without
photocrosslinking to pAbBD-D25-S11. Calcein fluorescenceretention
is only seen with photocrosslinked QCRL3, indicating that
photocrosslinking is necessary for delivery. (E and F) A549 cell
sensitivity to doxorubicin (E)or vincristine (F) following
treatment with 20 μM MK571, 500 nM cytosolically delivered
mIgG2a-(pAbBD-D25-S11)2, or 500 nM cytosolically delivered
QCRL3-(pAbBD-D25-S11)2. Data are mean ± SEM, n = 4.
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to induce uptake into desired cell populations, or be capable
ofpromoting escape from the endosome–lysosome system with ade-quate
efficiency to impart a therapeutic effect in vivo.In summary, we
have designed and rigorously validated a
modular cytosolic IgG delivery platform that allows
previouslydeveloped IgG collections to now be used as intracellular
tools.We believe that this capability will significantly expand the
waysin which intracellular pathways can be perturbed and will
shedunique insight into cell function in both health and
disease.
MethodspAbBD Expression and Purification. All pAbBD variants
were purified viaproximity-based sortase-mediated ligation (PBSL)
(47). Using PBSL to purifyproteins consists of 2 steps: 1) PBSL
resin preparation and 2) target proteinpurification. PBSL resin was
prepared as previously described with 2 minormodifications due to
the use of SpyCatcher-SrtA-His12 (47). Pellets werelysed by
resuspending in lysis buffer (PBS + 1% wt/vol
N-octyl-β-D-1-thio-glucopyranoside [OTG] + 200 μg/mL lysozyme + 4
μg/mL DNaseI + EDTA-freeprotease inhibitor mixture [Roche]) and
rotating for 30 min at room tem-perature (RT). Following binding,
the resin was washed with PBS + 20 mMimidazole 3 times followed by
PBS once.
Plasmids encoding for various pAbBD variants were transformed in
con-junction with pEVOL-pBpF into T7 express competent Escherichia
coli cells(New England Biolabs). Starter cultures were grown in LB
+ 100 μg/mL am-picillin (amp) + 25 μg/mL chloramphenicol (cam) at
37 °C with shaking untilOD600 ∼ 0.6. The starter culture was added
at a 1:1,000 dilution to auto-induction media (Formedium AIMLB0210
autoinduction media LB brothbase including trace elements
supplemented with 0.6% vol/vol glycerol and
100 μg/mL amp) further supplemented with 25 μg/mL cam + 0.1%
wt/volarabinose + 3.33 μM 4-benzoyl-L-phenylalanine (BPA, Bachem).
All pAbBDvariants were grown at 37 °C with shaking for 24 h, except
for pAbBD-D30-S11and pAbBD-E30-S11, which were grown at 25 °C with
shaking for 48 h. Ex-pression cultures were then pelleted and
stored at −20 °C.
Frozen pellets were lysed by resuspending in lysis buffer for 30
min at RT.Afterward, lysates were frozen at −80 °C and then thawed
in a 37 °C waterbath. The lysates were clarified by centrifuging
for 15 min at ≥14,000 × gand discarding the pellet. Clarified
lysates were incubated with theSpyCatcher-SrtA-His12 resin prepared
above while rotating for 25 min at RT.Following binding, the resin
was transferred to a Poly-Prep chromatographycolumn (Bio-Rad) and
washed with 1 column volume (CV) of PBS, 1 CV of PBS +20 mM
imidazole, and 1 CV of PBS + 1 M NaCl + 20 mM imidazole.
pAbBDvariants were then eluted from the resin by adding PBS + 250
μMCaCl2 + 2mMGly-Gly-Gly (triglycine) and incubating at 25 °C for 3
h. Following elution,pAbBD variants were buffer exchanged into PBS
and concentrated to≥0.5mg/mLvia a 10k MWCO Amicon Ultra centrifugal
filter (MilliporeSigma). The finalprotein was analyzed by SDS/PAGE
for purity, tested for splitGFP comple-mentation, stored at −80 °C,
and tolerated freeze–thaw cycles well.
See SI Appendix, Supplementary Methods, for details on plasmid
gener-ation, splitGFP(1–10) purification, and splitGFP
complementation assays.
Photocrosslinking pAbBD Variants to IgGs. For photocrosslinking,
pAbBDvariants were added to IgGs at a 2:1 molar ratio in PBS. IgG
concentration waskept at ≤5 μM and the pAbBD-IgG
uncrosslinkedmixture was aliquoted in 2 mLclear polypropylene
microcentrifuge tubes. The mixture was then placedin an ice bath
and irradiated for 3 h with 365 nm UV light using a UVP CL-1000L UV
crosslinker placed in a 4 °C cold room. After
photocrosslinking,IgG-pAbBD2 conjugates were washed with PBS 3
times and then concentrated
Fig. 6. Cytosolic anti-RelA IgG delivery inhibits NFκB. (A)
Schematic of NFκB inhibition. Anti-RelA IgGs inhibit NFκB
transcriptional activity by preventing itsnuclear translocation
following TNFα stimulation. (B and C) Representative
immunofluorescence images (B) and quantification (C) of RelA
nuclear trans-location following delivery of the indicated 150 nM
IgG-(pAbBD-D25-S11)2 antibody and TNFα treatment. Only delivery of
anti-RelA IgGs reduced RelAnuclear translocation. Data are mean ±
SEM, n = 3, ***P < 0.001 (1-way ANOVA). (D) A549 cells were
transiently transfected with a NFκB-driven fireflyluciferase
reporter plasmid. NFκB transcriptional activity was detected by
luminescence following delivery of the indicated 150 nM
IgG-(pAbBD-D25-S11)2antibody and TNFα treatment. Only delivery of
anti-RelA IgGs inhibited NFκB transcriptional activity. Data are
mean ± SEM, n = 3; *P < 0.05, **P < 0.01,***P < 0.001
(1-way ANOVA).
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to ≥10 μM via a 100k MWCO Amicon Ultra centrifugal filter to
remove anyuncrosslinked pAbBD. SDS/PAGE was used to confirm that
>95% of IgGheavy chains were photocrosslinked and that any
excess pAbBD was re-moved. The final protein was then tested for
splitGFP complementation andstored at 4 °C for short durations
(