A syn he ic biology app oach o consis en p oduc ion o
plan -made ecombinan polyclonal an ibodies agains
snake enom oxins
Jose Manuel Jul e Pa e~
no
1
, Es e an
ıa Hue
1
, Asun Fe n
andez-del-Ca men
1
, Al a o Segu a
2
, Micol Ven u i
1
,
An oni Gand
ıa
1
, Wei-song Pan
1
, I ene Albaladejo
1
, Ja ie Fo men
1
, Da inia Pla
3
, And 
es Wigdo o i z
4
,
Juan J. Cal e e
3
, Ca los Gu i
e ez
5
, Jos
e Ma 
ıa Gu i
e ez
2
, An onio G anell
1,
* and Diego O z
aez
1,
*
1
Ins i u o de Biolog
ıa Molecula y Celula de Plan as (IBMCP), Agencia Es a al Consejo Supe io de In es igaciones Cien 
ı icas, Uni e sidad Poli 
ecnica de Valencia,
Valencia, Spain
2
Ins i u o Clodomi o Picado, Facul ad de Mic obiolog
ıa, Uni e sidad de Cos a Rica, San Jos
e, Cos a Rica
3
Ins i u o de Biomedicina de Valencia, Agencia Es a al Consejo Supe io de In es igaciones Cien 
ı icas, Valencia, Spain
4
Ins i u o de Vi olog
ıa, CICVyA, INTA, Hu lingham Buenos Ai es, A gen ina
5
Resea ch Ins i u e o Biomedical and Heal h Sciences, Uni e si y o Las Palmas de G an Cana ia, A ucas, Las Palmas, Cana y Islands, Spain
Recei ed 30 May 2017;
e ised 3 Augus 2017;
accep ed 23 Augus 2017.
*Co espondence (Tel +34 963879933;
ax +34 963877873; email
[email p o ec ed]s (AG);
[email p o ec ed] (DO))
Keywo ds: ecombinan polyclonal
an ibodies, molecula pha ming, snake
an i enoms.
Abs ac
An i enoms de eloped om he plasma o hype immunized animals a e he only e ec i e
ea men a ailable agains snakebi e en enoma ion bu sho age o supply con ibu es o he
high mo bidi y and mo ali y oll o his opical disease. We desc ibe a syn he ic biology
app oach o a o dable and cos -e ec i e an i enom p oduc ion based on plan -made
ecombinan polyclonal an ibodies ( e med plu ibodies). The s a egy akes ad an age o i us
supe in ec ion exclusion o induce he o ma ion o soma ic exp ession mosaics in ag oin il a ed
plan s, which enables he exp ession o complex an ibody epe oi es in a highly ep oducible
manne . Plu ibodies de eloped using oxin-binding gene ic in o ma ion cap u ed om pe iphe al
blood lymphocy es o hype immunized camels ecapi ula ed he o e all binding ac i i y o he
immune esponse. Fu he mo e, an imp o ed plan -made an i enom (plan i enom) was
o mula ed using an in i o selec ed plu ibody agains Bo h ops aspe snake enom oxins and
has been shown o neu alize a wide ange o oxin ac i i ies and p o ide p o ec ion agains
le hal enom doses in mice.
In oduc ion
Passi e immuniza ion (PI) wi h polyclonal an ibodies (pAbs)
pu i ied om animal se um was i s adop ed o ea diph he ia
in he 19 h cen u y (G aham and Amb osino, 2015). An ibio ics
and monoclonal an ibodies ha e p og essi ely subs i u ed an is-
e a-based he apies. Howe e , pAbs-based an i enoms emain
he only a ailable ea men agains complex oxin mix u es such
as hose p esen in snake enoms. The global enom bu den has
been es ima ed a 2.5 million en enomings and 125 000 dea hs
pe yea (Chippaux, 1998). Despi e s eady imp o emen s (Gu ie -
ez, 2014b; Gu ie ez e al., 2011), an i enom-based snakebi e
ea men s a e no ee o side e ec s as a consequence o he
exposu e o he e ologous an ibody cons an egions. Fu he -
mo e, an i enom supply is a cons an isk due o high manu ac-
u ing cos s and he low income le el o a ge popula ions
(A nold, 2016; Ha ison e al., 2009). An i enoms made o
ecombinan human o humanized an ibody cock ails would be
a highly desi able al e na i e as hey would educe he ad e se
e ec s while acili a ing p oduc s anda diza ion and ep o-
ducibili y. Un o una ely, manu ac u ing cos s o complex an i-
body cock ails a e s ill high because o ensu e consis ency hey
o en equi e pa allel p oduc ion lines o each componen o he
cock ail. Con e sely, simpli ying he cock ail composi ion may
comp omise i s e icacy due o he high di e si y o oxins
comp ising enoms. Fu he mo e, i is di icul o econcile mo es
owa ds cock ail simpli ica ion wi h a majo end in an i enom
o mula ion, namely o widen he spec um o snake enoms
co e ed by a single ea men (La onas, 2012; S ock e al., 2007).
To o e come hese limi a ions, we ha e de eloped a syn he ic
biology s a egy o an i enom p oduc ion based on he exp es-
sion o ecombinan pAbs in plan s. Plan bio ac o ies p o ide
a o dable al e na i e pla o ms o ecombinan an ibody p o-
duc ion (usually known as ‘plan ibodies’) (S oge e al., 2002) due
o hei scalabili y, high p oduc ion yields, he low isk o
con amina ion wi h ad en i ious pa hogens and lowe equi e-
men s o p oduc ion acili y building (Paul and Ma, 2011; Paul
e al., 2015). Ou p oposed s a egy uses he plan chassis as a
p oduc ion pla o m and akes ad an age o an in insic p ope y
o many plan i uses known as supe in ec ion exclusion (SE) o
ensu e ep oducibili y. SE p e en s he supe in ec ion o cells by a
second i us when hey a e al eady in ec ed wi h a closely ela ed
esiden i us (Jul e e al., 2013; Solle and Eps ein, 1965; Sylle ,
2012). An impo an consequence o SE is he ac ha
popula ion a ian s in plan i us in ec ions a e no uni o mly
dis ibu ed bu s uc u ed in a mosaic-like pa e n. Mos impo -
an ly, his dis ibu ion ensu es he main enance and p opaga ion
o popula ion di e si y as compa ed o he i ness-d i en quasis-
pecies-like dis ibu ions ypical o uns uc u ed popula ion
dynamics (Elena e al., 2011). We ha e in es iga ed he use o
i us-based exp ession sys ems o he induc ion o soma ic
exp ession mosaics in plan lea es, whe e each ‘ ile’ in he mosaic
ª2017 The Au ho s. Plan Bio echnology Jou nal published by Socie y o Expe imen al Biology and The Associa ion o Applied Biologis s and John Wiley & Sons L d.
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use,
dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
727
Plan Bio echnology Jou nal (2018) 16, pp. 727–736 doi: 10.1111/pbi.12823
unc ions as an independen monoclonal mic o-p oduc ion line,
oge he leading o he p oduc ion o ecombinan polyclonal
cock ails. In his s udy, we show how his s a egy ensu es he
simul aneous p oduc ion o high yields o ecombinan pAbs
comp ising hund eds o idio ypes wi h ou s anding ba ch- o-
ba ch ep oducibili y, independen ly o he complexi y o he
polyclonal composi ion. Mos impo an ly, we show ha his
no el s a egy enables he exp ession o a selec ed po ion o a
mammalian immunized an ibody epe oi e in a ansien ly
mul i ansgenic plan in a highly ep oducible manne . We also
show ha his s a egy can be used o syn he ize a complex plan -
made an ibody cock ail able o neu alize a wide ange o oxins
in he Bo h ops aspe enom –medically he mos impo an
snake in Cen al Ame ica (Gu ie ez, 2014a) – hus opening he
doo o he o mula ion o e icien and a o dable plan -made
ecombinan an i enoms.
Resul s
Cha ac e iza ion o soma ic exp ession mosaics in
Nico iana ben hamiana
The spa io- empo al dynamics o he mosaic-like exp ession
pa e ns we e moni o ed in Nico iana ben hamiana lea es a e
ag oin il a ion o h ee i us-based in ec i e clones encoding
GFP, BFP and DsRed luo escen p o eins (Figu e 1a). To be e
unde s and he dynamics o mosaic o ma ion, we c ea ed a 2D
compu e simula ion in Ne Logo (see Figu e 1b and suppo ing
in o ma ion) and compa ed he p edic ed compu e ou pu s wi h
he expe imen al mosaic pa e ns ob ained by he simul aneous
ag oin il a ion o h ee i al clones encoding luo escen p o-
eins. Based on he compu e simula ions, we an icipa ed ha , o
a gi en se o ini ial pa ame e s, mosaic (colou ) composi ion
would be a highly ep oducible ou come (Figu e 1c) and ha
ep oducibili y would inc ease wi h he numbe o ini ial in ec ion
oci. Time-cou se co-in il a ion analysis expe imen ally co obo-
a ed his p edic ion (Figu e 1d). F om hese obse a ions, we
hypo hesized ha he (mosaic) dis ibu ion pa e n o i al clones
imposed by SE o e s a compe i ion-limi ed mic oen i onmen
ha ensu es he su i al o low- i ness clones. Mo eo e , wi h a
su icien ly high ini ial numbe o in ec i e oci, SE will also
p o ec he sys ems om luc ua ions in popula ion dynamics,
p o iding highly ep oducible ou comes.
Tes ing exp ession mosaics o he p oduc ion o
ecombinan pAbs: Plu ibody echnology
In a case s udy o a mul ip o ein plan bio ac o y, we a emp ed
o de e mine whe he his pla o m could p oduce high yields o
ecombinan an ibody mixes while p ese ing he di e si y o he
composi ion, ensu ing high ba ch- o-ba ch ep oducibili y and
yielding a inal p oduc ha was unc ionally equi alen o
adi ional animal an i enom. To assess di e si y p ese a ion, we
i s assayed he in plan a exp ession o an an ibody lib a y
ob ained om camel pe iphe al mononuclea blood cells (PMBC),
comp ising a ac ion o he an ibody epe oi e p esen in he
Figu e 1 P inciples o supe in ec ion exclusion.
(a) N. ben hamiana lea es showing soma ic
exp ession mosaics p oduced by h ee magnICON
i al clones encoding DsRed, g een and blue
lo escen p o eins ag oin ec ed simul aneously a
OD
600
=0.01. Mosaics a e obse ed ei he wi h
ed/g een il e s o DsRed (le ) o wi h UV ligh
( igh ). Below, de ail o he mosaic in he amed
lea a ea. (b) Ne Logo compu e simula ion o a
iple i al co-in ec ion ( ed, g een and blue)
subjec ed o SE in a i ual a ea comp ising
62 500 i ual cells. The i ual i ness (VF) o
each clone was adjus ed o 1.0 : 1.0 : 0.7 (R:G:
B), and i ual OD
600
(VOD) was adjus ed o 300
(uppe image) and 3000 (lowe image) a bi a y
uni s o ma ch he na u al in ec ion on (a, d) (see
Me hods o de ailed de ini ions o VF and VOD).
(c) Repea ed Ne Logo simula ions o he e olu ion
o iple R-G-B in ec ions using VOD alues o 300
(up) and 3000 (down), espec i ely.
(d) Expe imen al da a e olu ion o iple R-G-B
in ec ions in N. ben hamiana lea es we e
pe o med a wo di e en OD
600
and eco ded
daily up o day 7 pos in il a ion. E olu ion o
clone expansion is ep esen ed as pe cen age o
o al lea a ea occupied by each clone SD.
ª2017 The Au ho s. Plan Bio echnology Jou nal published by Socie y o Expe imen al Biology and The Associa ion o Applied Biologis s and John Wiley & Sons L d., 16, 727–736
Jose Manuel Jul e Pa e~
no e al.728
ci cula ing B cells o an indi idual d omeda y camel. Camel
an ibodies we e p e e ed because camelids display la ge immune
epe oi es in single chain an ibody o ma (Hcab), a ea u e ha
g ea ly simpli ies lib a y cons uc ion and subsequen p oduc ion
in plan s using i al ec o s. Hcab a iable an ibody egions we e
PCR-ampli ied om PMBC cDNA and cloned en masse in o he
pGV
H
H-His ec o –a modi ied e sion o obacco-mosaic i us
(TMV)-de i ed bina y magnICON ec o con aining an in- ame
his idine ag o de ec ion (Figu e 2a) . The pGV
H
H-His lib a y was
hen mobilized o Ag obac e ium ume aciens cells and om he e
o N. ben hamiana lea es using acuum ag oin il a ion. A e
7 days’ incuba ion, lea apoplas luid was eco e ed om
de ached lea es and he V
H
H-His we e pu i ied by a ini y
ch oma og aphy, esul ing in a pu i ied p o ein band o he
expec ed a e age size (15 kDa, Figu e 2b). As an icipa ed, a wo-
dimensional elec opho e ic sepa a ion esol ed he unde lying
p o ein complexi y and e ealed a pa e n o >200 dis inguishable
spo s, indica i e o he polyclonal na u e o he sample (Fig-
u e 2c). The esul ing plan -made mul iple ecombinan an ibody
p oduc s a e he ea e e e ed o as plu ibody samples. To
es ima e he size o he epe oi es ha can be exp essed using
his sys em, a GFP clone was co-in il a ed oge he wi h he
pGV
H
H-His lib a y a di e en a ios. I was calcula ed ha one
g am o esh N. ben hamiana lea es can p oduce up o 13 500
exp ession iles using an Ag obac e ium lib a y inoculum a
OD
600
=0.1 (Figu e 2c).
Plu ibody echnology ensu es ba ch- o-ba ch
ep oducibili y
We hen in es iga ed he abili y o he sys em o ep oduce he
composi ion and eac i i y o mammalian immune sub epe oi es.
To his end, new an ibody lib a ies in pGV
H
H-His we e
cons uc ed s a ing om PBMC samples ob ained om h ee
d omeda y camels immunized wi h he same enom cock ail
de i ed om h ee snake species, C o alus simus,C o alus
scu ula us and Bo h ops aspe . To a ou ep oducibili y and
ensu e he p ese a ion o he o iginal di e si y in any subsequen
analysis, he lib a y cons uc ion p ocedu e was s anda dized as
shown in Fig. S1 (de ailed in Supplemen a y me hods). Wo king
cell bank (WCB) aliquo s we e i s used o e alua e he
p oduc ion s a egy in e ms o ep oducibili y, by compa ing
di e en V
H
H-His plu ibody p epa a ions. Fi s , h ee plu ibody
samples de i ed om he same immune epe oi e bu p epa ed
om di e en WCBs (PIM_1, PIM_2 and PIM_3) we e labelled
wi h di e en luo opho es and esol ed by 2D-DIGE analysis. The
almos pe ec o e lapping o DIGE images indica ed ha all h ee
complex p o ein samples we e ema kably simila (Figu e 3a). In a
sepa a e DIGE expe imen , wo immune plu ibodies (PIM_1 and
PIM_2) we e compa ed wi h a hi d p epa a ion de i ed om a
nonimmune epe oi e (PPI_1). As he co esponding simila i y
plo s show (Figu e 3b), he compa ison o he p o ein spo s om
equi alen immune plu ibody samples (PIM_1 s PIM_2) p oduced
a na owly cen ed olume a io dis ibu ion indica i e o a highly
ep oducible composi ion. In con as , compa isons be ween
samples de i ed om di e en immune epe oi es (PPI_1 s
PIM_1; PPI_1 s PIM_2) showed clea de ia ions in olume a ios.
Fu he mo e, deep sequencing o he CDR3 egions in V
H
H
mRNAs om ag oin il a ed plan s showed he ema kable
conse a ion o sequence dis ibu ion be ween equi alen sam-
ples (PPI1 s PPI2 and PIM1 s PIM2), wi h only e y mino
di e ences obse ed in he mos abundan CDR3 sequences
(Figu e 3c). Finally, when he enom-binding ac i i ies o PPI and
PIM samples we e compa ed, he immune plu ibodies consis-
en ly showed eac i i y agains all h ee enoms employed o
Figu e 2 Assessmen o an ise um di e si y.
(a) Schema o he cloning and ansien exp ession
p ocedu es used o he p oduc ion o polyclonal
V
H
H an ibodies. (b) Fi s and second dimension
elec opho esis sepa a ion o V
H
H-His clones
pu i ied om N. ben hamiana lea es using Ni-
NTA a ini y columns. (c) Exp ession o a GFP clone
as a i al subpopula ion wi hin a V
H
H -His lib a y.
Images we e aken a e 10 dpi, and he a e age
ile size was eco ded and used o es ima e ile
densi y and i s dependence wi h OD600 ( igh ).
A B, si e-speci ic ecombina ion si e; SP, signal
pep ide; NTR, non ansla ed egion; Tnos,
e mina o o nopaline syn hase.
ª2017 The Au ho s. Plan Bio echnology Jou nal published by Socie y o Expe imen al Biology and The Associa ion o Applied Biologis s and John Wiley & Sons L d., 16, 727–736
Plan -made ecombinan an i enom 729
Figu e 3 Assessmen o plu ibody ep oducibili y. (a) Indi idual and me ged 2D DIGE images o h ee independen hype immune plu ibody p epa a ions in
V
H
H-His o ma (PIM_1, PIM_2 and PIM_3). (b) Compa ison o he ela i e abundance o indi idual an ibody clones in plan samples ag oin il a ed wi h inocula
om independen WCBs de i ed om he same MCB. An ibody clones we e iden i ied by deep sequencing o V
H
H CDR3 egion. PPI_1 and PP_2 co espond o
equi alen p e-immune WCBs. PIM_1 and PIM_2 co espond o equi alen immune polyclonal WCBs. In each compa ison, he same colou s co espond o
iden ical an ibody clones. (c). DIGE compa ison o PPI and PIM pu i ied plu ibody p epa a ions. Cen ed do s ep esen olume a ios close o one. (d) ELISA es
showing he enom-binding ac i i y o h ee nonequi alen PIM p epa a ions each ob ained om a di e en indi idual camel and analysed agains each o he
h ee enoms employed in he immuniza ion. (e) Speci ici y es o PIM enom binding, compa ing eac i i y agains C o alus simus and cob a enoms.
ª2017 The Au ho s. Plan Bio echnology Jou nal published by Socie y o Expe imen al Biology and The Associa ion o Applied Biologis s and John Wiley & Sons L d., 16, 727–736
Jose Manuel Jul e Pa e~
no e al.730
immuniza ions (Figu e 3d), and no binding ac i i y was obse ed
agains BSA o non ela ed Naja enoms (Naja nubiae and Naja
mossambica) (Figu e 3e).
Fo mula ion o a snake plan i enom using Plu ibody
echnology
In con as o adi ional an i enoms ob ained om hype immune
animal plasma, ou plu ibodies de i e om PMBC pools. Hence,
he gene ic in o ma ion e ie ed only co esponds o a mino
ac ion o he eac i e B cells made up o ci cula ing memo y
cells on hei way o he bone ma ow. The e o e, in o de o
c ea e a unc ional plan -made an i enom (he ea e e med
plan i enom), we decided o en ich ou plu ibody epe oi es in
oxin binde s by in oducing an in e media e in i o selec ion
s ep in o he p oduc ion low cha (see Fig. S1). PCR-ampli ied
immune sub epe oi es we e i s cloned in a phage display ec o
and hen subjec ed o h ee successi e ounds o selec ion agains
B. aspe enom. To maximize di e si y and a oid dominan
single-an igen selec ion bias, phage panning was conduc ed
agains di e en ac ions o he enom ( ou in o al, oughly
co esponding o g oups o oxin ac i i ies, Figu e 4a). A e
selec ion, all ou sublib a ies we e pooled and he a iable
an ibody egions we e ampli ied and ans e ed in bulk o he
pGV
H
H_IgGH1 ec o (Figu e 4b) ca ying he cons an egions o
human IgGH1 o c ea e an en iched polyclonal (EP) mas e cell
bank (MCB) wi h inc eased eac i i y and po en ially less likely o
p oduce hype sensi i i y eac ions. EP_MCB was subsequen ly
dis ibu ed in WCBs and ag oin il a ed in N. ben hamiana. The
PEP_1 o mula ion showed a 2xlog inc ease in o al binding
ac i i y when compa ed wi h he nonen iched plu ibody in ELISA
(Figu e 5A), demons a ing ha en ichmen s in polyclonal com-
posi ions ob ained by in i o selec ion can be success ully
ans e ed o he plan , whe e hey globally ep oduce he
eac i i y gains.
Finally, in o de o p oduce he i s i e a ion o a plan i enom
candida e, we decided o p oduce a plu ibody inco po a ing 36
indi idual clones ha showed maximum binding ac i i ies. The
clone composi ion o he esul ing en iched oligoclonal o mula-
ion (EO_1, see Fig. S1) is de ailed as supplemen a y in o ma ion
(Table S1). An EO_WCB was employed o scale up an ibody
p oduc ion using one kilog am o ag oin il a ed N. ben hamiana
lea es and yielding 0.2 g o ecombinan polyclonal an ibody
collec ed om he co esponding lea apoplas ic luid. Following
a ini y pu i ica ion (Figu e 5A), an an i enom solu ion in PBS
(PEO_1) was p epa ed a a concen a ion o 46.5 mg p o ein/mL,
simila o he ypical concen a ion o equi alen comme cial
an i enoms (55 mg/mL), and used o u he cha ac e iza ion
(summa ized in Table 1). As expec ed, he PEO_1 solu ion
showed en iched an i enom-binding ac i i y when assayed in
ELISA agains B. aspe enom (Figu e 5B, C). Figu e 5D showed
ha phospholipases (PLA
2
), and o a lesse ex en se ine
p o einases (SP), P-I and P-III snake enom me allop o einases
(SVMP) and L-amino acid oxidase (LAO) molecules, we e e i-
cien ly e ained by he immobilized plan i enom. Howe e , he
a ini y ma ix was poo in cap u ing disin eg ins (DISI). Such
pa e n o immuno ecogni ion mi o s he immunological p o ile
o monospeci ic (B. aspe ) an i enoms (Gu ie ez e al., 2010),
which we e shown o quan i a i ely immunocap u e mos P-III
SVMPs, se ine p o einases, L-amino acid oxidase, bu only o a
lesse ex en medium-sized disin eg ins, PLA
2
molecules, some
Figu e 4 S a egy o plu ibody o mula ion
en ichmen . (a) Ch oma og aphic p o ile and
elec opho esis sepa a ion o Bo h ops aspe
enom employed in an i enom plu ibody
en ichmen , showing he ou g oups o oxins
employed in phage display selec ion o educe
an igen d i . (b) Cloning p ocedu e o V
H
H
a iable egions om PBMC cDNA o pHEN2-
de i ed pSwo d phage display ec o and om
he e o bina y ec o o plan exp ession adap ed
om magnICON decons uc ed sys em.
ª2017 The Au ho s. Plan Bio echnology Jou nal published by Socie y o Expe imen al Biology and The Associa ion o Applied Biologis s and John Wiley & Sons L d., 16, 727–736
Plan -made ecombinan an i enom 731

se ine p o einases and P-I SVMP BaP1. La e , he abili y o PEO_1
o neu alize di e en B. aspe oxic and enzyma ic ac i i ies was
analysed. The PEO_1 plan i enom was able o neu alize enom
haemo hagic, PLA
2
, p o einase and le hal ac i i ies (Table 1).
Figu e 5C illus a es he neu aliza ion o haemo hagic ac i i y in
he mouse skin es . The plan i enom did no neu alize in i o
coagulan ac i i y on plasma a he maximum concen a ion
es ed (372 mg plan i enom/mg enom) (Table 1). Finally, we
es ed he capaci y o PEO_1 o neu alize le hali y induced by
B. aspe enom in mice, using 2 LD
50
s o enom as ‘challenge
dose’. PEO_1 p e en ed le hali y in all injec ed mice a a a io o
61.24 mg plan i enom/mg enom (suppo ing in o ma ion) wi h
an es ima ed median e ec i e dose (ED
50
) o 43 mg plan i enom/
mg enom (95% con idence limi s: 33–56 mg/mg) (Table 1). All
PEO_1 neu aliza ion ac i i ies we e assayed in pa allel wi h a
comme cial ho se-de i ed an i enom, and he co esponding
neu alizing ac i i ies a e also summa ized in Table 1. Ho se-
de i ed an i enom was e ec i e in he neu aliza ion o all
enzyma ic and oxic e ec s assayed.
Discussion
Global an i enom sho age has p omp ed se e al lines o esea ch
aimed a inding solu ions o he main p oduc ion bo lenecks.
The use o immuniza ion wi h DNA o wi h ecombinan -
exp essed epi opes o ele an oxins is an example o his,
aimed a ci cum en ing he sho age o an igens in species wi h
low enom yield, such as co al snakes (Ramos e al., 2016). In
compa ison, ully ecombinan s a egies can p o ide a mo e
sa is ac o y solu ion in he long e m, add essing no only he
sho age o an igens, bu also o he p oblems a ising om
cu en he apies such as ad e se eac ions and ep oducibili y
(Laus sen e al., 2016). In heo y, a ionally designed cock ail
o mula ions made o ecombinan mAbs could ou pe o m
animal plasma-de i ed an i enoms, as he composi ion o he
la e comp ises a la ge ac ion (≥80%) o immunologically
i ele an an i oxin an ibodies. In con as , by adjus ing he
an i enom composi ion o a minimal numbe o selec ed unc-
ional componen s, he sa e y p o ile can be imp o ed and
manu ac u ing cos s can be educed. Minimal ecombinan
an ibody cock ails comp ising only h ee mAbs an ibodies agains
he main Bo h ops oxins we e shown o neu alize he le hal
oxici y o he enom (F auches e al., 2013), indica ing ha
oligoclonal cock ails a e sui able an i enom o mula ions a leas
in some cases. No wi hs anding, a po en cock ail would p obably
equi e mo e complex combina ions, inco po a ing an ibodies
agains mino oxins, which could ac alone o syne gis ically wi h
o he oxins, o ensu e he co e age o in a- and in e speci ic
Figu e 5 Assessmen o plan i enom unc ionali y (A) Coomassie gel o an en iched oligoclonal plan i enom (PEO_1) p oduc ion and pu i ica ion s eps:
lane1, c ude apoplas ic luid; lane 2, lea c ude ex ac ; lane 3 con ol apoplas ic luid om unin ec ed lea ; lane 4 cla i ied apoplas ic luid; lane 5, p o ein A
low h ough; line 6, i s wash; lanes 7 and 8, elu ed plan i enom. (B) Compa ison o ELISA binding ac i i ies agains B. aspe enom o sequen ial
plan i enom en ichmen s eps. PPI, p e-immune plan i enom; PIM, immune plan i enom; PEP, en iched polyclonal plan i enom de i ed om second and
hi d phage display selec ion ounds. (C) Compa ison o binding ac i i ies be ween PEP and PEO_1. (D) An i enomic p o ile o PEO_1 plan i enom. Uppe
(a) and lowe (b) panels display, espec i ely, ch oma og aphic p o iles o whole Cos a Rican B. aspe (Paci ic popula ion) enom, and he enom ac ion
nonimmuno e ained in he immobilized PEO_1 a ini y column. (E) Neu aliza ion o B. aspe enom haemo hagic ac i i y in i o wi h PEO_1 plan i enom
p epa a ion (46.5 mg p o ein/mL). The igu e shows he abdominal su ace o mouse skin a e 2-h injec ion wi h a cons an dose o ull enom and
di e en amoun s o plan i enom. (a) haemo hagic spo appea ing in mice injec ed in ade mally wi h 15 lg (5 MHD) o enom; (b) inhibi ion o 5 MHD
haemo hagic spo wi h an an i enom dose o 250 lL PEO_1/mg enom; (c) 500 lL PEO_1/mg enom; (d) 750 lL PEO_1/mg enom; (e) nega i e con ol
(plan i enom dilu ed in PBS).
ª2017 The Au ho s. Plan Bio echnology Jou nal published by Socie y o Expe imen al Biology and The Associa ion o Applied Biologis s and John Wiley & Sons L d., 16, 727–736
Jose Manuel Jul e Pa e~
no e al.732
a ia ion. Complex cock ail composi ion imposes se ious con-
s ain s on cu en manu ac u ing sys ems. Indeed, whe eas he
o mula ion o imp o ed cock ails seems an accessible objec i e
wi h cu en an ibody selec ion echniques, he lack o sui able
p oduc ion sys ems o hese combina ions has p o ed a p ac ical
limi a ion, discou aging u he ad ances. The plu ibody s a egy
is in line wi h o he ecen s a egies based on mammalian cell
cul u es aimed o p oduce a o dable an ibody cock ails (Laus -
sen, 2016; Rasmussen e al., 2012), which can os e he
de elopmen o imp o ed ecombinan o mula ions. The employ
o plan i us-based exp ession sys ems o e s u he ad an ages
associa ed wi h he use o plan s as bio ac o ies. As an example,
he decons uc ed i us s a egy (Ma illonne e al., 2004, 2005)
was success ully used o p oduce he Z-Mapp an i-Ebola p o ec-
i e an ibody cock ail (Qiu e al., 2014) among o he examples.
Reco d yields up o 4.8 g/kg FW o an i-idio ype an ibodies ha e
been epo ed using magnICON (Bendandi e al., 2010). Wi h ou
cu en , nonop imized yields, a 1000 sqm g eenhouse acili y
g owing 15-kg biomass/m
2
/yea (Walwyn e al., 2015) could
p oduce up o 3000 1.0 g am doses pe yea . Fu he imp o e-
men s in yield and o mula ion could each 15 doses/m
2
/yea as a
easonable scena io.
The syn he ic di e si y o e ed by soma ic mosaicism can be
exploi ed o quickly ans e ‘in mass’ la ge immune mammalian
sub epe oi es, as exempli ied wi h he PIM o mula ions.
Al hough ai h ul ep oduc ion o he mammalian epe oi e
canno be gua an eed, we shown he e ha he an i enom-
binding p o iles o la ge plan -made epe oi es la gely esemble
ha o hei co esponding an ise a, and he same occu s du ing
he successi e in i o en ichmen s eps, indica ing ha any
possible exp ession bias does no a ec subs an ially he binding
p o ile o he inal p oduc . In he u u e, sampling o lymphoid
o gans and/o cell-so ing p ocedu es could be inco po a ed o
en ich he composi ion o polyclonal mix u es. Al e na i ely,
addi ional in i o selec ion s eps as hose shown he e can be
used o e ine composi ion, p oducing op imized oligoclonal
mix u es. In his line, he decision o conduc ou inal e icacy
es s wi h an oligoclonal o mula ion was aken wi h a double
objec i e: i s o maximize di e si y, as o some an igens la e
phage display ounds could end up selec ing s ong monoclonal
binde s only and second o acili a e he dissec ion o he
neu aliza ion esul s, as he indi idual cha ac e iza ion o he
an ibodies in he cock ail will acili a e imp o emen s in subse-
quen o mula ions ia inco po a ion o new (and dis inc )
indi idual clones.
The PEO_1 cock ail desc ibed he e showed a easonable
spec um o neu alizing ac i i ies wi h ela i ely li le e o in
an ibody selec ion; howe e , u he e o s will be equi ed o
each e icacy le els o cu en comme cial an i enoms. En en-
omings by Bo h ops aspe a e cha ac e ized by se e al pa holog-
ical and pa hophysiological al e a ions, among which al e a ions
in he coagula ion a e impo an . Thus, any an i enom o be
conside ed o use in en enomings by B. aspe should be able o
neu alize he coagulan e ec o he enom. This should p omp
u he e o s o inc ease he neu alizing capaci y o he
plan i enom and o gene a e neu aliza ion o he coagulan
ac i i y. Wi h consis ency and ep oducibili y ensu ed wi h he
plu ibody s a egy, he oppo uni ies o o mula ion
imp o emen s h ough he implemen a ion o successi e
design–build– es cycles a e mani old. The neu aliza ion o
coagulan ac i i y can be imp o ed wi h he in oduc ion o
new se ine p o ease neu alizing clones om imp o ed an ibody
selec ion p ocedu es, syn he ic epe oi es o al e na i e single-
cell selec ion me hods (Bee li and Rade , 2010). A s eamlined
o mula ion should also in ol e he elimina ion o clones wi h
o e lapping o non-neu alizing ac i i ies and, ul ima ely, he
inco po a ion o neu alizing ac i i ies agains o he snake en-
oms om he same egion. Ou esul s should be ollowed by a
mo e in dep h p eclinical assessmen o he plan i enom, using
a ious animal and in i o models. Once a plan i enom wi h he
capaci y o neu alize all oxic ac i i ies o he enom is
de eloped, he nex s ep would be o conside he implemen a-
ion o clinical ials and o s ablish clinical doses. The dose o
an i enom o be adminis e ed in clinical cases canno be di ec ly
ex apola ed om he p eclinical neu aliza ion es s. I has been
oughly es ima ed ha an adul B. aspe specimen may injec
50 mg o enom in a bi e, al hough his a ies om bi e o bi e
acco ding o many a iables (size o he snake, olume o enom
a ailable in he gland, s eng h o he bi e, e c.). Once an
an i enom has demons a ed e icacy in p eclinical es s, clinical
ials in ol e an ini ial dose- inding phase, in which he op imal
dose o an i enom is selec ed. This dose is hen used o mo e
ex ensi e clinical ials wi h a highe numbe o pa ien s.
The manipula ion o SE o manu ac u ing pu poses is pi o al in
he plu ibody app oach. SE ensu es he independence o each
mic o-p oduc ion line, esul ing in highly ep oducible inal p od-
uc s. The esul ing s uc u ed popula ion dis ibu ion simpli ies he
modelling o he p oduc ion p ocess, as he whole sys em can be
ega ded as he sum o housands o independen mic o-
p oduc ion lines (exp ession iles) wo king in pa allel. This wo k
also unde sco es he po en ial o plan syn he ic biology o o e he
wo ld new bio- ab ica ion solu ions. Plu ibody echnology combi-
nes gene ic elemen s om biological sou ces as dis an as camels,
humans and plan i uses o cap u e he immune epe oi e o
hype immunized camels and ecapi ula e his wi hin he plan
chassis, c ea ing plan i enoms able o neu alize a wide ange o
oxin ac i i ies ha p o ide p o ec ion agains le hal enom doses in
mice. Beyond an i enoms, we belie e ha he abili y o o mula e
plu ibodies 
a la ca e could p o ide a new boos o passi e
immuno he apy, acili a ing he o mula ion and manu ac u ing o
new, a o dable an i oxin and an imic obial p oduc s.
Table 1 Neu aliza ion o oxic ac i i ies o B. aspe enom by
plan i enom and equine-de i ed an i enom
Toxic and enzyma ic
ac i i ies o B. aspe
enom
ED
50
Plan i enom
(mg/mg enom)
ED
50
Equine-de i ed
(mg/mg enom)
Haemo hagic 22.2 0.65 6.9 0.01
Phospholipase A
2
224.1 13.2 37.4 0.4
P o eoly ic 120.5 4.2 43.0 2.0
Pe i oneal le hali y 43.2 (33.4–56.0) 3.1 (1.2–8.4)
Coagulan on
human plasma
≫372 11.3 0.13
Neu aliza ion o haemo hagic, phospholipase A
2
, p o einase and le hal
ac i i ies is exp essed as median e ec i e dose (ED
50
), whe eas neu aliza ion
o in i o coagulan ac i i y is exp essed as e ec i e dose (ED), in e ms o mg
plan i enom o an i enom/mg enom p o ein (see Ma e ials and me hods o
de ails). Resul s a e p esen ed as mean SD (n=3) o neu aliza ion o
haemo hagic, phospholipase A
2
, p o einase and coagulan e ec s. In he case
o neu aliza ion o le hal e ec , he 95% con idence limi s a e included in
pa en hesis.
ª2017 The Au ho s. Plan Bio echnology Jou nal published by Socie y o Expe imen al Biology and The Associa ion o Applied Biologis s and John Wiley & Sons L d., 16, 727–736
Plan -made ecombinan an i enom 733
Ma e ials and me hods
De ailed in o ma ion abou he expe imen al me hods can be
ound in SI. All p ocedu es in ol ing expe imen al mice mee he
equi emen s o he Guiding P inciples o Biomedical Resea ch
In ol ing Animals (CIOMS, 1985) and we e app o ed by he
Ins i u ional Commi ee o he Ca e and Use o Labo a o y
Animals o Uni e sidad de Cos a Rica (app o al numbe 82-08).
The camel immuniza ion p o ocol ollowed animal expe imen a-
ion guidelines published by he egional go e nmen o he
Cana y Islands (Spain) and was app o ed by he E hics Commi -
ee, Ve e ina y Medicine Se ice, Las Palmas de G an Cana ia
Uni e si y Founda ion (Re .: 009/2011)
Snake enoms and enom ac ions
Venoms we e ob ained om specimens o B. aspe ,C. scu ula us
scu ula us and C. simus a he Se pen a ium o he Ins i u o
Clodomi o Picado (Uni e si y o Cos a Rica, San Jos
e). Fo enom
ac iona ion, B. aspe enom p o eins we e sepa a ed by
e e se-phase HPLC; he esol ed ch oma og aphic peaks (48)
we e collec ed sepa a ely and he elu ed ac ions pooled in ou
g oups: g oup 1 was mainly composed o disin eg ins; g oup 2 o
phospholipases; g oup 3 mainly con ained se ine p o eases and
PLA2; and g oup 4 was en iched in me allop o einases.
Camel immuniza ion, o al RNA isola ion and
ampli ica ion o V
H
H sequences
Th ee camels (Camelus d omeda ius) we e immunized wi h a
cock ail con aining equal amoun o each o h ee snake enoms
(C. scu ula us,C. simus and B. aspe ). Pe iphe al blood lympho-
cy es (PBLs) ob ained om ‘p e-immune’ and ‘immune’ samples
we e used o isola e RNA and syn hesize cDNA o be used as
empla e o PCR ampli ica ion o V
H
H coding sequences (see
supplemen a y ma e ials and me hods and Table S2 o he lis o
p ime s used).
V
H
H Phage Display lib a y cons uc ion and selec ion
The ampli ied V
H
H sequences we e di ec ly cloned as PCR
p oduc s in o he pSwo d phagemid ec o and he liga ion
p oduc s ans o med in o E. coli. In a i s ound, enom binde s
we e selec ed using B. aspe ull enom as he an igen. Subse-
quen selec ion ounds we e ca ied ou sepa a ely agains each
one o he ou an igen g oups ob ained by enom ac iona ion.
Moni o ing o he lib a y en ichmen was ca ied ou by
polyclonal phage ELISA on B. aspe ull enom. Monoclonal
phage ELISA was ca ied ou in he same way om indi idual
clones o he hi d ound o selec ion; clones showing he highes
binding a ini y o each indi idual an igen we e selec ed o
o mula e an oligoclonal mix: wo clones om g oup 1; eigh
om g oup 2; six om g oup 3; and 20 om g oup 4 (SI).
V
H
H plan exp ession lib a ies
V
H
H sequences ampli ied om PBL cDNA we e di ec ly cloned as
PCR p oduc s in o pGV
H
H-His. Selec ed V
H
H phage display
lib a ies and indi idual clones we e cloned in pGV
H
H-IgG ec o .
pGV
H
H-His and pGV
H
H-IgG a e adap a ion o he magnICON
plan i al exp ession ec o pICH7410 (ICON Gene ics) buil on a
pDGB1 ec o backbone (Sa ion-Pe digones e al., 2011). Liga-
ion p oduc s we e ans o med in o E. coli cells and pla ed on
solid LB aga pla es. Fo lib a ies, bac e ia we e sc aped and
s o ed in 1 mL aliquo s as E. coli lib a y s ocks. One- en h o he
lib a y was used o plasmid DNA isola ion. Fo indi idual clones, a
single colony was used o inocula e a esh cul u e ha was
a e wa ds used o plasmid DNA isola ion, he emaining cul u e
being s o ed in 1 mL aliquo s as E. coli glyce ol s ocks. The
isola ed plasmid DNA was ans o med in o he Ag obac e ium
ume aciens GV3101 s ain. The ans o ma ion was sp ead on LB
pla es. Bac e ia we e sc aped and s o ed in 500 lL aliquo s as
lib a y s ocks o c ea e a mas e cell bank (MCB). MCB aliquo s
we e g own O/N and dis ibu ed in wo king cell banks (WCB)
used o ag oin il a ion.
Es ima ion o he polyclonal di e si y o he soma ic
mosaic
To de e mine a e age ile size in a ypical polyclonal expe imen , a
pGV
H
H-His an ibody lib a y was mixed wi h a pGGFP clone in
di e en a ios (1%, 5% and 10%) and co-in il a ed a di e en
OD600s (0.1, 0.03, 0.01 and 0.003) in N. ben hamiana lea es.
Images o each expe imen al poin aken a days 5 and 10 p.i
we e analysed wi h ImageJ so wa e (h p:// sb.in o.nih.go /ij/),
and he numbe o iles and a e age ile size we e calcula ed
using he ‘Analyse pa icle’ unc ion. An expe imen al su ace/
weigh con e sion a e o 35.2 cm
2
/g FW in N. ben hamiana
lea es was applied o calcula e he numbe o exp ession iles pe
biomass.
T ansien exp ession in Nico iana ben hamiana
Fo polyclonal and oligoclonal ag oin il a ions, an aliquo o he
WCB collec ion was used o inocula e an app op ia e olume o
LB supplemen ed wi h an ibio ics (1 : 250 subcul u e); cul u es
we e g own o 8 h a 28 °C. Cul u es we e collec ed by
cen i uga ion (10 min, 3000 g), e-suspended in ag oin il a ion
medium (10 mMMES pH 5.6, 10 mMMgCl
2
, 200 lMace osy-
ingone) o an op ical densi y a 600 nm o 0.05 and incuba ed
o 2 h a oom empe a u e on a ho izon al olling mixe . Be o e
in il a ion, he bac e ial suspensions ha bou ing V
H
H-6xHis o
V
H
H-Fc (30-p o ec o modules) we e mixed wi h equal olumes o
he pICH14011 (In eg ase) and pICH17388 (50-p o ec o module)
suspensions. Fo ag oin il a ion, 5- o 6-week-old N. ben hami-
ana plan s we e subme ged in he ag oin il a ion solu ion, a
acuum was applied o a p essu e o 0.9 ba (Vacuum
Degassing Chambe DP118, Applied Vacuum Enginee ing,
Tho nbu y, UK), held o 1 min and hen slowly eleased. Samples
o p o ein ex ac ion we e collec ed 7–10 days pos in il a ion.
Analysis o di e si y and ep oducibili y a DNA le el
Sequencing lib a ies we e p epa ed om V
H
H-6xHis immune and
p e-immune lib a y ag oin il a ed N. ben hamiana lea es (see
supplemen a y ma e ials and me hods and Table S2 o he lis o
p ime s used). Lib a ies we e sequenced on an Ion To en PGM a
Li e Sequencing (Valencia, Spain). Sequence quali y imming, de
no o assembly and pos e io analysis we e conduc ed using
cus om Py hon sc ip s and iAssemble so wa e (Zheng e al.,
2011). Unigenes composed o a leas wo membe s we e used o
lib a y compa ison by BLASTN using he Blas Use DB ool a h p://
nbc11.biologie.uni-kl.de/.
Compu e simula ion o Soma ic exp ession mosaics
Simula ion o exp ession mosaic o ma ion was pe o med wi h
Ne Logo 6.0 agen -based modelling so wa e (Wilensky, 1999),
wi h a sc ip designed o simula e cell- o-cell mo emen o
exclusion-enabled i al clones on wo-dimensional hexagonal g id
(Wilensky, 2007) (h p://pgb.ibmcp.csic.es/ne logo/ne logo.h ml
and SI).
ª2017 The Au ho s. Plan Bio echnology Jou nal published by Socie y o Expe imen al Biology and The Associa ion o Applied Biologis s and John Wiley & Sons L d., 16, 727–736
Jose Manuel Jul e Pa e~
no e al.734
An i enomics
A ini y ch oma og aphy-based an i enomic (Pla e al., 2012) was
employed o add ess he immunocap u ing abili y o PEO_1
owa ds B. aspe enom componen s. Columns we e p epa ed by
immobilizing plan i enom on cyanogen b omide-ac i a ed
Sepha ose 6 MB (SIGMA, S . Louis, USA), ollowing he manu-
ac u e ’s ins uc ions. The column was hen loaded wi h B. aspe
enom pooled om B. aspe specimens om he Paci ic e san
o Cos a Rica. Bound and non e ained ac ions we e analysed by
HPLC. Ch oma og aphic uns o whole B. aspe enom dissol ed
in PBS we e used as con ols.
Neu aliza ion o oxic ac i i ies
The abili y o plan i enom o neu alize p o einase, phospholipase
A2 (PLA2), hemo hagic, coagulan , and le hal ac i i ies was
de e mined as p e iously desc ibed (Bolanos, 1972; Gene e al.,
1989; Gu ie ez e al., 1985; Gu ie ez e al., 1986; Gu ie ez e
al., 2008; Theaks on and Reid, 1983; Wang e al., 2004).
Neu aliza ion was exp essed as median e ec i e dose (ED
50
) o
le hal, haemo hagic, p o einase and PLA
2
ac i i ies (plan i enom/
enom, o equine an i enom/ enom, a io in which he e ec
was inhibi ed by 50% (Segu a e al., 2010)), o as e ec i e dose
(ED) o coagulan ac i i y ( he plan i enom/ enom, o equine
an i enom/ enom, a io in which he clo ing ime was p olonged
h ee imes as compa ed o enom incuba ed wi h PBS ins ead o
an ibodies (Gene e al., 1989).
Acknowledgmen s
We hank B ian J. Robinson o his ca e ul e ision o he
manusc ip . The wo k ca ied ou a IBMCP-CSIC ecei ed
inancial suppo om MINECO and ERDF (p ojec g an s IPT-
2011-0720-010000, BIO2013-42193-R and BIO2016-78601-R).
Resea ch a IBV-CSIC was suppo ed by g an BFU2013-42833-P
om he Spanish Minis y o Economy and Compe i i eness,
Mad id, Spain. The au ho s decla e no con lic o in e es .
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