Specific in vivo knockdown of protein function by intrabodies

Figures & data

Figure 1. The intrabody approach for the generation of protein interference phenotypes. Antibody fragments containing the antigen binding domains (V regions) specific for a particular protein can be selected from antibody phage display libraries or other in vitro selection systems like bacterial, yeast, mammalian or ribosomal display libraries. Alternatively, the variable region genes can be obtained from hybridoma antibodies by PCR with consensus primers, RACE or PCR with adaptor ligated cDNA. Antibody fragments, typically in scFv format, are then cloned into a specific targeting vector allowing expression of the intrabody in the nucleus, cytoplasm or ER.

Figure 2. Differences between cytoplasmic/nuclear intrabodies and ER intrabodies. Via their retention signal KDEL, ER intrabodies (A) retain antigens passing the ER by binding to them. As antibodies are naturally produced in the ER, no particular selection for special folding/stability properties is required. In contrast, cytoplasmic/nuclear intrabodies (B) need to fold correctly in the reducing milieu of the cytoplasm. Further, they need to be tested and screened to identify antibodies which are capable, in addition to binding, to neutralise or inactivate their target's activity in the cytoplasmic biochemical milieu.

Table 1. Strategies for the selection of functional cytoplasmic intrabodies

Technique	Outcome	Reference
1) In vivo selection of intrabodies in the absence/presence of antigen in yeast and in E.coli
Antigen independent selection for cytoplasmic expression in yeast and mammalian cells using interaction between transcription activator domain coupled to scFv and DNA binding domain	Selection of stable frameworks from a human naïve antibody library	^Citation103
Antigen independent intrabody selection after export into the cytoplasm mediated by Tat (ISELATE).	Isolation of soluble scFv variants from an insoluble parental sequence.	^Citation106
Modification of ISELATE method. Selection will be performed by analyzing inner membrane anchored Tat-scFv fusions of transfected spheroblasts with anti-FLAG antibody.	Selection and affinity maturation of scFvs using error prone PCR resulted in scFvs with higher affinity than the original clones.	^Citation107
Intracellular Antibody Capture Technology based on an antigen-dependent 2 hybrid system. Cotransfection of antigen fused to DNA binding domain lexA and scFv fused to transactivator domain VP16 leads to selection of cytoplasmic active intrabodies.	Selection of intrabodies against Tau, amyloid-β peptide,α-synuclein, proNGF, gephyrin, Syk, EGFR, BCR-ABL, caspase 3, aggrecanase-2, NSP5 from rotavirus	^Citation33,10,^Citation9,96,^Citation97,98,^Citation11,13,99,101,27
Antigen dependent selection of intrabodies in the E.coli cytoplasm based on co-expression of Tat signalpeptide-scFv and antigen-β-lactamase fusion	Selection of antigen specific binders from a library of randomized CDR-H3 sequences.	^Citation108
2) In vivo selection of stable intrabody fusion proteins
Generation of scFv-protein fusions	Fusion to MBP, GFP or the Fc domain are the most reliable strategies.	^Citation41,39,37,40,42,38
3) CDR grafting or introduction of synthetic CDRs into stable frameworks
Transplantation of CDRs from one antibody to a different stable antibody framework. Introduction of synthetic CDR loops into a stable human framework. Construction of a novel human VH domain antibody library with restricted randomizations at fixed positions in the CDR2 and CDR3, respectively.	Generation of a framework stabilized version of an anti-GCN4 intrabody. Grafting several groups of residues in the CDRs important for antigen binding of scFv D1.3 into stable framework of scFvF8 Construction of a synthetic antibody library based on the human framework of scFv13R4 by introducing synthetic CDR3 loops. Construction of a synthetic single human VH domain antibody library based on the framework of antibody HEL4. Selection of thermodynamically stable anti-lysozyme clones expressed in E.coli.	^Citation46, ^Citation43, ^Citation45^Citation44
4) Cytoplasmic intrabodies in the single domain format
Generation of Camelid VH single domain antibodies from naïve or immune-camelid antibody repertoires Selection of stable human VH domains from naïve or synthetic VH domain antibody repertoires Selection of human VL domains from naïve or synthetic VL domain antibody repertoires	Camelid VH single domain antibodies against: F-actin capping protein CapG, β2-adrenergic receptor, Clostridium botulinum neurotoxin (BoNT) proteases, core antigen of HBV (HBcAg), 15-acetyldeoxynivalenol, caspase-3, Bax, nuclear polyA-binding protein, HIV-1 Vif Most of the VH domains isolated from a human germ-line VH library by the ISELATE method resulted in soluble clones expressed in E.coli. Construction of a large human VH domain antibody repertoire by combinatorial assembly of CDR building blocks from a smaller repertoire comprising aggregation-resistant clones Selection of a human VL domain against Etk kinase from a large VL domain phage display library, derived from a single human framework of light chain Construction of a naïve VL domain library and isolation of clones recognizing B cell super-antigen protein L. Selection of an α-hungtingtin human VL domain intrabody from an original non-functional scFv. Affinity maturation of the original scFv was performed using error prone PCR and yeast surface display.	^Citation14,100, ^Citation29,28, ^Citation6,122,^Citation123,30,^Citation124,2 ^Citation90 ^Citation34 ^Citation12 ^Citation91 ^Citation8
5) Selection of scFvs without disulfide bonds	Isolation of cysteine free scFvs by phage display. After DNA-shuffling and Error prone PCR cysteine free variants of an α-levan antibody could be expressed functional in the cytoplasm of E.coli. Functional cysteine free VL domains recognizing hungtingtin protein could be selected by yeast surface display after Error prone PCR.	^Citation47 ^Citation7

Table 2. ER intrabodies applied

Function targeted	Targets
Oncogenic receptors	VEGFR 2,^Citation87,153,198,88 Tie-2,^Citation87,88,131 ErbB-2,^Citation145,147,150,155,^Citation199-202 EGFR,^Citation203 metalloproteinases MMP-1 and MMP-9,^Citation204 extracellular matrix metalloproteinase inducer,^Citation205,206 human α folate receptor,^Citation207 cathepsin L^Citation208 oncoprotein E7^Citation209
Virus proteins to prevent virus assembly	HIV-1 gp120,^Citation210,211 HIV-1 gp 160,^Citation212 HBV precore antigen,^Citation213 HCV ApoB,^Citation214 HCV core protein,^Citation215 gp46 of maedivisna virus^Citation216
Knockdown of cellular virus receptors to block virus entry	CCR5,^Citation217-219 CXCR4.^Citation220,221
Receptors of the immune system	MHC I,^Citation222-224 integrins,^Citation225-227 VCAM-1,^Citation134,149 NCAM,^Citation56 TLR2,^Citation4 TLR9,^Citation5 IL-2,^Citation148,151,228 CD147,^Citation229 IL-6^Citation230
Nervous system	Neurotrophin Receptor p75,^Citation75 β-amyloid protein,^Citation132 β-amyloid precursor protein,^Citation154 cellular prion protein^Citation231,232

Table 3. A. Reports of ER intrabody mediated knockdown

Target	Cell line	Biochemical knockdown detection	Physiological knockdown readout	Ref.
ErbB2	ErbB2 transformed NIH/3T3 (clone 3.7)	Cell surface staining (Flow cytometry)	Phosphotyrosine analysis (WB), analysis of growth inhibition and morphology	Beerli 1994b^150
ErbB2	ErbB2 transformed NIH/3T3 (clone 3.7)	Cell surface staining (Flow cytometry)	Phosphotyrosine analysis (WB), analysis of growth inhibition and morphology	Beerli 1994b^150
EGFR	EGFR transformed NIH/3T3	No cell surface staining performed, effect of intrabodies on total cellular amount of target was analyzed	EGF induced colonies in soft agar	Beerli 1994a^155
ErbB2	T47D cells	Cell surface staining (Flow cytometry)	ErbB2 tyrosine phosphorylation abolished after EGF treatment (no phosphorylated ErbB2 detectable in WB), 46% reduction of EGFR phosphotyrosine following EGF treatment, following NDF treatment ErbB-3 phosphotyrosine reduced by 89%. Phosphorylation states of various proteins. Reduction of NDF induced soft agar growth	Graus-Porta 1995^147
IL-2 Rα	Jurkat cells with induced IL-2 Rα expression	Cell surface staining with the same antibody clone like the ER-intrabody (Flow cytometry), pulse chase experiment – detection of only the immature target in the presence of the ER-intrabody, endoglycosidase H sensitivity of target (consistent with localization in pre- or early Golgi compartment)	None	Richardson 1995^148
Anti-Integrin VLA4	RD rhabdomyosarcoma cells, Jurkat cells	Cell surface staining and flow cytometry	Number of adhering cells as functional assay, cell adhesion, cell spreading	Yuan 1996^152
EGFR	EGFR transformed NIH/3T3 fibroblast cells (NE1), IL-3 dependent FDC-P1 hematopoietic cell line (FE5) (note: activation of EGFR replaces IL-3 dependent growth)	Not determined for scFv225R	Not determined for scFv255R , EGF dependent cell growth reduced by 80% (FE5 cells) or 50% (NE1 cells) by scFv255S	Beerli 1996^141
ErbB2	ErbB2 transformed NIH/3T3 (clone 3.7)	No cell surface staining performed	Anchorage independent growth, ability to form tumors in nude mice	Beerli 1996^141
IL-2 Rα	T cell line Kit225 HTLV-I transformed cell lines (C8166–45 and HUT102) Jurkat	Cell surface staining with the same antibody clone like the ER-intrabody. Cell surface stainings were also repeated with an antibody that recognizes a different epitope than the ER intrabody.	Proliferative response	Richardson 1997^151
alphaV integrin	Saos-2	Cell surface staining and analysis by flow cytometry	Cell adhesion, cell spreading	Koistinen 1999^225
CCR5	HEK293T Cos 7 PM1	Cell surface staining and analysis by flow cytometry	CCR5 dependent cell fusion was prevented by the ER intrabody, protection of PM1 cells from R5 HIV-1 infection by the ER intrabody	Steinberger 2000^217
Procathepsin L	Human melanoma cell line A375SM	Immunoblots of cell culture supernatant (target is secreted)	Complement lysis assay	Guillaume-Rousselet 2002^208
Folate receptor	IGROV-1, SKOV3	Cell surface staining and analysis by flow cytometry	Cell proliferation, morphology, cell adhesion	Figini 2003^207
CCR5	SupT cells stably transfected with the target, PM1 cells, primary monocyte derived macrophages (MDMs) and microglial cells	Cell surface staining and analysis by flow cytometry	HIV infection assays	Cordelier 2004^219
MHC I	HEK293, Primary rat keratinocytes	Cell surface staining and analysis by flow cytometry	Cell lysis	Busch 2004^223
VEGF-R2	PAE VEGR-R2	Cell surface staining and analysis by flow cytometry	Angiogenesis	Böldicke 2005^153
VEGF-R2 and Tie-2	in vivo gene delivery into nude mice, HUVEC, HMEC-1	Cell surface staining and analysis by flow cytometry	Cell proliferation (in vitro), tumor growth (in vivo)	Jendreyko 2005^88
Tie-2	in vivo gene delivery into nude mice, HUVEC	Cell surface staining and analysis by flow cytometry	In vitro cell proliferation assay, Analysis of vessel density (87% reduction of vessel density upon ER intrabody treatment)	Popkov 2005^131
CCR5	THP-1, PM1, primary CD4+ T cells	Cell surface staining and analysis by flow cytometry	Susceptibility of cells to HIV-1 infection	Swan 2006^218
vIL-6	Cos-7	Comparison of target content in cell lysate and cell culture supernatant, analysis for colocalization of ER intrabody and target by microscopy	Neutralization of the target signaling in the ER analyzed by monitoring STAT3 phosphorylation.	Kovaleva 2006^230
TfR	MCF-7	Cell surface staining and analysis by flow cytometry	Analysis of proliferation and cell cycle	Peng 2007^233
ApoB	HepG2	Analysis of the cell culture supernatant for the presence of secreted target, Co-IP	Analysis of albumin secretion into the cell culture medium	Liao 2008^214
CD147	HEK293A	Cell surface staining and analysis by flow cytometry	—	Tragoolpua 2008^229
CD147	HeLa	Cell surface staining and analysis by flow cytometry	—	Intasai 2009^234
VCAM1	HEK293::VCAM-YFP	Cell surface staining and analysis by flow cytometry	Cell adhesion assessed by microscopy	Strebe 2009^149
TLR2	HEK293, RAW264.7, primary macrophages	Cell surface staining and analysis by flow cytometry	TNF-alpha release	Kirschning 2010^4
HBV precore antigen	pTRE AD38 HepG2.2.15	Analysis of ER intrabody and target localization by fluorescence microscopy	Reduction of extracellular HBeAg in pTRE precore and HepG2.2.15 cells.	Walsh 2011^213
p75NTR	PC12 and NSC19 cells	Cell surface staining and analysis by flow cytometry	Bcl^-xL mRNA expression, cell differentiation (neurite outgrowth)	Zhang 2012^75
TLR9	HEK293, RAW264.7	Co-IP, analysis by microscopy for colocalization with an ER marker	Analysis of TLR9 challenge by NF-kappa B reporter assay, TNF-alpha response upon treatment with TLR agonists analyzed by ELISA and intracellular flow cytometry	Reimer 2013^5
VCAM1	in vivo, mouse strain C57BL/6N	Cell surface staining and analysis by flow cytometry	analysis of B-cell distribution over blood and bone marrow	Marschall 2014a^134
Aβ oligomers	7PA2 fADcells (CHO cells which express a fADhumanV717FAPP mutant) 7WD4 cells	Co-immunoprecipitation	Dephosphorylation of ERK1/2 and CREB is reduced in hippocampal neurons treated with conditioned media of the ER intrabody expressing 7PA2-A13K cells. Effect of the ER intrabody on the mTor pathway in 7PA2-A13K cells, which plays a role in homeostasis and autophagy.	Meli 2014^235
Z α antitrypsin	Cos-7	Co-immunoprecipitation	-	Ordonez 2015

¹Transient expression (T), Stable expression (S), Endogenous expression (E).

²pool (polyclonal cells).

³of oncogenically activated form of target: ErbB2*.

Table 3. B. ER intrabody-mediated knockdown: methodological aspects

Target	expression target T/S/E*	expression ER intrabody T/S*	origin of binders	Affinity	Expression levels/degradation levels/half lives	Accumulation or accelerated degradation?	Knockdown efficiency	Ref.
ErbB2	S	S	Hybridoma, clone FRP5	4.2 nM for scFv(FRP5)-ETA construct (Wels et al. 1995), apparent affinity FRP5 (IgG) 0.82 nM (Harwerth et al. 1993)	Higher intracellular levels of retained antibodies than secreted antibodies, half-life of ErbB2* ∼1.5h, ErbB2 >7h (Reinman et al. 2003)	Accumulation of ER intrabodies in ER	Cell surface expression qualitatively analyzed ,NIH/ErbB2* cells: 95% growth inhibition	Beerli 1994b^150
ErbB2	S	S	Hybridoma, clone FWP51	Apparent affinity FWP51 (IgG) 1.3 nM (Harwerth et al. 1993)	Higher intracellular levels of retained antibodies than secreted antibodies, half-life of ErbB2* ∼1.5h, ErbB2 >7h (Reinman et al. 2003)	Accumulation of ER intrabodies in ER	Cell surface expression qualitatively analyzed ,NIH/ErbB2* cells: 80% growth inhibition	Beerli 1994b^150
EGFR	S	S	Hybridoma, mab225	Nanomolar (Sato et al. 1983)	1–2 x10^5 EGFR receptors per cell, scFv255 expression not detectable (potentially due to unavailability of optimal detection antibody)	Authors conclude retained scFv255 might accelerate degradation	Cell surface expression not analyzed, 31% EGFR reduction by secreted scFv225S, 9% EGFR reduction by retained scFv255R	Beerli 1994a^155
ErbB2	E	S	Hybridoma, clone FRP5	4.2 nM for scFv(FRP5)-ETA construct (Wels et al. 1995), apparent affinity FRP5 (IgG) 0.82 nM (Harwerth et al. 1993)	qualitatively assessed by WB, NDF induced stimulation of cell growth 2.8 fold in control cells, 1.5 fold stimulation in ER-intrabody transfected cells	(Not quantified but WB seems to indicate similar levels of ErbB2 in presence and absence of ER-intrabody → there seems to be neither accumulation nor accelerated degradation)	Cell surface expression qualitatively analyzed	Graus-Porta 1995^147
IL-2 Rα	induced in Jurkat	S	Hybridoma HD245–332: anti-Tac, Hybridoma	Not determined in this paper	Half-life of scFv-KDEL more than 30h (pulse chase)	Antibody/IL-2Ra complexes are degraded by a nonlysosomal mechanism (= accelerated degradation)	Cell surface expression qualitatively analyzed, 16 of 16 cell clones stably transfected with ER-intrabody showed complete inhibition of target induction, 3of 15 cell clones stably transfected with secreted antibody version also showed downregulation of target but incomplete (remaining 12 clones showed normal or even high levels of the target)	Richardson 1995^148
Anti-Integrin VLA4	S	S	Humanized form (hHP1/2, unpublished) of the mouse mAb mAbHP1/2) (hybridoma)	EC50 - 0.015 nM (Hanf et al. 2014)	Not quantified	Not determined	52% of stably transfected RD cell clones and 8% of stably transfected Jurkat cells displayed reduced surface expression of the target. Target reduction on the surface of selected RD clones was ∼80%, on the surface of selected Jurkat cells 65–100% (MFI levels used for quantification of knockdown efficiency). 90% of mock transfected but < 1% of ER-intrabody transfected RD cells were spread.	Yuan 1996^152
EGFR	S	S^2	Hybridoma, mab225	Nanomolar (Sato et al. 1983)	Expression levels of scFv225 were much lower than scFv-FRP5 in Cos-1 cells. scFv225 was mainly in the insoluble fraction.	Not determined for scFv225R	Not determined for scFv225R, EGF dependent cell growth reduced by 80% (FE5 cells) or 50% (NE1 cells) by scFv225S	Beerli 1996^141
ErbB2	S^3	S ^2	Hybridoma, clone FRP5	4.2 nM for scFv(FRP5)-ETA construct (Wels et al. 1995), apparent affinity FRP5 (IgG) 0.82 nM (Harwerth et al. 1993)	Expression levels of scFv-FRP5 were much higher than scFv255 in Cos-1 cells.	Not determined	97% growth inhibition in soft agar, 55% reduced tumor volume after 13 d of growth in nude mice	Beerli 1996^141
IL-2 Rα	S	S and inducible	Hybridoma HD245–332: anti-Tac, Hybridoma	Not determined in this paper	Half-life of IL2Ralpha is greater than 24h (Levin et al. 1997), Full induction of scFv after 24h of tetracycline withdrawal.IL2Ralpha persisted for up to 72h at the cell surface after tetracycline withdrawal. Cell lines normally express more than 200 000 target molecules per cell.	While mature form (55 kDa) of the target vanishes, the immature (40kDa) form of the target accumulates	2 or more than 2 log units of mean fluorescence intensity (cell surface staining) in all cell lines tested	Richardson 1997^151
alphaV integrin	E	S	Hybridoma, L230	—	Not determined	Not determined	70–100% decrease	Koistinen 1999^225
CCR5	TTT	TTS	ST6 from phage display of a Fab rabbit immune library, converted to scFv format	2.7 nM for ST6 8.5 nM for ST6/34 (humanized version of ST6) ST6 binds to a linear epitope in the first extracellular domain of CCR5^236	ST6 expressed at higher levels than its humanized version but the humanized version of ST6 led to a slightly higher knockdown efficiency (results from experiments with HEK293T cells transiently cotransfected with target and ER intrabody).^236	Not determined	Decrease from 66% target positive cells of controls to 5.9% target-positive cells for ST6 ER intrabody expressing cells or 2.7% target-positive cells for ST6/34 ER intrabody expressing cells.^236	Steinberger 2000^217
Procathepsin L	E	T	Hybridoma clone 3D8	5×10^–8 M	Not quantified	Intracellular accumulation of target	Preliminary studies suggested susceptibility to complement lysis was 50% increased if ER intrabody was expressed	Guillaume-Rousselet 2002^208
Folate receptor	E	S	Hybridoma clone MOV19 Mab	7.1×10^–8 M by displacement study, 1.8×10^–8 M by scatchard analysis	mRNA levels for the ER intrabody were highest in the IGROV-1 clone with the most efficient target knockdown	Accelerated degradation of the target upon ER intrabody expression (determined by western blot of whole cell lysates)	IGROV-1 clones with 60 (=DM60), 85 (=DM85) and 99% (=DM99) reduction, ER intrabody transfected SKOV3 were negative for surface target. Reduction of cell surface target remained stable over 1 y of culture in DM99, but spontaneous decrease in target expression was observed in DM60 and DM85	Figini 2003^207
CCR5	SE	S	Hybridoma, CTC8, antibody 2C7	—	Very high expression in SupT/CCR5 cells, target expression in <75% of PM1 cells, target expression in <50% of MDMs	Not quantified	50–60% reduction of surface target	Cordelier 2004^219
MHC I	E	ST	Hybridoma, clone OX-18	—	With increasing ER intrabody expression, the cell surface expression of the target decreased (indicated by flow cytometric analysis). Down-regulation of cell surface target was only observed at ER intrabody expression levels of >200 U green fluorescence.	Intracellular accumulation of target	Reduction of specific cell lysis from more than 80% to less than 30% of cells.	Busch 2004^223
VEGF-R2	S	S	Phage display,scFvA2, scFvA7	3.8 nM (Böldicke et al. 2001)	—	—	Not quantified	Böldicke 2005^153
VEGF-R2 and Tie-2	E	S	Phage display from an immune library, clone S08b (Popkov et al. 2003) and VR05 (Popkov et al. 2004)	2S08b: 1nM(Popkov et al. 2003)VR05: 3 nM (Popkov et al. 2004)	Not determined	Not determined	98.6% reduction of VEGF-R2 surface expression and 91.2% reduction of Tie-2 surface expression in HUVEC ; 92.1% reduction of VEGF-R2 and 92.5% reduction of Tir-2 cell surface expression in HMEC-1 cells	Jendreyko 2005^88
Tie-2	E	S	Phage display from an immune library, clones 1S05 and 2S03 (Popkov et al. 2003)	—	Not determined	Not determined	80–90% reduction of cell surface expression of the target	Popkov 2005^131
CCR5	E	S	Phage display from an immune library, clone ST6/34	8.5 nM (Steinberger et al. 2000)	Not determined	Not determined	Not quantified	Swan 2006^218
vIL-6	T	T	Phage display with naïve library (Tomlinson), monoclona l anti-vIL-6 (MAV)	340 nM	Not quantified	Not quantified	30% reduction or 69% reduction of phospho-STAT3 if 0.5 μg or 0.25 μg of target-vector DNA were transfected.	Kovaleva 2006^230
TfR	E	T	hybridoma	—	Not determined	Not determined	83% reduced surface expression of the target at 72h after transfection with the ER intrabody	Peng 2007^233
ApoB	E	S	Hybridoma clone 1D1 and 1C4	—	Not quantified	Not determined	Not quantified	Liao 2008^214
CD147	E	T	Hybridoma, clone M6–1B9	—	Not determined	Not determined	13% of ER intrabody transduced cells were positive for the target while 28% of untransduced and 29% of mock-transduced cells were positive for the target (36h after transduction)	Tragoolpua 2008^229
CD147	E	T	Hybridoma, clone M6–1B9	—	not determined	Not determined	Not quantified	Intrasai 2009^234
VCAM1	S	T	Hybridoma clone 6C7.1	—	not determined	Not determined	43% of cells were negative for VCAM1 48h after transfection with the ER intrabody, 94 % after 96h	Strebe 2009^149
TLR2	TEE	TSS	Hybridoma clone T2.5	—	—	Intracellular accumulation of target	Complete knockdown	Kirschning 2010^4
HBV precore antigen	S	T	Immunoglobulin new antigen receptor (IgNAR) single domain antibody VNAR H6 isolated by phage display from a shark library	53 nM for HBeAg	Not quantified	Slight intracellular accumulation of p25/p17 in AD38 cells, slight reduction of intracellular p25 in pTRE precore and HepG2.2.15 cells	In pTRE precore cells ER intrabody expression resulted in 0.9 fold reduced intracellular p25 levels and 0.3 fold reduction of p17 HBeAg. In secretion incompetent AD38 cells, ER intrabody expression led to a slight increase (1.1-1.2 fold) in p25. In HepG2.2.15 intracellular p25 was reduced 0.6-0.8 fold and secretion of p17 HBeAg was reduced 0.5 fold upon ER intrabody expression.	Walsh 2011^213
p75NTR	E	T	phage display from a naïve library, clones SH325-A11, SH325-B6, SH325-G7	SH325-A11: 6.5×10^–9SH325-B6: 3.9×10^–9SH325-G7:3.9×10^–8	SH325-A11 and SH325-B6: 5–10 ng ER intrabodies per 10^6 cells, SH325-G7:>15 ng ER intrabodies per 10^6 cells	Not determined	56% reduction of cell surface target after 96h (maximal knockdown efficiency was reached 96h after transfection with the ER intrabody plasmid)	Zhang 2012^75
TLR9	T and E	T	Hybridoma clone 5G5	Not determined	Not determined	Not determined	Not determined	Reimer 2013^5
VCAM1	E	S	Hybridoma clone 6C7.1	Not determined	Not quantified	Not determined	Cell surface stainings suggested complete depletion of cell surface VCAM1 in bone marrow cells but absence of the lethal phenotype expected for the complete knockout of VCAM1 hinted at the potential presence of traces of residual VCAM1cell surface expression	Marschall 2014a^134
Aβ oligomers	S	S	scFvA13 selected by IACT in yeast	Not determined	ER intrabody levels were unchanged after treatment with proteasome inhibitor for 6h suggesting degradation does not occur via ERAD, but the ER intrabody had a short half-life, being almost completely degraded after 6 h	No intracellular accumulation of Aβ. Steady state levels of APP and APP C-terminal fragments were similar in control and ER intrabody expressing cells.	60–70% reduction of secreted Aβ dimers and trimers upon expression of the ER intrabody (detected in concentrated conditioned media), 70–80% reduction of secreted oligomeric conformers, increase in monomeric species was observed	Meli 2014^235
Z α antitrypsin	T	T	Hybridoma clone mAb4B12	Not determined	ER Intrabodies were expressed at level comparable to those of the target	Target accumulates upon ER intrabody expression (compared to cells that express a secreted antibody). No marked ER stress was found upon ER intrabody expression and degradation of the ER-intrabody-target complex by proteasomal or autophagic pathways was not observed.	Intracellular polymerization of Z α antitrypsin was reduced by 60%, secretion of Z α antitrypsin was reduced in scFv4B12-KDEL expressing cells and secretion was increased in scFv4B12 expressing cells.	Ordonez 2015

¹Transient expression (T), Stable expression (S), Endogenous expression (E).

²pool (polyclonal cells).

³of oncogenically activated form of target: ErbB2*.

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