Plant Biotechnology Journal (2010) 8, pp. 529–563 doi: 10.1111/j.1467-7652.2009.00494.

Review article
Production of antibodies in plants: status after twenty
years
Benoit De Muynck, Catherine Navarre and Marc Boutry*
Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 5 ⁄ 15, B-1348 Louvain-la-Neuve, Belgium

Received 11 June 2009; Summary

revised 3 September 2009;
accepted 7 September 2009.
*Correspondence (fax 32 10 473872;
e-mail marc.boutry@uclouvain.be)
Keywords: monoclonal antibody,
plant, proteolysis, promoter, subcellu-
lar localization, transformation.
Thanks to their potential to bind virtually all types of molecules; monoclonal antibod-
ies are in increasing demand as therapeutics and diagnostics. To overcome the over-
loading of current production facilities, alternative expression systems have been
developed, of which plants appear the most promising. In this review, we focus on
the expression of monoclonal IgG or IgM in plant species. We analyse the data for
32 different antibodies expressed in various ways, differing in DNA construction,
transformation method, signal peptide source, presence or absence of an endoplas-
mic reticulum retention sequence, host species and the organs tested, together
resulting in 98 reported combinations. A large heterogeneity is found in the quantity
and quality of the antibody produced. We discuss in more detail the strategy used to
express both chains, the nature of the transcription promoters, subcellular localiza-
tion and unintended proteolysis, when encountered.

priate expression host. Plants have appeared as an inter-

Introduction
Since the establishment of monoclonal antibody produc-
tion (Kohler and Milstein, 1975), antibodies have become
important tools in research, as well as in therapy and diag-
nostics. For human therapy, there were initial limitations
because of the non-human origin of monoclonal antibod-
ies. Rodent antibodies are immunogenic in humans and
show variable ability to initiate antibody-dependent cellular
cytotoxicity or complement-dependent cytotoxicity in
humans. This problem was overcome thanks to molecular
engineering and the progressive introduction of chimeric
(Morrison et al., 1984) and humanized (Jones et al., 1986)
antibodies. Besides full antibodies, antibody derivatives,
such as single-chain, single-domain or bispecific antibod-
ies, minibodies as well as protein- or toxin-conjugated
antibodies were also developed (Jain et al., 2007).
In view of the growing demand for recombinant anti-
bodies, existing facilities might rapidly reach saturation, so
there is a need for the development of alternative means
of production to the classical mammalian expression sys-
tems (Stoger et al., 2005b). Because of their complex gly-
cosylation and their assembly from four polypeptides
linked by disulphide bridges, antibodies require an appro-
esting alternative, even though mammalian and plant-type
glycosylations are not identical. Two advantages often
mentioned for plant-produced antibodies are their lower
cost of production compared to mammalian cells and, for
therapeutic purposes, the absence of contamination by
mammalian pathogens. Concerning the former, it is not
obvious that this statement is true, considering the rela-
tively low expression of antibodies in most plant systems
described so far. However, recent progress in expression
systems (e.g. transient expression, see in the following
paragraphs) might change this view. Moreover, an advan-
tage of plant-produced antibodies might be the possibility
of their glycoengineering and, as a result, the production
of improved pharmaceutical proteins (bio-betters). Further-
more, if whole transgenic plants are used instead of cul-
ture cells, antibody production is only limited by land area,
and harvesting can be performed using existing agronomi-
cal facilities (Schillberg et al., 2003).
Since the first report of antibody production in N. taba-
cum plants (Hiatt et al., 1989), antibodies have been
expressed in moss (for review, see Decker and Reski, 2008),
algae (for review, see Franklin and Mayfield, 2005) and vari-
ous dicot and monocot species (Table 1 and references

ª 2010 The Authors

Journal compilation ª 2010 Blackwell Publishing Ltd 529
 Table 1 Expression of antibodies in plants and the strategy used

Signal peptide ⁄ Signal peptide ⁄

sequence sequence Construct Host Expression
Antibody Isotype* Promoter H† Promoter L† tag Hà tag Là type§ species Organ level– Reference

6D4 mIgG1 CaMV 35S Murine ⁄ none T-DNA H and N. tabacum Leaves 1.3% TP; 95% (Hiatt et al.,
T-DNA L, assembly 1989)
530 Benoit De Muynck et al.

None ⁄ none cross-pollination 0% TP; 0%

assembly
S. cerevisiae Native ⁄ none 0.83% TP (Hein et al.,
a-mating 1991)
factor ⁄ none
S. cerevisiae None ⁄ none 0
a-mating
factor ⁄ none
S. cerevisiae 0.77% TP
a-mating
factor ⁄ none
Native ⁄ none S. cerevisiae 0.83% TP
a-mating
factor ⁄ none
None ⁄ none S. cerevisiae 0
a-mating
factor ⁄ none
B 1-8 mIgMk pNOS mas1’2’ Barley Single T-DNA, N. tabacum Plant-derived Not (During et al.,
a-amylase ⁄ fi fi callus tissue, mentioned 1990)
none leaves, stems
MAK33 mIgG1j CaMV 35S A. thaliana T-DNA H and N. tabacum Callus 1.265% TSP (De Neve et al.,
2S2 storage T-DNA L, Leaves 0.055%TSP 1993)
protein ⁄ none co-transformation A. thaliana Leaves 1.013% TSP
A. thaliana Leaves 10.906% TSP (De Wilde
of apoplast et al., 1996)
0.400% TSP
Solanum Leaves 0.1–0.2% TSP (De Wilde
tuberosum Tubers 0.1% TSP et al., 2002)
(in the field)
A. thaliana A. thaliana Leaves 0.2–0.75%
2S2 storage 2S2 storage TSP
protein ⁄ none protein ⁄ DIKDEL Tubers 0.17–0.55% TSP
(in the field)

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
ª 2010 The Authors
 Table 1 Continued

Signal peptide ⁄ Signal peptide ⁄

sequence sequence Construct Host Expression
Antibody Isotype* Promoter H† Promoter L† tag Hà tag Là type§ species Organ level– Reference

ª 2010 The Authors

G1 ⁄ A Chimeric: CaMV 35S Native ⁄ none T-DNA H and N. tabacum Leaves 1.5 lg ⁄ ml (Ma et al., 1994)
var cc1 T-DNA L, plant extract
ca2 ca3 cross-pollination
Guy’s 13 mIgG1j CaMV 35S Murine ⁄ none T-DNA H and N. tabacum Leaves 7.7 lg ⁄ ml plant
T-DNA L, extract
cross-pollination Hairy root 1.0–1.8% TSP (Wongsamuth and
cultures (intracellular) Doran, 1997)
13–18 lg total
ab ⁄ mL culture
3.2 lg ⁄ mL
(extracellular)
530 lg ⁄ g
(intracellular, 7 d)
1.99 lg ⁄ mL
(extracellular, 7 d)
Hairy root 730 lg ⁄ g DW (Sharp and
cultures Doran, 1999)
Suspension 110 lg ⁄ g DW
cells
Hairy root 7.0 lg total ab ⁄ mL (Sharp and

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
cultures culture Doran, 2001a; b)
1100 lg ⁄ g DW
2.4% TSP
Extracellular ab: 26%
of total ab
Shooty 3.2 lg total ab ⁄ mL
teratoma culture
cultures 280 lg ⁄ g DW
1.2% TSP
Suspension 7.5 lg total ab ⁄ mL
cells culture
6.5% TSP
Extracellular ab :
72% of
total ab
1200 lg ⁄ g DW
Antibodies in plants: twenty years later 531
 Table 1 Continued

Signal peptide ⁄ Signal peptide ⁄

sequence sequence Construct Host Expression
Antibody Isotype* Promoter H† Promoter L† tag Hà tag Là type§ species Organ level– Reference

Leaves 1.27% TSP (Nuttall et al.,

2002)
532 Benoit De Muynck et al.

Roots 11.7 lg ⁄ g (Drake et al.,

(rhizosecretion) root DW ⁄ day 2003)
Native ⁄ TM Native ⁄ none Leaves 1.1% TSP with (Vine et al., 2001)
1% Nonidet
P40
21C5 mIgG1j CaMV 35S+ mas1’2’ Murine ⁄ none Single T-DNA, N. tabacum Roots 0.6% TSP (van Engelen
fi fi et al., 1994)
Single T-DNA, 1.1% TSP
‹ fi
mAb24 mIgG2bj CaMV 35S+ Native ⁄ none Single T-DNA, N. tabacum Leaves F0: 0.04–0.16% (Voss et al.,
fi fi TP; F1: 0.07– 1995)
0.3% TP
Suspension 15–16 lg ⁄ g wet (Fischer et al.,
cells cell weight or 1999)
0.3%TSP
6D4 mIgMj CaMV 35S Murine ⁄ none T-DNA H and N. tabacum Leaves 0.01% TP (Baum et al.,
T-DNA L, Roots 0.003% TP 1996)
cross-pollination
Not Humanized CaMV 35S Tobacco extensin ⁄ Two plasmids Soybean Whole plants Not (Zeitlin et al.,
mentioned IgG1j none (H and L), biolistic mentioned 1998)
CO17-1A mIgG2a TMV promoter Native ⁄ His6, Native ⁄ none Inoculation with N. benthamiana Leaves Not (Verch et al.,
KDEL in vitro transcripts mentioned 1998)
of two recombinant
TMV 30B viral
vectors (H and L)
CaMV 35S+ Pin2 Native ⁄ none Single T-DNA, N. tabacum Leaves 0.9 lg ⁄ g FW (Ko et al., 2005)
fi fi or 0.02% TSP
C5-1 mIgGj CaMV 35S Native ⁄ none T-DNA H and T-DNA Medicago sativa Leaves 0.13–1.0% TSP (Khoudi et al.,
L, cross-pollination 1999)
CaMV 35S and CPMV Native ⁄ none Agroinfiltration with two N. benthamiana Leaves 0.4% TSP or 19 (Sainsbury
promoter full length CPMV RNA-2 lg ⁄ g FW et al., 2008)
vectors (H and L), CPMV
RNA-1 and HcPro (silencing
suppressor) vectors, (tr.)

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
ª 2010 The Authors
 Table 1 Continued

Signal peptide ⁄ Signal peptide ⁄

sequence sequence Construct Host Expression
Antibody Isotype* Promoter H† Promoter L† tag Hà tag Là type§ species Organ level– Reference

ª 2010 The Authors

Agroinfiltration with two 0.75% TSP
CPMV delRNA-2 vectors
(H and L), CPMV RNA-1
and HcPro (silencing
suppressor) vectors, (tr.)
Native ⁄ SEKDEL Native ⁄ none Agroinfiltration with two 0.2% TSP
full length CPMV RNA-2
vectors (H and L), CPMV
RNA-1 and HcPro (silencing
suppressor) vectors, (tr.)
Agroinfiltration with two 1.9% TSP or
CPMV delRNA-2 vectors 74 lg ⁄ g FW
(H and L), CPMV RNA-1
and HcPro (silencing
suppressor) vectors, (tr.)
Native ⁄ none Native ⁄ SEKDEL Agroinfiltration with two Not mentioned
full length CPMV RNA-2
vectors (H and L), CPMV
RNA-1 and HcPro (silencing

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
suppressor) vectors, (tr.)
Agroinfiltration with two Not mentioned
CPMV delRNA-2 vectors
(H and L), CPMV RNA-1
and HcPro (silencing
suppressor) vectors, (tr.)
Native ⁄ SEKDEL Agroinfiltration with two 0.3% TSP
full length CPMV RNA-2
vectors (H and L), CPMV
RNA-1 and HcPro (silencing
suppressor) vectors, (tr.)
Agroinfiltration with two 1.7% TSP
CPMV delRNA-2 vectors
(H and L), CPMV RNA-1
and HcPro (silencing
suppressor) vectors, (tr.)
Antibodies in plants: twenty years later 533
 Table 1 Continued

Signal peptide ⁄ Signal peptide ⁄

sequence sequence Construct Host Expression
Antibody Isotype* Promoter H† Promoter L† tag Hà tag Là type§ species Organ level– Reference

Medicago sativa Native ⁄ none Single plasmid, N. benthamiana Leaves 106 lg ⁄ g FW (Vezina et al., 2009)
plastocyanin fi fi , (tr.) [558 lg ⁄ g FW]
promoter Native ⁄ KDEL Native ⁄ none [co-expression 211 lg ⁄ g FW
534 Benoit De Muynck et al.

with HcPro [757 lg ⁄ g FW]

(silencing
suppressor)]
cT84.66 Chimeric IgG1 CaMV 35S+ Murine Murine Two plasmids N. tabacum Leaves 1 lg ⁄ g FW (Vaquero et al.,
(optimized) ⁄ (optimized) ⁄ (H and L), (tr.) 1999)
KDEL none
rAb29 mIgG1j CaMV 35S+ None ⁄ none Single T-DNA, N. tabacum Leaves 0.02 lg ⁄ g FW (Schillberg et al.,
Native ⁄ none fi fi 8.5 lg ⁄ g FW, 1999)
0.14% TSP
MGR48 mIgG1j CaMV 35S+ mas1’2’ Murine ⁄ none Single T-DNA N. tabacum Leaves 30–60 lg ⁄ g FW (Stevens et al.,
(top leaves) 2000)
5–15 lg ⁄ g FW
(bottom leaves)
GAN4B.5 hIgG1 CaMV 35S Murine ⁄ none T-DNA H and A. thaliana Whole plants 0.6% TSP (Bouquin et al.,
T-DNA L, 2002)
cross-pollination
CaMV 35S Single T-DNA, 0.3% TSP
fi fi
mas1’2’ Single T-DNA, 0.12% TSP
‹ fi
cPIPP Chimeric CaMV 35S+ Murine ⁄ KDEL Two plasmids N. tabacum Leaves 20–40 lg (Kathuria et al.,
IgG1j (H and L), (tr.) purified ⁄ g FW 2002b)
Murine Two plasmids 20–40 lg (Kathuria et al.,
(optimized) ⁄ KDEL (H and L), (tr.) purified ⁄ g FW 2002a)
Murine (optimized) ⁄ T-DNA H and Not mentioned (Sriraman et al.,
VDGGGGSAA T-DNA L, 2004)
ARGSEKDEL cross-pollination
Murine (optimized) ⁄ Two plasmids 0.02%–0.1% total
none (H and L), (tr.) extracted protein
Murine (optimized) ⁄ Two plasmids Physalis Not mentioned
VDGGGGSAAAR (H and L), (tr.) alkekengi
GSEKDEL
CB-Hep.1 mIgG2bj CaMV 35S Sweet potato Single T-DNA, N. tabacum Leaves 60 lg ⁄ g FW or (Ramirez et al.,
sporamin ⁄ KDEL fi fi 0.5% TSP 2003)

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
ª 2010 The Authors
Seedlings 17 lg ⁄ g DW (Triguero et al., 2005)
 Table 1 Continued

Signal peptide ⁄ Signal peptide ⁄

sequence sequence Construct Host Expression
Antibody Isotype* Promoter H† Promoter L† tag Hà tag Là type§ species Organ level– Reference

ª 2010 The Authors

Leaves 31.7 lg ⁄ g DW
Stems 1.8 lg ⁄ g DW
Whole 19.6 lg ⁄ g DW
plants
Leaves 25 lg purified ⁄ g FW (Valdes et al.,
(9 weeks plants) 2003)
11 lg purified ⁄ g FW
(10 weeks plants)
SO57 hIgG1k CaMV 35S+ Pin2 Native ⁄ KDEL Native ⁄ none Single T-DNA, N. tabacum Leaves 3 lg ⁄ g FW or (Ko et al., 2003)
fi fi 0.07% TSP
Suspension 10–30 lg ⁄ g DW (Girard et al.,
cells 2006)
CL4 hIgG1j CaMV 35S Native ⁄ none Single T-DNA, fi fi N. tabacum Suspension 0.2% TSP (Yano et al., 2004)
N. plumbaginifolia cells 0.2–0.6% TSP;
calreticulin ⁄ none up to 16.2 lg
purified ⁄ mL
culture
Barley hordothionin ⁄ none 0.25% TSP
hOAT Humanized IgG1j 2,4-D inducible Tomato SBT1 Two plasmids Lactuca sativa Leaves No data (Negrouk et al.,
(OCS)3Mas subtilase ⁄ none (H and L), (tr.) published 2005)

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promoter Single plasmid, 18.1 lg ⁄ g FW
fi fi (tr.)
IQ6E4 hIgGj CaMV 35S Native ⁄ none Two plasmids N. benthamiana Leaves No data (Hull et al.,
(H and L), (tr.) published 2005)
TheraCIM Humanized IgG1j CaMV 35S Sweet potato Single plasmid, N. tabacum Leaves 1.2 lg purified ⁄ g (Rodriguez et al.,
sporamin ⁄ KDEL fi fi , (tr.) FW 2005)
Not IgG4 mas2’(Leung N. plumbaginifolia Single T-DNA, N. tabacum Roots 8.2 lg ⁄ g DW (Komarnytsky
mentioned et al., 1991) calreticulin ⁄ none fi ‹ with et al., 2006)
non-secreted
BBI
21.8 lg ⁄ g DW
with secreted
BBI
14D9 mIgG1j CaMV 35S+ Murine ⁄ none T-DNA H and N. tabacum Leaves 2.9% TSP (Petruccelli
T-DNA L, et al., 2006)
cross-pollination
Antibodies in plants: twenty years later 535
 Table 1 Continued

Signal peptide ⁄ Signal peptide ⁄

sequence sequence Construct Host Expression
Antibody Isotype* Promoter H† Promoter L† tag Hà tag Là type§ species Organ level– Reference

Seeds  0.2% TSP

Murine ⁄ Leaves 5.2% TSP
SEKDEL Seeds 0.41%TSP
536 Benoit De Muynck et al.

7S Murine ⁄ none Seeds  0.05% TSP

Murine ⁄ 1.08% TSP
SEKDEL
BR55-2 mIgG2aj CaMV 35S+ Native ⁄ KDEL Native ⁄ none Single T-DNA, N. tabacum Leaves 31 lg ⁄ g FW (Brodzik et al.,
‹ fi 2006)
A5 hIgG1k CaMV 35S Native ⁄ none Two plasmids (H and L), (tr.) N. benthamiana Leaves 10–15 (Giritch et al.,
lg ⁄ g FW 2006)
Pact and CaMV 35S Agroinfiltration with two TMV 500
TMV and PVX provector modules (5¢ and 3¢ lg ⁄ g FW
promoter promoter H), two PVX provectors
modules (5¢ and 3¢ L) and two
auxiliary plasmids (recombinase
and PVX CP), (tr.)
CaMV 35S and Pact and Agroinfiltration with two TMV 200–300
PVX promoter TMV provector modules (5¢ and 3¢ L), lg ⁄ g FW
promoter two PVX provectors modules (5¢
and 3¢ H) and two auxiliary
plasmids (recombinase and
PVX CP), (tr.)
Not Chimeric CaMV 35S+ A. thaliana basic A. thaliana Single T-DNA, N. tabacum Leaves 4 lg purified ⁄ g FW (McLean et al.,
mentioned IgG1j chitinase ⁄ basic chitinase ⁄ none fi fi 2007)
6His+KDEL)
2G12 hIgG1 gt-1 Native ⁄ none Three plasmids Zea mays Seeds 75 lg ⁄ g DW (Ramessar et al.,
(H, L and selectable 2008)
marker), biolistic
CaMV 35S+ Single T-DNA, fi fi A. thaliana Leaves 0.2% TSP (Schahs et al., 2007)
gt-1 Native ⁄ SEKDEL Two plasmids Zea mays Seeds F1: max 30 lg ⁄ g (Rademacher
(H and L), biolistic DW; F3: 40–60 et al., 2008)
lg ⁄ g DW
CaMV 35S Native ⁄ none Agroinfiltration with N. benthamiana Leaves 47 lg ⁄ g FW (Sainsbury and
two CPMV delRNA-2 Lomonossoff,
vectors (H and L) and 2008)
P19 (silencing
suppressor), (tr.)

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
ª 2010 The Authors
 Table 1 Continued

Signal peptide ⁄ Signal peptide ⁄

sequence sequence Construct Host Expression
Antibody Isotype* Promoter H† Promoter L† tag Hà tag Là type§ species Organ level– Reference

ª 2010 The Authors

Agroinfiltration with two 117 lg ⁄ g FW
CPMV delHTRNA-2
vectors (H and L) and
P19 (silencing
suppressor), (tr.)
Native ⁄ HDEL Native ⁄ none Agroinfiltration with 120 lg ⁄ g FW
two CPMV delRNA-2
vectors
(H and L) and P19
(silencing suppressor),
(tr.)
Agroinfiltration with 325 lg ⁄ g FW
two CPMV delHTRNA-2
vectors (H and L) and
P19 (silencing
suppressor), (tr.)
2F5 hIgG1 CaMV 35S+ Native ⁄ SEKDEL Single T-DNA, N. tabacum Suspension 6.4 lg (Sack et al.,
fi fi cells purified ⁄ g 2007)
FW
CaMV 35S Native ⁄ c-myc, Native ⁄ c-myc, T-DNA H and Leaves 0.1% TSP (Floss et al.,

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
KDEL KDEL T-DNA L, 2008)
Native ⁄ c-myc, cross-pollination 0.3% TSP
ELP, KDEL
Native ⁄ c-myc, Native ⁄ c-myc, 0.2% TSP
ELP, KDEL KDEL
Native ⁄ c-myc, 0.6% TSP
ELP, KDEL
Not mentioned mIgG2a CaMV 35S with Native ⁄ none T-DNA H and N. tabacum Leaves Not (Hong et al.,
transcriptional T-DNA L, mentioned 2007)
and translational cross-pollination
enhancer regions
(Kwon et al., 2003)
H10 hIgG1k CaMV 35S Murine ⁄ none T-DNA H and N. tabacum Leaves 0.6–1.1 lg (Villani et al.,
T-DNA L, purified ⁄ g FW 2009)
cross-pollination
Two plasmids N. benthamiana Leaves 10 lg
(H and L), (tr.) purified ⁄ g FW
Antibodies in plants: twenty years later 537
 Table 1 Continued

Signal peptide ⁄ Signal peptide ⁄

538 Benoit De Muynck et al.

sequence sequence Construct Host Expression

Antibody Isotype* Promoter H† Promoter L† tag Hà tag Là type§ species Organ level– Reference

LO-BM2 Chimeric IgG1j En2pPMA4 Native ⁄ none Single T-DNA, fi ‹ N. tabacum Suspension cells 0 (De Muynck
Leaves 0 et al., 2009)
Single T-DNA, fi fi Suspension cells 0.22% TSP (cell
extract)
0.2 lg ⁄ mL
(extracellular
medium)
Leaves 0.29% TSP of
whole leaf
1% of leaf ab in
the apoplast

*m, murine; h, human; j, kappa light chain; k, lambda light chain; chimeric: var cc1 ca2 ca3, chimeric antibody containing a mixed gamma ⁄ alpha heavy chain;
†
H, heavy chain; L, light chain; when a single promoter is mentioned, it was used to drive transcription of both heavy and light chain genes; CaMV 35S, cauliflower mosaic virus 35S; CaMV 35S+, cauliflower mosaic
virus 35S with duplicated enhancer (Kay et al., 1987); NOS, nopaline synthase gene of Agrobacterium tumefaciens; mas1’2’, mannopine synthase genes 1 and 2 of Agrobacterium tumefaciens (organized as divergent
transcription units) (Velten et al., 1984); TMV, tobacco mosaic virus; CPMV, cowpea mosaic virus; Pin2, potato proteinase inhibitor II gene (Korth and Dixon, 1997); (OCS)3Mas promoter, promoter of the mannopine
synthase gene of Agrobacterium tumefaciens reinforced by a trimer of the upstream activating sequence of the octopine synthase gene (also from Agrobacterium tumefaciens) (Gelvin et al., 1999); 7S, a’ subunit of
b-conglycinin (7S) soybean storage protein promoter (Fujiwara et al., 1992); Pact, Arabidopsis thaliana actin 2 promoter; PVX, potato virus X; gt-1, rice glutelin-1 promoter; En2pPMA4, promoter of the Nicotiana
plumbaginifolia plasma membrane ATPase4 gene reinforced by two copies of the cauliflower mosaic virus 35S enhancer (Zhao et al., 1999);
à
When a single signal peptide is mentioned, it was used to drive targeting of both heavy and light chains; Sequence tag: KDEL, c-myc, His6, TM (transmembrane sequence), ELP (elastin-like peptides);
§
Single T-DNA, T-DNA bearing both heavy and light chain coding sequences; T-DNA H, T-DNA bearing the heavy chain coding sequence; T-DNA L, T-DNA bearing the light chain coding sequence; the arrows refer to
the relative orientation of the transgenes; TMV, tobacco mosaic virus; CPMV delRNA-2, cowpea mosaic virus RNA-2 with the region encoding the movement protein and both coat proteins deleted (Canizares et al.,
2006); (tr.), transient expression; CP coat protein; CPMV delHTRNA-2, hypertranslatable cowpea mosaic virus RNA-2 lacking AUG 161 and with the region encoding the movement protein and both coat proteins
deleted;
–
TP, total proteins; TSP, total soluble proteins; ab, antibody; total ab, antibody extracted from both the biological material and the culture medium; d, day; DW, dry weight; FW, fresh weight; F0, transformed plant;
F1 (F3): first (third) generation obtained after sexual crossing; BBI, Bowman–Birk serine peptidases inhibitor.

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
ª 2010 The Authors

herein, Stoger et al. (2005a)). Different expression strategies
have been successfully used, i.e. transient or stable expres-
sion and the use of vector-dependent (Agrobacterium or
viral) or vector-free transformation. However, the wide
experience gained of expression of antibodies in plants indi-
cates that there are still problems regarding expression, sub-
cellular localization and proteolytic degradation. Close
inspection of the literature shows that these problems vary
depending on the antibody, host system or expression sys-
tem used. In this review, we compare the data obtained for
32 different IgGs or IgMs expressed in either monocot or
dicot species. We focused on whole antibodies because
they require expression of two genes, an additional problem
compared to the expression of single-chain fragments.
The majority were expressed in various ways, differing in
the gene construction and transformation method used,
the nature of the signal peptide, the presence or absence of
an endoplasmic reticulum (ER) retention sequence, the host
species and the organs tested, resulting in 98 reported
combinations (Table 1). Using these data, we discuss in
more detail the type of molecular constructs used, the
nature of the transcription promoters, subcellular localiza-
tion and unintended proteolysis, when encountered.
A single or two independent insertion events?
Because antibodies contain two distinct polypeptides, two
possible insertion strategies are conceivable, the insertion of
a single DNA construct (usually a T-DNA insert) bearing both
the heavy and light chain coding sequences (irrespective of
their relative arrangement) or two independent insertion
events. In the latter, two different approaches have been
used: 1 - the generation of independent plants, each con-
taining one transgene (heavy or light chain cassette) fol-
lowed by cross-pollination of individuals to obtain plants
Table 2 Influence of the transgene insertion strategy on the expression level
Antibody Insertion strategy*
21C5 Single T-DNA, tandem orientation
Single T-DNA, inverted divergent orientation
GAN4B.5 T-DNA H and T-DNA L, cross-pollination
Single T-DNA, tandem orientation
LO-BM2 Single T-DNA, inverted convergent orientation
Single T-DNA, tandem orientation
*
Single T-DNA, T-DNA bearing both heavy and light chain coding sequences; T-DNA H, T-DNA bearing the heavy chain coding sequence; T-DNA L, T-DNA
bearing the light chain coding sequence;
†
TSP, total soluble proteins;
à
values for expression in the plant.
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
Antibodies in plants: twenty years later 539
expressing both the heavy and light chains (described here-
after as the cross-pollination method); 2 - the concomitant
co-transformation with both DNA constructs. Concomitant
co-transformation with two different plasmids resulted, in
some cases, in the insertion of both transgenes in the same
locus and, sometimes, in complex insertion patterns involv-
ing transgene repeats (De Block and Debrouwer, 1991; De
Neve et al., 1997, 1999; Ramessar et al., 2008). This might
thus be considered as a single DNA construct-like configura-
tion. It is rather difficult to draw any clear conclusion about
the best transgene(s) insertion strategy (Table 1), because
the data were obtained for different antibodies, different
host plants and different vectors. However, Bouquin et al.
(2002) compared the expression of GAN4B.5 using either
transformation with a single T-DNA bearing both the heavy
and light chain coding sequences in tandem or the cross-
pollination method, and, having tested approximately 20
homozygous lines for each construct, found better expres-
sion with the latter (Table 2). Other reports showed that the
accumulation of mRNAs for heavy and light chains occurred
independently, even though they were controlled by identi-
cal regulatory elements (Voss et al., 1995; Law et al., 2006)
and within a single T-DNA (Voss et al., 1995). It is therefore
unlikely to find a plant in which both transgenes are tran-
scribed at a particularly high level. In conclusion, cross-polli-
nation seems to be the most promising method. However,
it was not used in most cases reported (Figure 1a), probably
because it is more time-consuming. Furthermore, it cannot
be used when suspension cells or vegetatively-reproduced
plants (e.g. some varieties of potato) are transformed.
Transgene design
Most of the single T-DNA constructs tested so far were in
the tandem orientation. However, although only poorly
Expression level
(% TSP†) Host species Reference
0.6 N. tabacum (van Engelen et al., 1994)
1.1
0.6 A. thaliana (Bouquin et al., 2002)
0.3
 0à N. tabacum (De Muynck et al., 2009)
0.29à
540 Benoit De Muynck et al.

(a) (b)

(c) (d)

Figure 1 Frequency of expression strategies used (a), organs tested (b), combinations of promoters used (c) and species transformed (d), to
express monoclonal antibodies in plants among the 98 combinations reported in Table 1. Abbreviations are as in Table 1. In (c), when a single pro-
moter is mentioned, this promoter was used for both heavy and light chain gene transcription; When two promoters are mentioned, the first one
was used to drive heavy chain gene transcription and the second one to drive light chain gene transcription.

investigated, the relative arrangement of the two transg-
enes seems to be of upmost importance (Table 2). The
expression of antibody 21C5 was nearly twice as high in
lines in which the two transgenes were arranged in a
divergent orientation than in lines in which they were in
tandem. This difference could not be attributed to the
influence of neighbouring plant DNA, as the same result
was observed in transient expression assays (van Engelen
et al., 1994). The authors suggested that the two cauli-
flower mosaic virus (CaMV) 35S promoters used in this
case acted as transcription enhancers for each other in a
distance-dependent way, as shown by Kay et al. (1987). In
another study, expression of the antibody LO-BM2 using
an inverted convergent configuration of the transgenes
was very poor compared to that using the tandem config-
uration (De Muynck et al., 2009). These authors hypothe-
sized that leaky transcription termination would result in
the synthesis of transcripts encompassing both genes, thus
generating double-stranded RNA (dsRNA), which might
catalyse RNA silencing (De Wilde et al., 2000; De Muynck
et al., 2009). Note that the inverted convergent orienta-
tion does not always lead to poor results, as exemplified
by the yield of 8.2 lg antibody ⁄ g root dry weight per day
obtained by Komarnytsky et al. (2006). However, in the
transformation vector used in their study, the heavy and
light chain genes were separated by root proliferation
genes. An inverted divergent orientation of two reporter
genes had previously been shown to be a better arrange-
ment than an inverted convergent orientation (Padidam
and Cao, 2001). The authors attributed this result to tran-
scriptional interference, in which RNA polymerases
recruited by the two promoters interfere, because of leaky
terminators (Shearwin et al., 2005). Given these results,
the orientation of the selectable marker relative to the
other transgene(s) should also be taken into account.
While obtaining high expression levels during plant
screening is an important factor, maintaining it during
subsequent generations is also critical. De Neve et al.

ª 2010 The Authors

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563

(1999) studied instability of antibody production in con-
nection with gene silencing. In particular, they showed
that, in three lines obtained after co-transformation and
showing transcriptional gene silencing, the transgenes
were linked to each other at a single locus and oriented
convergently. They also showed that, in two lines in which
the transgenes were located at different loci, post-tran-
scriptional gene silencing was somehow triggered during
development by the presence of the light chain gene
insert. Another striking observation is that plants trans-
formed using Agrobacterium sometimes display sequences
originating from outside the right and left borders of the
binary vector (Kononov et al., 1997; De Wilde et al.,
2000). Some authors (Iglesias et al., 1997; Matzke and
Matzke, 1998; De Wilde et al., 2000; Agrawal et al.,
2005) suggested that vector backbone sequences, because
of their prokaryotic origin (and thus their different nucleo-
tide composition compared to plant DNA), may trigger
de novo DNA methylation, resulting in transgene silencing.
According to Matzke and Matzke (1998), the apparent
tendency of multicopy transgene loci to become silenced
might actually reflect a higher probability that binary vec-
tor sequences outside the T-DNA are present in complex
inserts. An alternative method for overcoming this phe-
nomenon might be biolistic plant transformation using
minimal cassettes (i.e. promoter, transgene and terminator
in the form of linear DNA, following restriction digestion
of the plasmid) (Altpeter et al., 2005).
Whatever the transformation method used, a key fea-
ture for high antibody expression might be a clean trans-
formation, i.e. irrespective of the copy number or
repeated elements, a transformation event in which the
transferred sequences are intact, not rearranged and free
of vector backbone (Agrawal et al., 2005). Moreover, Igle-
sias et al. (1997) showed that out of four independent
transgenic tobacco lines, the two that displayed stable
expression in the homozygous state over several genera-
tions had their insert in the vicinity of the telomeres,
whereas, in the other two, the insert was in intercalary
and paracentromeric locations. Although the number of
transgenic plants studied was not sufficient to draw a gen-
eral conclusion, this observation underlines the necessity
for a detailed study of the insertion locus.
Another strategy allowing increased transgene expres-
sion consists of flanking the transgene with matrix attach-
ment regions (MARs), such as the tobacco Rb7. This
strategy has been used by some authors to improve anti-
body yield (Sack et al., 2007; Schahs et al., 2007; Rade-
macher et al., 2008; Ramessar et al., 2008). MARs are
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
Antibodies in plants: twenty years later 541
DNA sequences that bind to the proteinaceous network
within the nucleus (Spiker and Thompson, 1996). It was
postulated that transgenes flanked by MARs adopt a
three-dimensional conformation that is prone to transcrip-
tion (Spiker and Thompson, 1996), allowing increased
transcription by an order of magnitude (Streatfield, 2007).
MARs might also act by preventing both the spreading of
silent chromatin into the transgene (Abranches et al.,
2005) and the RNA-mediated silencing in cis because of
transcriptional read-through into the regions flanking the
transgene (De Wilde et al., 2000; Abranches et al., 2005).
This is in agreement with the previously mentioned tran-
scriptional interference and dsRNA-induced silencing.
Transcription promoters
The choice of transcription promoters to express hetero-
multimeric proteins, such as antibodies, has to take into
account not only the high expression level expected, but
also the coordinated expression of both the heavy and
light chain genes. This latter point explains the widespread
use of identical promoters to drive heavy and light chain
gene transcription (see Table 1 and Figure 1c). Neverthe-
less, one should carefully weigh the need for the coordi-
nated expression of both genes against the risk of
homology-dependent gene silencing inherent in the use of
identical regulatory elements. van Engelen et al. (1994)
observed the production of similar amounts of 21C5
heavy and light chain mRNAs using two different promot-
ers in the same T-DNA (see Table 1). Furthermore, they
showed that levels of heavy and light chain mRNAs covar-
ied in different independent transformants. In contrast,
Voss et al. (1995) and Law et al. (2006) reported different
levels of heavy and light chains mRNAs despite using the
same transcription promoter. These conflicting results
highlight the difficulty in guaranteeing the coordinated
expression of the two genes.
Various strategies have been used to further improve
the final yield. These include adding enhancers of tran-
scription [e.g. the amplification-promoting sequence iso-
lated from the non-transcribed spacer region of tobacco
ribosomal DNA (Borisjuk et al., 2000)] or translation (e.g.
the X translational enhancer sequence from tobacco
mosaic virus) and ⁄ or mRNA-stabilizing 5¢ and 3¢ untrans-
lated regions (UTRs) (e.g. those from the chalcone syn-
thase gene). This latter strategy not only increases the
transcription rate, but also favours correct mRNA process-
ing, resulting in increased mRNA stability (Streatfield,
2007).
542 Benoit De Muynck et al.
It is noteworthy that only thirteen different promoters
were used to drive heavy and light chain gene transcrip-
tion of the different antibodies listed in Table 1. This prob-
ably reflects the low number of strong plant transcription
promoters available. Furthermore, of these thirteen pro-
moters, only six are directly derived from plants, the others
being derived from either Agrobacterium tumefaciens
T-DNA genes or the CaMV 35S transcript, by far the most
widely used (see Figure 1c). Of special interest is the Medi-
cago sativa plastocyanin promoter used by Vezina et al.
(2009) to transiently express the C5-1 antibody in Nicoti-
ana benthamiana leaves. Plastocyanin is a protein involved
in electron transfer during photosynthesis and is therefore
highly expressed in the palisade layer and spongy paren-
chyma, where initial agroinfection takes place during tran-
sient expression. Using this promoter and the
corresponding 5¢ and 3¢ UTRs to drive heavy and light
chain transcription in a tandem cassette, as much as
757 lg antibody ⁄ g leaves fresh weight (FW) was obtained
[antibody retained in the ER and using the co-expressed
silencing suppressor HcPro (Vezina et al., 2009)]. Seed-
specific promoters offer an interesting option, as seeds are
generally considered a stable storage compartment for
recombinant antibodies. This is particularly the case if
accumulation occurs just prior to desiccation (Streatfield,
2007). Moreover, the seed endosperm has a triploid gen-
ome, thus increasing transgene dosage (zygosity level) and
thus the antibody content in mature seeds, as reported by
Law et al. (2006). In contrast, the number of inserted
transgenes following a single transformation event did not
correlate with antibody levels (Law et al., 2006). Inducible
transcription promoters might also be useful in preventing
toxicity of antibody expression (although, to our knowl-
edge, this has never been reported) or transgene silencing.
In this respect, the development of new constitutive and
inducible strong promoters would probably represent
important progress in heterologous expression.
Besides the transcription rate, RNA stability is a major
factor in ensuring high expression (Green, 1993). Much
less is known about the role of the 5¢ and 3¢ UTRs.
Although such regions taken from plant transcripts have
been added to recombinant antibody-coding sequences,
their exact role is still unclear, especially because the
replacement of the original plant-coding sequences by
antibody-coding sequences might considerably modify the
3-D structure of these chimeric transcripts and lead to the
loss of the potential positive effects of these UTRs.
Although not related to transcription or transcript stability,
we should also mention the growing interest in optimizing
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
protein translation. Optimization of the translation initia-
tion codon context is easy, as it involves only a few nucle-
otides. On the other hand, adapting the whole coding
sequence to the host codon usage is much more time-
consuming and expensive. Interestingly, Batard et al.
(2000) improved the expression of a P450 oxygenase by
limiting codon optimization to the first 40 amino acid
region. This strategy allowed a fivefold increase in enzy-
matic activity. Nevertheless, if optimization of the whole
sequence is preferred, putative cis-acting sequence motifs
(e.g. cryptic splice sites, RNA-destabilizing sequence ele-
ments) can be removed at the same time as the codon
bias is adapted.
Transient expression
Transient expression classically consists in the Agrobacte-
rium tumefaciens-mediated delivery of binary transforma-
tion vector into the plant cell following leaf infiltration.
This method was generally used either to test the efficacy
of an expression cassette or to produce small amount of
proteins. Nevertheless, recent progress such as the devel-
opment of new promoters (e.g. Vezina et al. (2009), men-
tioned earlier and Table 1) or the introduction of viral
vector-based expression systems have revolutionized the
field of transient expression. This method now allows the
rapid and high-yield production of heterologous proteins.
Viral vector-based expression systems were developed
to express multimeric proteins, such as antibodies (see
Table 1 and Figure 1a,c). Initial attempts were made by
co-infecting N. benthamiana plants with in vitro synthe-
sized transcripts of two recombinant tobacco mosaic
viruses (TMV) modified to encode either the antibody
heavy or light chain (Verch et al., 1998). However, Giritch
et al. (2006) showed that using two TMV-based vectors to
express two different proteins rapidly led to a spatial sepa-
ration of the two distinct TMV populations in the infil-
trated tissues. To overcome this limitation, Giritch et al.
(2006) used non-competing viral vectors [TMV and potato
virus X (PVX)] to drive antibody expression. This innovative
method was used together with magnifection technology
(Marillonnet et al., 2005), which is based on the replica-
tion of viral vectors delivered to the plant by Agrobacte-
rium tumefaciens infiltration. In this case, the plant
promoter placed at the 5¢ end of the modified viral gen-
ome mainly acts to produce the first viral-like mRNA, as
the subsequent replication steps are carried out by the
viral RNA-dependant RNA polymerase. Sainsbury et al.
(2008) took advantage of the bipartite RNA cowpea
ª 2010 The Authors

mosaic virus (CPMV) to express the C5-1 antibody via ag-
roinfection. In this system, the RNA-1 vector encoding the
replicase was co-infiltrated with two modified RNA-2 vec-
tors encoding either the heavy or light chain. While the
infiltrated tissues showed co-expression of both chains,
the modified RNA-2 molecules segregated during the sys-
temic spread of CPMV. As the advantage of systemic
spread was lost, a deleted version of RNA-2 (delRNA-2), in
which the region encoding the movement protein and
both coat proteins had been removed, but the elements
needed for RNA-1-mediated replication were retained,
was tested to allow the cloning of larger inserts and avoid
the bio-containment problem. Different combinations of
full length and deleted versions of RNA-2 molecules
encoding heavy and light chains were used to express C5-
1 and, in all cases, a higher yield (up to 1.9% of the total
soluble protein, corresponding to 74 lg antibody ⁄ g FW)
was obtained with the deleted version (see Table 1) (Sains-
bury et al., 2008). In a more recent version of this
method, Sainsbury and Lomonossoff (2008) developed a
delRNA-2-based expression system without using RNA-1-
mediated replication. Transcription is therefore under the
control of a plant promoter, and high expression is con-
ferred by the viral 5¢ and 3¢ UTR. This vector was further
improved by deleting an in-frame AUG codon [referred to
as hypertranslatable deleted RNA-2 (delHTRNA-2)]. Up to
325 lg antibody ⁄ g FW was obtained (ER-accumulated
antibody). Note that, in the CPMV-based expression
system reported earlier, a silencing suppressor was
co-expressed.
Antibody glycosylation
In mammals, both the heavy and light chains are synthe-
sized with a peptide signal, which targets the nascent pro-
teins to the ER lumen, where the antibody is assembled.
Complex glycosylation takes place in the ER and the Golgi
before the antibody is secreted. Plant and animal glycosy-
lations are similar in the early steps but differ in the later
steps. However, plants have been genetically engineered
to mimic the typical animal glycosylation pattern and so
prevent potential side effects and rapid clearance from the
blood stream of plant-made pharmaceuticals (Saint-Jore-
Dupas et al., 2007). In recent years, glycoengineering has
been addressed in two different ways: inactivating endog-
enous glycosyltransferases and ⁄ or expressing heterologous
glycosyltransferases.
Koprivova et al. (2004) disrupted the genes for a1,3-
fucosyltransferase (Fuc-T) and b1,2-xylosyltransferase (Xyl-T)
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
Antibodies in plants: twenty years later 543
in Physcomitrella patens by homologous recombination.
Plant growth and morphology were not impaired. Mass
spectrometry analysis of N-glycans from the double knock-
out plant showed complete absence of a1,3-fucosyl and
b1,2-xylosyl residues. Moreover, secretion of the human
glycosylated growth factor VEGF121 was shown to be as
effective in double knockout than in WT plants.
Cox et al. (2006) used a single T-DNA construct to sta-
bly express an antibody and an RNA interference cassette
aiming at silencing both Fuc-T and Xyl-T in the aquatic
plant Lemna minor. GnGn was shown to make up 95.8%
of the total N-glycans harboured by the antibody and no
a1,3-fucosyl and b1,2-xylosyl residues were found.
The high glycosylation homogeneity offered by this system
undoubtedly constitutes an advantage compared to
cultured mammalian cells that generally produce hetero-
geneous N-glycosylation. Moreover, the plant glycoengi-
neered antibody was shown to exhibit higher binding
affinity for human Fc receptors, resulting in a 20- to
35-fold increase in biological activity compared to its
CHO-produced counterpart.
Instead of inactivating Fuc-T and Xyl-T expression,
Vezina et al. (2009) rather expressed a chimeric form of
the human b1,4-galactosyltransferase (Gal-T). The chimeric
protein consisted in a protein fusion between the N-termi-
nal part (cytosolic tail and transmembrane domain) of
A. thaliana N-acetylglucosaminyltransferase I (GNTI) and
the catalytic domain of human Gal-T. The transmembrane
and cytosolic domains of GNTI allowed targeting of the
Gal-T activity in the ER and the cis-Golgi apparatus, i.e.
upstream from Fuc-T and Xyl-T activity in the plant secre-
tory pathway. Transient co-expression of this chimeric con-
struct together with genes encoding C5-1 antibody heavy
and light chains in N. benthamiana leaves resulted in high-
yield production of the antibody containing no detectable
(<1%) a1,3-fucose and b1,2-xylose.
Beside sensu stricto plant glycoengineering, other strate-
gies were used to alleviate plant-made N-glycosylation lim-
itations in antibody production. These include mutation of
N-glycosylation sites to produce aglycosylated antibodies
(Rodriguez et al., 2005) and the addition of ER retention
signal to prevent the addition of complex-type glycans
(see Table 1). Nevertheless, both methods are not desir-
able as N-glycans structure is determinant in antibody
functional activity and half-life (Saint-Jore-Dupas et al.,
2007).
To conclude this part, it is clear that glycosylation engi-
neering is effective in adapting plant-made antibodies to
therapeutic applications.
544 Benoit De Muynck et al.
Subcellular localization
In the first attempt to express an antibody in plants, several
different constructs were tested: one in which both the
heavy and light chain cDNAs retained their native signal
peptide sequence, one in which this sequence was
removed from both cDNAs and two other combinations in
which only one of the two chains retained its signal pep-
tide (Hiatt et al., 1989; Hein et al., 1991). The results
showed that, as in mammalian systems, both the heavy
and light chains had to be directed to the secretory path-
way to allow antibody assembly and accumulation. It was
also shown that the native signal peptide could be replaced
by a plant signal peptide (During et al., 1990) (i.e. that
from barley a-amylase) or a yeast signal peptide (Hein
et al., 1991) [i.e. the pre-pro sequence from the Saccharo-
myces cerevisiae a-mating factor (Kurjan and Herskowitz,
1982)] without affecting antibody secretion in N. tabacum.
Once in the secretory pathway, antibodies can be
retained in the ER if they are provided with a KDEL or
HDEL retention signal. ER sequestration generally allows
increased antibody accumulation and also prevents the
addition of non-mammalian glycosylation types added in
the late-Golgi apparatus which might promote an immune
response if the antibody had to be used parenterally.
However, ER-retained antibodies end up with high man-
nose structures which are not desirable from a pharmaco-
kinetic viewpoint. Furthermore, in some cases, antibodies
without a retention signal have been found in intracellular
compartments. We will restrict our discussion to antibod-
ies for which an in situ subcellular localization has been
reported (Table 3). We must stress that only antibodies
retaining their antigenic epitopes can be identified in this
way and that a fraction of the proteins might be partly
degraded in some compartments and so escape detection.
The subcellular localization of an antibody (B1-8)
expressed in plants was first determined by During et al.
(1990) using immunogold labelling and electron micros-
copy. This analysis was performed on callus and stem tis-
sues generated from transgenic tobacco plants by
wounding and hormone application. Both chains were
provided with the signal peptide of barley a-amylase. The
free light chain was found in the cytosol, while the assem-
bled antibody was found in the ER and also in the chlo-
roplasts. A chloroplast localization does not seem to have
been reported for any other antibody.
Rademacher et al. (2008) showed, by immunofluore-
scence and electron microscopy, that 2G12, a human
IgG1, accumulated in the prolamin bodies of maize endo-
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
sperm when placed under the control of the gt-1 pro-
moter and with both chains retaining their native signal
peptide and each fused to a SEKDEL ER retention
sequence. This result is in agreement with the facts that
prolamin bodies are derived from the ER (Chrispeels and
Herman, 2000) and that, in maize, prolamins are retained
within the ER lumen (Shewry et al., 1995).
Petruccelli et al. (2006) assessed, by immunogold label-
ling and electron microscopy, the accumulation in leaves
or seeds of N. tabacum of 14D9, a mouse IgG1, in which
both chains retained their native mouse signal sequence,
under the control of two different promoters [CaMV
35S+, i.e. the cauliflower mosaic virus 35S promoter with
duplicated enhancer (Kay et al., 1987) or 7S, i.e. the pro-
moter for the gene coding for the a’ subunit of the b-con-
glycinin (7S) soybean storage protein] and with or without
an ER retention signal. When the heavy and light chains
were under the control of the CaMV 35S+ promoter and
fused to a SEKDEL ER retention signal, 14D9 accumulated
in the ER lumen in leaf tissues and in both the protein
storage vacuole (PSV) matrix and the apoplast in seeds
(Table 3). A similar construct in which the CaMV 35S+
promoter was replaced by the 7S promoter gave identical
results, with an accumulation of 14D9 in the seed PSV
matrix and apoplast. When both the heavy and light
chains lacked an ER retention signal, 14D9 still accumu-
lated in the PSV matrix of the seeds, irrespective of the
promoter used. In leaves, the construct with the CaMV
35S+ promoter and without any retention signal on the
heavy and light chains led to 14D9 accumulation in the
apoplast (note that this result was not assessed by micro-
scopy, but inferred from the glycosylation pattern).
Together, these results indicate that the ER retention sig-
nal was efficient in leaves, but did not prevent the trans-
port of 14D9 to later Golgi compartments in the seeds of
N. tabacum, where it was found in the PSV and apoplast
(Petruccelli et al., 2006). It is noteworthy that these
authors also showed that the SEKDEL-bearing antibody
was immunogenic in BALB ⁄ c mice, whereas the secreted
antibody was not. As BALB ⁄ c mice are not immuno-sensi-
tive to plant glycosylation, this suggests that the SEKDEL
tag itself and ⁄ or an antibody conformational change
induced by the typical ER glycosylation pattern may be
responsible for this immunoreactivity (Petruccelli et al.,
2006).
Four other antibodies (21C5, mAb24, MAK33 and
unspecified) were reported to be strictly apoplastic (van
Engelen et al., 1994; Voss et al., 1995; De Wilde et al.,
1996; Komarnytsky et al., 2006). They were all devoid of
ª 2010 The Authors
 Table 3 Subcellular localization of antibodies expressed in plants

Heavy chain Light chain

Antibody Promoter Signal peptide Retention signal Promoter Signal peptide Retention signal Cell type Subcellular localization Reference

ª 2010 The Authors

B 1-8 pNOS Barley a-amylase ⁄ mas1’2’ dual Barley a-amylase ⁄ N. tabacum callus Endoplasmic reticulum (During et al., 1990)
promoter in and stem Chloroplast
the 1’ position
21C5 CaMV 35S+ Murine (native) ⁄ mas1’2’ dual Murine (native) ⁄ N. tabacum roots Apoplast (van Engelen et al., 1994)
promoter in
the 2’ position
mAb24 CaMV 35S+ Murine (native) ⁄ CaMV 35S+ Murine (native) ⁄ N. tabacum leaves Apoplast (Voss et al., 1995)
MAK33 CaMV 35S A. thaliana 2S2 ⁄ CaMV 35S A. thaliana 2S2 ⁄ A. thaliana leaves Apoplast (De Wilde et al., 1996)
storage protein storage protein Apoplast, Endoplasmic (De Wilde et al., 1998)
reticulum
Guy’s 13 CaMV 35S Murine (native) Native CaMV 35S Murine (native) ⁄ N. tabacum leaves Plasma membrane (Vine et al., 2001)
transmembrane bound
sequence
14D9 CaMV 35S+ Murine (native) ⁄ CaMV 35S+ Murine (native) ⁄ N. tabacum leaves Apoplast (Petruccelli et al., 2006)
N. tabacum seeds Protein storage
vacuole matrix
SEKDEL SEKDEL N. tabacum leaves Endoplasmic
reticulum
N. tabacum seeds Apoplast, Protein
storage vacuole

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
matrix
7S ⁄ 7S ⁄ N. tabacum seeds Protein storage
vacuole matrix
SEKDEL SEKDEL N. tabacum seeds Apoplast, Protein
storage vacuole
matrix
Not reported mas2’ N. plumbaginifolia ⁄ mas2’ N. plumbaginifolia ⁄ N. tabacum roots Apoplast (Komarnytsky et al., 2006)
calreticulin calreticulin
2G12 gt-1 Human (native) SEKDEL gt-1 Human (native) SEKDEL Zea mays seeds Prolamin bodies (Rademacher et al., 2008)
LO-BM2 En2pPMA4 Rat (native) ⁄ En2pPMA4 Rat (native) ⁄ N. tabucum BY-2 cells Endoplasmic (De Muynck et al., 2009)
reticulum
N. tabucum leaves Pre-vacuolar
compartment

CaMV 35S+, Cauliflower Mosaic Virus 35S promoter with duplicated enhancer; En2pPMA4, promoter of the Nicotiana plumbaginifolia plasma membrane ATPase4 gene reinforced by two copies of the cauliflower
mosaic virus 35S enhancer; mas1’2’, promoters of the mannopine synthase genes 1 and 2 of Agrobacterium tumefaciens (organized as divergent transcription units); mas2’, promoter of the mannopine synthase
genes 2 of Agrobacterium tumefaciens; CaMV 35S, Cauliflower Mosaic Virus 35S promoter; 7S, a’ subunit of b-conglycinin (7S) soybean storage protein promoter; gt-1, rice glutelin-1 promoter.
Antibodies in plants: twenty years later 545
546 Benoit De Muynck et al.
any ER retention signal and directed by either their native
murine signal peptide (van Engelen et al., 1994; Voss
et al., 1995) or a plant signal peptide (De Wilde et al.,
1996; Komarnytsky et al., 2006) and were expressed in
N. tabacum roots (van Engelen et al., 1994; Komarnytsky
et al., 2006), N. tabacum leaves (Voss et al., 1995) or
A. thaliana leaves (De Wilde et al., 1996) (Table 3). In a
subsequent study, De Wilde et al. (1998) showed that
MAK33 antibody was localized not only to the intercellular
space of A. thaliana leaves, but also to the cytoplasm in a
thread-like structure which might be the ER. The same
pattern was observed in roots. Furthermore, they detected
antibodies in leaf xylem vessel elements and therefore
hypothesized that, following secretion, antibody might be
transported by the transpiration stream to accumulate at
preferential sites.
Vine et al. (2001) expressed a membrane-bound variant
of the Guy’s 13 mouse antibody in N. tabacum leaves.
Both the heavy and light chains retained their native mur-
ine signal peptide. Only the heavy chain contained a
native transmembrane sequence and this was sufficient to
obtain a membrane-bound assembled antibody, as both
chains were co-localized at the cell surface.
De Muynck et al. (2009) expressed LO-BM2, a chimeric
rat ⁄ human antibody, in both N. tabacum plants and BY-2
cells. Both the heavy and light chains retained their native
(rat) signal peptide. The authors reported that, in BY-2
cells, LO-BM2 was localized to the ER, while, in N. taba-
cum plants, it was localized to a post-Golgi compartment,
most probably a pre-vacuolar compartment, in leaf pro-
toplasts and epidermal cells. This unexpected localization
was not totally a surprise, because Irons et al. (2008),
using fluorescent reporter proteins fused to the light and
heavy chains, showed that an antibody transiently
expressed in leaf epidermal cells was located in punctuate
structures in close association with the ER and partly over-
lapping with a pre-vacuolar compartment marker. In BY-2
cells, the ER-localized LO-BM2 antibody, which was
expected to be secreted, might represent only part of the
total amount synthesized. Indeed, De Muynck et al.
(2009) showed that antibody in the extracellular medium
was resistant to endoglycosidase H, indicating that it bears
complex glycans typical of the late-Golgi. This strongly
suggests that at least part of the antibody was secreted.
From this survey, it is clear that some antibodies were
found to be localized in the expected subcellular compart-
ment, while others seemed to be, in part, diverted from
the expected accumulation site (During et al., 1990;
Petruccelli et al., 2006; De Muynck et al., 2009) or at least
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
delayed in their transport (De Muynck et al., 2009)
(Table 3). Moreover, the very same antibody, encoded by
the same transgene construct, could end up in different
subcellular localizations, depending on the tissue in which
it is expressed (Petruccelli et al., 2006; De Muynck et al.,
2009). At this stage, it is difficult to propose any coherent
explanation for these variable results, because they were
obtained with different antibodies, different signal pep-
tides, different transcription promoters and different host
species and analysed in different tissues (see Figure 1b, c
and d). From a practical point of view, heterogeneous sub-
cellular localization might result in heterogeneity and ⁄ or
reduced recovery of the final product, which represents a
problem for the industrial production of large quantities of
homogenous antibodies. Thus, effort should be directed
towards a better understanding of the trafficking of heter-
ologous proteins in plants to improve this limiting step.
In conclusion, although it is possible to address antibod-
ies to different subcellular localizations, partial mistarget-
ing sometimes occurs, which might result in antibody
structural heterogeneity. As discussed later, choosing the
final destination should also take into consideration costs
of processing as well as the purpose (e.g. therapeutics,
diagnostics).
Antibody degradation
A major drawback to antibody production in plants is the
loss of material because of proteolytic degradation. This
problem is not specific to this protein-host combination,
because it is typically found in many cases of heterologous
expression in various organisms. The presence of addi-
tional and smaller than expected antibody fragments can
be noted in the reports on 22 of the 32 different antibod-
ies listed in Table 1 (for the other 10 antibodies, smaller
bands were not reported or no electrophoretic analysis
was provided). It is noteworthy that this occurred in all of
the analysed tissues (leaf, callus, tuber, hairy root, suspen-
sion cell, shooty teratoma, root and seed). As reported by
Sharp and Doran (2001a), antibody fragments in plants
have been variously explained as antibody assembly inter-
mediates or the consequence of extracellular peptidase
activity after secretion or the activity of peptidases
released during sample homogenization. Although it is
likely that all three phenomena occur in some cases, it has
become clear that extracellular peptidase activity is a major
factor to consider in antibody production in plants (van
Engelen et al., 1994; Sharp and Doran, 2001a,b;
Komarnytsky et al., 2006; De Muynck et al., 2009).
ª 2010 The Authors

Despite the numerous reports on antibody degradation
in plants (Table 4), it is not possible to draw any general
rules concerning the degradation pattern. This is mainly
because of the diversity of the host species and antibodies
tested. De Neve et al. (1993), for instance, showed that
MAK33 antibody displayed many more degradation prod-
ucts when expressed in N. tabacum than in A. thaliana.
Furthermore, it is likely that fragments resulting from a
single cleavage might be further processed by exopeptid-
ases into smaller fragments. It is also important to bear in
mind that the detection of antibodies by Western blotting
may not reveal all generated fragments, depending on the
antibody used for detection, whether antigenic epitopes
are still present in the fragments and whether the frag-
ment size is large enough to be seen on SDS-PAGE. It is
therefore possible that degradation is largely underesti-
mated in some cases.
In three cases (all human IgG1 antibodies, but expressed
in different plant species), degradation fragments of the
heavy chain were analysed by either mass spectrometry
(MS) (Ramessar et al., 2008; Villani et al., 2009) or Edman
degradation (De Muynck et al., 2009) to localize the cleav-
age(s) site(s). Villani et al. (2009) identified two major deg-
radation products derived from cleavage occurring close to
the hinge region. The exact cleavage point could not be
precisely determined because a small region on both sides
of the hinge was not identified by MS, reflecting either
the absence of this region (and thus its degradation by
peptidases) or the failure to detect the corresponding pep-
tides by MS. Furthermore, De Muynck et al. (2009) deter-
mined the sequence of the N-terminal part of a heavy
chain degradation product as KTHTCPPCP, a sequence
localized in the hinge region. These results pinpoint the
antibody hinge and closely related regions as the most
sensitive to proteolytic activity. However, other susceptible
regions must exist, because products other than those
resulting from cleavage in the hinge region are found. For
instance, Ramessar et al. (2008) established a continuous
MS peptide map of a heavy chain degradation product
encompassing the CH1, hinge, CH2 and CH3 regions.
Sharp and Doran (2001a) extensively studied the degra-
dation patterns of a mouse IgG1 antibody (Guy’s 13) pro-
duced in different N. tabacum expression systems and
suggested that antibody degradation in plants is likely to
occur in the extracellular medium (apoplast or culture
medium) and also during secretion, between the ER and
the Golgi. However, it is clear (Table 4) that antibodies
with an ER retention KDEL sequence are also often sub-
jected to degradation when expressed in plants. This
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
Antibodies in plants: twenty years later 547
observation might be explained either by the antibody
escaping from the ER or, more puzzling, by the fact that
the ER might not be as safe a storage compartment for
heterologous proteins as generally accepted. A case study
is the 14D9 antibody fused to a KDEL sequence and
expressed in N. tabacum, which was correctly retained
within the ER in leaves, but further transported to protein
storage vacuoles in seeds, where it underwent proteolytic
cleavage (Petruccelli et al., 2006).
Attempting to overcome the peptidase issue, Komarnyt-
sky et al. (2006) showed that an antibody expressed in
N. tabacum was less subject to degradation and showed
higher accumulation (a nearly threefold increase) when
co-expressed with a secreted form of the Bowman–Birk
serine peptidase inhibitor than when co-expressed with a
cytosolic form of this inhibitor. It has also been reported
that addition of gelatin as a substitution substrate for pep-
tidases increased total antibody levels by 68% in N. taba-
cum hairy roots (Wongsamuth and Doran, 1997) or by
300% during N. tabacum rhizosecretion (Drake et al.,
2003) (Table 4). Furthermore, De Muynck et al. (2009)
showed, using zymography, that the extracellular medium
of N. tabacum (i.e. culture medium from suspension cells
or leaf apoplasm) contains several peptidases able to
degrade human IgG in vitro.
To reduce proteolytic processing of plant-produced
pharmaceuticals, other strategies were also used such as
confining transgene expression to specific tissues, target-
ing to cellular organelles or fusing stabilizing partners
(Benchabane et al., 2008). Stoger et al. (2000) showed
that an scFv antibody expressed in seeds of wheat and rice
could be stored for at least 6 months at room tempera-
ture without significant lost of amount and activity. Simi-
larly, Artsaenko et al. (1998) showed that half of the
amount of an scFv antibody expressed in potato tubers
remained detectable after 1.5 years of tuber storage at
4 C and that the specific activity did not decreased dur-
ing tuber storage. These results highlight the advantage of
specific tissues presenting a reduced metabolic activity as
storage compartments for recombinant proteins (Bencha-
bane et al., 2008). Recombinant proteins have been
expressed in, or targeted to, different subcellular compart-
ments (for review, see Benchabane et al. (2008); Streat-
field (2007)). Nevertheless, full-size immunoglobulins need
to be addressed to the secretory pathway for assembly
and glycan processing. Fusion of recombinant proteins to
a stabilizing partner has been shown to be an effective
strategy to improve the final yield as well as to facilitate
protein recovery. For instance, Floss et al. (2008) showed
 Table 4 Putative degradation products noted on PAGE of plant-produced antibodies

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References
548 Benoit De Muynck et al.

MAK33 Nicotiana Callus  35 kDa (es) Recognized by anti-mouse No ⁄ ⁄ (De Neve et al.,
tabacum 40 kDa IgG antibodies 1993)
44 kDa Recognized by anti-mouse
IgG antibodies (may be
partially degraded HC, LC
dimer, or Fab-like fragment)
65 kDa Recognized by anti-mouse
88 kDa (es) IgG antibodies
110 kDa (es)
126 kDa (es)
139 kDa (es)
144 kDa (es)
Arabidopsis Callus 35 kDa (es) Recognized by 45 kDa (es) Recognized by anti-mouse ⁄
thaliana 88 kDa (es) anti-mouse IgG antibodies (possibly
126 kDa (es) IgG antibodies aglycosylated HC or
139 kDa (es) degraded HC)
144 kDa (es)
A. thaliana Leaves 2 bands > 110 Recognized by No (p) ⁄ ⁄ (De Wilde et al.,
kDa (p) Fc-specific 1996)
anti-mouse
antibodies
Solanum Tubers (X) Not mentioned ⁄ 28 kDa (p) Coomassie blue-stained ⁄ (De Wilde et al.,
tuberosum 34 kDa Recognized by anti-mouse 2002)
IgG antibodies
G1 ⁄ A N. tabacum Leaves 44 kDa (es) Recognized by 31 kDa (es) Recognized by anti-mouse ⁄ (Ma et al., 1994)
56 kDa (es) anti-mouse alpha chain antiserum
86 kDa (es) kappa chain
116 kDa (es) antibodies
Guy’s 13 N. tabacum Leaves 44 kDa (es) Recognized by 40 kDa (es) Recognized by ⁄ (Ma et al., 1994)
72 kDa (es) anti-mouse anti-mouse
116 kDa (es) kappa chain gamma chain
antibodies antiserum

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
ª 2010 The Authors
 Table 4 Continued

ª 2010 The Authors

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References

Hairy root 40 kDa (es) Recognized by 40 kDa (es) Recognized by Total antibody levels (Wongsamuth and
cultures (biomass) anti-mouse (biomass) anti-mouse increased by up to Doran, 1997)
50 kDa (es) (biomass) antibodies antibodies 90% with 0.1%
60 kDa (es) (biomass) Recognized by anti-mouse (w ⁄ v) nitrate, 4 mg
90 kDa (es) (biomass) antibodies (proposed to antibody ⁄ L in the medium
115 kDa (es) (biomass) be assembly intermediates) Total antibody levels
140 kDa (es) (biomass) increased by 20% to
90% in cultures with
PVP, 11 mg antibody ⁄ L
in the medium
supplemented with
2 g ⁄ L PVP
Total antibody levels
increased by 14% to
68% with gelatin, 9.5
mg antibody ⁄ L in the

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
medium supplemented
with 9 g ⁄ L gelatin
Hairy root 40 kDa (es) (biomass) Recognized by 40 kDa Recognized by 100 lg ⁄ mL bacitracin had (Sharp and Doran, 1999)
cultures 45 kDa (es) (biomass) anti-mouse anti-mouse no significant effect on
antibodies antibodies antibody loss from the
50 kDa (biomass Recognized by anti-mouse (proposed to culture medium
and medium) antibodies (suggested to be HC with a
90 kDa (biomass be assembly intermediates different
and medium) or antibody fragments) glycosylation
120 kDa (biomass Recognized by anti-mouse pattern)
and medium) antibodies (suggested to
140 kDa (biomass be assembly intermediates)
and medium)
Antibodies in plants: twenty years later 549
 Table 4 Continued

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
550 Benoit De Muynck et al.

Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References

Hairy root 40 kDa (es) Recognized by anti-mouse 33 kDa Recognized by Medium supplemented (Sharp and
cultures (biomass) IgG, anti-mouse Fab, (biomass) anti-mouse IgG with 1.5 g ⁄ L PVP: Doran, 2001a),
45 kDa (es) anti-mouse IgG-gamma1 and anti-mouse prevents antibody loss (Sharp and
(biomass) heavy chain, and anti-mouse IgG-gamma1 at the end of the culture, Doran, 2001b)
kappa light chain antibodies heavy chain maximum total antibody
50 kDa (biomass Recognized by anti-mouse IgG, antibodies levels of 4% TSP,
and medium) anti-mouse Fab, anti-mouse maximum antibody
80 kDa (biomass IgG-gamma1 heavy chain, 43 kDa (biomass Recognized by accumulation in the
and medium) and anti-mouse kappa light and medium) anti-mouse IgG medium ± 4 times
chain antibodies, endo H-resistant and anti-mouse higher than in controls
120 kDa (biomass Recognized by anti-mouse IgG, IgG-gamma1 Periodic antibody recovery
and medium) anti-mouse Fab, anti-mouse Fc, heavy chain from the medium using
anti-mouse IgG-gamma1 heavy antibodies, and hydroxyapatite: maximum
chain, and anti-mouse kappa light biotinylated total antibody levels 20%
chain antibodies, endo H-sensitive concanavalin A higher than without
135 kDa (biomass Recognized by anti-mouse IgG, 150% air saturation in a
and medium) anti-mouse Fab, anti-mouse Fc, recirculation bioreactor:
anti-mouse IgG gamma1 heavy biomass antibody content
chain, and anti-mouse kappa 52% higher at 1.6 mg ⁄ g
light chain antibodies, endo DW (negligible amount in
H-sensitive (biomass) the medium)
Plant-derived 50 kDa (biomass) Recognized by anti-mouse IgG 43 kDa Recognized by ⁄
shooty 80kDa (biomass) antibodies (biomass) anti-mouse IgG
teratoma 120 kDa (biomass) antibodies
cultures 135 kDa (biomass)

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
ª 2010 The Authors
 Table 4 Continued

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References

ª 2010 The Authors

Suspension 135 kDa (biomass Recognized by anti-mouse
cells and medium) IgG antibodies, reduced
intensity after BFA treatment
(performed only on biomass),
endo H-sensitive (biomass,
medium), unaffected by
swainsonine (performed
on biomass only), unaffected
by benzylamine
120 kDa (biomass Recognized by anti-mouse IgG
and medium) antibodies, reduced intensity
after BFA treatment (performed
only on biomass), endo H-sensitive
(biomass only, medium resistant),
sensitive to swainsonine
(performed only on biomass),
unaffected by benzylamine
80 kDa (biomass Recognized by anti-mouse IgG
and medium) antibodies, reduced intensity
after BFA treatment (performed
No ⁄ Medium supplemented with
1.5 g ⁄ L PVP: maximum total
antibody levels of 12% TSP,
maximum antibody accumulation
in the medium 2.5 times higher
than in controls
Negative effect of tunicamycin
(10 lg ⁄ mL) on antibody content
(biomass )35%, medium )49%)
No significant effect of
castanospermine (100 lg ⁄ mL)
on antibody levels in the biomass,
antibody content 74% lower in
the medium of treated cells
compared to the controls
Medium supplemented with BFA
(20 lM): antibody levels in the
biomass increased to 1.4–2.7
times that in untreated cultures
Periodic antibody recovery from

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
only on biomass), endo H-resistant, the medium using hydroxyapatite:
unaffected by benzylamine maximum total antibody levels 21%
50 kDa (medium) Recognized by anti-mouse IgG higher with antibody recovery than
antibodies, endo H-resistant, without
unaffected by benzylamine
Roots 20 kDa Recognized by anti-mouse IgG1 Not mentioned ⁄ Medium supplemented with (Drake et al., 2003)
(rhizosecretion) 60 kDa (es) antiserum 8 g ⁄ L gelatin: percentage
110 kDa (es) of intact IgG to total antibody
125 kDa (es) about 4 times higher than in
140 kDa (es) controls
21C5 N. tabacum Roots 110 kDa Binds antigen, Not mentioned ⁄ ⁄ (van Engelen et al.,
recognized 1994)
by anti-light chain,
anti-heavy chain, and
anti-Fab, but not anti-Fc
antibodies (suggested
to be F(ab’)2)
Antibodies in plants: twenty years later 551
 Table 4 Continued

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References

mAb24 N. tabacum Suspension cells Not mentioned ⁄ 35 kDa (p) Recognized by anti-mouse IgG Medium supplemented (Fischer et al., 1999)
(heavy + light chain-specific) with essential and
antibodies non-essential amino
552 Benoit De Muynck et al.

acids 10h prior to cell

harvest resulted in a
transient threefold
increase in antibody
expression
6D4 N. tabacum Leaves Not mentioned ⁄ 50 kDa Recognized by anti-(mouse ⁄ (Baum et al., 1996)
mu chains) antibodies
CO17-1A N. tabacum Leaves Not mentioned ⁄  25 kDa (p) (es) Recognized by anti-mouse ⁄ (Ko et al., 2005)
F(ab’)2 fragment-specific
antibodies, but not anti-mouse
Fcc fragment-specific antibodies
C5-1 Medicago sativa Leaves HL Recognized by No ⁄ ⁄ (Khoudi et al., 1999)
H2 + H2 (p) anti-mouse IgG
H2L + H2L (p) antibodies (heavy +
light chains)
Nicotiana Leaves (X) 35 kDa Recognized by Not mentioned ⁄ Use of the deleted RNA-2 (Sainsbury et al.,
benthamiana 50 kDa anti-mouse IgG vector increased antibody 2008)
60 kDa antibodies (heavy + accumulation
75 kDa (es) light chains)
95 kDa
130 kDa
Leaves 130 kDa Recognized by anti-mouse IgG Not mentioned ⁄
antibodies (heavy + light chains)
Leaves 100 kDa Recognized by anti-mouse IgG No (p) ⁄ Co-expression with HcPro (Vezina et al., 2009)
antibodies (heavy + light chains) silencing suppressor
and anti-mouse heavy chain increased antibody
antibodies (proposed to be accumulation by 5.3-fold
assembly intermediate)
135 kDa Recognized by anti-mouse IgG
antibodies (heavy + light
chains) and anti-mouse heavy
chain antibodies, binds antigen
(suggested to be H2L)
90 kDa (p) Binds antigen

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
ª 2010 The Authors
120 kDa (p)
 Table 4 Continued

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References

ª 2010 The Authors

Leaves (X) 100 kDa Recognized by anti-mouse IgG No (p) Co-expression with HcPro
antibodies (heavy + light silencing suppressor
chains) and anti-mouse heavy increased antibody
chain antibodies (suggested to accumulation by 3.6-fold
be assembly intermediate)
135 kDa Recognized by anti-mouse IgG
antibodies (heavy + light chains),
anti-mouse heavy chain, binds
antigen
75 kDa (p) Binds antigen
90 kDa (p)
100 kDa (p)
120 kDa (p)
cT84.66 N. tabacum Leaves (X) Not mentioned ⁄ 32 kDa (p) (es) Recognized by anti-sc ⁄ (Vaquero et al.,
FvT84.66 antibodies 1999)
43 kDa (p) (es) Recognized by anti-KDEL
antibody
44 kDa (p) (es) Recognized by anti-KDEL
antibody
50 kDa (p) (es) Recognized by anti-sc

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
FvT84.66 antibodies
MGR48 N. tabacum Leaves 44 kDa (p) Coomassie blue-stained 28 kDa (p) (same Coomassie blue-stained Maximum antibody levels (Stevens et al.,
gel (suggested to be mobility as light and recognized by at 15C, high light (275 2000)
Fab-like fragment) chain) anti-mouse IgG (heavy + lmol m)2s)1, 16 h ⁄ d)
65 kDa (p) Coomassie blue-stained gel light chains) antibodies
125 kDa (p) Coomassie blue-stained gel
160 kDa (p) Coomassie blue-stained gel
(suggested to be F(ab’)2-like
fragment)
GAN4B.5 A. thaliana Leaves 50 kDa Recognized by a mixture of Not mentioned ⁄ ⁄ (Bouquin et al.,
anti-human kappa and 2002)
gamma chain antibodies
(suggested to be assembly
intermediate)
63 kDa (es) Recognized by a mixture of
anti-human kappa and
gamma chain antibodies
Antibodies in plants: twenty years later 553
 Table 4 Continued

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References

78 kDa (es) Recognized by a mixture of

554 Benoit De Muynck et al.

anti-human kappa and

gamma chain antibodies
125 kDa Recognized by a mixture of
anti-human kappa and
gamma chain antibodies
(suggested to be assembly
intermediate)
cPIPP N. tabacum Leaves Not ⁄ 36 kDa Coomassie blue-stained ⁄ (Sriraman et al.,
mentioned (p) (es) and recognized by anti-human 2004)
IgG1 Fc-specific antiserum, and
concanavalin A
Physalis alkekengi Leaves (X) Not ⁄ 36 kDa Recognized by anti-human IgG1 ⁄
mentioned (p) (es) Fc-specific antiserum and
concanavalin A
CB-Hep.1 N. tabacum Leaves (x) Yes, not Not 10–15 kDa Not recognized by anti-mouse IgG Maximum (Valdes et al.,
shown mentioned (p) antibodies (possibly a plant antibody levels 2003)
contaminant) obtained with
35 kDa (p) Recognized by anti-mouse IgG 9-week
antibodies plants
SO57 N. tabacum Leaves (X) Not ⁄ 22 kDa (es) Recognized by anti-human F(ab’)2 ⁄ (Ko et al.,
mentioned fragment-specific antibodies, but 2003)
not anti-human Fcc fragment-
specific antibodies
Suspension Not ⁄  60 kDa Recognized by anti-rabies virus ⁄ (Girard et al.,
cells (X) mentioned (biomass) monoclonal antibody light and 2006)
heavy chains, but not anti-heavy
chain-specific antibody (suggested
to be LC dimer)
75 kDa Recognized by anti-rabies virus
(biomass) monoclonal antibody light and heavy
chains and anti-heavy chain-specific
antibody (suggested to be LC-HC
dimer)

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ª 2010 The Authors
 Table 4 Continued

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References

ª 2010 The Authors

CL4 N. tabacum Suspension Not ⁄ 33 kDa (p) (es) Coomassie blue-stained (either ⁄ (Yano et al.,
cells mentioned 37 kDa (p) (es) nonspecific 2004)
39 kDa (p) (es) binding or degradation)
41 kDa (p) (es)
Not N. tabacum Roots 30 kDa (es) Recognized by anti-human Not mentioned ⁄ Co-expressed with secreted (Komarnytsky
mentioned (rhizosecretion) 60 kDa IgG (heavy and light BBI peptidase inhibitor et al., 2006)
120 kDa chains) antibodies
12 kDa (es) Recognized by anti-human Not mentioned ⁄ Co-expressed with non-secreted
30 kDa (es) IgG (heavy and light chains) BBI peptidase inhibitor
50 kDa (es) antibodies
60 kDa (es)
77 kDa (es)
93 kDa (es)
120 kDa (es)
14D9 N. tabacum Seeds Not mentioned ⁄ 40 kDa (p) Recognized by anti-mouse ⁄ (Petruccelli et al.,
IgG, anti-b1,2-Xyl, and 2006)
anti-a1,3-Fuc antibodies
Seeds (X) Not mentioned ⁄ 40 kDa (p) Coomassie blue-stained, ⁄
recognized by anti-mouse
IgG, anti-b1,2-Xyl, and

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anti-a1,3-Fuc antibodies
and concanavalin A, not
recognized by anti-KDEL
antibody
Leaves 75 kDa (p) (es) Recognized by anti-mouse No (p) ⁄ ⁄
118 kDa (p) (es) IgG antibodies (possibly
aggregation forms of
heavy and light chains)
Leaves (x) 118 kDa (p) (es) Recognized by anti-mouse IgG ⁄
antibodies (possibly aggregation
form of heavy and light chains)
A5 N. benthamiana Leaves L2 Recognized by anti-human IgG <25 kDa Recognized by anti-human ⁄ (Giritch et al., 2006)
(LC-specific) antibodies, gG (LC-specific) antibodies
Coomassie blue-stained
H Recognized by anti-human IgG
(HC-specific) antibodies
Antibodies in plants: twenty years later 555
 Table 4 Continued

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References

HL Recognized by anti-human
IgG (HC-specific), anti-human
556 Benoit De Muynck et al.

IgG (LC-specific) antibodies

H2 Recognized by anti-human
IgG (HC-specific) antibodies,
Coomassie blue-stained
110 kDa Recognized by anti-human
IgG (HC-specific) and
anti-human IgG (LC-specific)
antibodies
H2L Recognized by anti-human
IgG (HC-specific) and anti-human
IgG (LC-specific) antibodies,
Coomassie blue-stained
125 kDa (p) (es) Coomassie blue-stained
170 kDa (p) (es)
2G12 Zea mays Seeds Not mentioned ⁄ 40 kDa (p) Coomassie blue-stained, does 100 lg antibody ⁄ g (Ramessar et al.,
not bind antigen, cleavage DW of seeds of 2008)
presumably located between plants regenerated
G119 and L123, a continuous from dedifferentiated
fragment from L123 to K472 immature zygotic
was fully covered by MS embryos
peptides
A. thaliana Leaves No (p) ⁄ 30 kDa (es) Recognized by anti-human IgG ⁄ (Schahs et al.,
40 kDa (es) antibodies (constant heavy and 2007)
light chain-specific)
Not mentioned N. tabacum Leaves Not mentioned ⁄ 29 kDa (es) Recognized by anti-mouse IgG ⁄ (Hong et al.,
antibodies (heavy and light 2007)
chain-specific)
H10 N. tabacum Leaves 50 kDa (p) Coomassie blue-stained, 30 kDa Recognized by anti-human ⁄ (Villani et al.,
recognized by 40 kDa (es) gamma chain antibodies 2009)
anti-human lambda
chain antibodies,
binds antigen (suggested
to be Fab fragment)

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ª 2010 The Authors
 Table 4 Continued

Organ ⁄ ER Additional bands Reactivity of additional Additional bands Additional bands Special
Host retention under non-reducing bands (non-reducing under reducing (reducing conditions) conditions ⁄
Antibody species signal (X) conditions conditions) conditions reactivity Improvements References

ª 2010 The Authors

N. benthamiana Leaves 50 kDa (p) Coomassie blue-stained, 25 kDa (p) (es) Coomassie blue-stained, degradation 50–100 mg purified
binds antigen, products of heavy chain occurring antibody ⁄ kg FW when
recognized by around the hinge region, having co-expressed with p19
anti-human lamda either a correctly processed C-terminal silencing suppressor
chain antibodies peptide (the most N-terminal intact of artichoke mottled
(suggested to be peptide DTLMISR) or a correctly crinkle virus (AMCV)
Fab fragment) processed N-terminal peptide (the
100 kDa (p) Coomassie blue-stained 27 kDa (p) (es) most C-terminal intact peptide
32 kDa (p) (es) STSGGTAALGCLVK), together
with some minor degradation
products of LC
LO-BM2 N. tabacum Suspension cells Not mentioned ⁄ 13 kDa (medium) Recognized by anti-human IgG ⁄ (De Muynck
(Fc-specific) antibodies et al., 2009)
13kDa (es) (biomass) Recognized by anti-human IgG
(Fc-specific) and anti-human
kappa light chain antibodies
19 kDa (es) (medium) Recognized by anti-human
kappa light chain antibodies
35 kDa (es) (biomass) Recognized by anti-human IgG

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
antibodies (Fc-specific),
35 kDa (p) (biomass) Coomassie blue-stained, N-terminal
residues KTHTCPPCP (Edman
degradation performed on purified
antibody)
35 kDa (medium) Recognized by anti-human IgG
antibodies (Fc-specific)
39 kDa (es) (biomass) Recognized by anti-human
IgG antibodies (Fc-specific)
39 kDa (p) (es) Coomassie blue-stained
Leaves Not mentioned ⁄ 13 kDa (apoplast) Recognized by anti-human IgG ⁄
(Fc specific) and anti-human kappa light chain antibodies
35 kDa (es) (apoplast) Recognized by anti-human IgG antibodies (Fc-specific)

(es), our size estimation from authors’ PAGE; (p), purified antibody; BFA, brefeldin A; PVP, Polyvinylpyrrolidone; TSP, total soluble proteins; DW, dry weight; BBI, Bowman–Birk serine peptidases inhibitor; FW, fresh
weight
Antibodies in plants: twenty years later 557
558 Benoit De Muynck et al.
that fusion of elastin-like peptides (ELPs) to the C-terminal
of the 2F5 antibody enhanced its stability while keeping
its binding properties unchanged compared to the CHO-
produced counterpart lacking ELPs. Nevertheless, if
removal of the fusion partner is mandatory for recombi-
nant protein activity, this step might be either performed
in vivo taking advantage of identified recognition sites of
endogenous peptidases or in vitro, but therefore adding
to downstream processing costs (Streatfield, 2007;
Benchabane et al., 2008). Gene knockout or silencing of
plant peptidases might also serve to increase recombinant
protein stability (Streatfield, 2007; Benchabane et al.,
2008). This approach is only feasible if the target peptidases
are not essential for plant growth. In addition, it only
works when antibody is degraded by a single or few pep-
tidases. This might turn out not to be true considering the
number of peptidases found, e.g. in the tobacco apoplasm
(Delannoy et al., 2008).
Downstream processing
Recently, Hassan et al. (2008) have examined different
extraction methods for monoclonal antibodies targeted to
different subcellular compartments in transgenic N. taba-
cum plants. Extraction methods were knowingly chosen as
simple as possible because they are more likely to be scal-
able and financially viable at an industrial scale (Hassan
et al., 2008). Antibodies were targeted to the apoplasm,
within the ER, or, when fused to a membrane span, to
the plasma membrane. Their results show that while a
grinding step was necessary for ER-retained and mem-
brane-bound antibody recovery, secreted antibody was
efficiently recovered by a simple freeze-thaw technique
consisting in freezing the leaves at )20 C and thawing
them for 10 min at room temperature before adding an
extraction buffer. This technique is also very convenient as
the freezing step allows storage of leaves prior to extrac-
tion procedure (Hassan et al., 2008). Together with glyco-
sylation considerations that have already been mentioned
in this review, this result brings a new and major point in
favour of secreted antibodies, particularly if they are
expressed for therapeutic purposes. Secreted antibodies
are also appropriate for suspension cells secretion or plant
rhizosecretion as antibodies can be purified directly from
the liquid culture medium without grinding. Nevertheless,
purification in those cases might be made difficult depend-
ing on the complexity of the medium used and on the
endogenous secreted compounds (e.g. carbohydrates).
Expression of antibodies in storage organs such as seeds
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
or tubers was shown to be advantageous from a storage
point of view (Artsaenko et al., 1998; Stoger et al., 2000).
Nevertheless, this strategy would invariably lead to the
need of a grinding step that is economically unfavourable,
except if recombinant protein accumulation is large
enough to compensate for the extra costs of grinding. The
high expression levels obtained recently for antibodies
transiently expressed in N. benthamiana leaves (Sainsbury
and Lomonossoff, 2008; Vezina et al., 2009) suggest that,
to date, this system coupled with the extraction procedure
described by Hassan et al. (2008) constitute the most
promising way for expressing therapeutic antibodies in
plants at an industrial scale. It is noteworthy that expres-
sion strategies should always be weighted according to
the particular use intended for the recombinant protein.
For instance, while it is clear that membrane-bound IgG
are not recommended for further purification and thera-
peutic use, they might be particularly well suited for phy-
toremediation purposes.
A limited number of antibodies expressed in plants have
reached the clinical development stage. For instance,
CaroRx, an antibody that specifically binds Streptococcus
mutans (the bacteria that causes tooth decay) was
expressed in N. tabacum (Ma et al., 1994) and is licensed
in Europe. In addition, three cases are expected to soon
enter Phase I clinical trials: BLX-301, an anti-CD20 opti-
mized antibody for the treatment of non-Hodgkin’s B-cell
lymphoma expressed in the aquatic plant Lemna minor
(http://www.biolex.com); MAPP66, a combination of sev-
eral antibodies expressed in N. benthamiana by the Mag-
nIcon technology and used as a HSV ⁄ HIV microbicide
(http://www.mappbio.com); and 2G12, an HIV-neutralizing
antibody expressed in N. tabacum or maize (http://
www.pharma-planta.org). There are also other antibodies
developed by several companies that are currently being
involved in preclinical development. Finally, CIGB is pro-
ducing in N. tabacum plants the antibodies CB-Hep.1 and
HB-01 (http://www.cigb.edu.cu) that have been used for
several years in Cuba for the manufacturing process of a
Hepatitis B vaccine.
Concluding remarks
The survey of several publications dealing with antibody
expression in plants indicates that this is an appropriate
heterologous expression system. We did not discuss the
activity of the antibodies produced in plants, because,
although binding activity was shown in most cases, there
was usually no quantitative comparison of the binding
ª 2010 The Authors

activity of the plant- and animal-produced antibody, and
the biological activity of the antibody was seldom
addressed. Nevertheless, it is clear that, when activity was
reported in the literature, plant-produced antibodies were
functional. The data also clearly show important variations
in the yield, subcellular localization and proteolytic degra-
dation profile of antibodies. This is, in part, because of the
use of different transcription promoters, the presence or
absence of retention signals, the transformation of differ-
ent host species and the analysis of different organs. There
are also indications that different antibodies might not
behave in the same way. In this respect, comparing the
expression of different antibodies in the same expression
system would be very informative.
Two recent advances have moved antibody expression
closer to industrial application. Indeed, high antibody
expression has been obtained recently using transient
expression and this system is expected to become predom-
inant. In addition, glycosylation engineering of antibodies
has shown to be functional and represents an interesting
tool for adapting the antibody structure to therapeutic
use.
The drawback of proteolytic degradation is still particu-
larly annoying, as, besides decreasing the yield of func-
tional antibodies, it also necessitates costly steps to
remove the partly degraded antibodies. In this respect, it
would be interesting to develop extracellular peptidase-
free host plants or to engineer antibodies displaying higher
resistance to peptidases while maintaining their activity. In
addition to the scientific aspects, there are economical
considerations that were not covered here, but are of
prime interest when moving from the laboratory to the
industrial production. Most of the articles dealing with
antibody expression in plants do not take into account the
costs and pitfalls of downstream processing (For review,
see Basaran and Rodriguez-Cerezo (2008); Stoger et al.
(2005b)).
Beside full antibodies that were covered in this review,
more and more antibody derivatives are considered for
therapeutic applications. A nice illustration is the recent
production of scFv idiotypic vaccines in Nicotiana benth-
amiana plants using a viral tobacco mosaic virus vector
(McCormick et al., 2003, 2008). This system allowed the
production of personalized vaccines (derived from each
patient’s tumour) in even less time than required with
some animal cell-based system and showed to be effective
and safe to administer to patients (McCormick et al.,
2008).
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 529–563
Antibodies in plants: twenty years later 559
Finally, while plant expression systems are expected to
be further improved, there are already several plant-pro-
duced therapeutic antibodies that are in the pipeline for
medical evaluation. This is probably the best indication
that plant-produced antibodies have a future.
Acknowledgements
The work carried out in this laboratory was supported
financially by a grant from the European Community
(PHARMA-PLANTA integrated Project), the Région Wall-
onne, the Inter-university Attraction Poles Program-Belgian
Science Policy and the Belgian Fund for Scientific Research.
BDM was the recipient of a fellowship from the Fonds
pour la Formation à la Recherche dans l’Industrie et dans
l’Agriculture (Belgium).
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